WO2006107708A1 - Treatment of disease caused by excess free hemoglobin - Google Patents

Abstract

The present invention relates to methods and compositions useful for treating diseases or conditions associated with excess free hemoglobin or heme in circulation. The methods and compositions may also be useful in treating diseases or conditions associated with a reduced level of nitric oxide in circulation.

Description

TREATMENT OF DISEASE CAUSED BY EXCESS FREE HEMOGLOBIN

RELATED APPLICATION

This application claims the benefit of and priority to U.S. Provisional Application No. 60/668,049, filed on April 4, 2005, the disclosure of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Several diseases result in the lysis of red blood cells resulting in the presence of high levels of free hemoglobin within the bloodstream. High levels of free hemoglobin overload the body's natural hemoglobin removal mechanisms, e.g., by haptoglobin binding of the free hemoglobin, and the excess free hemoglobin consumes nitric oxide resulting in a lack or reduced level of nitric oxide. Nitric oxide plays a major role in vascular homeostasis and has been shown to be a critical regulator of basal and stress-mediated smooth muscle relaxation and vasomotor tone, endothelial adhesion molecule expression, and platelet activation and aggregation. Additionally, hemoglobin itself, at least in part due to free heme, exerts direct cytotoxic, inflammatory, and pro-oxidant effects that adversely affect endothelial function. As a result of the lysis of red blood cells and concomitant excess of free hemoglobin, free heme levels increase and nitric oxide levels decrease and several disorders ensue.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides methods and compositions useful for treating the clinical sequelae resulting from an excess of free hemoglobin in the plasma or bloodstream of a mammal, especially a human.

In certain embodiments, the present invention provides methods and compositions that are useful in hastening, facilitating, or aiding in the removal of free hemoglobin or free heme in circulation (or in the bloodstream) and thereby treating diseases or conditions associated with such free hemoglobin or free heme in circulation. The methods and compositions include an agent, such as for example an antibody or antibody fragment, that binds hemoglobin or heme, which facilitates or assists in the removal of the hemoglobin or heme from circulation. The increased rate of removal of hemoglobin can be via, among other means, macrophages and monocytes via the reticuloendothelial system. For example, the binding of an antibody or fragment thereof, preferably an antibody or antibody fragment with an Fc region, to hemoglobin will aid in the removal of hemoglobin from the plasma thereby decreasing the amount of free hemoglobin in the plasma. In certain embodiments, the present invention also provides methods and compositions that are useful in increasing or maintaining nitric oxide levels in circulation and thereby treating diseases or conditions associated with a reduced level or depletion of nitric oxide, for example, caused by excess free hemoglobin or free heme in circulation. For example, methods and compositions are presented for removing (or hastening, facilitating, or aiding in the removal of) free hemoglobin present in the bloodstream, or inhibiting or blocking the ability of hemoglobin to oxidize nitric oxide, such that the hemoglobin's ability to act as a sump for nitric oxide by oxidizing nitric oxide to nitrate is reduced or eliminated. This will help maintain physiologically desirable levels of nitric oxide in the plasma in mammals, especially humans, which have excess free hemoglobin or heme in circulation, e.g., as a result of hemolysis.

Another aspect of the invention is to treat mammals, especially humans, with an agent that reduces or inhibits the ability of nitric oxide to react with hemoglobin. For example, the agent reduces or inhibits the ability of hemoglobin to convert or oxidize nitric oxide to nitrate. In certain embodiments, the agent is a protein or protein analog, e.g., an antibody or antibody fragment. In certain embodiments, the agent is a small molecule, a nucleic acid or nucleic acid analog, a macromolecule (e.g., lipids or fatty acids) that is not a protein or nucleic acid, or a peptidomimetic. These agents include, but are not limited to, small organic molecules, RNA aptamers, L-RNA ap tamers, speigelmers, antisense compounds, double stranded RNA, small interfering RNA, locked nucleic acid inhibitors, and peptide nucleic acid inhibitors. Another aspect of the invention is to treat mammals, especially humans, who have excess free heme in their plasma or bloodstream, with an agent that binds free heme, preferably wherein the agent is a protein and more preferably wherein the protein is an antibody or antibody fragment. Treatment with such an agent will inhibit or prevent the results which can occur due to the presence of excess free heme in the plasma, such results including, but not being limited to, inflammation, oxidative stress, and toxic effects on endothelial cells.

An antibody or antibody fragment of the invention may be a chimerized or chimeric, humanized, deimmunized or fully human antibody or antibody fragment, a single chain antibody, a single-chain peptide such as for example a small modular immunopharmaceutical (or SMIP™), or a Fab or F(ab')₂.

The methods and compositions of the present inventions are useful for treating mammals, especially humans, who are subject to hemolysis of red blood cells. Those who may benefit include, but are not limited to, mammals who suffer from paroxysmal nocturnal hemoglobinuria, sickle cell disease, thalassemias, hereditary spherocytosis and stomatocytosis, microangiopathic hemolytic anemias, pyruvate kinase deficiency, ABO mismatch transfusion reaction, paroxysmal cold hemoglobinuria, severe idiopathic autoimmune hemolytic anemia, infection-induced anemia, malaria, dialysis, cardiopulmonary bypass, and mechanical heart valve- induced anemias. The mammals suitable for and subject to the treatment methods of the invention may suffer from any combination of two or more of those diseases or conditions.

The methods and compositions of the present inventions are useful for treating mammals, especially humans, who are subject to the clinical sequelae resulting from excess red blood lysis in a mammal. These sequelae result from the lysis releasing excess free hemoglobin into the plasma and nitric oxide levels decreasing as a result of nitric oxide reacting with hemoglobin and being converted to nitrate. These sequelae include dystonias (for example but not limited to, dystonias involving the gastrointestinal, cardiovascular, pulmonary and/or urogenital systems), clotting disorders, pulmonary and systemic hypertension, gastrointestinal contractions, abdominal pain, sternal pain, erectile dysfunction, inflammation, esophageal spasm and dysphagia, thrombosis, decreased organ perfusion, platelet activation, death, or any combination of two or more of these diseases or conditions. Other sequelae concern the quality of life, including but not limited to global health status, physical functioning, emotional functioning, cognitive functioning, role functioning, social functioning, fatigue, pain, dyspnea, appetite loss and insomnia.

The treatments contemplated herein include but are not limited to chronic treatments for recurring or chronic conditions, use on an as needed basis for recurring or chronic conditions, short term treatment for limited time illness, and one time use such as for patients undergoing a surgical procedure. The treatments contemplated herein can be conducted through various routes of administration. For example, a therapeutically effective amount of an agent, e.g., an antibody or antibody fragment, of the invention may be administered systemically by injection, e.g., intravenously. Therapeutic effectiveness of an agent of the invention can be determined by various conventional procedures. A further aspect of the invention contemplates the uses of an agent herein in the manufacture of medicaments for treating a disease or condition associated with hemolysis, excess free hemoglobin or heme in circulation, and/or a reduced level of or depletion of nitric oxide in the bloodstream in a mammal.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to methods of treating or inhibiting the sequelae of excess amounts of free hemoglobin or free heme in the plasma of mammals, especially humans. These sequelae can be inhibited or prevented by administration of an agent which facilitates or assists in the removal of free hemoglobin from the plasma and/or inhibits or blocks the free hemoglobin from reacting with nitric oxide, thereby maintaining a normal or more close to normal level of nitric oxide than would occur in the absence of the agent. One agent capable of hastening or aiding in the removal of free hemoglobin and/or preventing the reaction between hemoglobin and nitric oxide is an antibody or antibody fragment that binds hemoglobin. Preferably, antibodies or fragments thereof will react with free hemoglobin in the bloodstream but will not appreciably bind to hemoglobin still sequestered in the red blood cells, since the antibodies or fragments thereof will have limited access to the interior of red blood cells.

Administration of an agent to facilitate or aid in removing free hemoglobin from the plasma and/or to inhibit or block the interaction between hemoglobin and nitric oxide should prevent or inhibit the sequelae associated with or caused by an excess of free hemoglobin and decreased level of nitric oxide in circulation. Additionally, administration of an agent to aid in removing free heme from the plasma should prevent or inhibit the undesired effects caused by free heme, hi certain embodiments, administration of such an agent or agents is performed on patients, e.g., human patients, prior to their undergoing a procedure which may result in increased levels of free hemoglobin (and therefore increased levels of free heme and decreased levels of nitric oxide) in the plasma. Additionally, administration of such an agent or agents will be useful in patients, e.g., human patients, who are subject to having periods of excess free hemoglobin in their plasma, for example but not limited to, patients suffering from paroxysmal nocturnal hemoglobinuria or sickle cell disease. Such persons may be administered an agent at intervals which will maintain an adequate level of the inhibitor to block or inhibit the effects which would otherwise occur as a result of the excess free hemoglobin reacting with nitric oxide thereby decreasing the nitric oxide levels in the plasma below a physiologically desirable level and/or the effects due to excess free heme, e.g., as would occur during a paroxysm in which numerous blood cells lyse. Administration of such an agent or agents will additionally be useful in mammals in which excess free hemoglobin is already present in the plasma, regardless of the cause. Thus, administration of such an agent may be either prophylactic to prevent sequelae (e.g., dystonia, hypertension, etc.) or can be used once the sequelae have already occurred, such use being to limit the extent of the symptoms and to resolve the symptoms more quickly.

The term "treating" as used herein includes prophylactic and/or therapeutic treatments. The term "prophylactic or therapeutic" treatment is art-recognized and includes administration to the patient, e.g., a human patient, of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted condition of the patient) then the treatment is prophylactic (i.e., it protects the patient against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof). Interaction between Hemoglobin and Nitric Oxide, and Clinical Implications

Hemoglobin is a highly conserved molecule found in species ranging from single-cell organisms to mammals, but the role of hemoglobin in different organisms varies, hi mammals, hemoglobin primarily serves a respiratory function in the delivery of oxygen and removal of carbon dioxide. Based on the recent discovery that nitric oxide is a critical regulator of vasodilation and vascular homeostasis, the interactions of nitric oxide with hemoglobin in mammals has drawn increasing interest. Because the reaction of nitric oxide with the vast amounts of intravascular oxyhemoglobin (16 g/dL) is fast (10⁷ M^-1S^""1) and irreversible, nitric oxide produced by endothelium would be immediately scavenged by hemoglobin and would therefore be incapable of paracrine diffusion from endothelium to vascular smooth muscle. See, e.g., Rother et al., JAMA 2005 Apr 6; 293(13):1653-62; Patel and Gladwin, Free Radic Biol Med. 2004 Feb 15 ; 36(4) :399-401 ; Schechter and Gladwin, NEngl JMed. 2003 Apr 10; 348(15):1483-5. However, the ability of hemoglobin to react with nitric oxide produced by endothelium is limited by compartmentalization of hemoglobin inside the erythrocyte. Thus, the evolution of the erythrocyte may be considered as a mechanism of reducing toxicity while ensuring separation of the critical respiratory transport machinery needed for efficient oxygen delivery from the endothelium. Moreover, multiple systems have evolved to control the level of cell-free hemoglobin in the plasma during normal physiological hemolysis, presumably to curtail the deleterious effects of plasma hemoglobin on nitric oxide bioavailability and endothelial function.

During intravascular hemolysis, cell-free plasma hemoglobin may overwhelm homeostatic systems in place to remove it. Tabbara IA, Med Clin North Am. 1992; 76:649-668. Hemolytic conditions with substantial intravascular hemolysis include paroxysmal nocturnal hemoglobinuria (PNH), sickle-cell disease (SCD), thalassemias, hereditary spherocytosis and stomatocytosis, microangiopathic hemolytic anemias, pyruvate kinase deficiency, ABO mismatch transfusion reaction, paroxysmal cold hemoglobinuria, severe idiopathic autoimmune hemolytic anemia, infection-induced anemia, malaria, cardiopulmonary bypass, mechanical heart valve-induced anemia, and chemical-induced anemias. Although the various hemolytic diseases each have unique symptoms, they often share hemoglobinemia- related sequelae, hi addition, observations from the clinical administration of artificial, purified, and recombinant hemoglobin solutions have provided further support for the causal relationship between excess cell-free hemoglobin in the bloodstream, symptoms, and cardiovascular events. E.g., Savitsky JP et al., Clin Pharmacol Titer. 1978; 23:73-80; Carmichael FJ et al., Crit Care Med. 2000;

28:2283-2292; Lamy ML et al., Anesthesiology 2000; 92:646-656; Viele MK et al., Anesthesiology 1997; 86:848-858; Saxena R et al., Stroke 1999;j30:993-996; Murray JA et al., Gastroenterology 1995; 109:1241-1248; Przybelski RJ et al., Crit Care Med. 1996; 24:1993-2000; Lamuraglia GM, et al., J Vase Surg. 2000; 31:299-308. Nitric oxide scavenging by excess plasma hemoglobin has been implicated in various clinical manifestations of intravascular hemolysis. Nitric oxide is a regulator of smooth muscle tone and platelet activation, and reductions in nitric oxide plasma levels lead to smooth muscle dystonias, including hypertension, gastrointestinal contractions, and erectile dysfunction, as well as clot formation. Murray JA et al., Gastroenterology 1995; 109: 1241-1248; Przybelski RJ et al., Crit Care Med. 1996; 24: 1993-2000; Erhart SM et al., Artif Cells Blood Substit Immobil Biotechnol. 2000; 28:385-396; Deem S et al., Circ Res. 2002; 91:626-632; Cannon RO III et al., J Clin Invest. 2001 ; 108:279-287; Gladwin MT et al., N Engl J Med. 2004; 350:886-895; Moyo VM et al., Br J Haematol. 2004; 126:133-138; Radomski MW et al., Lancet 1987; 2:1057-1058; Olsen SB et al., Circulation 1996; 93:327- 332; Schafer A et al., Circulation 2004; 109:1819-1822. Hemoglobin also exerts direct cytotoxic, inflammatory, and pro-oxidant effects that adversely affect endothelial function. Wagener et al., Blood 2001; 98:1802-1811.

Hence, the present invention relates, in part, to the mechanism that the release of hemoglobin during intravascular hemolysis results in excessive consumption of nitric oxide, subsequent reduction in guanylate cyclase activity, smooth muscle contraction, vasoconstriction, and platelet activation/aggregation. To illustrate, during intravascular hemolysis, hemoglobin is released into the plasma where it is normally cleared by the hemoglobin scavengers haptoglobin, CD 163, and hemopexin. Haptoglobin-hemoglobin complexes bind to CD 163 on the surface of macrophages/monocytes initiating endocytosis and degradation of the complex. Hemoglobin also releases ferric heme on oxidation, which is bound by hemopexin and degraded by hepatocytes in the liver. Excessive hemolysis saturates and depletes these hemoglobin removal systems and leads to a buildup of hemoglobin and heme in the plasma. Plasma hemoglobin and heme mediate direct proinflammatory, proliferative, and pro-oxidant effects on vessel endothelial cells. NO is normally generated from L-arginine in vessel endothelial cells by the enzyme nitric oxide synthase (NOS). NO maintains smooth muscle relaxation and inhibits platelet activation and aggregation, thereby regulating vessel tone and promoting organ system homeostasis. During intravascular hemolysis, NO availability can be severely limited by its reaction with oxyhemoglobin (NO scavenging) and by the breakdown of the substrate for NO synthesis, L-arginine, by the red cell enzyme arginase, despite elevated levels of NOS (decreased NO synthesis). NO depletion results in decreased activation of guanylate cyclase, an enzyme required for the generation of cyclic guanine monophosphate (cGMP). Decreased cGMP levels disrupt regulation of smooth muscle tone resulting in dystonias, including systemic and pulmonary hypertension, erectile dysfunction, dysphagia, and abdominal pain. Decreased cGMP levels through the depletion of NO can also lead to platelet activation and aggregation, promoting clot formation. Some other clinical sequelae of excess hemoglobin or heme in the bloodstream and consequent decrease in NO include, but are not limited to, global health status, physical functioning, emotional functioning, cognitive functioning, role functioning, social functioning, fatigue, pain, dyspnea, appetite loss and insomnia. Quality of life can be measured by the EORTC QLQ-C30 instrument (Aaronson et al, J Natl Cancer Inst. 1993; 85:365- 76) or by the FACIT-Fatigue instrument (Yellen et al., J Pain Symptom Manage. 1997; 13:63-74). Stroma-free hemoglobin, cross-linked human or bovine hemoglobin, purified human hemoglobin, or recombinant human hemoglobin preparations have been administered to patients, human volunteers, and animals. As summarized in the Table entitled "Clinical Signs and Symptoms Associated With the Administration of Hemoglobin Solutions" in Rother et al., supra, intravascular plasma hemoglobin is associated with a dose-dependent increase in adverse clinical signs and symptoms, including hemoglobinuria, abdominal pain, sternal pain, esophageal spasm, and dysphagia, as well as increases in blood pressure, platelet activation, creatine kinase level, and mortality.

In several disease states or as a result of accidents or medical intervention, excess lysis of red blood cells occurs releasing large amounts of hemoglobin. The excess hemoglobin results in a decreased level of nitric oxide in the plasma which in turn results in a variety of physiological disorders, including but not limited to, dystonias (for example but not limited to, dystonias involving the gastrointestinal, cardiovascular, pulmonary and urogenital systems), clotting disorders, pulmonary and systemic hypertension, gastrointestinal contractions, abdominal pain, sternal pain, erectile dysfunction, inflammation, esophageal spasm and dysphagia, thrombosis, decreased organ perfusion, platelet activation and death. Additionally, at least in part due to free heme, hemoglobin exerts direct cytotoxic, inflammatory, and pro-oxidant effects that adversely affect endothelial function.

Hemoglobinuria is one of the most prominent clinical signs of excessive intravascular hemolysis and is commonly associated with the administration of hemoglobin solutions. Plasma hemoglobin is normally filtered through the glomerulus and actively reabsorbed in proximal tubule cells where it is catabolized with release of iron in the form of hemosiderin. When the kidney's reabsorption capacity is exceeded, clinically significant hemoglobinuria occurs. Acute renal failure may occur during severe episodes of hemoglobinuria. Persistent severe hemoglobinuria is also associated with substantial proximal tubule hemosiderin deposition, Fanconi syndrome (defective renal reabsorption of small molecules leading to hyperaminoaciduria, glycosuria, hyperphosphaturia, and bicarbonate and water loss), and chronic renal failure.

Administration of hemoglobin preparations to healthy human volunteers is associated with dose-dependent gastrointestinal symptoms, including abdominal pain, esophageal spasms, and dysphagia. Increasing doses of recombinant hemoglobin result in increases in the duration of esophageal contractions. Further, acute episodes of intravascular hemolysis in patients undergoing long-term dialysis with plasma hemoglobin levels ranging from approximately 0.3 to 2.1 g/dL have also been associated with abdominal pain. Hemoglobin-induced esophageal spasms are most likely attributable to nitric oxide consumption, as inhibition of this molecule in healthy human volunteers results in an increase in esophageal peristaltic amplitude and velocity (spasms) and a decrease in gastric distention-triggered transient lower esophageal sphincter relaxation. Indeed, it has been shown that augmentation of the downstream effect of nitric oxide via inhibition of phosphodiesterase type 5 (PDE5) with sildenafil relieves spasms in patients with esophageal motor disorders.

The administration of cell-free hemoglobin solutions to healthy volunteers and patients is commonly associated with a dose-dependent increase in systolic and diastolic blood pressure, which is reversed by the administration of the nitric oxide donor, sodium nitroprusside, confirming the importance of nitric oxide scavenging in vasoregulation. Plasma hemoglobin and erythrocytes also augment hypoxic pulmonary vasoconstriction by scavenging nitric oxide, with plasma hemoglobin demonstrating an approximately 1000-fold greater nitric oxide scavenging potency in these models. Pulmonary arterial hypertension is an increasingly recognized complication of chronic hereditary and acquired hemolytic anemias, including SCD, thalassemia intermedia and major, PNH, hereditary spherocytosis and stomatocytosis, microangiopathic hemolytic anemias, and pyruvate kinase deficiency. There are a number of pathophysiological features shared by these disparate disorders, including intravascular hemolysis, iron overload, a propensity toward thrombosis, and surgical or autosplenectomy. Markers of hemolysis, including anemia, bilirubin, lactate dehydrogenase (LDH), and aspartate aminotransferase (but not liver-specific alanine aminotransferase), were associated with pulmonary hypertension. In addition to limiting nitric oxide bioavailability via hemoglobin-based nitric oxide scavenging and dysregulated arginine metabolism, hemolysis is associated with activation of downstream adhesion, prothrombotic, and pro-oxidant pathways that may further contribute to endothelial dysfunction and vasculopathy. Other mechanisms may also contribute to the development of pulmonary hypertension, including chronic thromboembolism and in situ thrombosis, asplenia, pulmonary fibrosis, liver cirrhosis secondary to iron overload and hepatitis C, and induction of hypoxia- inducible factor 1 -dependent factors such as vascular endothelial growth factor, endothelin 1, and erythropoietin.

Excessive plasma hemoglobin may contribute to platelet activation and thrombosis. The infusion of cross-linked hemoglobin increases platelet aggregation and adhesion in vivo on prothrombotic surfaces such as an injured vessel wall. Additionally, administration of heme in healthy volunteers is associated with thrombophlebitis, demonstrating that heme can cause vascular inflammation followed by vascular obstruction in vivo. The addition of cell-free hemoglobin to human serum at concentrations of 0.2 to 2.0 g/dL causes a dose-dependent inhibition of the metalloprotease ADAMTS 13, an enzyme critical in limiting platelet thrombus formation. The major untoward effects of plasma hemoglobin on platelet function are most likely mediated by the scavenging of nitric oxide. Nitric oxide has been shown to inhibit platelet aggregation, induce disaggregation of aggregated platelets, and inhibit platelet adhesion through increasing cyclic guanine monophosphate (cGMP) levels. In fact, nitric oxide donor drugs (S-nitrothiols) that increase systemic levels of nitric oxide have been shown to inhibit platelet aggregation. Conversely, nitric oxide scavenging by hemoglobin or the reduction of nitric oxide generation by the inhibition of arginine metabolism results in an increase in platelet aggregation.

Nitric oxide interacts with components of the coagulation cascade to downregulate clot formation. For example, nitric oxide has been shown to chemically modify and inhibit factor XIII, which suggests that nitric oxide deficiency would enhance clot stability and reduce clot dissolution, hi animal models, reduction of nitric oxide causes increases in fibrin split products and thrόmbin-antithrombin complexes leading to significant fibrin deposition and thrombus formation. Moreover, in a patient with L-arginine deficiency, reduced nitric oxide production is associated with increased thrombin-antithrombin complexes and fibrin split products, while reversal of nitric oxide deficiency with L- arginine causes a reduction in intravascular coagulopathy.

Hemoglobin release during intravascular hemolysis has been implicated in the pathogenesis of erectile dysfunction in patients with PNH, presumably through the scavenging of nitric oxide. It has been well established that local nitric oxide deficiency due to decreased synthesis, impaired release, or premature destruction is one of the factors responsible for erectile dysfunction. The capacity of PDE5 inhibitors such as sildenafil to improve erectile dysfunction via the accumulation of cGMP is dependent on the availability of nitric oxide. Plasma hemoglobin and heme may possess inflammatory properties. The presence of large amounts of vascular heme results in inflammatory infiltrates in various organs in mice and induction of neutrophil activation and migration in vitro. Heme stimulates the expression of the adhesion molecules ICAM-I (intracellular adhesion molecule 1), VCAM-I (vascular cell adhesion molecule 1), and E-selectin on endothelial cells in vitro. Heme and hemoglobin blood substitutes are associated with significant increases in vascular permeability. Plasma hemoglobin promotes formation of the biologically hazardous hydroxyl-radical, a process that may be regulated by the hemoglobin scavenger haptoglobin.

Many of the proinflammatory effects of plasma hemoglobin and heme may involve consumption of nitric oxide. Studies have shown that nitric oxide inhibits cytokine-induced induction of VCAM-I, ELAM-I (endothelial leukocyte adhesion molecule 1), and ICAM-I resulting in an anti-inflammatory effect. The consumption of nitric oxide by hemoglobin circumvents the anti-inflammatory properties of nitric oxide. Agents and Assays

The invention provides various therapeutic agents. One agent is an antibody or antibody fragment to hemoglobin. Antibodies can be screened to obtain one or more antibodies which bind to hemoglobin and result in a more rapid clearance of free hemoglobin from the plasma than will occur in the absence of the antibody. Antibodies may be made by any conventional method, e.g., using hemoglobin or a fragment thereof as an antigen to immunize a host animal. Alternatively, antibodies and/or fragments thereof that bind hemoglobin or its fragment may be selected from a library of antibodies or antibody fragments. The anti-hemoglobin antibodies and/or fragments thereof can then be screened for their ability to facilitate or aid in the removal of free hemoglobin from the plasma. Alternatively, another agent can inhibit or prevent free hemoglobin from reacting with nitric oxide to produce nitrate. Such an agent may be an antibody or antibody fragment that reacts with or binds to a hemoglobin or an interface between hemoglobin and nitric oxide, thereby inhibiting or preventing free hemoglobin from reacting with nitric oxide to produce nitrate. Alternatively, the agent that can inhibit or prevent free hemoglobin from reacting with nitric oxide to produce nitrate is a small molecule (up to 6,000 Da in molecular weight), a nucleic acid or nucleic acid analog, a peptidomimetic, or a macromolecule that is not a nucleic acid or a protein. These agents include, but are not limited to, small organic molecules, RNA aptamers, L-RNA aptamers, speigelmers, antisense compounds, double stranded RNA, small interfering RNA, locked nucleic acid inhibitors, and peptide nucleic acid inhibitors.

Yet another useful agent is an agent, preferably, an antibody or antibody fragment, that binds free heme. The binding of such an agent to heme inhibits or blocks the undesired effects of free heme in circulation, including, but not being limited to, inflammation, oxidative stress, and toxic effects on endothelial cells.

Antibodies are found in multiple forms, e.g., IgA, IgG, IgM, etc. Additionally, antibodies can be engineered in numerous ways. They can be made as single-chain antibodies (including small modular immunopharmaceuticals or SMIPs™), Fab and F(ab')₂ fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications set forth the many types of antibodies and the methods of engineering such antibodies. For example, see U.S. Patent Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and 5,260,203.

This invention provides fragments of hemoglobin or heme antibodies, which may comprise a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')₂, and Fv fragments; diabodies; linear antibodies (Zapata et al, Protein Eng.1995; 8(10): 1057-1062); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

"Fv" usually refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V_H-V_L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although likely at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')₂ antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

This disclosure also provides monoclonal hemoglobin or heme antibodies. A monoclonal antibody can be obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are often synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. Monoclonal antibodies may also be produced in transfected cells, such as CHO cells and NSO cells. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and does not require production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al., Nature 1975; 256:495, or may be made by recombinant DNA methods {see, e.g., U.S. Patent Nos. 4,816,567 and 6,331,415). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 1991; 352:624- 628 and Marks et al., J. MoI. Biol.1991; 222:581-597, for example.

Other antibodies specifically contemplated are "oligoclonal" antibodies. As used herein, the term "oligoclonal" antibodies" refers to a predetermined mixture of distinct monoclonal antibodies. See, e.g., PCT publication WO 95/20401; U.S. Patent Nos. 5,789,208 and 6,335,163. hi one embodiment, oligoclonal antibodies consisting of a predetermined mixture of antibodies against one or more epitopes are generated in a single cell. In other embodiments, oligoclonal antibodies comprise a plurality of heavy chains capable of pairing with a common light chain to generate antibodies with multiple specificities (e.g., PCT publication WO 04/009618). Oligoclonal antibodies are particularly useful when it is desired to target multiple epitopes on a single target molecule (e.g., hemoglobin or heme). In view of the assays and epitopes disclosed herein, those skilled in the art can generate or select antibodies or mixture of antibodies that are applicable for an intended purpose and desired need. hi certain embodiments that include a humanized and/or chimeric antibody, one or more of the CDRs are derived from an anti-human hemoglobin or heme antibody. In a specific embodiment, all of the CDRs are derived from an anti- human hemoglobin or heme antibody. In another specific embodiment, the CDRs from more than one anti-human hemoglobin or heme antibodies are mixed and matched in a chimeric antibody. For instance, a chimeric antibody may comprise a CDRl from the light chain of a first anti-human hemoglobin or heme antibody combined with CDR2 and CDR3 from the light chain of a second anti-human hemoglobin or heme antibody, and the CDRs from the heavy chain may be derived from a third anti-human hemoglobin or heme antibody. Further, the framework regions may be derived from one of the same anti-human hemoglobin or heme antibodies, from one or more different antibodies, such as a human antibody, or from a humanized antibody. "Single-chain Fv" or "scFv" antibody fragments comprise the V_H and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and V_L domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds. (Springer-Verlag: New York, 1994), pp. 269-315.

SMlPs are a class of single-chain peptide engineered to include a target binding region, effector domain (CH2 and CH3 domains). See, e.g., U.S. Patent Application Publication No. 20050238646. The target binding region may be derived from the variable region or CDRs of an antibody, e.g., an anti-hemoglobin or anti-heme antibody of the invention. Alternatively, the target binding region is derived from a protein that binds hemoglobin or heme, such as for example haptoglobin.

The term "diabodies" refers to small antibody fragments with two antigen- binding sites, which fragments comprise a heavy-chain variable domain (V_H) connected to a light-chain variable domain (V_L) in the same polypeptide chain (VH- V_L). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993). An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In specific embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, or greater than 99% by weight, (2) to a degree that complies with applicable regulatory requirements for administration to human patients (e.g., substantially pyrogen-free), (3) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (4) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step, for example, an affinity chromatography step, an ion (anion or cation) exchange chromatography step, or a hydrophobic interaction chromatography step.

It is well known that the binding to a molecule (or a pathogen) of antibodies with an Fc region assists in the processing and clearance of the molecule (or pathogen). The Fc portions of antibodies are recognized by specialized receptors expressed by immune effector cells. The Fc portions of IgGl and IgG3 antibodies are recognized by Fc receptors present on the surface of phagocytic cells such as macrophages and neutrophils, which can thereby bind and engulf the molecules or pathogens coated with antibodies of these isotypes (C. A. Janeway et al., Immunobiology 5th edition, page 147, Garland Publishing (New York, 2001)).

The present invention provides agents, particularly antibodies or fragments thereof, that can bind hemoglobin and facilitate or aid in the removal of free hemoglobin from the bloodstream. The present invention also provides various assays that are useful for determining such activity of the agents. For example, a hemoglobin test system available from HemoCue® can be used to determine the level of hemoglobin in the plasma, serum, or other aqueous solution, and White-light spectroscopy has also been used to measure the level of hemoglobin. Conventional plasma hemoglobin assays can be classified as either (1) direct optical techniques (e.g., quantification based on oxyhemoglobin's absorbance characteristics at 415 nm or 575-578 nm; differential optical analysis as described in U.S. Patent No. 5,277,181), or (2) added chemical techniques (e.g. cyanmethemoglobin, tetramethylbenzidine). These conventional methods can be employed to determine the level of hemoglobin in the plasma in the presence or absence of a candidate agent of the invention. NO consumption assays (e.g., Reiter et al., Nat. Med. 2002; 8:1383-1389) are also contemplated to determine the activity of candidate agents that are evaluated based on their ability to inhibit or block the interaction between free hemoglobin and nitric oxide, and/or to inhibit or block hemoglobin's ability to convert nitric oxide into nitrate.

Pharmaceutical Formulations and Uses

Methods of administration of therapeutic agents, particularly antibody therapeutics, are well-known to those of skill in the art. To achieve the desired inhibition, the anti-hemoglobin or anti-heme antibodies (or fragments thereof) can be administered in a variety of unit dosage forms. The dose will vary according to the particular antibody. For example, different antibodies may have different masses and/or affinities, and thus require different dosage levels. Antibodies prepared as Fab fragments will also require differing dosages than the equivalent intact immunoglobulins, as they are of considerably smaller mass than intact immunoglobulins, and thus require lower dosages to reach the same molar levels in the patient's blood. The dose will also vary depending on the manner of administration, the particular symptoms of the patient being treated, the overall health, condition, size, and age of the patient, and the judgment of the prescribing physician. Dosage levels of the antibodies for human subjects are generally between about 1 mg per kg and about 100 mg per kg per patient per treatment, and preferably between about 5 mg per kg and about 50 mg per kg per patient per treatment, hi terms of plasma concentrations, the antibody concentrations are preferably in the range from about 25 μg/mL to about 500 μg/mL. However, greater amounts may be required for extreme cases and smaller amounts may be sufficient for milder cases. For example, plasma hemoglobin levels in patients with paroxysmal nocturnal hemoglobinuria (PNH) are commonly in the range of 0.05 to 0.2 g/dL and can exceed 1.0 g/dL during severe hemolytic episodes. Hemoglobin is a tetramer of two α and two β chains with a total molecular weight of approximately 64 kDa. Following release from the red blood cell hemoglobin exists in an equilibrium between a tetramer and dimer. Each hemoglobin tetramer has four active sites for reacting with nitric oxide while hemoglobin dimers have two active sites. A typical whole antibody has a molecular weight of approximately 150 IcDa with two active binding sites. Therefore, one antibody molecule could theoretically remove two hemoglobin tetramers or two hemoglobin dimers. Since the antibody is roughly twice the molecular weight of a hemoglobin tetramer, but contains two binding sites for the molecule, it would require approximately 0.1 g/dL of whole antibody to remove 0.1 g/dL of free hemoglobin tetramer. The amount of antibody administered will depend upon the severity of the condition as well as the binding affinity of the antibody for hemoglobin. Clearance of free plasma hemoglobin can be easily monitored by conventional clinical assays. Administration of the anti-hemoglobin or anti-heme antibodies will generally be performed by an intravascular route, e.g., via intravenous infusion by injection. Other routes of administration may be used if desired but an intravenous route will be the most preferable. Formulations suitable for injection are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). Such formulations must be sterile and non-pyrogenic, and generally will include a pharmaceutically effective carrier, such as saline, buffered (e.g., phosphate buffered) saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions, and the like. The formulations may contain pharmaceutically acceptable auxiliary substances as required, such as, tonicity adjusting agents, wetting agents, bactericidal agents, preservatives, stabilizers, and the like.

Formulations particularly useful for antibody-based therapeutic agents are also described in U.S. Patent App. Publication Nos. 20030202972, 20040091490 and 20050158316. hi certain embodiments, the liquid formulations of the invention are substantially free of surfactant and/or inorganic salts, hi another specific embodiment, the liquid formulations have a pH ranging from about 5.0 to about 7.0. In yet another specific embodiment, the liquid formulations comprise histidine at a concentration ranging from about 1 niM to about 100 mM. hi still another specific embodiment, the liquid formulations comprise histidine at a concentration ranging from 1 mM to 100 mM. It is also contemplated that the liquid formulations may further comprise one or more excipients such as a saccharide, an amino acid (e.g., arginine, lysine, and methionine) and a polyol. Additional descriptions and methods of preparing and analyzing liquid formulations can be found, for example, in PCT publications WO 03/106644, WO 04/066957, and WO 04/091658.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical compositions of the invention.

In certain embodiments, formulations of the subject antibodies are pyro gen- free formulations which are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are confined inside microorganisms and are released when the microorganisms are broken down or die. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, it is advantageous to remove even low amounts of endotoxins from intravenously administered pharmaceutical drag solutions. The Food & Drag Administration ("FDA") has set an upper limit of 5 endotoxin units (EU) per dose per kilogram body weight in a single one hour period for intravenous drag applications (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic proteins are administered in amounts of several hundred or thousand milligrams per kilogram body weight, as can be the case with monoclonal antibodies, it is advantageous to remove even trace amounts of endotoxin.

Formulations of the subject antibodies include those suitable for oral, dietary, topical, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), ophthalmologic (e.g., topical or intraocular), inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops), rectal, and/or intravaginal administration. Other suitable methods of administration can also include rechargeable or biodegradable devices and controlled release polymeric devices. Stents, in particular, may be coated with a controlled release polymer mixed with an agent of the invention. The pharmaceutical compositions of this disclosure can also be administered as part of a combinatorial therapy with other agents (either in the same formulation or in a separate formulation).

The amount of the formulation which will be therapeutically effective can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The dosage of the compositions to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making those determinations include the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. For example, the actual patient body weight may be used to calculate the dose of the formulations in milliliters (mL) to be administered. There may be no downward adjustment to "ideal" weight. In such a situation, an appropriate dose may be calculated by the following formula:

Dose (mL) = [patient weight (kg) x dose level (mg/kg)/ drug concentration (mg/mL)]

EXAMPLES

EXAMPLE 1. PROPHYLACTIC TREATMENT OF A PNH PATIENT WITH AN ANTIBODY TO HEMOGLOBIN WHICH AIDS IN CLEARING HEMOGLOBIN

PNH patients have episodes of hemolysis in which large amounts of red blood cells are lysed and hemoglobin is released free into the plasma. Because of the recurring nature of this disease, such patients will be treated prophylactically by administering anti-hemoglobin antibodies at regular intervals such that active antibody is present in the plasma at any time and can bind to free hemoglobin and hasten its removal from the plasma. Preferably the antibody will comprise an Fc region to aid in removal via interaction with macrophages and/or monocytes or the activation of complement and subsequent clearance of hemoglobin through C3b- mediated events. The amount of antibody required will depend upon the severity of the disease. If a PNH patient typically has episodes which result in levels of 0.1 g/dL of free hemoglobin, such a patient will regularly be administered anti- hemoglobin antibody to keep the equivalent of at least 0.1 g/dL of active whole antibody present in the plasma. A person typically has on the order of 7 liters of plasma. Antibodies are degraded over time and must be replaced. As an example, PNH patients are administered an anti-C5 antibody approximately once every two weeks to maintain an active level of that antibody (Hillmen et al., N. Engl. J. Med. 350:552-559 (2004)) to prevent lysis of red blood cells. The amount delivered depends upon the form of the antibody and its in vivo half-life. Alternatively, such patients may also be treated with an antibody which binds to hemoglobin and prevents the reaction between hemoglobin and nitric oxide.

EXAMPLE 2. TREATMENT OF A PATIENT UNDERGOING CARDIOPULMONARY ARTERY BYPASS GRAFT SURGERY WITH AN ANTIBODY WHICH PREVENTS REACTION BETWEEN FREE HEMOGLOBIN AND NITRIC OXIDE

In contrast to PNH patients who will benefit from chronic treatment with an agent to inhibit or prevent hemoglobin from reacting with nitric oxide, some persons will benefit from a single treatment of such an agent. For example, persons undergoing cardiopulmonary artery bypass graft surgery will often have excess lysis of red blood cells as a result of the surgery, likely due to the passage of blood through extracorporeal machinery. This could lead to the types of physiological orders discussed above, e.g., dystonias and hypertension. To prevent such a result, a person about to undergo this surgery will be treated with an agent that inhibits or prevents free hemoglobin from reacting with nitric oxide. Preferably such an agent will be an anti-hemoglobin antibody. Alternatively, such persons can be treated with an antibody to hemoglobin which hastens the removal of free hemoglobin from the plasma. A single dose should be sufficient to last until the body has removed the excess free hemoglobin.

In a less preferable mode, persons undergoing cardiopulmonary artery bypass graft surgery can first undergo the surgery and then be monitored for lysis of red blood cells either during and/or after the surgery. Those patients who show excess lysis of red blood cells leading to excess free hemoglobin in their plasma can then be treated with the agent to inhibit reaction of the free hemoglobin with nitric oxide.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

EQUIVALENTS

While the above description contains many specific details of methods and compositions in accordance with this invention, these specific details should not be construed as limitations on the scope of the invention, but merely as exemplifications of preferred embodiments thereof. Those skilled in the art will envision many other possible variations that fall within the scope and spirit of the invention as defined by the claims appended hereto.

Claims

CLAIMS:

1. A method for facilitating the removal of free hemoglobin from the blood plasma of a mammal, said method comprising treating said mammal with a therapeutically effective amount of an antibody or antibody fragment that binds hemoglobin.

2. The method of claim 1 , wherein said antibody or antibody fragment is selected from the group consisting of a polyclonal antibody, a monoclonal antibody or antibody fragment, a diabody, a chimerized or chimeric antibody or antibody fragment, a humanized antibody or antibody fragment, a deimmunized human antibody or antibody fragment, a fully human antibody or antibody fragment, a single chain antibody, an Fv, an Fab, an Fab', and an F(ab')₂.

3. The method of claim 1 , wherein said mammal is a human.

4. The method of claim 1, wherein said mammal suffers from a disease or condition selected from the group consisting of: paroxysmal nocturnal hemoglobinuria, sickle cell disease, thalassemia, hereditary spherocytosis, hereditary stomatocytosis, microangiopathic hemolytic anemia, pyruvate kinase deficiency, ABO mismatch transfusion reaction, paroxysmal cold hemoglobinuria, severe idiopathic autoimmune hemolytic anemia, infection- induced anemia, malaria, dialysis, cardiopulmonary bypass, mechanical heart valve-induced anemia, and any combination thereof.

5. The method of claim 1 , wherein said treating is chronic.

6. The method of claim 1, wherein said treating comprises a one time treatment.

7. The method of claim 1 , wherein said mammal is treated by administering said protein intravenously.

8. The method of claim 1, wherein said mammal is treated to inhibit or prevent a disease or undesirable physiological condition resulting from a lack or reduced level of nitric oxide in the plasma of said mammal.

9. The method of claim 8, wherein said disease or undesirable physiological condition is selected from the group consisting of dystonia, a clotting disorder, pulmonary hypertension, systemic hypertension, gastrointestinal contractions, abdominal pain, sternal pain, erectile dysfunction, inflammation, esophageal spasm, dysphagia, thrombosis, decreased organ perfusion, platelet activation, death, global health status, physical functioning, emotional functioning, cognitive functioning, role functioning, social functioning, fatigue, dyspnea, appetite loss, insomnia, and any combination thereof.

10. A method of inhibiting or blocking nitric oxide depletion in the plasma of a mammal, wherein said depletion results from reaction of nitric oxide with hemoglobin, said method comprising treating said mammal with a therapeutically effective amount of an agent that inhibits or blocks the reaction between hemoglobin and nitric oxide such that the nitric oxide is not converted to nitrate.

11. The method of claim 10, wherein said agent is an antibody or antibody fragment.

12. The method of claim 11 , wherein said antibody or antibody fragment is selected from the group consisting of a polyclonal antibody, a monoclonal antibody or antibody fragment, a diabody, a chimerized or chimeric antibody or antibody fragment, a humanized antibody or antibody fragment, a deimmunized human antibody or antibody fragment, a fully human antibody or antibody fragment, a single chain antibody, an Fv, an Fab, an Fab', and an F(ab')₂.

13. The method of claim 10, wherein the agent is selected from the groups consisting of: a small molecule, a nucleic acid or nucleic acid analog, a protein, a macromolecule that is not a nucleic acid or protein, a peptidomimetic, small organic molecules, RNA aptamers, L-RNA aptamers, speigelmers, antisense compounds, double stranded RNA, small interfering RNA, locked nucleic acid inhibitors, and peptide nucleic acid inhibitors.

14. The method of claim 10, wherein said mammal is a human.

15. The method of claim 10, wherein said mammal suffers from a disease or condition selected from the group consisting of: paroxysmal nocturnal hemoglobinuria, sickle cell disease, thalassemia, hereditary spherocytosis, hereditary stomatocytosis, microangiopathic hemolytic anemia, pyruvate kinase deficiency, ABO mismatch transfusion reaction, paroxysmal cold hemoglobinuria, severe idiopathic autoimmune hemolytic anemia, infection- induced anemia, malaria, dialysis, cardiopulmonary bypass, mechanical heart valve-induced anemia, and any combination thereof.

16. The method of claim 10, wherein said treating is chronic.

17. The method of claim 10, wherein said treating comprises a one time treatment.

18. The method of claim 10, wherein said mammal is treated by administering said agent intravenously.

19. A method of inhibiting or preventing a disease or undesirable physiological condition in a mammal resulting from a lack or reduced level of nitric oxide in the plasma of said mammal, said method comprising treating said mammal with a therapeutically effective amount of an agent that inhibits or blocks the reaction between hemoglobin and nitric oxide such that the nitric oxide is not converted to nitrate.

20. The method of claim 19, wherein said disease or undesirable physiological condition is selected from the group consisting of dystonia, a clotting disorder, pulmonary hypertension, systemic hypertension, gastrointestinal contractions, abdominal pain, sternal pain, erectile dysfunction, inflammation, esophageal spasm, dysphagia, thrombosis, decreased organ perfusion, platelet activation, death, global health status, physical functioning, emotional functioning, cognitive functioning, role functioning, social functioning, fatigue, dyspnea, appetite loss, insomnia, and any combination thereof.

21. The method of claim 20, wherein said dystonia is selected from a group consisting of a gastrointestinal dystonia, a cardiovascular dystonia, pulmonary dystonia, a urogenital dystonia, and any combination thereof.

22. The method of claim 19, wherein said mammal is a human.

23. The method of claim 19, wherein said agent is an antibody or antibody fragment.

24. The method of claim 23, wherein said antibody or antibody fragment is selected from the group consisting of a polyclonal antibody, a monoclonal antibody or antibody fragment, a diabody, a chimerized or chimeric antibody or antibody fragment, a humanized antibody or antibody fragment, a deimmunized human antibody or antibody fragment, a fully human antibody or antibody fragment, a single chain antibody, an Fv, an Fab, an Fab', and an F(ab')₂.

25. The method of claim 19, wherein the agent is selected from the groups consisting of: a small molecule, a nucleic acid or nucleic acid analog, a protein, a macromolecule that is not a nucleic acid or protein, a peptidomimetic, small organic molecules, RNA aptamers, L-RNA aptamers, speigelmers, antisense compounds, double stranded RNA, small interfering RNA, locked nucleic acid inhibitors, and peptide nucleic acid inhibitors.

26. The method of claim 19, wherein said mammal suffers from a disease or condition selected from the group consisting of: paroxysmal nocturnal hemoglobinuria, sickle cell disease, thalassemia, hereditary spherocytosis, hereditary stomatocytosis, microangiopathic hemolytic anemia, pyruvate kinase deficiency, ABO mismatch transfusion reaction, paroxysmal cold hemoglobinuria, severe idiopathic autoimmune hemolytic anemia, infection- induced anemia, malaria, dialysis, cardiopulmonary bypass, mechanical heart valve-induced anemia, and any combination thereof.

27. The method of claim 19, wherein said treating is chronic.

28. The method of claim 19, wherein said treating comprises a one time treatment.

29. The method of claim 19, wherein said mammal is treated by administering said agent intravenously.

30. A method for facilitating or aiding in the removal of free heme from the blood plasma of a mammal, said method comprising treating said mammal with a therapeutically effective amount of an antibody or antibody fragment that binds heme.

31. The method of claim 30, wherein said antibody or antibody fragment is selected from the group consisting of a polyclonal antibody, a monoclonal antibody or antibody fragment, a diabody, a chimerized or chimeric antibody or antibody fragment, a humanized antibody or antibody fragment, a deimmunized human antibody or antibody fragment, a fully human antibody or antibody fragment, a single chain antibody, an Fv, an Fab, an Fab', and an F(ab')₂.

32. The method of claim 30, wherein said mammal is a human.

33. The method of claim 30, wherein said mammal suffers from a disease or condition selected from the group consisting of: paroxysmal nocturnal hemoglobinuria, sickle cell disease, thalassemia, hereditary spherocytosis, hereditary stomatocytosis, microangiopathic hemolytic anemia, pyruvate kinase deficiency, ABO mismatch transfusion reaction, paroxysmal cold hemoglobinuria, severe idiopathic autoimmune hemolytic anemia, infection- induced anemia, malaria, dialysis, cardiopulmonary bypass, mechanical heart valve-induced anemia, and any combination thereof.

34. The method of claim 30, wherein said treating is chronic.

35. The method of claim 30, wherein said treating comprises a one time treatment.

36. The method of claim 30, wherein said mammal is treated by administering said antibody or antibody fragment intravenously.

37. A method of inhibiting or preventing an undesirable physiological condition in a mammal resulting from free heme in the plasma of said mammal, said method comprising treating said mammal with a therapeutically effective amount of an antibody or antibody fragment that binds free heme.

38. The method of claim 37, wherein said undesirable physiological condition is selected from the group consisting of inflammation, oxidative stress, a toxic effect on endothelial cells, and any combination thereof.

39. The method of claim 37, wherein said mammal is a human.

40. The method of claim 37, wherein said antibody or antibody fragment is selected from the group consisting of a polyclonal antibody, a monoclonal antibody or antibody fragment, a diabody, a chimerized or chimeric antibody or antibody fragment, a humanized antibody or antibody fragment, a deimmunized human antibody or antibody fragment, a fully human antibody or antibody fragment, a single chain antibody, an Fv, an Fab, an Fab', and an F(ab')₂.

41. The method of claim 37, wherein said mammal suffers from a disease or condition selected from the group consisting of: paroxysmal nocturnal hemoglobinuria, sickle cell disease, thalassemia, hereditary spherocytosis, hereditary stomatocytosis, microangiopathic hemolytic anemia, pyruvate kinase deficiency, ABO mismatch transfusion reaction, paroxysmal cold hemoglobinuria, severe idiopathic autoimmune hemolytic anemia, infection- induced anemia, malaria, dialysis, cardiopulmonary bypass, mechanical heart valve-induced anemia, and any combination thereof.

42. The method of claim 37, wherein said treating is chronic.

43. The method of claim 37, wherein said treating comprises a one time treatment.

44. The method of claim 37, wherein said mammal is treated by administering said agent intravenously.