Hemoglobin-Based Oxygen Carriers as Red Cell Substitutes and Oxygen Therapeutics

Hemoglobin (Hb), a tetrameric protein, is responsible for the transport of oxygen in red blood cells (Perutz

1980

). Attempts to use hemolysate (Amberson

1937

) and stroma-free Hb as Hb based oxygen carrier (Rabiner et al.

1967

) resulted in nephrotoxicity and cardiovascular adverse effects (Savitsky et al.

1978

). Thus, nanobiotechnological modification is needed before it can be used (Chang

1964

)

This chapter will provide a basic summary of the fundamentals whereby oxygen is delivered from the atmosphere to its ultimate site of utilization, the mitochondria, and the means whereby this delivery is regulated and optimized for efficiency and the “survival” of the organism as a whole. This occurs in four sequential steps comprising 1

. convective

mass transport exchange of air into and out of the alveolar spaces; 2.

diffusive

exchange of oxygen and carbon dioxide between alveoli and blood in pulmonary capillaries; 3.

convective

mass transport of blood from the pulmonary to the systemic circulation; and 4.

diffusive

exchange of carbon dioxide and oxygen in the systemic capillaries between blood and tissue cells. The critical link between the mass convective and diffusive movements in each of the respiratory and systemic circulations is hemoglobin with its important functional properties that amplifies the respiratory gas transport and assures efficient delivery of oxygen to all tissue cells. Those physiological processes will be summarized which enable the organism to respond to a wide range of metabolic demands for oxygen in an efficient way. A few examples of pathological entities will also be described that may compromise the efficiency of the process and of dysregulation that may result in cellular, tissue and and/or organ hypoxia and consequent dysfunction.

Human hemoglobin A (Hb) is the main protein component of red blood cells, making up to 97 % of their dry content. Hb plays a crucial role in vertebrates, as it carries oxygen from the lungs to the tissues for their oxidative metabolism.

It is a generalized perception that blood products are needed when oxygen (O

2

) delivery capacity is jeopardized by the decrease of blood’s intrinsic O

2

carrying capacity due to the decrease of hematocrit (Hct) or blood hemoglobin.

Shock may be defined broadly as a condition in which metabolic energy production is limited either by the supply or utilization of oxygen, manifest by derangement of the normal oxygen supply–demand balance, accumulation of the byproducts of anaerobic metabolism, and dysfunction of one or more organ system. In the case of massive hemorrhage, shock is due specifically to impaired oxygen delivery (VO

2

) secondary to both hypovolemia and anemia. Restoration of tissue perfusion, termed resuscitation, proceeds systematically, is based upon the underlying etiology of shock, terminates upon achievement of clearly defined endpoints, and requires frequent re-evaluation. Resuscitation has been refined substantially over the previous decade, with a resultant improvement in the outcomes of critically ill patients (Brun-Buisson et al.

2004

; Martin et al.

2003

). Major changes have included improved accuracy of the assessment of intravascular volume status, recognition of the detrimental effects of both allogeneic blood product transfusion and excessive volume expansion, and timely, goal-directed treatment of shock and its complications. This chapter will focus on the diagnosis of shock, differentiation into hemorrhagic shock, the benefits and limitations of various measurements used to determine the adequacy of resuscitation, general resuscitative strategies, and complications of resuscitation.

Blood transfusion is an essential treatment for patients suffering blood loss associated with trauma and surgery. This chapter discusses the goal and rationale for allogeneic red cell transfusion in surgical and traumatic hemorrhage in a hospital setting. A large number of patients have now been enrolled in clinical trials. This experience provides high quality evidence to inform the transfusion decision. A restrictive transfusion has been established to be safe in adult and pediatric patients in many clinical settings. The criteria used for making the decision to transfuse and current clinical practice guidelines and evidence-based recommendations are provided.

Blood substitutes such as hemoglobin-based oxygen carriers (HBOC) have tremendous potential to improve pre hospital fluid resuscitation in both civilian and military populations. This chapter reviews goals and current recommendations of pre hospital resuscitation, the historical development of HBOC and initial clinical trials, and future developments that may improve pre hospital resuscitation.

Research and development in the field of oxygen therapeutics has made substantial progress in the past two decades. However, significant challenges have been encountered, as highlighted at an NIH/FDA/HHS workshop in 2008.

Oxygen therapeutics (OT) consist of hemoglobin-based oxygen carriers (HBOCs) and fluorocarbons. They have been developed to deliver oxygen (O

2)

to tissues, mainly for treatment of shock due to blood loss. These products are being developed in order to treat blood loss when red blood cells are not available, as may occur on the battlefield or in civilian life when trauma occurs.

We have produced several recent reviews related to the technology of chemical stabilization of haemoglobin and a brief view of the future of HBOCs [

1, 2

]. This chapter is intended to provide a broader perspective of the topic of chemical stabilization for the production of HBOCs.

Enhancing the molecular size/volume Hb was introduced, as a simple molecular approach to overcome or attenuate the extravasation mediated NO scavenging activity of Hb in vivo. Conjugation of Hb, as a chemical tool to achieve this objective, has now advanced beyond this initial concept. It requires us now to recognize that engineering of resuscitation fluids like properties to Hb is a novel concept and simple approach to neutralize/attenuate the in vivo hypertensive activity of acellular Hb. Development of conjugated hemoglobins is reviewed here as it lead to the development of this concept along with future perspectives. Extension Arm chemistry, that integrates the concepts of click chemistry into the various conjugation approaches of relevance to this aspect of blood substitute research is discussed. Bridging these approaches to increase the efficacy of engineering resuscitation fluid like properties to Hb is suggested as the future direction of designing conjugated Hbs as oxygen therapeutics. The integration of site directed mutagenesis to tame the molecular properties of Hb with the extension arm facilitated PEGylation to attenuate the impact of conjugation chemistry and/or conjugated polymer on the tertiary/quaternary structure of the Hb molecule is discussed in detail as applied to PEGylation of Hb as the conjugation approach of choice to generate nonhypertensive Hb as oxygen therapeutics. We conclude that such a synergistic approach will result in the attenuation of heme degradation product dependent and oxidative stress mediated in vivo toxicity as well as facilitating an increase in the oxygen delivering capacity of the conjugated Hb.

Hemoglobin (Hb) is the most abundant protein in blood (12–15 g/dL). Nevertheless, various side effects of stroma-free Hb have emerged during the long development of Hb-based oxygen carriers (HBOCs). The physiological significance of the RBC structure is undergoing reconsideration. Fundamentally, excessive native Hb molecules are toxic, but encapsulation can shield this toxic effect. Cellular type HBOCs of various kinds are developed using liposome and polymer membranes that mimic the RBC cellular structure. Even though many cell-free HBOCs are undergoing clinical trials, cellular type HBOCs remain in the preclinical stage. Encapsulation of Hb can shield the toxic effects of Hb, but encapsulation presents its own obstacles to realization, such as efficient encapsulation, biocompatibility, and biodegradability.

Three recombinant octameric mutants of human normal adult hemoglobin (Hb A), rHb (αN78C), rHb (αN78C/L29F), and rHb (αN78C/L29W), were expressed in our

Eschericia coli

expression system and purified. They were used as resuscitation fluids in our unique mouse model of traumatic brain injury (TBI) combined with severe hemorrhagic shock (HS). A sulfhydryl group was introduced onto the surface of the α-subunits of rHb A by substituting Asn78 with cysteine. The rHb (αN78C) form octamers by linking the two tetramers with 2 intermolecular disulfide bonds. Nuclear magnetic resonance (NMR) spectroscopic studies indicate that rHb (αN78C) has the same quaternary and tertiary structures as those of Hb A. Furthermore, the oxygen-binding activity (as measured by

P

50

) and the cooperativity of the oxygenation process (as measured by the Hill coefficient) of this mutant have not been altered compared to Hb A. The Leu29 residue on the α-subunits of the octamers was then mutated into either phenylalanine (F) or tryptophan (W) to yield rHb (αN78C/L29F) and rHb (αN78C/L29W), respectively. Compared to Hb A, rHb (αN78C/L29F) has high- while rHb (αN78C/L29W) has low-oxygen affinity. Both mutants are cooperative in their oxygen-binding properties, but lower than that observed for Hb A. They maintain their quaternary structure as those of Hb A, but exhibit perturbation of their tertiary structure at or near the heme pockets as detected by NMR measurements. Although all octameric rHbs would be expected to have reduced nitric oxide (NO) binding based on their size, rHb (αN78C/L29F) and rHb (αN78C/L29W) may have a further reduction in NO binding as a result of introducing a bigger aromatic amino acid residue at the distal heme pocket of the α-chain. These three rHbs were used as resuscitation solutions in mice after TBI combined with HS. TBI was induced by a controlled cortical impact (CCI) to the left parietal cortex. Blood was then withdrawn (2.4 mL/100 g body weight) over 15 min to induce HS. A pressure-controlled model was used with the mean arterial pressure (MAP) maintained at 25–27 mm Hg for an additional 20 min. At the end of the HS Phase (35 min total), i.e., the beginning of Pre-Hosptial Phase, lactated Ringers (LR) or rHb (120 mg/mL) solutions were administered to the mice at 2 mL/100 g. Additional LR or rHb solution was given if needed at 1 mL/100 g to maintain the MAP at > 70 mm Hg for the next 90 min. The shed blood was then returned simulating definitive care with transfusion in a “Hospital Phase”, lasting 15 min. The mice were then recovered, returned to cages and observed for 24 h before sacrificing for neuropathology. A marked difference in the fluid requirements was observed between the LR and the rHb groups. At the end of the Pre-Hospital Phase, the LR group received >4 times more resuscitation fluid (21.5 ± 0.75 mL/100 g) than the rHb groups (5.0 ± 0 mL/100 g;

P

< 0.05), while the arterial Hb level in rHb groups were ~3 g/dL higher than that of the LR group. More importantly, the LR group exhibited persistent refractory hypotension during the Pre-Hospital Phase. The MAP of rHb groups stayed near baseline level over the entire Pre-Hospital Phase and Hospital Phase. However, an initial elevation in MAP above baseline followed by gradual diminution was observed only for the rHb (αN78C) group (

P

< 0.05). Brain tissue oxygen (PbtO

2

) levels in the hippocampus ipsilateral to the site of CCI did not differ significantly between groups, although the LR group showed deterioration in PbtO

2

during late Pre-Hospital Phase. Numerically, the normal oxygen affinity rHb (αN78C) group and the high oxygen affinity rHb (αN78C/L29F) groups exhibited the highest and lowest PbtO

2

, respectively. Surprisingly, the high oxygen affinity rHb conferred a neuroprotective effect in the CA1 region of the selectively vulnerable region of the hippocampus vs. all other groups. No difference in neuronal survival was seen between groups in the CA3 hippocampus. Our present results suggest that these novel octameric rHbs have the potential to develop into a small-volume resuscitation fluid for treatment of TBI combined with HS.

While the use of liposome-encapsulated hemoglobin (LEH) as artificial oxygen carriers (AOCs) has long been studied, its complicated manufacturing process and large production costs have hindered the progress in its development and, subsequently, its advancement to practical applications. However, we have been persistently developing LEH as an analogue of red blood cells (RBCs) for its use as AOCs, and to this end we have constructed a Good Manufacturing Practice (GMP) facility where LEH can be manufactured as biologics under quality assurance systems as well as establishing the manufacturing processes of LEH at this facility. We have also clarified the physicochemical properties and stability of LEH, and further confirmed the fundamental efficacy and safety of LEH as AOCs. Although the technology of LEH has already reached the stage of clinical testing, its high manufacturing costs remains an unsolved issue if LEH is to be used as a medicinal product.

Zero-link Hb (OxyVita

®

, OXYVITA, Inc., New Windsor, New York), a new generation hemoglobin-based-oxygen-carrier (HBOC), is produced using a modified zero-linked polymerization mechanism that employs chemical activators to incorporate inter-dimerically cross-linked bovine hemoglobin tetramers into “super-polymeric” macromolecules (Average M. wt. = 17 MDa) for oxygen delivery when whole blood or packed red cells are not available. This molecular design pathway was developed to address several basic biochemical and physiological concerns associated with earlier generations of HBOCs. Observations made during pre-clinical (various animal models) and clinical studies provided evidence these earlier generation acellular HBOCs gave evidence of reduced retention times within the circulatory system, extravasation across endothelial tissue membranes due to their small molecular size leading to arterial and veinous vasoconstriction that was coupled with rapid increases in mean arterial pressure (MAP) upon infusion. Zero-link Hb’s increased molecular size and structural stability was developed in direct response to these serious concerns that accompanied the evolution of HBOC development within the past several decades. The distinct nature of the zero-linked synthetic route eliminates the need for chemical linkers within the product, eliminates side reaction concerns: such as reversibility and decomposition due to weak and non-specific chemical bonding, changes in temperature and/or pressure within the circulatory system, and residual toxicity. The incorporation of these molecular design characteristics within OxyVita Hb have been responsible for significant findings as determined by the pre-clinical studies that have been carried out by many independent investigators between 2002 to 2012. This chapter will present: (1) a short overview of previous generation HBOCs; (2) the experimental development of OxyVita Hb (liquid and powder forms); (3) the physiochemical properties and functional behavior of OxyVita Hb; and (4) the implications of the molecular design properties for pre-clinical studies that involved issues of extravasation and vasoconstriction, increases in MAP, regulation of oxygen delivery, and effects on coagulatory functions.

Polynitroxylated pegylated hemoglobin (PNPH, VitalHeme™, SynZyme Technologies LLC, Irvine, CA) is a pegylated hemoglobin covalently labeled with catalytic nitroxides, aka caged nitric oxide (cNO). PNPH serves as a hyperoncotic biopolymer coated and redox transformed hemoglobin acting in the plasma phase as a neurovascular protective multifunctional nanomedicine. This drug is for the correction or prevention of inadequate regional and/or global blood flow without oxidative stress under normal or hypovolemic states in critical care and transfusion medicine. Specific clinical indications for which PNPH has shown efficacy are traumatic brain injury complicated by hemorrhagic shock, stroke, and sickle cell disease. PNPH may also be an ideal alternative to aged red blood cells and a bridge to transfusion.

The quest to develop a viable substitute for human blood has been ongoing for many decades. Many attempts to create an effective blood substitute fall short because of toxicity issues and at present no product is commercially available. Armed with an awareness of the problems associated with the performance of the first generation blood substitute products, which are linked to hemoglobin’s (Hb) intrinsic toxicity that causes hypertension, oxidative and inflammatory reactions, gastrointestinal disorders and even heart attack and stroke, researchers at Texas Tech University Health Sciences Center created a novel concept of pharmacological cross-linking of Hb with ATP, adenosine and reduced glutathione (GSH) that resulted in the non-toxic and effective ATP-ADO-GSH-Hb product. This novel blood substitute possesses vasodilatory, anti-oxidant, anti-inflammatory and erythropoietic properties; all of which are beneficial to the patients suffering from acute and chronic anemias, sickle cell disease, ischemic diseases including heart attack and stroke, and cancer. ATP-ADO-GSH-Hb and its manufacturing technology have been extensively tested including viral and prion clearance validation studies and various non-clinical pharmacology, toxicology, genotoxicity and efficacy tests. The effects of ATP-ADO-GSH-Hb on appropriate physiological measures in human cell systems, normal animals and disease models have also been determined. The clinical proof-of-concept was carried out in sickle cell anemia patients. The obtained results confirmed that pharmacologic cross-linking of Hb molecules with ATP, adenosine and GSH is highly effective in designing a viable blood substitute for a wide array of therapeutic uses.

Human serum albumin (HSA) is the most prominent plasma protein in the circulatory system and has a remarkable capability to bind a broad range of hydrophobic molecules. Protoheme IX released from hemoglobin (Hb) is also captured by HSA with a high binding constant. This strong affinity of HSA for heme has stimulated efforts to develop albumin as an artificial hemoprotein which is capable of mimicking the O

2

transport of Hb. In this chapter, we report on our representative results of albumin-heme O

2

carriers; HSA incorporating synthetic heme, its poly(ethylene glycol) conjugate, and recombinant HSA mutant complexing protoheme IX. These protein hybrids might be of great medical importance not only for RBC alternatives, but also for O

2

-providing therapeutic reagents.

Hemoglobin (Hb) can produce reactive oxygen species, including superoxide anions, which are intrinsically toxic. Superoxide dismutase (SOD) is present in red blood cells (RBCs) and provides important protection against such oxidative stress. Upon hemolysis, Hb becomes released from the RBCs and the normal protection systems involving SOD and catalase become very inefficient. In these instances, both the Hb protein in itself and its cellular surroundings may be exposed to severe oxidative damage. In order to generate less toxic Hb variants, we have produced fusion proteins between SOD and HbA. The fusion protein has been prepared by coexpressing the human Hb α-chain linked to the manganese SOD gene as a chimeric construct together with the native β-chain Hb gene in

Eschericia coli

. The engineered SOD-Hb fusion protein retains the oxygen-binding capacity and, moreover, decreases cytotoxic ferrylHb (HbFe(4 +)) formation when exposed to superoxide radicals. The SOD-Hb fusion protein is also substantially less prone to autoxidation. Our findings not only provide insight into the synergistic functions between SOD and Hb when they are closely and spatially organized, but could also potentially be used to develop therapeutic blood substitutes with more efficient oxygen carrying capabilities.

A great deal of research has focused on methods to produce red blood cells (RBCs) industrially in order to reduce reliance on the supply from human donations. Among the possible candidates for artificial blood, the most perfect substitute would be actual human RBCs derived from erythroid progenitor cells grown in vitro just as occurs in vivo.

Artificial oxygen carriers were originally developed as a substitute for red blood cells (RBC) for transfusion. As the blood demand–supply balance becomes critical, it is reasonable to explore artificial oxygen (O

2

) carriers.

Hemoglobin vesicles (HbV), cellular-type artificial oxygen carriers, are human hemoglobin encapsulated within a phospholipid bilayer membrane. Because HbV are injected intravenously, the biocompatibility of the HbV with blood components is extremely important to ensure their safety for clinical use. We evaluated this biocompatibility by particularly examining the influence of HbV on human blood cells and on plasma proteins in vitro. (1) Regarding influences to platelets that are involved not only in hemostasis but also in inflammation, HbV themselves neither activate platelets nor show aberrant effect on agonist-induced platelet activation. (2) HbV do not affect the agonist-induced activation of neutrophil functions that play important roles in innate immunity. (3) HbV do not affect hematopoietic progenitor activity. (4) The HbV examined herein (containing DHSG) do not activate the complement system, but old-type HbV (containing DPPG, no PEG modification) do so to a marked degree. (5) Neither coagulation nor the kallikrein–kinin cascade was affected by the current type of HbV. (6) Multiple HbV infusions in rats cause a transiently slight decrease in the complement titer only after first infusion, with no allergic reaction. No anaphylactic shock was observed in rats administered multiple empty vesicles without hemoglobin. (7) Infusion of HbV can “transiently” render HbV phagocytized-splenic cells immunosuppressive in

ex vivo culture condition.

Results of our in vitro and in vivo investigations show that HbV are highly biocompatible with human blood cells, human plasma proteins and rat immune systems.

The inherent limitations of traditional blood transfusion have launched substantial efforts to develop blood substitutes, most of which are hemoglobin-based oxygen carriers (HBOCs). Polymerized human placenta hemoglobin (PolyPHb) is a promising HBOC developed in China. Based on its excellent oxygen transporting capacity, alternative uses of PolyPHb have been explored from several year ago. This review will summarize the results of animal studies about PolyPHb and discuss the potential alternative uses of PolyPHb in clinical situations.

Current management strategies for hemorrhaging trauma patients recommend fluid resuscitation with relatively small or restricted fluid volumes. Because most recommended fluids are essentially plasma expanders with limited oxygen carrying capacity, hemoglobin [Hb]-based oxygen-carrying compounds (HBOCs) have been suggested as an adjunct to improve oxygen delivery to the tissues. We review evidence from clinical to animal trials as to the efficacy of three competing low volume fluid resuscitation strategies (LVFR), and the potential in this context for HBOCs as oxygen therapeutic adjuncts. Unfortunately data are too limited in quantity and quality to allow any reliable conclusions as to the efficacy and safety of HBOC-augmented LVFR. There is an imperative need for higher quality and adequately-powered animal trials and clinical randomized controlled trials to clarify the efficacy and safety of both LVFR and adjunct HBOCs for traumatic hemorrhage.

Experimental studies indicate that hemoglobin-based oxygen carriers (HBOCs) can be effective in rescuing ischemic tissue with residual collateral blood flow after occlusion of a major artery supply. Much of the work showing positive effects of HBOC transfusion has been performed in middle cerebral artery occlusion models of stroke, although some evidence supports their use during ischemia in heart and peripheral tissue. Strategies for improving convective oxygen transport through collateral arteries with HBOCs include (1) boosting blood oxygen carrying capacity, (2) decreasing hematocrit and blood viscosity while limiting the decrease in oxygen carrying capacity, and (3) augmenting dilation of collateral arteries. In addition, plasma-based HBOCs may enhance the delivery of oxygen from the microcirculation to the ischemic parenchymal cells by (1) increasing oxygen flux through individual capillaries that have a disproportionately low flux of red blood cells but persistent plasma flow, (2) filling the capillary spaces between red blood cells with an oxygen source that increases the effective capillary surface area for oxygen diffusion, and (3) facilitating oxygen transport from the red blood cell to the endothelium through the plasma. Newer formulations of HBOCs with carbon monoxide, S-nitrosylation, and superoxide dismutase–mimetic activity endowed by polynitroxylation can be effective at relatively low plasma concentrations, possibly because they may serve to stabilize endothelial function and limit inflammation and reperfusion injury. Future development of HBOCs as a therapeutic for ischemic organs will require determining the precise mechanisms of action of each type of HBOC and the optimal dosage and timing for the specific application.

The pre-clinical evaluation of hemoglobin (Hb) based oxygen carriers (HBOCs) consists of both proof of concept studies and studies to support safe use in clinical subjects. Certain HBOCs have demonstrated problematic safety profiles in human subjects, particularly in later phases of clinical trials when complicated and/or pre-existing disease states occur together. The mechanisms leading to adverse events in humans are likely related to nitric oxide (NO) interactions with Hb, but may also be associated oxidative toxicity in tissue. While the effects of NO on hypertension and vasculopathy are well established, limited data exist to support the role between Hb and oxidative tissue injury. The primary focus areas of this chapter are to (1) outline existing approaches to proof of concept studies for HBOCs and good laboratory practice (GLP) based studies to establish safe use in humans (2) suggest novel supportive approaches to pre-clinical evaluation using pre-existing disease state animal models and HBOC administration (3) propose novel markers of early tissue injury that may be beneficial in the study of HBOC safety and allow for additional understanding of human subject risk.

In response to the terrorist attacks in the United States on September 11, 2001 and the subsequent advent of conflicts in Afghanistan and Iraq, the Naval Medical Research Center (NMRC, Silver Spring, MD) initiated a search for an oxygen-carrying resuscitation fluid that could be used for combat trauma and in situations with wide-spread civilian casualties. The ability to supply blood to mass casualties or far-forward combat trauma victims in austere environments with long evacuation times is wrought with logistical complications.

Hemoglobin-vesicles (HbV,

d

= 251 nm) are an artificial oxygen carrier encapsulating a concentrated Hb solution in polyethylene glycol-modified phospholipid vesicles (liposomes). Their safety and efficacy as artificial red blood cells and a fluid for oxygen therapeutics have been extensively studied in various rodent models. We have previously reported the efficacy of HbV as a resuscitation fluid in canine hemorrhagic shock model. However, long term safety was not yet studied. In this chapter, we report the results of efficacy and safety study in Beagle dog using hemorrhagic shock model. We observed the animals for one year. Twenty-six male beagle dogs were used. For acute study, a dog was anesthetized by 2.0–2.5 % sevoflurane and spontaneous respiration was maintained. A thermodilution catheter was introduced into pulmonary artery for measurement of cardiovascular parameters. Fifty percent of estimated blood volume was withdrawn from right femoral artery, and the shock state was stabilized for 1 h. The dogs were resuscitated either with isovolemic 5 % recombinant human serum albumin alone (rHSA) HbV suspended in rHSA (HbV/rHSA), or shed autologous blood (SAB). HbV/rHSA group showed immediate recovery of hemodynamic and blood gas parameters as did SAB, and no abnormal hypertension was observed. HbV contributed to about 34-38 % of the total delivery of oxygen and 26–29 % of total consumption of oxygen for 4 h. For chronic phase study, the beagle dogs resuscitated with HbV/rHSA or SAB were caged for up to 1 year. All dogs survived and gained weight. Results of hematological and plasma biochemical analyses and pathological examination showed no notable abnormalities except the transient accumulation of HbV in reticuloendothelial system and a release of cholesterol derived from HbV. The present results using beagle dogs reassure that HbV suspended in rHSA shows a similar resuscitative ability and safety to that of SAB.

Synopsis: Hemoglobin based oxygen carriers (HBOC) are used for management of acute surgical and medical anemia. In this chapter, the clinical application, dosing, administration, side effects and toxicities of three HBOC that have undergone recent Phase III clinical trials are discussed. Side effects include gastrointestinal (nausea, vomiting, diarrhea, abdominal pain and bloating), skin rashes, jaundice without elevated bilirubin, fever, and plasma hemoglobin interference with laboratory assays, and methemoglobinemia. Toxicities of these three HBOC include elevated blood pressure, hemostatic effects, myocardial infarction and in the elderly, stroke. HBOC because of side effects, are not ideal resuscitation fluids from acute anemia, but have advantages when blood is unavailable or refused. HBOC could be useful in the pre-hospital environment, military conflicts, disasters, and in the developing world, and in other situations where blood is not readily accessible. HBOC management of acute anemia is not without risk, but should be used when the benefits outweigh such risks, as HBOC toxicities are transient, readily reversible, and less than 1 % result in fatalities.

From the author’s experience in the HBOC field of basic sciences and commercial product development some critical comments are offered on some of the negative aspects of the major human clinical trials. In particular in trials conducted in the emergency trauma setting the flaws in the execution of the trials have seriously affected the achievement of both efficacy and safety endpoints. In elective surgery trials the safety profile of the HBOC products has been the principal negative outcome. It is proposed that reducing unintentional bias is important and it be accomplished by achieving a balanced subject profile of the trial arms.

One of the most elusive goals in the modern medical era has been to design a safe and effective asanquinous oxygen therapeutic (OT). After 80 years of research and dozens of clinical trials, most of the 17 billion people in the world are still without an alternative to blood; South Africa and Russia are currently the rare exceptions (Amberson

1933

;

www.opkbiotech.com

, 2012).

A key impediment to regulatory approval of HBOCs appears to be an undesirable BP elevation (hypertension) often observed following IV administration in preclinical and clinical studies. The HBOC-mediated hypertension, as it occurs without increased cardiac output, appears to be primarily mediated by systemic vasoconstriction. In fact, a recent statement by FDA indicates that all HBOC products reviewed are vasoactive at the doses proposed for clinical use. Acute BP elevation could cause serious adverse effects (AEs) in vulnerable patients. Because vasoconstriction can cause suboptimal blood flow to affected organs, a crucial question is whether HBOC-mediated vasoconstriction is directly or indirectly responsible for some of the adverse events observed in recent clinical trials. There are many theories and hypotheses but little evidence that definitively links HBOC to observed adverse events. The pathophysiology involved in trauma, hemorrhage, ischemia and reperfusion is extremely complex and etiology of HBOC treatment associated AEs has not been elucidated. In this chapter, key currently proposed mechanisms for the HBOC-mediated BP elevation/vasoconstriction are reviewed. A most widely accepted mechanism for most HBOC-mediated vasoconstriction cases is Hb inactivation of endothelial NO, a potent vasodilator constitutively produced in the vascular endothelium. However, other mechanisms that may also play a more prominent role or multiple mechanisms may participate in concert under certain clinical conditions. In addition, a variety of factors that contribute to the manifestation of HBOC-mediated vasoactivity are also discussed.

Increased incidence of myocardial infarction (MI) associated with the use of hemoglobin based oxygen carriers (HBOCs) is a concern arising from the clinical evaluation of these formulations. Many characteristics of HBOCs may affect cardiac performance, as will specifics of treatment protocols. The vasoactivity of HBOCs affects the hemodynamic response to their infusion, but tissue oxygenation and blood flow to the heart is maintained or enhanced in a variety of circumstances, even when cardiac output is decreased. HBOC infusions are protective in several preclinical models of cardiac ischemia/reperfusion injury and supportive or restorative of cardiac function in models of hemodilution and resuscitation from hemorrhagic shock. Microscopic cardiac lesions observed during the preclinical testing of some HBOCs are a consequence of nitric oxide scavenging, but this mechanism is unlikely to be clinically significant unless humans are unusually sensitive. Understanding the reason(s) for the observed clinical imbalance in MI is hampered by the lack of detailed information concerning the physiologic and biochemical responses of the human cardiovascular system to HBOC infusion and appreciation for the degree to which suboptimal patient management in the presence of these unique solutions has contributed to the observed results.

There have been at least two major concerns with use of HBOCs as resuscitation fluids for traumatic hemorrhage: potential coagulopathy and interference with conventional clinical laboratory coagulation measurements.

Cell-free hemoglobin (Hb) outside the natural protective environment of the red blood cell (RBC) can undergo uncontrolled redox transformations to non-functional and/or toxic protein-bound and unbound species. Understanding the impact of Hb redox activity on vascular endothelial integrity and function remains a critical factor in the development of safe and efficacious acellular Hb-based oxygen therapeutics. This chapter will provide an overview of Hb redox reactions and their propensity to generate oxidative stress in the vascular system. Specific emphasis will be placed on studies from our laboratory that link oxidative stress with the development of endothelial dysfunction in cell culture systems and in animal models.

Volume loading solutions used therapeutically (albumin, dextrans, modified gelatins and hydroxylstarches) are simple plasma substitutes and cannot ensure oxygen transport. The search for blood substitute initially seemed utopian, but this goal is now within our reach, in the form of hemoglobin-based oxygen carriers (HBOC) for example.

Hemoglobin (Hb)-based oxygen (O

2

) carriers (HBOCs) are continuously being optimized as red blood cell (RBC) substitutes in order to overcome the well-known side-effects that are associated with transfusion of cell-free Hb (Alayash

1999

,

2004

). These side-effects include vasoconstriction, systemic hypertension and oxidative tissue toxicity (Alayash

1999

,

2004

). It is hypothesized that these side-effects are due to HBOC extravasation into the tissue space and the NO scavenging ability of cell-free Hb (Suaudeau et al.

1979

; Nakai et al.

1998

; Dull et al.

2004

; Liu et al.

1998

; Liao et al.

1999

). Polymerized HBOCs (PolyHbs) were thus engineered with molecular sizes larger than that of cell-free Hb, with the goal of reducing extravasation of the HBOC from the vascular space into the tissue space (Alayash

2004

; Palmer

2006

). This approach should reduce the proximity of the HBOC to the endothelium, which in turn should reduce/eliminate the aforementioned side-effects (Alayash

2004

; Palmer

2006

). In order to address this need for large sized HBOCs compared to cell-free Hb, the following commercial PolyHbs were initially developed: Oxyglobin® (OPK Biotech LLC, Cambridge, MA), Hemopure® (OPK Biotech LLC, Cambridge, MA), PolyHeme® (Northfield Laboratories Inc., Evanston, IL) and Hemolink™ (Hemosol Inc., Toronto, Canada) (OPK Biotech

2012

; Rentko et al.

2006

; Day

2003

; Adamson and Moore

1998

). However, these commercial products still elicited the aforementioned side-effects and are coincidentally close together in molecular weight (MW) (Rentko et al.

2006

; Day

2003

; Adamson and Moore

1998

; Tsai et al.

2006

; Butt et al.

2010

,

2011

; Kasper et al.

1996

; LaMuraglia et al.

2000

; Jahr et al.

2008

; Freilich et al.

2009

; Yu et al.

2010

; Handrigan et al.

2005

; Cheng et al.

2004

; Greenburg and Kim

2004

; Hill et al.

2002

; Lieberthal et al.

1999

). In the case of Oxyglobin®, its’ side-effects were directly attributed to PolyHb extravasation from the blood vessel lumen into the tissue space Butt et al. (

2010

;

2011

). Therefore, the ultrahigh MW HBOC Oxyvita™ (OXYVITA Inc., New Windsor, NY) was developed, which did not extravasate and induce significant vasoconstriction or hypertension (OXYVITA Inc.

2012

; Matheson et al.

2002

; Bucci et al.

2007

; Jia and Alayash

2009

). However, Oxyvita® has a high O

2

affinity so it is unclear how well it will deliver O

2

in vivo, despite its lack of vascular side-effects (Matheson et al.

2002

; Jia and Alayash

2009

). It is interesting to note that these commercial PolyHbs differ in MW by several orders of magnitude, and until recently there has been no systematic study of PolyHb MW on its’ in vivo safety profile (Rentko et al.

2006

; Day

2003

; Adamson and Moore

1998

; Matheson et al.

2002

; Bucci et al.

2007

; Jia and Alayash

2009

). To address this deficit in the literature, recent studies have shed light on the relationship between PolyHb molecular size and its’ in vivo safety profile (Cabrales et al.

2009

; Cabrales et al.

2010

; Baek et al.

2012

). These studies demonstrate that increasing the molecular size of tense (T)-state bovine PolyHb reduces vasoconstriction, systemic hypertension, and oxidative tissue toxicity (Cabrales et al.

2009

,

2010

; Baek et al.

2012

). Therefore, it is expected that further work in this area will lead to the optimization of PolyHb size in order to improve its in vivo safety profile for eventual human use.

Hemoglobin-based oxygen carriers (HBOCs) are currently in late phase clinical development as potential red blood cell substitutes. However, in recent clinical trials, most HBOC products have elicited vasoconstriction and blood pressure elevation. Mechanisms of the HBOC-mediated vasoactivity have not been fully elucidated. However, regardless of mechanisms involved, observations from preclinical and clinical studies indicate that the HBOC-mediated vasoconstriction/blood pressure elevation can be attenuated by conventional and newer anti-hypertensive agents and vasodilators. These include calcium channel blockers, nitrovasodilators, ACE inhibitors, selective PDE inhibitors and inhalation of gaseous NO or nebulized nitrites. There is little information available regarding the safety and effectiveness of these agents when used for treatment of HBOC-mediated vasoconstriction and BP elevation. In this review we identify some potentially useful pharmacologic agents and discuss potential issues involved in their use with HBOCs.

Today, allogeneic donor blood transfusion has evolved as a life-saving treatment for many acute anemic conditions. In developed countries, safe donor blood supply is generally adequate for routine clinical demands.