Forum: therapeutic applications of reactive oxygen and nitrogen species in human disease
Pyridoxalated hemoglobin polyoxyethylene: a nitric oxide scavenger with antioxidant activity for the treatment of nitric oxide-induced shock

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Abstract

Hemoglobins modified for therapeutic use as either hemoglobin-based oxygen carriers or scavengers of nitric oxide are currently being evaluated in clinical trials. One such product, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), is a human-derived and chemically modified hemoglobin that has yielded promising results in Phase II clinical trials, and is entering a pivotal Phase III clinical trial for the treatment of shock associated with systemic inflammatory response syndrome (SIRS). Shock associated with SIRS is a NO-induced shock. PHP, a new mechanism-based therapy, has been demonstrated in clinical trials to have the expected hemodynamic activity of raising blood pressure and reducing catecholamine use, consistent with its mechanism of action as a NO scavenger. PHP is conjugated with polyoxyethylene, which results in a surface-decorated molecule with enhanced circulation time and stability as well as in attachment of soluble red blood cell enzymes, including catalase and superoxide dismutase. PHP thus contains an antioxidant profile similar to the intact red blood cell and is therefore resistant to both initial oxidative modification by oxidants such as hydrogen peroxide and subsequent ferrylhemoglobin formation. These studies suggest both that the redox activity of modified hemoglobins can be attenuated and that modified hemoglobins containing endogenous antioxidants, such as PHP, may have reduced pro-oxidant potential. These antioxidant properties, in addition to the NO-scavenging properties, may allow the use of PHP in other indications in which excess NO, superoxide, or hydrogen peroxide is involved, including ischemia-reperfusion injury and hemorrhagic shock.

Introduction

Hemoglobin-based therapeutics are currently under development for a wide range of applications. These include oxygen-carrying fluids intended for blood replacement during surgery and hemorrhagic shock, enhancers of radiation and chemotherapy, and scavengers of nitric oxide [1], [2], [3]. Hemoglobin-based therapeutics are produced by modification of ultrapurified or stroma-free hemoglobin using a variety of intermolecular or intramolecular reactions. The class of hemoglobin molecules can be characterized by the type of modification and can be grouped into one of the following categories: cross-linked, conjugated, or polymerized [4], [5], [6]. In addition, liposomal encapsulation of unmodified or modified hemoglobin is under development as a hemoglobin-based oxygen carrier [3]. These modifications are generally designed to stabilize the protein and modulate oxygen binding (P50) to acceptable physiological levels, as well as to decrease renal toxicity. Due to the presence of both the heme and iron moieties, potential toxicities have been postulated with in vivo use of these products [7], [8]. These toxicities include rapid autoxidation of cell-free hemoglobin following free radical interactions, hemoglobin-mediated vasoconstriction, and reactions of hemoglobins with cellular oxidants such as hydrogen peroxide, resulting in production of both methemoglobin (Fe3+) and ferrylhemoglobin (Fe4+), a strong oxidant reported to cross-link proteins and peroxidize lipids. In addition, the complex interplay of reactive species including superoxide, nitric oxide, and peroxynitrite, whose levels will be influenced by reaction with hemoglobin, can potentially perturb the balance between pro- and antioxidant processes normally present in the vasculature [9].

Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) is a chemically modified, human-derived hemoglobin currently in clinical trials as a nitric oxide (NO) scavenger for the treatment of shock associated with systemic inflammatory response syndrome (SIRS), a subset of NO-induced shock [10]. NO-induced shock occurs in proinflammatory states that can be the result of infection or other etiologies such as burns, pancreatitis, hemodialysis, and cytokine therapies. These proinflammatory disorders are collectively referred to as systemic inflammatory response syndrome (SIRS) [11]. NO-induced shock is characterized by clinical hypotension (acute low blood pressure) and contributes to significant morbidity (multiple organ failure) and high mortality [12]. The role of NO in various forms of shock [13] and sepsis [14] has been the subject of recent reviews. In addition to overproduction of NO, patients with septic shock, a subset of NO-induced shock, have decreased antioxidant status [15], increased lipid peroxidation [15], and increased xanthine oxidase activity [16], suggesting a role for oxidative stress in shock and multiple organ failure.

These findings, in conjunction with concerns regarding hemoglobin-mediated production of or participation in oxygen radical-mediated events, suggest that a hemoglobin-based NO scavenger with antioxidant activity would be a candidate for treatment of the hypotension associated with NO-induced shock. In this regard, the chemical modification described below results in a surface-decorated hemoglobin, PHP, which contains endogenous catalase-superoxide dismutase (SOD) activities [17], [18], [19], [20], [21]. PHP, in several in vitro models, has been demonstrated to display decreased reactivity toward hydrogen peroxide (H2O2)-mediated oxidations when compared to unmodified HbA or αα-crosslinked hemoglobin [17], [18], [19], [20]. These results indicate that the redox reactivity of hemoglobin preparations can be manipulated and, in addition, suggest that modified hemoglobins containing endogenous antioxidants may reduce the pro-oxidant potential of hemoglobin.

Section snippets

Chemical characterization

PHP was initially developed in the 1980s as an oxygen carrier [22], [23]. PHP is produced from erythrocyte lysate obtained from outdated human red blood cells. The purification method used for production provides for separation of the hemoglobin from red cell stroma and potential adventitious agents, but not from several of the enzymes normally associated with hemoglobin in red blood cells [21]. The purified hemoglobin is then subjected to pyridoxalation in order to reduce the oxygen affinity.

Conclusion

The pro-oxidant potential of modified hemoglobins is currently the focus of intense interest as these products are being evaluated in clinical trials as hemoglobin-based oxygen carriers and nitric oxide scavengers. One such product, PHP, is a human-derived and chemically modified hemoglobin preparation currently in clinical trials as a NO scavenger for the treatment of shock associated with SIRS. This new mechanism-based therapy has yielded promising results in Phase II clinical trials. PHP, in

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    Christopher T. Privalle obtained his Ph.D. in Pharmacology from the University of Wisconsin, Madison. Following a postdoctoral fellowship in the laboratory of Irwin Fridovich, he became a research assistant professor in the Department of Biochemistry at Duke University. He joined Apex Bioscience, Inc. in 1993 and is currently the Director of Research, Biochemistry.

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