Polynitroxyl hemoglobin: a pharmacokinetic study of covalently bound nitroxides to hemoglobin platforms
Introduction
Hemoglobin (Hb) is both an oxygen carrier and a pro-oxidant participating in complex redox processes that involve heme iron oxidation state changes. The oxidation of iron centers generates ferric iron contained in methemoglobin (Hb FeIII) and potentially cytotoxic agents, including ferrylhemoglobin (Hb FeIV), hemichromes, heme degradation products, free heme, and iron [1]. Additionally, the process of Hb iron oxidation triggers cascades that result in the sequential overproduction of reactive oxygen species/reactive nitrogen species (ROS/RNS) such as superoxide radical (O2·−), hydrogen peroxide (H2O2), hydroxyl radical (OH·), and peroxynitrite (ONOO−), which is the result of O2·− reacting with nitric oxide (NO·) [2]. Accumulation of non-oxygen-carrying Hb FeIII readily generates a highly reactive Hb FeIV species resulting from a pseudoperoxidase cycle driven by H2O2. Persistence of Hb FeIV can lead to heme release, which is capable of mediating further cellular damage, particularly toward the vascular endothelium [3]. Hb autoxidation rates may be aggravated by differing chemical modifications. For example, glutaraldehyde-polymerized bovine Hb accounted for as much as 33% conversion of Hb to Hb FeIII in the plasma of sheep 24 h post-exchange transfusion [4].
Cellular Hb exists in a confined physiological milieu consisting of enzymes present to protect Hb from internal oxidative insult as well as to protect the external physiological environment from the pro-oxidative nature of Hb. Methemoglobin reductase, superoxide dismutase (SOD), catalase, and glutathione peroxidase function in concert within red blood cells (RBCs) to maintain an acceptable level of safety through redox cycling between ferrous Hb (Hb FeII) and Hb FeIII and to neutralize ROS, including O2·− and H2O2 [2], [3]. In contrast hemoglobin-based oxygen carriers (HBOCs) are cell-free Hb’s, chemically modified via various methods of intramolecular cross-linking, intermolecular polymerization, and surface conjugation, and are generally absent of RBC protective enzymes [5], [6].
Extensive efforts have been directed at the removal of tetrameric Hb (64 kDa) from HBOC preparations to prevent extravasation, limit endothelial layer interactions, and ultimately minimize the HBOC/NO· interaction. Several HBOC products are in various stages of clinical trial for critical care applications; however, safety concerns have been voiced by regulatory authorities and government agencies about whether they are safe enough for use in very sick, elderly, or stressed (e.g., trauma) individuals. In vitro data suggest that transfusion of HBOCs may predispose patients to irreversible oxidative processes, especially in situations of ischemia followed by HBOC tissue reperfusion [7], [8], [9], [10], thus leading to a potentially complex scenario in which HBOC administration contributes to an amplified cascade of ROS generation [11]. ROS, including O2·−, OH·, and ONOO−, and oxidative heme iron complexes such as Hb FeIV lead to peroxidation of lipids, carbohydrates, nucleic acids, proteins, and, ultimately, irreversible tissue damage [12], [13], [14] with probable contribution to early and late multiple organ failure.
The current clinical development of HBOCs ultimately involves hypoxia/resuscitation, or ischemia/reperfusion (I/R), during which tissues are exposed to significant levels of oxidative stress. Preclinical in vitro and in vivo models of I/R indicate that certain HBOCs may lead to an unrecoverable cascade of oxidative events and a failure of therapeutic intent [15], [16]. The failure in early and late phase human trials with diaspirin cross-linked hemoglobin in the treatment of ischemic stroke and traumatic injury raised significant safety concerns [17], [18].
To avoid the potential of HBOC-mediated oxidative stress and I/R injury, copolymerized SOD and catalase with HBOCs is being developed [19], [20], [21], [22]. An alternative to this approach is covalently linking antioxidant enzyme mimetic nitroxides to HBOCs. Nitroxides are a class of synthetic stable free radicals which demonstrate varying degrees of SOD mimetic activity [23], [24]. In addition to SOD activity, it has been shown that nitroxides and heme iron together demonstrate catalase-like activity [25]. Furthermore, nitroxides possess additional catalytic activities in the inhibition of nitration induced by ONOO− [26], [27]. In addition to antioxidant mimetic activities, certain nitroxides demonstrate significant hemodynamic effects caused in part by systemic vascular dilation [28]. Thus, covalent linking of nitroxides (i.e., polynitroxylation) to HBOCs could provide a broad spectrum of I/R protective activity similar to that of copolymerized SOD-catalase-HBOCs described above. We have previously demonstrated that polynitroxylation of HBOCs contributed to the reduction of HBOC-induced mean arterial pressure and peripheral vascular resistance elevation leading to increased cardiac index and tissue perfusion, all of which may have contributed to observed increases in duration of resuscitative action in our rodent model of hemorrhagic shock/resuscitation [29], [30].
The present study provides pharmacokinetic (PK) data in support of pharmacodynamic (PD) observations made after administration of polynitroxylated Hb’s. In the current study we have (1) examined how in vivo vascular exposure of the nitroxides is affected by covalent binding versus physically mixing them with HBOCs and (2) evaluated the influence of HBOC molecular size on covalently bound nitroxides (in which one HBOC is void of tetrameric Hb and the other is predominantly tetrameric Hb). Results are presented to emphasize that the covalent binding and not the size of the HBOC is the major contributing factor to prolonged in vivo vascular exposure of the nitroxide. Therapeutic potential of HBOC polynitroxylation is discussed.
Section snippets
Preparation of chemically modified hemoglobin
Stroma-free hemoglobin (SFHb) was isolated and purified from outdated human red blood cells obtained from the American Red Cross (Los Angeles, CA, USA). Intramolecular cross-linking of SFHb was subsequently performed according to Walder et al. [31] using the bifunctional linking agent bis(3,5-dibromosalicyl) fumarate (DBBF) generously provided by The Blood Research Detachment of the Walter Reed Army Institute of Research (Washington, DC, USA). DBBF was reacted with SFHb in 50 mM phosphate
Characterization of HBOC solutions
The physicochemical properties of modified hemoglobin solutions utilized in experimentation are presented in Table 1. XLHb and PNH represent, predominantly, tetrameric hemoglobin with PNH bearing 12 mM covalently bound AcTPO. Determinations of Mw by size-exclusion chromatography coupled to light scattering indicate a small fraction of intermolecular cross-linked product within the XLHb and PNH samples (Mw 78.8 ± 13 and 82.2 ± 9.0 kDa, respectively). The deviation from the MW of SFHb (64 kDa)
Discussion
Current HBOC development is directed toward the reduction of Hb extravasation by cross-linking, polymerization, and conjugation to form-modified Hb of increased molecular size. However, the intrinsic intravascular toxicity of cell-free Hb is still a concern. By itself, pro-oxidant heme lacks the naturally occurring enzymatic mechanisms to regulate ROS generation; the antioxidants that maintain systemic equilibrium and prevent overproduction of ROS during periods of resuscitation/reperfusion are
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