A Hemoglobin-Based Multifunctional Therapeutic: Polynitroxylated Pegylated Hemoglobin

Polynitroxylated pegylated hemoglobin (PNPH) as a multifunctional therapeutic takes advantage of the ability of hemoglobin (Hb) to transport oxygen, the antioxidative stress activities from the redox coupling of nitroxide and heme iron, and the hypercolloid properties of pegylation. The published preclinical data demonstrating that PNPH acts as a neurovascular protective multifunctional therapeutic in an animal model simulating prehospital resuscitation of traumatic brain injury (TBI) with hemorrhagic shock (HS) are reviewed. Preliminary results on the potential utility of PNPH for neurovascular protection in thrombolytic stroke therapy and for correction of vascular dysfunction through transfusion in sickle-cell disease (SCD) are also discussed. We hypothesize that with PNPH, Hb has more than been tamed--it has become a therapeutic and not just a nontoxic extracellular oxygen carrier--and that successful PNPH development as a multifunctional therapeutic that protects the neurovasculature and reduces oxidative stress may represent a paradigm shift in transfusion and critical care medicine, which may meet a number of unmet medical needs resulting from oxidative stress and inadequate blood flow, such as HS, TBI, SCD, and stroke

Polynitroxyl Albumin and Albumin Therapy After Pediatric Asphyxial Cardia Arrest: Effects on Cerebral Blood Flow and Neurologic Outcome

Postresuscitation cerebral blood flow (CBF) disturbances and generation of reactive oxygen species likely contribute to impaired neurologic outcome after pediatric cardiac arrest (CA). Hence, we determined the effects of the antioxidant colloid polynitroxyl albumin (PNA) versus albumin or normal saline (NS) on CBF and neurologic outcome after asphyxial CA in immature rats. We induced asphyxia for 9 minutes in male and female postnatal day 16 to 18 rats randomized to receive PNA, albumin, or NS at resuscitation from CA or sham surgery. Regional CBF was measured serially from 5 to 150 minutes after resuscitation by arterial spin-labeled magnetic resonance imaging. We assessed motor function (beam balance and inclined plane), spatial memory retention (water maze), and hippocampal neuronal survival. Polynitroxyl albumin reduced early hyperemia seen 5 minutes after CA. In contrast, albumin markedly increased and prolonged hyperemia. In the delayed period after resuscitation (90 to 150 minutes), CBF was comparable among groups. Both PNA- and albumin-treated rats performed better in the water maze versus NS after CA. This benefit was observed only in males. Hippocampal neuron survival was similar between injury groups. Treatment of immature rats with PNA or albumin resulted in divergent acute changes in CBF, but both improved spatial memory retention in males after asphyxial CA

Polynitroxylated Pegylated Hemoglobin: A Novel Neuroprotective Hemoglobin for Acute Volume-Limited Fluid Resuscitation After Combined Traumatic Brain Injury and Hemorrhagic Hypotension in Mice

Objective: Resuscitation of hemorrhagic hypotension after traumatic brain injury is challenging. A hemoglobin-based oxygen carrier may offer advantages. The novel therapeutic hemoglobin-based oxygen carrier, polynitroxylated pegylated hemoglobin (PNPH), may represent a neuroprotective hemoglobin-based oxygen carrier for traumatic brain injury resuscitation.Hypotheses: 1) PNPH is a unique non-neurotoxic hemoglobin-based oxygen carrier in neuronal culture and is neuroprotective in in vitro neuronal injury models. 2) Resuscitation with PNPH would require less volume to restore mean arterial blood pressure than lactated Ringer\u27s or Hextend and confer neuroprotection in a mouse model of traumatic brain injury plus hemorrhagic hypotension.Design: Prospective randomized, controlled experimental study.Setting: University center.Measurements and Main Results: In rat primary cortical neuron cultures, control bovine hemoglobin was neurotoxic (lactate dehydrogenase release; 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyltetrazolium bromide assay) at concentrations from 12.5 to 0.625 [mu]M, whereas polyethylene glycol-conjugated hemoglobin showed intermediate toxicity. PNPH was not neurotoxic (p \u3c .05 vs. bovine hemoglobin and polyethylene glycol hemoglobin; all concentrations). PNPH conferred neuroprotection in in vitro neuronal injury (glutamate/glycine exposure and neuronal stretch), as assessed via lactate dehydrogenase and 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyltetrazolium bromide (all p \u3c .05 vs. control). C57BL6 mice received controlled cortical impact followed by hemorrhagic hypotension (2 mL/100 g, mean arterial blood pressure ~35-40 mm Hg) for 90 min. Mice were resuscitated (mean arterial blood pressure \u3e50 mm Hg for 30 min) with lactated Ringer\u27s, Hextend, or PNPH, and then shed blood was reinfused. Mean arterial blood pressures, resuscitation volumes, blood gasses, glucose, and lactate were recorded. Brain sections at 7 days were examined via hematoxylin and eosin and Fluoro-Jade C (identifying dying neurons) staining in CA1 and CA3 hippocampus. Resuscitation with PNPH or Hextend required less volume than lactated Ringer\u27s (both p \u3c .05). PNPH but not Hextend improved mean arterial blood pressure vs. lactated Ringer\u27s (p \u3c .05). Mice resuscitated with PNPH had fewer Fluoro-Jade C positive neurons in CA1 vs. Hextend and lactated Ringer\u27s, and CA3 vs. Hextend (p \u3c .05).Conclusions: PNPH is a novel neuroprotective hemoglobin-based oxygen carrier in vitro and in vivo that may offer unique advantages for traumatic brain injury resuscitation

Polynitroxylated Hemoglobin as a Multifunctional Therapeutic for Critical Care and Transfusion Medicine

Polynitroxylation (PN) technology is a chemical modification process, which covalently links multiple nitroxides to various macromolecules, including hemoglobin-based oxygen carriers (HBOCs). Like free nitroxides, PN products possess superoxide dismutase mimetic activity. In addition, when redox coupled with heme centers, polynitroxylated hemoglobins have catalase and peroxidase mimetic activities. PN of HBOCs allows the therapeutic benefits of nitroxides to be target delivered intravascularly. More significantly, it corrects the intrinsic toxicity of HBOCs and adds therapeutic neurovascular protective, anti-oxidant, and anti-inflammatory activities to these products. Results from multiple pre-clinical efficacy studies suggest that PN improves oxygen delivery by HBOCs through the correction of inadequate blood flow via prevention of both nitric oxide depletion and reperfusion/inflammation injuries after ischemia. PN of conjugated hemoglobins with hyper-colloid effects, such as polyethylene glycol or dextran conjugated hemoglobins, converts these HBOCs into small volume multifunctional hemodynamic therapeutics. Polynitroxylated pegylated hemoglobin (PNPH), in particular, has the potential to be a high-therapeutic-index next-generation HBOC for treatment of civilian un-met medical needs and military combat casualty care. Efficacy of PNPH has been demonstrated in models of traumatic brain injury with hemorrhagic shock, ischemic stroke, and sickle cell disease. This chapter traces the research leading to the development of PNPH. Read More: http://www.worldscientific.com/doi/abs/10.1142/9789814472869_000

Polynitroxylated Pegylated Hemoglobin (PNPH) A Nanomedicine for Critical Care and Transfusion

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

Use of Antimetastatic SOD3-Mimetic Albumin as a Primer in Triple Negative Breast Cancer

Of the deaths attributed to cancer, 90% are due to metastasis. Treatments that prevent or cure metastasis remain elusive. Low expression of extracellular superoxide dismutase (EcSOD or SOD3) has been associated with poor outcomes and increased metastatic potential in multiple types of cancer. Here, we characterize the antimetastatic therapeutic mechanisms of a macromolecular extracellular SOD3-mimetic polynitroxyl albumin (PNA, also known as VACNO). PNA is macromolecular human serum albumin conjugated with multiple nitroxide groups and acts as an SOD-mimetic. Here we show that PNA works as a SOD3-mimetic in a highly metastatic 4T1 mouse model of triple negative breast cancer (TNBC). In vitro, PNA dose dependently inhibited 4T1 proliferation, colony formation, and reactive oxygen species (ROS) formation. In vivo, PNA enhanced reperfusion time in the hypoxic cores of 4T1 tumors as measured by ultrasound imaging. Furthermore, PNA enhanced ultrasound signal intensity within the cores of the 4T1 tumors, indicating PNA can increase blood flow and blood volume within the hypoxic cores of tumors. Lung metastasis from 4T1 flank tumor was inhibited by PNA in the presence or absence of doxorubicin, a chemotherapy agent that produces superoxide and promotes metastasis. In a separate study, PNA increased the survival of mice with 4T1 flank tumors when used in conjunction with three standard chemotherapy drugs (paclitaxel, doxorubicin, and cyclophosphamide), as compared to treatment with chemotherapy alone. In this study, PNA-increased survival was also correlated with reduction of lung metastasis. These results support the hypothesis that PNA works through the inhibition of extracellular superoxide/ROS production leading to the conversion of 4T1 cells from a metastatic tumorigenic state to a cytostatic state. These findings support future clinical trials of PNA as an antimetastatic SOD3-mimetic drug to increase overall survival in TNBC patients

Unusual Peroxidase Activity of Polynitroxylated Pegylated Hemoglobin: Elimination of H\u3csub\u3e2\u3c/sub\u3eO\u3csub\u3e2\u3c/sub\u3e Coupled with Intramolecular Oxidation of Nitroxides

Polynitroxylated hemoglobin (Hb(AcTPO)12) has been developed as a hemoglobin-based oxygen carrier. While Hb(AcTPO)12 has been shown to exert beneficial effects in a number of models of oxidative injury, its peroxidase activity has not been characterized thus far. In the blood stream, Hb(AcTPO)12 undergoes reduction by ascorbate to its hydroxylamine form Hb(AcTPOH)12. Here we report that Hb(AcTPOH)12exhibits peroxidase activity where H2O2 is utilized for intramolecular oxidation of its TPOH residues to TPO. This represents an unusual redox-catalytic mechanism whereby reduction of H2O2 is achieved at the expense of reducing equivalents of ascorbate converted into those of Hb(AcTPOH)12, a new propensity that cannot be directly associated with ascorbate

Normoxic versus Hyperoxic Resuscitation in Pediatric Asphyxia Cardiac Arrest: Effects on Oxidative Stress

Objective: To determine the effects of normoxic vs. hyperoxic resuscitation on oxidative stress in a model of pediatric asphyxial cardiac arrest.Design: Prospective, interventional study.Setting: University research laboratory.Subjects: Postnatal day 16-18 rats (n = 5 per group).Interventions: Rats underwent asphyxial cardiac arrest for 9 min. Rats were randomized to receive 100% oxygen, room air, or 100% oxygen with polynitroxyl albumin (10 mL[middle dot]kg-1 intravenously, 0 and 30 min after resuscitation) for 1 hr from the start of cardiopulmonary resuscitation. Shams recovered in 100% oxygen or room air after surgery.Measurements and Main Results: Physiological variables were recorded at baseline to 1 hr after resuscitation. At 6 hrs after asphyxial cardiac arrest, levels of reduced glutathione and protein-thiols (fluorescent assay), activities of total superoxide dismutase and mitochondrial manganese superoxide dismutase (cytochrome c reduction method), manganese superoxide dismutase expression (Western blot), and lipid peroxidation (4-hydroxynonenal Michael adducts) were evaluated in brain tissue homogenates. Hippocampal 3-nitrotyrosine levels were determined by immunohistochemistry 72 hrs after asphyxial cardiac arrest. Survival did not differ among groups. At 1 hr after resuscitation, Pao2, pH, and mean arterial pressure were decreased in room air vs. 100% oxygen rats (59 +/- 3 vs. 465 +/- 46 mm Hg, 7.36 +/- 0.05 vs. 7.42 +/- 0.03, 35 +/- 4 vs. 45 +/- 5 mm Hg; p \u3c .05). Rats resuscitated with 100% oxygen had decreased hippocampal reduced glutathione levels vs. sham (15.3 +/- 0.4 vs. 20.9 +/- 4.1 nmol[middle dot]mg protein-1; p \u3c .01). Hippocampal manganese superoxide dismutase activity was significantly increased in 100% oxygen rats vs. sham (14 +/- 2.4 vs. 9.5 +/- 1.6 units[middle dot]mg protein-1, p \u3c .01), with no difference in protein expression of manganese superoxide dismutase. Room air and 100% oxygen plus polynitroxyl albumin groups had hippocampal reduced glutathione and manganese superoxide dismutase activity levels comparable with sham. Protein thiol levels were unchanged across groups. Compared with all other groups, rats receiving 100% oxygen had increased immunopositivity for 3-nitrotyrosine in the hippocampus and increased lipid peroxidation in the cortex.Conclusions: Resuscitation with 100% oxygen leads to increased oxidative stress in a model that mimics pediatric cardiac arrest. This may be prevented by using room air or giving an antioxidant with 100% oxygen resuscitation