Inhibition of metastatic brain cancer in Sonic Hedgehog medulloblastoma using caged nitric oxide albumin nanoparticles.

Medulloblastoma is a tumor of the cerebellum that metastasizes to the leptomeninges of the central nervous system (CNS), including to forebrain and to spinal cord. The inhibitory effect of polynitroxylated albumin (PNA), a caged nitroxide nanoparticle, on leptomeningeal dissemination and metastatic tumor growth was studied in a Sonic Hedgehog transgenic mouse model. PNA treated mice showed an increased lifespan with a mean survival of 95 days (n = 6, P<0.05) compared with 71 days in controls. In primary tumors, proliferation was significantly reduced and differentiation was significantly increased (P<0.001) as shown by Ki-67+ and NeuN+ immunohistochemistry, while cells in spinal cord tumors appeared unaffected. Yet, histochemical analysis of metastatic tumor in spinal cord showed that the mean total number of cells in spinal cord was significantly reduced in mice treated with PNA compared to albumin vehicle (P<0.05). Examination of various levels of the spinal cord showed that PNA treated mice had significantly reduced metastatic cell density in the thoracic, lumbar and sacral spinal cord levels (P<0.05), while cell density in the cervical region was not significantly changed. The mechanism by which PNA may exert these effects on CNS tumors is discussed.

Polynitroxylated PEGylated hemoglobin protects pig brain neocortical gray and white matter after traumatic brain injury and hemorrhagic shock.

Polynitroxylated PEGylated hemoglobin (PNPH, aka SanFlow) possesses superoxide dismutase/catalase mimetic activities that may directly protect the brain from oxidative stress. Stabilization of PNPH with bound carbon monoxide prevents methemoglobin formation during storage and permits it to serve as an anti-inflammatory carbon monoxide donor. We determined whether small volume transfusion of hyperoncotic PNPH is neuroprotective in a porcine model of traumatic brain injury (TBI) with and without accompanying hemorrhagic shock (HS). TBI was produced by controlled cortical impact over the frontal lobe of anesthetized juvenile pigs. Hemorrhagic shock was induced starting 5 min after TBI by 30 ml/kg blood withdrawal. At 120 min after TBI, pigs were resuscitated with 60 ml/kg lactated Ringer's (LR) or 10 or 20 ml/kg PNPH. Mean arterial pressure recovered to approximately 100 mmHg in all groups. A significant amount of PNPH was retained in the plasma over the first day of recovery. At 4 days of recovery in the LR-resuscitated group, the volume of frontal lobe subcortical white matter ipsilateral to the injury was 26.2 ± 7.6% smaller than homotypic contralateral volume, whereas this white matter loss was only 8.6 ± 12.0% with 20-ml/kg PNPH resuscitation. Amyloid precursor protein punctate accumulation, a marker of axonopathy, increased in ipsilateral subcortical white matter by 132 ± 71% after LR resuscitation, whereas the changes after 10 ml/kg (36 ± 41%) and 20 ml/kg (26 ± 15%) PNPH resuscitation were not significantly different from controls. The number of cortical neuron long dendrites enriched in microtubules (length >50 microns) decreased in neocortex by 41 ± 24% after LR resuscitation but was not significantly changed after PNPH resuscitation. The perilesion microglia density increased by 45 ± 24% after LR resuscitation but was unchanged after 20 ml/kg PNPH resuscitation (4 ± 18%). Furthermore, the number with an activated morphology was attenuated by 30 ± 10%. In TBI pigs without HS followed 2 h later by infusion of 10 ml/kg LR or PNPH, PNPH remained neuroprotective. These results in a gyrencephalic brain show that resuscitation from TBI + HS with PNPH protects neocortical gray matter, including dendritic microstructure, and white matter axons and myelin. This neuroprotective effect persists with TBI alone, indicating brain-targeting benefits independent of blood pressure restoration.

Resuscitation with macromolecular superoxide dismutase/catalase mimetic polynitroxylated PEGylated hemoglobin offers neuroprotection in guinea pigs after traumatic brain injury combined with hemorrhage shock.

BACKGROUND: Polynitroxylated PEGylated hemoglobin (PNPH, aka SanFlow) possesses superoxide dismutase/catalase mimetic activities that may directly protect the brain from oxidative stress. Stabilization of PNPH with bound carbon monoxide prevents methemoglobin formation during storage and permits it to serve as a carbon monoxide donor. We determined whether small volume transfusion of hyperoncotic PNPH is neuroprotective in a polytrauma model of traumatic brain injury (TBI) plus hemorrhagic shock. Guinea pigs were used because, like humans, they do not synthesize their own ascorbic acid, which is important in reducing methemoglobin. RESULTS: TBI was produced by controlled cortical impact and was followed by 20 mL/kg hemorrhage to a mean arterial pressure (MAP) of 40 mmHg. At 90 min, animals were resuscitated with 20 mL/kg lactated Ringer's solution or 10 mL/kg PNPH. Resuscitation with PNPH significantly augmented the early recovery of MAP after hemorrhagic shock by 10-18 mmHg; whole blood methemoglobin was only 1% higher and carboxyhemoglobin was 2% higher. At 9 days of recovery, unbiased stereology analysis revealed that, compared to animals resuscitated with lactated Ringer's solution, those treated with PNPH had significantly more viable neurons in the hippocampus CA1 + 2 region (59 ± 10% versus 87 ± 18% of sham and naïve mean value) and in the dentate gyrus (70 ± 21% versus 96 ± 24%; n = 12 per group). CONCLUSION: PNPH may serve as a small-volume resuscitation fluid for polytrauma involving TBI and hemorrhagic shock. The neuroprotection afforded by PNPH seen in other species was sustained in a species without endogenous ascorbic acid synthesis, thereby supporting potential translatability for human use.

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.

Transfusion of Polynitroxylated Pegylated Hemoglobin Stabilizes Pial Arterial Dilation and Decreases Infarct Volume After Transient Middle Cerebral Artery Occlusion.

BACKGROUND: Polynitroxylation of hemoglobin confers superoxide dismutase-mimetic and peroxidase activity and may protect from reperfusion injury in addition to facilitating oxygen transport. We determined whether transfusion of polynitroxylated PEGylated hemoglobin (PNPH) is protective in the rat filament model of 2 hours of middle cerebral artery occlusion (MCAO). METHODS AND RESULTS: Transfusion of 10 mL/kg of PNPH at 20 minutes of MCAO reduced infarct volume by over 70% (n=10). To determine whether PNPH might act by promoting vasodilation, pial arteriolar diameter in the distal MCA border region was measured in closed cranial windows. With no transfusion, MCAO induced an initial dilation (36±2% ±SE) that subsided by 2 hours (5±4%; n=8). With PNPH transfusion at 20 minutes of MCAO, the initial dilation (31±3%) was better maintained at 2 hours (21±4%; n=7; P<0.02). Delaying PNPH transfusion until 90 minutes of MCAO increased perfusion in the border region from 48±6% of the preischemic baseline to 67±8% (n=8; P<0.005). The effect of PNPH transfusion after reperfusion was also tested. Compared with the control median hemispheric infarct volume of 22% (13% to 34% interquartiles; n=15), infarct volume was reduced to 7% (3% to 13%; n=14 P<0.05) when PNPH was transfused at 4 hours after MCAO (2 hours of reperfusion) but not significantly when transfused at 6 hours (8%; 3% to 35%; n=14) or at 8 hours (12%; 10% to 25%; n=14) after MCAO. CONCLUSIONS: PNPH transfusion has a significant therapeutic window for protection during and after transient MCAO and may act, in part, by stabilizing vascular function and improving collateral blood flow.

Polynitroxylated Pegylated Hemoglobin-A Novel, Small Volume Therapeutic for Traumatic Brain Injury Resuscitation: Comparison to Whole Blood and Dose Response Evaluation.

Resuscitation with polynitroxylated pegylated hemoglobin (PNPH), a pegylated bovine hemoglobin decorated with nitroxides, eliminated the need for fluid administration, reduced intracranial pressure (ICP) and brain edema, and produced neuroprotection in vitro and in vivo versus Lactated Ringer's solution (LR) in experimental traumatic brain injury (TBI) plus hemorrhagic shock (HS). We hypothesized that resuscitation with PNPH would improve acute physiology versus whole blood after TBI+HS and would be safe and effective across a wide dosage range. Anesthetized mice underwent controlled cortical impact and severe HS to mean arterial pressure (MAP) of 25-27 mm Hg for 35 min, then were resuscitated with PNPH, autologous whole blood, or LR. Markers of acute physiology, including mean arterial blood pressure (MAP), heart rate (HR), blood gases/chemistries, and brain oxygenation (PbtO2), were monitored for 90 min on room air followed by 15 min on 100% oxygen. In a second experiment, the protocol was repeated, except mice were resuscitated with PNPH with doses between 2 and 100 mL/kg. ICP and 24 h %-brain water were evaluated. PNPH-resuscitated mice had higher MAP and lower HR post-resuscitation versus blood or LR (p < 0.01). PNPH-resuscitated mice, versus those resuscitated with blood or LR, also had higher pH and lower serum potassium (p < 0.05). Blood-resuscitated mice, however, had higher PbtO2 versus those resuscitated with LR and PNPH, although PNPH had higher PbtO2 versus LR (p < 0.05). PNPH was well tolerated across the dosing range and dramatically reduced fluid requirements in all doses-even 2 or 5 mL/kg (p < 0.001). ICP was significantly lower in PNPH-treated mice for most doses tested versus in LR-treated mice, although %-brain water did not differ between groups. Resuscitation with PNPH, versus resuscitation with LR or blood, improved MAP, HR, and ICP, reduced acidosis and hyperkalemia, and was well tolerated and effective across a wide dosing range, supporting ongoing pre-clinical development of PNPH for TBI resuscitation.

Polynitroxylated-pegylated hemoglobin attenuates fluid requirements and brain edema in combined traumatic brain injury plus hemorrhagic shock in mice.

UNLABELLED: Polynitroxylated-pegylated hemoglobin (PNPH), a bovine hemoglobin decorated with nitroxide and polyethylene glycol moieties, showed neuroprotection vs. lactated Ringer's (LR) in experimental traumatic brain injury plus hemorrhagic shock (TBI+HS). HYPOTHESIS: Resuscitation with PNPH will reduce intracranial pressure (ICP) and brain edema and improve cerebral perfusion pressure (CPP) vs. LR in experimental TBI+HS. C57/BL6 mice (n=20) underwent controlled cortical impact followed by severe HS to mean arterial pressure (MAP) of 25 to 27 mm Hg for 35 minutes. Mice (n=10/group) were then resuscitated with a 20 mL/kg bolus of 4% PNPH or LR followed by 10 mL/kg boluses targeting MAP>70 mm Hg for 90 minutes. Shed blood was then reinfused. Intracranial pressure was monitored. Mice were killed and %brain water (%BW) was measured (wet/dry weight). Mice resuscitated with PNPH vs. LR required less fluid (26.0±0.0 vs. 167.0±10.7 mL/kg, P<0.001) and had a higher MAP (79.4±0.40 vs. 59.7±0.83 mm Hg, P<0.001). The PNPH-treated mice required only 20 mL/kg while LR-resuscitated mice required multiple boluses. The PNPH-treated mice had a lower peak ICP (14.5±0.97 vs. 19.7±1.12 mm Hg, P=0.002), higher CPP during resuscitation (69.2±0.46 vs. 45.5±0.68 mm Hg, P<0.001), and lower %BW vs. LR (80.3±0.12 vs. 80.9±0.12%, P=0.003). After TBI+HS, resuscitation with PNPH lowers fluid requirements, improves ICP and CPP, and reduces brain edema vs. LR, supporting its development.

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 cardiac 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's 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 µM, whereas polyethylene glycol-conjugated hemoglobin showed intermediate toxicity. PNPH was not neurotoxic (p<.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<.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>50 mm Hg for 30 min) with lactated Ringer's, 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's (both p<.05). PNPH but not Hextend improved mean arterial blood pressure vs. lactated Ringer's (p<.05). Mice resuscitated with PNPH had fewer Fluoro-Jade C positive neurons in CA1 vs. Hextend and lactated Ringer's, and CA3 vs. Hextend (p<.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.

Unusual peroxidase activity of polynitroxylated pegylated hemoglobin: Elimination of H(2)O(2) 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)(12) exhibits peroxidase activity where H(2)O(2) is utilized for intramolecular oxidation of its TPOH residues to TPO. This represents an unusual redox-catalytic mechanism whereby reduction of H(2)O(2) 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.

Resuscitation of traumatic brain injury and hemorrhagic shock with polynitroxylated albumin, hextend, hypertonic saline, and lactated Ringer's: Effects on acute hemodynamics, survival, and neuronal death in mice.

Outcome after traumatic brain injury (TBI) is worsened by hemorrhagic shock (HS), but the optimal resuscitation approach is unclear. In particular, treatment of TBI patients with colloids remains controversial. We hypothesized that resuscitation with the colloids polynitroxylated albumin (PNA) or Hextend (HEX) is equal or superior to resuscitation with the crystalloids hypertonic (3%) saline (HTS) or lactated Ringer's solution (LR) after TBI plus HS in mice. C57/BL6 mice (n = 30) underwent controlled cortical impact (CCI) and 90 min of volume-controlled HS (2 mL/100 g). The mice were randomized to resuscitation with LR, HEX, HTS, or PNA, followed by 30 min of test fluid administration targeting a mean arterial pressure (MAP) of >50 mm Hg. Shed blood was re-infused to target a MAP >70 mm Hg. At 7 days post-insult, hippocampal neuron counts were assessed in hematoxylin and eosin-stained sections to quantify neuronal damage. Prehospital MAP was higher, and prehospital and total fluid requirements were lower in the PNA and HEX groups (p < 0.05 versus HTS or LR). Also, 7-day survival was highest in the PNA group, but was not significantly different than the other groups. Ipsilateral hippocampal CA1 and CA3 neuron loss did not differ between groups. We conclude that the colloids PNA and HEX exhibited more favorable effects on acute resuscitation parameters than HTS or LR, and did not increase hippocampal neuronal death in this model.

Small volume resuscitation with tempol is detrimental during uncontrolled hemorrhagic shock in rats.

BACKGROUND: In a previous study, titration of a hypertonic saline (HTS) solution during severe uncontrolled hemorrhagic shock (UHS) failed to reduce mortality. In a separate study, a novel antioxidant, polynitroxylated albumin (PNA) plus tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), infused during shock increased long-term survival. We hypothesized that combining potent antioxidants with a hypertonic solution during UHS would preserve the logistical advantage of small volume resuscitation and improve survival. METHODS: An UHS outcome model in rats was used. UHS phase I (90 min) included blood withdrawal of 30 ml/kg over 15 min, followed by tail amputation for uncontrolled bleeding. At 20 min, rats were randomized to four groups (n=10 each) for hypotensive resuscitation from 20 to 90 min (mean arterial pressure [MAP] > or = 40 mmHg): HTS/starch group received 7.2% NaCl/10% hydroxyethyl starch; HTS/albumin group received 7.5% NaCl/20% albumin; HTS/PNA group received 7.5% NaCl/20% PNA; HTS/albumin+tempol group received 7.5% NaCl/20% albumin plus tempol. Resuscitation phase II (180 min) included hemostasis, return of shed blood and administration of fluids to restore MAP > or = 80 mmHg. Observation phase III was to 72 h. RESULTS: The total amount of fluid required to maintain hypotensive MAP during HS was low and did not differ between groups (range: 3.4+/-1.9 to 5.3+/-2.5 ml/kg). The rate of fluid administration required was higher in the HTS/albumin+tempol group compared to all other groups (p=0.006). Additional uncontrolled blood loss was highest in the HTS/PNA group (16.2+/-5.7 ml/kg [p=0.01] versus 10.4+/-7.9 ml/kg in the HTS/starch group, 7.7+/-5.2 ml/kg in the HTS/albumin group and 8.2+/-7.1 ml/kg in the HTS/albumin+tempol group). MAP after start of resuscitation in phase I was lower in the HTS/albumin+tempol group than the HTS/albumin or HTS/PNA groups (p<0.01). This group was also less tachycardic. Long-term survival was low in all groups (2 of 10 after HTS/starch and 1 of 10 after HTS/albumin, 3 of 10 after HTS/PNA, 1 of 10 after HTS/albumin+tempol). Median survival time was shortest in the HTS/albumin+tempol group (72 min [CI 34-190]) compared to all other groups (p=0.01). CONCLUSIONS: Despite its benefits in other model systems, free tempol is potentially hazardous when combined with hypertonic fluids. PNA abrogates these deleterious effects on acute mortality but may lead to increased blood loss in the setting of UHS.

Inhibition of sickle red cell adhesion and vasoocclusion in the microcirculation by antioxidants.

In sickle cell anemia (SCA), inflammatory (i.e., intravascular sickling and transient vasoocclusive) events result in chronic endothelial activation. In addition to sickling behavior, sickle (SS) red blood cells exhibit abnormal interaction with the vascular endothelium, which is considered to have an important role in initiation of vasoocclusion. Upregulation of endothelial adhesion molecules caused by oxidants (and cytokines) may lead to increased SS red cell adhesion. We hypothesize that endothelial activation is indispensable in SS red cell adhesion to the endothelium and that antioxidants will have an inhibitory effect on this interaction. We examined the effect of selected antioxidants in ex vivo mesocecum vasculature, a well-established model that allows measurement of hemodynamic parameters and, by intravital microscopy, can allow quantification of adhesion. We tested antioxidant enzymes (SOD and catalase) and an intravascular SOD mimetic, polynitroxyl albumin (PNA), in the presence of platelet-activating factor (PAF); the latter causes endothelial oxidant generation and endothelial activation, which characterize SCA. In ex vivo preparations, PAF not only induced marked endothelial oxidant generation, it also enhanced SS red cell adhesion, resulting in frequent blockage of small-diameter venules. The adhesion, inversely related to venular diameter, and vasoocclusion were markedly inhibited by antioxidants, resulting in improved hemodynamics. PNA, the most effective antioxidant, also abolished SS red cell adhesion in non-PAF-activated preparations. Thus SS red cell adhesion and related vasoocclusion may be ameliorated by antioxidant therapy with a stable and long-acting molecule (e.g., PNA).

Polynitroxyl albumin inhibits inflammation and vasoocclusion in transgenic sickle mice.

Individuals with sickle-cell disease (SCD) and transgenic sickle mice expressing human betaS globin exhibit enhanced reactive oxygen species (ROS) production, vascular inflammation, and episodic vasoocclusion. We hypothesize that reduction of ROS will reduce endothelial-cell activation and adhesion-molecule expression, thereby inhibiting vasoocclusion. To test this hypothesis, we measured endothelial-cell activation, adhesion-molecule expression, and vasoocclusion in sickle mice after administering i.v. polynitroxyl albumin (PNA), a superoxide dismutase and catalase mimetic. Untreated sickle mice, compared with normal mice, showed increased activation of nuclear factor-kappaB (NF-kappaB), an oxidant-sensitive transcription factor, in their lungs, livers, and skin. NF-kappaB activation was increased further in the livers and skin of sickle but not normal mice after hypoxia-reoxygenation. IV administration of PNA inhibited NF-kappaB activation by 60% (P < .01) in the lungs and by 33% (P < .05) in the livers of sickle mice after hypoxia-reoxygenation. PNA also reduced the expression of vascular cell-adhesion molecule-1 (VCAM-1) by 57% in lung (P < .05) and by 33% in liver (P < .05) and reduced the expression of intercellular-adhesion molecule-1 (ICAM-1) by 40% in lung (P < .05) and by 53% in liver (P < .05). PNA inhibited a hypoxia-reoxygenation-induced increase in leukocyte rolling (P < .01) and adhesion (P < .05) in venules of the dorsal skin. Most importantly, PNA completely inhibited hypoxia-reoxygenation-induced vasoocclusion (P < .001). Control albumin had no effect on NF-kappaB, VCAM-1, ICAM-1, rolling, adhesion, or vasoocclusion. We speculate that therapies to reduce oxidative stress will inhibit inflammation and vasoocclusion in SCD.

Polynitroxyl hemoglobin: a pharmacokinetic study of covalently bound nitroxides to hemoglobin platforms.

Adding antioxidant activities to hemoglobin-based oxygen carriers (HBOCs) represents a means of reducing cell-free hemoglobin-mediated oxidative cascades. We have covalently bound nitroxides, a class of antioxidant enzyme mimetics, to HBOCs. The objectives of this study were (1) to evaluate the pharmacokinetic (PK) effects of administering nitroxide covalently bound to HBOCs compared to those of free nitroxide coadministered with HBOCs and (2) to elucidate the effects of differing molecular weight HBOCs on the PK of bound nitroxide in a conscious guinea pig model of 25% blood exchange transfusion. Two HBOC platforms were used, intramolecular cross-linked hemoglobin (XLHb) and dextran polymerized/conjugated XLHb (PolyHb). Polynitroxylation was achieved by reacting 4-(2-bromoacetamido)-2,2,6,6,-tetramethylpiperidine-1-oxyl with XLHb or PolyHb to form polynitroxylated XLHb and polynitroxylated PolyHb, respectively, whereas a physical mixture of XLHb or PolyHb with 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl was prepared to reflect a molar equivalence to HBOC-bound nitroxide. Plasma concentrations of two redox states, nitroxide and hydroxylamine, were determined by electron paramagnetic resonance spectroscopy. Results are presented to illustrate the influence of covalent labeling and HBOC molecular weight on nitroxide PK. The therapeutic potential of polynitroxylation of HBOCs as it relates to observations from the current and previously reported studies is discussed.

Prevention of catecholaminergic oxidative toxicity by 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl and its recycling complex with polynitroxylated albumin, TEMPOL/PNA.

Reactive oxygen species (ROS) generated from dopamine and its oxidation products have been implicated in the pathogenesis and toxicity from treatment of Parkinson's disease-associated autonomic neuropathy, and antioxidant therapies have been proposed as treatment and prophylaxis for this disorder. However, many antioxidants are rapidly and, under physiological conditions, irreversibly oxidized, rendering them redox-inactive. We have examined the potential of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl and polynitroxylated albumin (TEMPOL/PNA), an antioxidant complex that facilitates recycling of inactivated antioxidant to its redox-active form, as a protective agent against the toxicity of the catecholaminergic ROS generator, 6-hydroxydopamine (6-OHDA). TEMPOL/PNA is more effective against depression of activity level by 6-OHDA than the non-recycling antioxidant, TEMPOL, in a murine model of catecholaminergic oxidative damage. TEMPOL/PNA is also less toxic than TEMPOL in mice, allowing administration of higher doses of antioxidant. Both TEMPOL and TEMPOL/PNA give rise to prevention of apoptosis and to translocation of NF-kappaB from the cytoplasm to the nucleus of PC12 cells treated with 6-OHDA, but in vivo, TEMPOL/PNA maintains redox-active blood levels of TEMPOL for almost 5 h, whereas administration of TEMPOL alone results in clearance of blood redox activity within 1 h. PNA enhances the therapeutic index of TEMPOL, and the recycling antioxidant that results from their adjunctive administration may prove useful in disorders involving oxidative stress.