Polynitroxyl Albumin Research Articles

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.

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.

DDIS-17. POLYNITROXYL ALBUMIN INHIBITS BRAIN TUMORIGENIC CASCADE THROUGH A REACTIVE OXYGEN SPECIES MECHANISM ACROSS THE BLOOD BRAIN BARRIER

This study seeks to develop a safe therapy with potential for a broad spectrum of brain tumors. For this pilot study, we have selected medulloblastoma (MB), the most common malignant pediatric brain cancer that arises from the cerebellum. MB can disseminate through the cerebrospinal fluid to the leptomeninges covering the brain and spinal cord. Leptomeningeal metastasis, a frequent finding at diagnosis and recurrence, is a serious complication for which innovative therapies are urgently needed to improve patient survival while reducing treatment-related toxicity. Polynitroxyl albumin (PNA) has been tested at Sanford Research and shown to reduce intracellular levels of reactive oxygen species (ROS) and inhibition of the tumorigenic cascade: proliferation, progression, and metastasis. This ROS inhibition appears to be responsive in multiple cancers, including triple negative breast cancer and two chemoradiation-resistant head & neck squamous cell carcinoma cell lines. Because intracellular ROS are readily membrane permeable, we have tested efficacy of PNA across the blood brain barrier (BBB) in treating MB. For this we used a metastatic mouse model of the Sonic Hedgehog (SHH) subgroup. PNA was administered i.p. on a thrice weekly schedule; an albumin-only control group was also treated. Survival analysis was obtained, along with histopathology of both brain and spinal cord; primary tumor, leptomeningeal dissemination, and secondary tumor growth were examined. In our preclinical model, a significant decrease in disseminated MB within the brain was detected (P < 0.05), along with secondary tumor growth along the spinal cord (P < 0.05), compared to the control group. Our results compliment PNA studies in other cancer models; PNA works across the BBB to decrease proliferation of tumor cells and inhibit metastatic growth. Follow up studies are warranted to compare the therapeutic index of intra- vs extra-cerebral PNA delivery to generate preclinical efficacy data in support of conjunctive MB clinical therapy.

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.

Normoxic versus hyperoxic resuscitation in pediatric asphyxial cardiac arrest: Effects on oxidative stress

To determine the effects of normoxic vs. hyperoxic resuscitation on oxidative stress in a model of pediatric asphyxial cardiac arrest. Prospective, interventional study. University research laboratory. Postnatal day 16-18 rats (n = 5 per group). 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·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. 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 < .05). Rats resuscitated with 100% oxygen had decreased hippocampal reduced glutathione levels vs. sham (15.3 ± 0.4 vs. 20.9 ± 4.1 nmol·mg protein⁻¹; p < .01). Hippocampal manganese superoxide dismutase activity was significantly increased in 100% oxygen rats vs. sham (14 ± 2.4 vs. 9.5 ± 1.6 units·mg protein⁻¹, p < .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. 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.

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

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. 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. 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). 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).

Oxidative Stress and Vaso-Occlusion in Sickle Cell Disease: Role of Activated Leukocytes and Redox Active Iron.

Sickle cell disease (SCD) is a disease of oxidative stress. We and others have demonstrated increased oxidative stress, inflammation, endothelial cell activation and white blood cell counts in human patients and transgenic murine models of SCD. Leukocytosis in SCD is associated with increases in the incidence of pain crisis, acute chest syndrome, stroke and mortality. We hypothesize that reactive oxygen species (ROS) derived from leukocytes and excess redox active iron promote vascular inflammation and vaso-occlusion. Leukocytes were activated in S+S-Antilles sickle mice compared to normal C57BL/6 control mice as measured by the percentage of leukocytes expressing CD11b on their surface in ambient air (25.4% vs. 19.3%, p<0.05) and after exposure of mice to hypoxia-reoxygenation (31.7% vs. 23.0%, p<0.05). In addition, resting leukocytes from S+S-Antilles mice produce 1.8-fold more H2O2 than normal mice (p<0.05) as measured by Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) fluorescence. These leukocyte oxidants are especially toxic in the presence of excess redox active iron. Histopathology of the lungs and livers of 10 week old S+S-Antilles and BERK sickle mice showed red blood cell (RBC) congestion compared to normal. In addition, the sickle livers had multiple areas of infarction and inflammatory leukocyte infiltration. The heme contents of S+S-Antilles sickle lungs and livers were increased by 37- and 4.9-fold, respectively, compared to normals (p<0.05 for both organs). Furthermore, there was significantly more chelatable iron that is potentially redox active as measured by Ferene-S in sickle lungs (21.0-fold, p<0.05) and livers (2.4-fold, p<0.05) compared to normals. Thus, these data demonstrate there is an explosive pro-oxidative environment in sickle mice. These excess oxidants lead to NF-kB activation, VCAM-1 and ICAM-1 expression, and increased oxidative injury, as seen histopathologically by nitro-tyrosine and dihydroethidium staining in organs. Hypoxia-reoxygenation, which induces RBC sickling and enhances ROS production in sickle mice, causes an increase in leukocyte rolling (4.4-fold, S+S-Antilles vs. normal, p<0.05) and adhesion (6.5-fold, p<0.05). Hypoxia-reoxygenation induces transient vaso-occlusion in 12% and 24% of the subcutaneous venules of S+S-Antilles and BERK mice respectively. No vessels become static in normal mice (p<0.05 sickle vs. normal). Hypoxia-reoxygenation-induced vaso-occlusion can be inhibited by antibodies to P-selectin, VCAM-1 or ICAM-1. Furthermore, scavenging ROS with the SOD and catalase mimetic, polynitroxyl albumin or the iron chelator Trimidox, inhibited hypoxia-reoxygenation-induced vaso-occlusion (p<0.05). We conclude that oxidative stress derived from activated leukocytes and excess redox active iron plays a critical role in promoting vaso-occlusion and organ injury in SCD. We speculate that iron chelators, leukocyte adhesion molecule blockade and anti-oxidants will modulate vaso-occlusion in patients with SCD.

Neuroprotective effects of TEMPOL in central and peripheral nervous system models of Parkinson's disease

TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine- N-oxyl) is a stable nitroxyl antioxidant. Previous studies have suggested that TEMPOL is protective in acute disorders thought to involve reactive oxygen species (ROS), such as ischemic stroke and cardiac reperfusion injury. Oxidized TEMPOL can be recycled to its redox-active reducing form by co-administration with polynitroxylated albumin, making it a candidate as a pharmacological “reservoir” for reducing potential of use in chronic disorders involving ROS. The present studies examine the efficacy of TEMPOL in cell culture and animal models of the central and peripheral dysfunction associated with Parkinson's disease, a disorder in the pathogenesis of which ROS generated from dopamine have been implicated. Antioxidants have been proposed as both preventive and symptomatic therapy for Parkinson's disease. TEMPOL protects MN9D dopaminergic mesencephalic cells in culture from 6-hydroxydopamine (6-OHDA)-induced apoptosis. Translocation of the p65 component of NF-κB to the nucleus accompanies protection by TEMPOL. In vivo, intraperitoneal TEMPOL protects mice from intrastriatal 6-OHDA-induced cell and dopamine metabolite loss in the striatum. TEMPOL also protects mice against the 6-OHDA-induced rotational behavior elicited by intrastriatal administration of d-amphetamine. In addition, TEMPOL protects mice from the ptosis, activity level decrement, and mortality induced by intraperitoneal administration of 6-OHDA, a model of autonomic dysfunction in Parkinson's disease. Adjunctive use of polynitroxylated albumin enhances the in vitro and in vivo effects of TEMPOL.

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 Albumin Prevents Vaso-Occlusion in Transgenic Sickle Mice.

Sickle cell disease is characterized by excessive oxidative stress. Sickle patients have enhanced rates of endogenous reactive oxygen species (ROS) production and impaired antioxidant defense mechanisms. We hypothesize that excessive production of ROS promotes activation of vascular endothelium and vaso-occlusion in sickle cell disease. Intravascular ROS production has been shown to trigger the activation of vascular endothelium and increase leukocyte-endothelium interactions. We investigated whether inhibition of intravascular ROS production by treatment with polynitroxyl albumin (PNA), a superoxide dismutase and catalase mimetic agent, could modulate endothelial cell activation and vaso-occlusion in S+S Antilles transgenic sickle mice after hypoxia-reoxygenation. Nuclear factor-kappa B (NF-kB), an oxidant sensitive transcription factor critical for endothelial cell activation, was elevated in lungs and livers of sickle mice and was reduced 2.5 (p=0.043) and 1.5 (p=0.043) fold respectively, by treatment with PNA after hypoxia-reoxygenation. Similarly, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were elevated 3 to 4.7 fold in the same organs (p<0.05). After treatment with PNA, VCAM-1 expression was reduced 2.3 fold in lungs (p=0.014) and 1.5 fold in livers (p=0.006), ICAM-1 expression decreased 1.7 fold in lungs (p=0.014) and 2 fold in livers (p=0.007). Control studies using human serum albumin had no effect on NF-kB activation or adhesion molecule expression in sickle mice. These anti-inflammatory effects of PNA on endothelium corresponded with changes in blood flow in dorsal skin venules. Intravital microscopy revealed that PNA significantly inhibited hypoxia-reoxygenation-induced leukocyte rolling (3.8 fold inhibition, p<0.001) and completely inhibited vaso-occlusion (p<0.001) in venules in the dorsal skin of transgenic sickle mice. We speculate that therapies that reduce ROS will result in improved organ perfusion in sickle cell disease.

Antioxidants Inhibit Sickle Cell Adhesion to Activated Endothelium in an Ex Vivo Preparation.

In sickle cell anemia (SS), intravascular sickling and attendant transient occlusive events are implicated in the generation of reactive oxygen species (ROS) and vascular endothelial activation. Oxidants (as well as cytokines) result in up-regulation of endothelial adhesion molecules that may lead to increased adhesion of sickle red blood cells (SS RBC) and contribute to vaso-occlusion. We hypothesize that endothelial activation will augment the expression of adhesion molecules involved in SS RBC-endothelium interaction, and that antioxidants will have an inhibitory effect on this interaction. To this end, we have tested selected antioxidants for their efficacy to inhibit SS RBC adhesion in the ex vivo mesocecum vasculature of the rat stimulated by platelet-activating factor (PAF). PAF, a potent inflammatory agent, results in endothelial ROS generation (Suematsu et al. Am. J. Physiol. 1993), as well as in increased adhesion of SS RBCs (Kaul et al. Blood, 2000). Here, we have tested the efficacy of superoxide dismutase (SOD), catalase and polynitroxylated albumin (PNA). While SOD and catalase remove superoxide (O2 ·−) and H2O2, respectively, nitroxides covalently attached to albumin (i.e., PNA) act as intra-vascular SOD mimetic and also facilitate heme-mediated catalytic removal of H2O2. To directly demonstrate the involvement of ROS (O2 •- and H2O2), the ex vivo vasculature was pre-treated with PAF (200 pg/ml in Ringer-albumin solution) followed by infusion with SOD (1500 U in 5 ml Ringer-albumin) or catalase (10,000 U in 5 ml) (each 15-min incubation). The control preparations were incubated with PAF and Ringer-albumin. For experiments involving PNA, the vasculature was first treated with PAF and followed by infusion of PNA or control human serum albumin (HSA) (each 33 mg/ml) diluted in Ringer-albumin solution (incubation 30 min). In PAF or PAF/HSA-treated preparations, infusion of SS RBCs (Hct 30 in Ringer-albumin) caused widespread adhesion of these cells exclusively in postcapillary venules. The adhesion was inversely correlated with vessel diameter, resulting in frequent blockage of small-diameter venules. In contrast, preparations treated with PAF/SOD or PAF/catalase showed a marked decrease in adhesion in venules of all diameters resulting in a significantly lower Y-intercept of the regression line as compared with the control group (p<0.00001). On the other hand, PNA caused almost complete inhibition of SS RBC adhesion in PAF-treated preparation, demonstrating even a greater anti-adhesive efficacy as evidenced by lower Y-intercept (p<0.001–0.0001 vs. SOD and catalase). The decrease in adhesion was accompanied by a significantly lower peripheral resistance (p<0.03–0.018). These results show that antioxidants inhibit SS RBC adhesion to endothelium stimulated by PAF, probably by scavenging and/or blocking PAF-generated oxidants. Thus, blockade of oxidant generation may constitute an effective therapeutic strategy to prevent SS RBC adhesion.

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

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

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-κB 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.

Early antioxidant therapy with Tempol during hemorrhagic shock increases survival in rats.

Hemorrhagic shock (HS) is associated with the generation of reactive oxygen species, which may contribute to delayed multiple organ system failure and death. Previous studies have shown that the antioxidant Tempol improved physiologic variables, although not necessarily outcome, in septic shock and HS. We hypothesized that the combination of free Tempol with polynitroxylated albumin (PNA)-bound Tempol (which prolongs half-life and decreases toxicity) improves outcome after HS in rats. In study 1, HS was induced by blood withdrawal of 3 mL/100 g over 15 minutes. Mean arterial pressure was maintained at 40 mm Hg with either infusion of normal saline or withdrawal of blood from 20 to 90 minutes. Resuscitation (90-270 minutes) was with infusion of shed blood. Observation was to 72 hours. At HS 45 min, albumin (ALB) (n = 10) or PNA + Tempol (n = 10) was infused slowly (1 mL/100 g/h) until 120 minutes. Study 2 was the same as study 1 (n = 6 per group), but terminated at 150 minutes. Study 3 was the same as study 1, but started with ALB or PNA + Tempol (n = 7 per group) at 20 minutes. The primary outcome variable in studies 1 and 3 was survival, whereas the primary outcome variables in study 2 were antioxidant reserve (ability of the serum or tissue homogenate to scavenge peroxyl radicals produced by 2,2'-azobis [2-aminodipropane]-dihydrochloride) in serum and small intestine, and low-molecular-weight thiols in tissues (liver, small intestine, and kidney). In study 1, 72-hour survival was 1 of 10 (ALB group) versus 2 of 10 (PNA + Tempol group). At 90 minutes, pH was lower in the ALB group versus the PNA + Tempol group (p = 0.02) and remained low. Arterial lactate increased to 8.9 +/- 3.2 (mean +/- SD) versus 6.5 +/- 1.8 mmol/L (p = 0.04) and base excess was -9.6 +/- 4.3 versus -5.2 +/- 3.2 mmol/L (p = 0.01) (ALB vs. PNA + Tempol groups, respectively). In study 2, antioxidant reserve in serum was lower in the ALB group versus the PNA + Tempol group (p = 0.002). There were no differences between groups in antioxidant reserve in the small intestine or low-molecular-weight thiols in liver, kidney, and small intestine. In study 3, 72-hour survival was zero of seven (ALB group) versus five of seven (PNA + Tempol group) (p = 0.02). Heart rate and systolic blood pressure during late HS were higher in the ALB group in studies 1 and 3 (p < 0.05). When infused early in HS, PNA + Tempol can increase survival. When given late, it significantly improves acid-base and serum antioxidant status, without an effect on survival. Additional studies will be required to determine whether early resuscitation with PNA + Tempol attenuates reactive oxygen species-mediated injury as the mechanism for preventing the progression toward multiple organ failure and death after HS. The results suggest that antioxidant therapy with Tempol deserves further study as a potential adjunct in the initial resuscitation from HS.

Delayed treatment with polynitroxyl albumin reduces infarct size after stroke in rats.

Nitroxides are antioxidants that are known to protect cells from oxidative damage. Polynitroxyl albumin (PNA) is a compound of human serum albumin covalently labeled with nitroxides that exhibits a prolonged half-life and an enhanced antioxidant activity. Adult male Sprague-Dawley rats were subjected to 90 min intraluminal middle cerebral artery occlusion and the drug was administered intravenously immediately or 2 h after reperfusion. The effects of the drug were evaluated 24 h after reperfusion. Infarct volume was significantly reduced in immediate (79% reduction) and delayed (53% reduction) PNA-treated groups. The efficacy of a single, delayed i.v. injection of PNA suggests that PNA has great promise in the treatment of acute human stroke.

Polynitroxyl albumin plus tempol attenuates liver injury and inflammation after hepatic ischemia and reperfusion

PNA+Tempol, albumin containing conjugated (polynitroxyl albumin; PNA) and free (4-hydroxyl-2,2,6,6-tetramethyl-piperidinyl-1-oxyl; Tempol) nitroxide may protect against injury caused by reactive oxygen species. Therefore, the actions of PNA+Tempol on liver injury and inflammation induced by hepatic ischemia and reperfusion (I/R) were examined. Rats were subjected to 1 h ischemia followed by 24 h reperfusion in the absence (I/R) or presence of PNA+Tempol (25%; 15 mL/kg, i.v.) (I/R+PNA+Tempol) or human serum albumin (23%; 13.5 mL/kg, i.v.) (I/R+HSA). Test solutions were administered prior to and for 2 h during reperfusion. Sham-operated rats underwent surgery with neither ischemia nor infusion. I/R+PNA+Tempol rats had significantly less liver injury and inflammation than I/R rats. I/R+PNA+Tempol livers exhibited focal lesions whereas I/R livers exhibited global necrosis. Likewise, plasma ALT activity was significantly lower in I/R+PNA+Tempol rats. PNA+Tempol reduced I/R-induced neutrophil accumulation and intercellular adhesion molecule-1 (ICAM-1) expression. HSA did not alter I/R-induced liver injury or inflammation. Sham-operated rats exhibited normal liver morphology and no inflammation. Attenuation of I/R liver injury by PNA+Tempol may be mediated by its effect on inflammation, the major contributor to I/R injury. Reduction of inflammation by PNA+Tempol is most likely due to the antioxidative nature of the nitroxides.

In vivo topical EPR spectroscopy and imaging of nitroxide free radicals and polynitroxyl-albumin.

Piperidine nitroxides have considerable clinical potential, both as antioxidant therapeutic compounds and contrast agents in magnetic resonance imaging. However, their development has thus far been limited by their rapid bioreduction in vivo. Recently, it was reported that polynitroxyl albumin (PNA) can reverse the bioreduction of the reduced 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol) in the rat heart, enabling the performance of high resolution EPR imaging for prolonged time (Kuppusamy et al., Biochemistry 35, 7051-7057 (1996)). In this report, the efficacy of PNA in maintaining Tempol concentrations in vivo in mice was demonstrated, using L-band (1.25 GHz) EPR spectroscopy and imaging. The EPR signal of intravenous Tempol had a half-life of 1.0+/-0.2 min and became undetectable within 6 min. Subcutaneous Tempol, however, decayed at a slower rate (half-life, 5.0+/-0.5 min) suggesting that Tempol had been bioreduced to the corresponding hydroxylamine form, Tempol-H. Subcutaneously injected PNA restored 20% of the Tempol signal in the vicinity of the PNA deposit. In vivo topical EPR imaging demonstrated that the Tempol signal was restored at the site of PNA injection, but not at locations remote from the PNA injection site. The ability of PNA to maintain Tempol in its paramagnetic state in vivo should enable a wide range of therapeutic and diagnostic applications of piperidinyl nitroxides.

Polynitroxyl albumin reduces infarct size in transient focal cerebral ischemia in the rat: potential mechanisms studied by magnetic resonance imaging.

Nitroxide free radicals are known to protect cells from oxidative damage. Diffusion-weighted and perfusion-weighted magnetic resonance imaging was used to evaluate the effects of polynitroxyl albumin (PNA) in a middle cerebral artery intraluminal suture model of transient focal cerebral ischemia in the rat. Three groups of Sprague-Dawley rats were investigated: (1) PNA (N=6), (2) human serum albumin (N =6), and (3) saline (N=7). The middle cerebral artery was occluded for 2 hours. Treatment was started 30 minutes after induction of ischemia. A total dose of 1% body weight (volume/weight) of PNA (23.5 mg/dL protein and 110 mmol/L nitroxide), albumin (23.5 mg/dL), or saline was injected intravenously at three time points: 0.5% at 0.5 hours, 0.25% at 2 hours (i.e., just before reperfusion), and 0.25% at 4 hours after occlusion. Six sets of diffusion- and perfusion-weighted magnetic resonance images were acquired throughout the 2 hours of ischemia and the 2 hours of reperfusion. The rats were killed at 24 hours, and the brains were stained with 2,3,5-triphenyltetrazolium chloride (TTC). Diffusion-weighted imaging showed that the growth of the ischemic lesion was suppressed in the PNA-treated group. The 4 hours diffusion-weighted imaging--derived hemispheric lesion volume in the PNA-treated group (25%+/-9%) was significantly smaller than that in the saline-treated (43%+/-13%; P=0.016) or albumin-treated groups (38%+/-6%; P=0.017). A larger difference was observed for the 24-hour TTC-derived lesion volumes in the PNA (8%+/-7%), saline (35%+/-8%; P < 0.001), and albumin (31%+/-6%; P < 0.001) groups. Perfusion-weighted imaging demonstrated a marked improvement in cerebral perfusion in the PNA-treated group during ischemia and reperfusion. In conclusion, treatment with PNA results in an improvement in perfusion and a reduction of infarct volume in a model of transient focal cerebral ischemia in the rat.