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Abstract: Renal ischemia/reperfusion (I/R) injury often occurs as a result of vascular surgery, organ procurement, or transplantation. We previously showed that renal I/R results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. There have been several reports that overexpression of MnSOD protects tissues/organs from I/R-related damage, thus a loss of MnSOD activity during I/R likely contributes to tissue injury. The present study examined the therapeutic benefit of a catalytic antioxidant, Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP5+), using the rat renal I/R model. This was the first study to examine the effects of MnTnHex-2-PyP5+ in an animal model of oxidative stress injury. Our results showed that porphyrin pretreatment of rats for 24 h protected against ATP depletion, MnSOD inactivation, nitrotyrosine formation, and renal dysfunction. The dose (50 μg/kg) used in this study is lower than doses of various types of antioxidants commonly used in animal models of oxidative stress injuries. In addition, using novel proteomic techniques, we identified the ATP synthase-β subunit as a key protein induced by MnTnHex-2-PyP5+ treatment alone and complex V (ATP synthase) as a target of injury during renal I/R. These results showed that MnTnHex-2-PyP5+ protected against renal I/R injury via induction of key mitochondrial proteins that may be capable of blunting oxidative injury. [Copyright &y& Elsevier]

Abstract: Peroxynitrite (ONOO−) formation in mitochondria may be favored due to the constant supply of superoxide radical () by the electron transport chain plus the facile diffusion of nitric oxide ( NO) to this organelle. Herein, a model system of submitochondrial particles (SMP) in the presence of succinate plus the respiratory inhibitor antimycin A (to increase rates) and the NO-donor NOC-7 was studied to directly establish and quantitate peroxynitrite by a multiplicity of methods including chemiluminescence, fluorescence and immunochemical analysis. While all the tested probes revealed peroxynitrite at near stoichiometric levels with respect to its precursor radicals, coumarin boronic acid (a probe that directly reacts with peroxynitrite) had the more straightforward oxidation profile from -forming SMP as a function of the NO flux. Interestingly, immunospintrapping studies verified protein radical generation in SMP by peroxynitrite. Substrate-supplemented SMP also reduced Mn(III)porphyrins (MnP) to Mn(II)P under physiologically-relevant oxygen levels (3–30μM); then, Mn(II)P were capable to reduce peroxynitrite and protect SMP from the inhibition of complex I-dependent oxygen consumption and protein radical formation and nitration of membranes. The data directly support the formation of peroxynitrite in mitochondria and demonstrate that MnP can undergo a catalytic redox cycle to neutralize peroxynitrite-dependent mitochondrial oxidative damage. [Copyright &y& Elsevier]

Abstract: Chemoresistance due to oxidative stress resistance or upregulation of Bcl-2 contributes to poor outcome in the treatment of hematological malignancies. In this study, we utilize the copper-chelator drug ATN-224 (choline tetrathiomolybdate) to induce cell death in oxidative stress-resistant cells and cells overexpressing Bcl-2 by modulating the cellular redox environment and causing mitochondrial dysfunction. ATN-224 treatment decreases superoxide dismutase 1 (SOD1) activity, increases intracellular oxidants, and induces peroxynitrite-dependent cell death. ATN-224 also targets the mitochondria, decreasing both cytochrome c oxidase (CcOX) activity and mitochondrial membrane potential. The concentration of ATN-224 required to induce cell death is proportional to SOD1 levels, but independent of Bcl-2 status. In combination with doxorubicin, ATN-224 enhances cell death. In primary B-cell acute lymphoblastic leukemia patient samples, ATN-224 decreases the viable cell number. Our findings suggest that ATN-224’s dual targeting of SOD1 and CcOX is a promising approach for treatment of hematological malignancies either as an adjuvant or as a single agent. [Copyright &y& Elsevier]

Abstract: The cationic, ortho Mn(III) N-alkylpyridylporphyrins (alkyl=ethyl, E, and n-hexyl, nHex) MnTE-2-PyP5+ (AEOL10113, FBC-007) and MnTnHex-2-PyP5+ have proven efficacious in numerous in vivo animal models of diseases having oxidative stress in common. The remarkable therapeutic efficacy observed is due to their: (1) ability to catalytically remove O2 •− and ONOO− and other reactive species; (2) ability to modulate redox-based signaling pathways; (3) accumulation within critical cellular compartments, i.e., mitochondria; and (4) ability to cross the blood–brain barrier. The similar redox activities of both compounds are related to the similar electronic and electrostatic environments around the metal active sites, whereas their different bioavailabilities are presumably influenced by the differences in lipophilicity, bulkiness, and shape. Both porphyrins are water soluble, but MnTnHex-2-PyP5+ is approximately 4 orders of magnitude more lipophilic than MnTE-2-PyP5+, which should positively affect its ability to pass through biological membranes, making it more efficacious in vivo at lower doses. To gain insight into the in vivo tissue distribution of Mn porphyrins and its impact upon their therapeutic efficacy and mechanistic aspects of action, as well as to provide data that would ensure proper dosing regimens, we conducted comprehensive pharmacokinetic (PK) studies for 24h after single-dose drug administration. The porphyrins were administered intravenously (iv), intraperitoneally (ip), and via oral gavage at the following doses: 10mg/kg MnTE-2-PyP5+ and 0.5 or 2mg/kg MnTnHex-2-PyP5+. Drug levels in plasma and various organs (liver, kidney, spleen, heart, lung, brain) were determined and PK parameters calculated (C max, C 24 h, t max, and AUC). Regardless of high water solubility and pentacationic charge of these Mn porphyrins, they are orally available. The oral availability (based on plasma AUCoral/AUCiv) is 23% for MnTE-2-PyP5+ and 21% for MnTnHex-2-PyP5+. Despite the fivefold lower dose administered, the AUC values for liver, heart, and spleen are higher for MnTnHex-2-PyP5+ than for MnTE-2-PyP5+ (and comparable for other organs), clearly demonstrating the better tissue penetration and tissue retention of the more lipophilic MnTnHex-2-PyP5+. [Copyright &y& Elsevier]

Abstract: Our recent studies showed that total body irradiation (TBI) induces long-term bone marrow (BM) suppression in part by induction of hematopoietic stem cell (HSC) senescence through reactive oxygen species (ROS). In this study, we examined if Mn(III) meso-tetrakis-(N-ethylpyridinium-2-yl) porphyrin (MnTE), a superoxide dismutase mimetic and potent antioxidant, can mitigate TBI-induced long-term BM injury in a mouse model. Our results showed that post-TBI treatment with MnTE significantly inhibited the increases in ROS production and DNA damage in HSCs and the reduction in HSC frequency and clonogenic function induced by TBI. In fact, the clonogenic function of HSCs from irradiated mice after MnTE treatment was comparable to that of HSCs from normal controls on a per-HSC basis, suggesting that MnTE treatment inhibited the induction of HSC senescence by TBI. This suggestion is supported by the finding that MnTE treatment also reduced the expression of p16Ink4a (p16) mRNA in HSCs induced by TBI and improved the long-term and multilineage engraftment of irradiated HSCs after transplantation. Therefore, the results from this study demonstrate that MnTE has the potential to be used as a therapeutic agent to mitigate TBI-induced long-term BM suppression by inhibiting ionizing radiation-induced HSC senescence through the ROS–p16 pathway. [Copyright &y& Elsevier]

Abstract: Chronic production of reactive oxygen and nitrogen species is an underlying mechanism of irradiation (IR)-induced lung injury. The purpose of this study was to determine the optimum time of delivery of an antioxidant and redox-modulating Mn porphyrin, MnTE-2-PyP5+, to mitigate and/or treat IR-induced lung damage. Female Fischer-344 rats were irradiated to their right hemithorax (28 Gy). Irradiated animals were treated with PBS or MnTE-2-PyP5+ (6 mg /kg/24 h) delivered for 2 weeks by sc-implanted osmotic pumps (beginning after 2, 6, 12, 24, or 72 h or 8 weeks). Animals were sacrificed 10 weeks post-IR. Endpoints were body weight, breathing frequency, histopathology, and immunohistochemistry (8-OHdG, ED-1, TGF-β, HIF-1α, VEGF A). A significant radioprotective effect on functional injury, measured by breathing frequency, was observed for all animals treated with MnTE-2-PyP5+. Treatment with MnTE-2-PyP5+ starting 2, 6, and 12 h but not after 24 or 72 h resulted in a significant decrease in immunostaining for 8-OHdG, HIF-1α, TGF-β, and VEGF A. A significant decrease in HIF-1α, TGF-β, and VEGF A, as well as an overall reduction in lung damage (histopathology), was observed in animals beginning treatment at the time of fully developed lung injury (8 weeks post-IR). The catalytic manganese porphyrin antioxidant and modulator of redox-based signaling pathways MnTE-2-PyP5+ mitigates radiation-induced lung injury when given within the first 12 h after IR. More importantly, this is the first study to demonstrate that MnTE-2-PyP5+ can reverse overall lung damage when started at the time of established lung injury 8 weeks post-IR. The radioprotective effects are presumably mediated through its ability both to suppress oxidative stress and to decrease activation of key transcription factors and proangiogenic and profibrogenic cytokines. [Copyright &y& Elsevier]

Abstract: MnTE-2-PyP5+ is a potent catalytic scavenger of reactive oxygen and nitrogen species, primarily superoxide and peroxynitrite. It therefore not only attenuates primary oxidative damage, but was found to modulate redox-based signaling pathways (HIF-1α, NF-κB, SP-1, and AP-1) and thus, in turn, secondary oxidative injury also. Cancer has been widely considered an oxidative stress condition. The goal of this study was to prove if and why a catalytic SOD mimic/peroxynitrite scavenger would exert anti-cancer effects, i.e., to evaluate whether the attenuation of the oxidative stress by MnTE-2-PyP5+ could suppress tumor growth in a 4T1 mouse breast tumor model. Tumor cells were implanted into Balb/C mouse flanks. Three groups of mice (n =25) were studied: control (PBS) and 2 and 15 mg/kg/day of MnTE-2-PyP5+ given subcutaneously twice daily starting when the tumors averaged 200 mm3 (until they reached ∼5-fold the initial volume). Intratumoral hypoxia (pimonidazole, carbonic anhydrase), HIF-1α, VEGF, proliferating capillary index (CD105), microvessel density (CD31), protein nitration, DNA oxidation (8-OHdG), NADPH oxidase (Nox-4), apoptosis (CD31), macrophage infiltration (CD68), and tumor drug levels were assessed. With 2 mg/kg/day a trend toward tumor growth delay was observed, and a significant trend was observed with 15 mg/kg/day. The 7.5-fold increase in drug dose was accompanied by a similar (6-fold) increase in tumor drug levels. Oxidative stress was largely attenuated as observed through the decreased levels of DNA damage, protein 3-nitrotyrosine, macrophage infiltration, and NADPH oxidase. Further, hypoxia was significantly decreased as were the levels of HIF-1α and VEGF. Consequently, suppression of angiogenesis was observed; both the microvessel density and the endothelial cell proliferation were markedly decreased. Our study indicates for the first time that MnTE-2-PyP5+ has anti-cancer activity in its own right. The anti-cancer activity via HIF/VEGF pathways probably arises from the impact of the drug on the oxidative stress. Therefore, the catalytic scavenging of ROS/RNS by antioxidants, which in turn suppresses cellular transcriptional activity, could be an appropriate strategy for anti-cancer therapy. Enhancement of the anti-cancer effects may be achieved by optimizing the dosing regime, utilizing more bioavailable Mn porphyrins (MnP), and combining MnP treatment with irradiation, hyperthermia, and chemotherapy. Mn porphyrins may be advantageous compared to other anti-cancer drugs, owing to their radioprotection of normal tissue and the ability to afford pain management in cancer patients via prevention of chronic morphine tolerance. [Copyright &y& Elsevier]

Abstract: We tested several classes of antioxidant manganese compounds for radioprotective effects using human lymphoblastoid cells: six porphyrins, three salens, and two cyclic polyamines. Radioprotection was evaluated by seven assays: XTT, annexin V and propidium iodide flow cytometry analysis, γ-H2AX immunofluorescence, the neutral comet assay, dichlorofluorescein and dihydroethidium staining, resazurin, and colony survival assay. Two compounds were most effective in protecting wild-type and A-T cells against radiation-induced damage: MnM x -2-PyP-Calbio (a mixture of differently N-methylated MnT-2-PyP+ from Calbiochem) and MnTnHex-2-PyP. MnTnHex-2-PyP protected WT cells against radiation-induced apoptosis by 58% (p =0.04), using XTT, and A-T cells by 39% (p =0.01), using annexin V and propidium iodide staining. MnTnHex-2-PyP protected WT cells against DNA damage by 57% (p =0.005), using γ-H2AX immunofluorescence, and by 30% (p <0.01), using neutral comet assay. MnTnHex-2-PyP is more lipophilic than MnM x -2-PyP-Calbio and is also >10-fold more SOD-active; consequently it is >50-fold more potent as a radioprotectant, as supported by six of the tests employed in this study. Thus, lipophilicity and antioxidant potency correlated with the magnitude of the beneficial radioprotectant effects observed. Our results identify a new class of porphyrinic radioprotectants for the general and radiosensitive populations and may also provide a new option for treating A-T patients. [Copyright &y& Elsevier]

Development of radiation therapy (RT)-induced lung injury is associated with chronic production of reactive oxygen and nitrogen species (ROS/RNS). MnTE-2-PyP5+ is a catalytic Mn porphyrin mimic of SOD, already shown to protect lungs from RT-induced injury by scavenging ROS/RNS. The purpose of this study was to compare MnTE-2-PyP5+ with a newly introduced analogue MnTnHex-2-PyP5+, which is expected to be a more effective radioprotector due to its lipophilic properties. This study shows that Fischer rats which were irradiated to their right hemithorax (28 Gy) have less pulmonary injury as measured using breathing frequencies when treated with daily subcutaneous injections of MnTE-2-PyP5+ (3 and 6 mg/kg) or MnTnHex-2-PyP5+ (0.3, 0.6, or 1.0 mg/kg) for 2 weeks after RT. However, at 16 weeks post-RT, only MnTE-2-PyP5+ at a dose of 6 mg/kg is able to ameliorate oxidative damage, block activation of HIF-1α and TGF-β, and impair upregulation of CA-IX and VEGF. MnTnHex-2-PyP5+ at a dose of 0.3 mg/kg is effective only in reducing RT-induced TGF-β and CA-IX expression. Significant loss of body weight was observed in animals receiving MnTnHex-2-PyP5+ (0.3 and 0.6 mg/kg). MnTnHex-2-PyP5+ has the ability to dissolve lipid membranes, causing local irritation/necrosis at injection sites if given at doses of 1 mg/kg or higher. In conclusion, both compounds show an ability to ameliorate lung damage as measured using breathing frequencies and histopathologic evaluation. However, MnTE-2-PyP5+ at 6 mg/kg proved to be more effective in reducing expression of key molecular factors known to play an important role in radiation-induced lung injury. [Copyright &y& Elsevier]

Superoxide plays a role in blood pressure regulation in certain vascular diseases, however, its involvement in regulating basal blood pressure is uncertain. Vascular superoxide concentrations are limited by extracellular superoxide dismutase (EC-SOD), which is highly expressed in the vasculature of most animal species. Metalloporphyrins are low molecular weight, synthetic, redox-active, catalytic antioxidants that act as SOD mimetics. We evaluated the effects of metalloporphyrins on blood pressure in different animal species. The metalloporphyrin AEOL10113 (5–10 μg/kg iv), but not native or polyethylene glycol-CuZnSOD, caused a dose-dependent reduction in blood pressure in anesthetized rats. AEOL10113 had no effect on blood pressure in mice (wild-type or EC-SOD knockouts), guinea pigs, dogs, or baboons at doses up to 5 mg/kg iv Structure-activity studies indicated that metalloporphyrins with high SOD activity were more effective in lowering rat blood pressure than low-activity analogs. The blood pressure effect of AEOL10113 was not attributable to the release of manganese, nor was it affected by inhibitors of nitric oxide synthase (L-NAME) and guanylate cyclase (ODQ, 8-bromo-cGMP, and methylene blue) or nitric oxide scavengers (HbAo). Chlorpheniramine attenuated the effect, suggesting that the blood pressure response in rats is related to histamine release rather than the protection of nitric oxide. [Copyright &y& Elsevier]