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Reactive oxygen species (ROS) cause oxidative stress by generating reactive aldehydes known as 4-hydroxynonenal (4-HNE). 4-HNE modifies protein via covalent adduction; however, little is known about the degradation mechanism of 4-HNE-adducted proteins. Autophagy is a dynamic process that maintains cellular homeostasis by removing damaged organelles and proteins. In this study, we determined the role of a superoxide dismutase (SOD) mimetic MnTnBuOE-2-PyP5+ (MnP, BMX-001) on rotenone-induced 4-HNE aggresome degradation in HL-1 cardiomyocytes. A rotenone treatment (500 nM) given for 24 h demonstrated both increased ROS and 4-HNE aggresome accumulation in HL-1 cardiomyocytes. In addition, cardiomyocytes treated with rotenone displayed an increase in the autophagy marker LC3-II, as shown by immunoblotting and immunofluorescence. A pre-treatment with MnP (20 µM) for 24 h attenuated rotenone-induced ROS formation. An MnP pre-treatment showed decreased 4-HNE aggresomes and LC3-II formation. A rotenone-induced increase in autophagosomes was attenuated by a pre-treatment with MnP, as shown by fluorescent-tagged LC3 (tfLC3). Rotenone increased tubulin hyperacetylation through the ROS-mediated pathway, which was attenuated by MnP. The disruption of autophagy caused HL-1 cell death because a 3-methyladenine inhibitor of autophagosomes caused reduced cell death. Yet, rapamycin, an inducer of autophagy, increased cell death. These results indicated that a pre-treatment with MnP decreased rotenone-induced 4-HNE aggresomes by enhancing the degradation process.

We have developed Mn porphyrins (MnPs) initially as mimics of superoxide dismutase (SOD) enzymes based on structure–activity relationships. Several cationic Mn porphyrins, being substituted with cationic ortho [Formula: see text]-alkyl- or alkoxyalkylpyridyl groups in meso positions of the porphyrin ring, have been identified as potential therapeutics based on their high SOD-like activity and high bioavailability. Two of those [Mn(III) meso-tetrakis([Formula: see text]-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP[Formula: see text] (BMX-010, AEOL10113) and Mn(III) meso-tetrakis(Nn-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP[Formula: see text] (BMX-001)] are now in five Phase II clinical trials. Studies of ours, and those of others, contributed to the understanding of the diverse activities of these compounds. With biologically compatible potentials and four biologically accessible oxidation states, Mn porphyrins interact with numerous reactive species, both as oxidants and reductants. Among those reactions, their abilities to (catalytically) oxidize [Formula: see text]-glutathionylate protein thiols may perhaps be their major in vivo mode of action. Via [Formula: see text]-glutathionylation, MnPs modulate actions of signaling proteins and, in turn, cellular apoptotic and proliferative pathways. During the major part of our stay in the USA, our lives have been dedicated to Mn porphyrins. Our families and especially our son and his three babies have been our inspiration not to give up on a life often burdened with hardship. It is thus our immense pleasure to see our compounds in clinical trials. Above all, we hope that our story will inspire future researchers to persevere — women in particular.

High levels of hyaluronic acid (HA) in tumors correlate with poor outcomes with several types of cancers due to HA-driven support of adhesion, migration and proliferation of cells. In this study we explored how to enhance the degradation of HA into low-molecular fragments, which cannot prevent the immune system to fight tumor proliferation and metastases. The physiological solution of HA was exposed to oxidative degradation by ascorbate and cupric ions in the presence of either one of three ortho isomeric Mn(III) substituted N-alkyl- and alkoxyalkylpyridylporphyrins or para isomeric Mn(III) N-methylpyridyl analog, commonly known as mimics of superoxide dismutase. The changes in hyaluronan degradation kinetics by four Mn(III) porphyrins were monitored by measuring the alteration in the dynamic viscosity of the HA solution. The ortho compounds MnTE-2-PyP5+ (BMX-010, AEOL10113), MnTnBuOE-2-PyP5+ (BMX-001) and MnTnHex-2-PyP5+ are able to redox cycle with ascorbate whereby producing H2O2 which is subsequently coupled with Cu(I) to produce the •OH radical essential for HA degradation. Conversely, with the para analog, MnTM-4-PyP5+, no catalysis of HA degradation was demonstrated, due to its inertness towards redox cycling with ascorbate. The impact of different Mn(III)-porphyrins on the HA decay was further clarified by electron paramagnetic resonance spectrometry. The ability to catalyze the degradation of HA in a biological milieu, in the presence of cupric ions and ascorbate under the conditions of high tumor oxidative stress provides further insight into the anticancer potential of redox-active ortho isomeric Mn(III) porphyrins.

Significance: After approximatelty three decades of research, two Mn(III) porphyrins (MnPs), MnTE-2-PyP(5+) (BMX-010, AEOL10113) and MnTnBuOE-2-PyP(5+) (BMX-001), have progressed to five clinical trials. In parallel, another similarly potent metal-based superoxide dismutase (SOD) mimic—Mn(II)pentaaza macrocycle, GC4419—has been tested in clinical trial on application, identical to that of MnTnBuOE-2-PyP(5+)—radioprotection of normal tissue in head and neck cancer patients. This clearly indicates that Mn complexes that target cellular redox environment have reached sufficient maturity for clinical applications. Recent Advances: While originally developed as SOD mimics, MnPs undergo intricate interactions with numerous redox-sensitive pathways, such as those involving nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), thereby impacting cellular transcriptional activity. An increasing amount of data support the notion that MnP/H(2)O(2)/glutathione (GSH)-driven catalysis of S-glutathionylation of protein cysteine, associated with modification of protein function, is a major action of MnPs on molecular level. Critical Issues: Differential effects of MnPs on normal versus tumor cells/tissues, which support their translation into clinic, arise from differences in their accumulation and redox environment of such tissues. This in turn results in different yields of MnP-driven modifications of proteins. Thus far, direct evidence for such modification of NF-κB, mitogen-activated protein kinases (MAPK), phosphatases, Nrf2, and endogenous antioxidative defenses was provided in tumor, while indirect evidence shows the modification of NF-κB and Nrf2 translational activities by MnPs in normal tissue. Future Directions: Studies that simultaneously explore differential effects in same animal are lacking, while they are essential for understanding of extremely intricate interactions of metal-based drugs with complex cellular networks of normal and cancer cells/tissues.

Novel synthetic compounds, known as manganese porphyrins (MnPs), have been designed to shift the redox status of both normal cells and cancer cells. When MnPs are coupled with cancer therapies, such as radiation, they have been shown to sensitize tumor cells to treatment and protect normal tissues from damage through the modulation of the redox status of various tissue types. Until now, our preclinical studies have focused on local effects of MnPs and radiation; however, we recognize that successful outcomes for cancer patients involve control of tumor cells throughout the body. In this study, using murine orthotopic mammary tumor models, we investigated how MnPs and radiation influence the development of distant metastasis. We hypothesized that the combination of MnP (MnP/RT), such as MnTnBuOE-2-PyP(5+) and radiation treatment (RT) would increase local tumor control via a shift in the intratumoral redox environment, leading to subsequent downregulation of HIF1 in the primary tumor. Secondarily, we hypothesized that these primary tumor treatment effects would result in a reduction in pulmonary metastatic burden. Balb/c mice with orthotopic 4T1 mammary carcinomas were treated with saline, MnP, RT or MnP/RT. We found MnP/RT did extend local tumor growth delay and overall survival compared to controls and was associated with increased intratumoral oxidative stress. However, the primary tumor growth delay observed with MnP/RT was not associated with a reduced pulmonary metastatic burden. Future directions to investigate the effects of MnP/RT on the development of distant metastasis may include modifications to the radiation dose, the experimental timeline or using a murine mammary carcinoma cell line with a less aggressive metastatic behavior. Clinical trials are underway to investigate the clinical utility of MnTnBuOE-2-PyP(5+) for patients undergoing radiotherapy for various tumor types. The promising preclinical data from this study, as well as others, provides support that MnP/RT has the potential to improve local tumor control for these patients.

Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin, (H2O)MnTnHex-2-PyP5+ (MnHex) carrying long hexyl chains, is a lipophilic mimic of superoxide dismutase (SOD) and a redox-active drug candidate. MnHex crosses the blood&ndash, brain barrier, and improved neurologic outcome and decreased infarct size and inflammation in a rat middle cerebral artery occlusion (MCAO) ischemic stroke model. Yet, the dose and the therapeutic efficacy of Mn porphyrin were limited by an adverse effect of arterial hypotension. An equally lipophilic Fe analog, (OH)FeTnHex-2-PyP4+ (FeHex), is as redox-active and potent SOD mimic in vitro. With different coordination geometry of the metal site, FeHex has one hydroxo (OH) ligand (instead of water) bound to the Fe center in the axial position. It has ~2 orders of magnitude higher efficacy than MnHex in an SOD-deficient E. coli model of oxidative stress. In vivo, it does not cause arterial hypotension and is less toxic to mice. We thus evaluated FeHex versus MnHex in a rodent MCAO model. We first performed short- and long-term pharmacokinetics (PK) of both porphyrins in the plasma, brain, and liver of rats and mice. Given that damage to the brain during stroke occurs very rapidly, fast delivery of a sufficient dose of drug is important. Therefore, we aimed to demonstrate if, and how fast after reperfusion, Fe porphyrin reaches the brain relative to the Mn analog. A markedly different plasma half-life was found with FeHex (~23 h) than with MnHex (~1.4 h), which resulted in a more than 2-fold higher plasma exposure (AUC) in a 7-day twice-daily treatment of rats. The increased plasma half-life is explained by the much lower liver retention of FeHex than typically found in Mn analogs. In the brain, a 3-day mouse PK study showed similar levels of MnHex and FeHex. The same result was obtained in a 7-day rat PK study, despite the higher plasma exposure of FeHex. Importantly, in a short-term PK study with treatment starting 2 h post MCAO, both Fe- and Mn- analogs distributed at a higher level to the injured brain hemisphere, with a more pronounced effect observed with FeHex. While a 3-day mouse MCAO study suggested the efficacy of Fe porphyrin, in a 7-day rat MCAO study, Mn-, but not Fe porphyrin, was efficacious. The observed lack of FeHex efficacy was discussed in terms of significant differences in the chemistry of Fe vs the Mn center of metalloporphyrin, relative to MnHex, FeHex has the propensity for axial coordination, which in vivo would preclude the reactivity of the Fe center towards small reactive species.

Cationic manganese(III) ortho N-substituted pyridylporphyrins (MnP) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnP can reach mitochondria offering protection against reactive species in different animal models of disease. Although an LC-MS/MS-based method for MnP quantitation and subcellular distribution has been reported, a direct method capable of evaluating both the uptake and the redox state of MnP in living cells has not yet been developed. In the present work we applied resonance Raman (RR) spectroscopy to analyze the intracellular accumulation of two potent MnP-based lipophilic SOD mimics, MnTnBuOE-2-PyP5+ and MnTnHex-2-PyP5+ within endothelial cells. RR experiments with isolated mitochondria revealed that the reduction of Mn(III)P was affected by inhibitors of the electron transport chain, supporting the action of MnP as efficient redox active compounds in mitochondria. Indeed, RR spectra confirmed that MnP added in the Mn(III) state can be incorporated into the cells, readily reduced by intracellular components to the Mn(II) state and oxidized by peroxynitrite. To assess the combined impact of reactivity and bioavailability, we studied the kinetics of Mn(III)TnBuOE-2-PyP5+ with peroxynitrite and evaluated the cytoprotective capacity of MnP by exposing the endothelial cells to nitro-oxidative stress induced by peroxynitrite. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. These data introduce a novel application of RR spectroscopy for the direct detection of MnP and their redox states inside living cells, and helps to rationalize their antioxidant capacity in biological systems. Fil: Carballal, Sebastián. Universidad de la República; Uruguay Fil: Valez, Valeria. Universidad de la República; Uruguay Fil: Álvarez Paggi, Damián Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Tovmasyan, Artak. University of Duke; Estados Unidos Fil: Batinic-Haberle, Ines. University of Duke; Estados Unidos Fil: Ferrer-Suetac, Gerardo. Universidad de la República; Uruguay Fil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Radi, Rafael. Universidad de la República; Uruguay

Ascorbate (Asc) as a single agent suppressed growth of several tumor cell lines in a mouse model. It has been tested in a Phase I Clinical Trial on pancreatic cancer patients where it exhibited no toxicity to normal tissue yet was of only marginal efficacy. The mechanism of its anticancer effect was attributed to the production of tumoricidal hydrogen peroxide (H2O2) during ascorbate oxidation catalyzed by endogenous metalloproteins. The amount of H2O2 could be maximized with exogenous catalyst that has optimized properties for such function and is localized within tumor. Herein we studied 14 Mn porphyrins (MnPs) which differ vastly with regards to their redox properties, charge, size/bulkiness and lipophilicity. Such properties affect the in vitro and in vivo ability of MnPs (i) to catalyze ascorbate oxidation resulting in the production of H2O2; (ii) to subsequently employ H2O2 in the catalysis of signaling proteins oxidations affecting cellular survival pathways; and (iii) to accumulate at site(s) of interest. The metal-centered reduction potential of MnPs studied, E1/2 of MnIIIP/MnIIP redox couple, ranged from −200 to +350 mV vs NHE. Anionic and cationic, hydrophilic and lipophilic as well as short- and long-chained and bulky compounds were explored. Their ability to catalyze ascorbate oxidation, and in turn cytotoxic H2O2 production, was explored via spectrophotometric and electrochemical means. Bell-shape structure-activity relationship (SAR) was found between the initial rate for the catalysis of ascorbate oxidation, vo(Asc)ox and E1/2, identifying cationic Mn(III) N-substituted pyridylporphyrins with E1/2 > 0 mV vs NHE as efficient catalysts for ascorbate oxidation. The anticancer potential of MnPs/Asc system was subsequently tested in cellular (human MCF-7, MDA-MB-231 and mouse 4T1) and animal models of breast cancer. At the concentrations where ascorbate (1 mM) and MnPs (1 or 5 μM) alone did not trigger any alteration in cell viability, combined treatment suppressed cell viability up to 95%. No toxicity was observed with normal human breast epithelial HBL100 cells. Bell-shape relationship, essentially identical to vo(Asc)ox vs E1/2, was also demonstrated between MnP/Asc-controlled cellular cytotoxicity and E1/2-controlled vo(Asc)ox. Magnetic resonance imaging studies were conducted to explore the impact of ascorbate on T1-relaxivity. The impact of MnP/Asc on intracellular thiols and on GSH/GSSG and Cys/CySS ratios in 4T1 cells was assessed and cellular reduction potentials were calculated. The data indicate a significant increase in cellular oxidative stress induced by MnP/Asc. Based on vo(Asc)ox vs E1/2 relationships and cellular cytotoxicity, MnTE-2-PyP5+ was identified as the best catalyst among MnPs studied. Asc and MnTE-2-PyP5+ were thus tested in a 4T1 mammary mouse flank tumor model. The combination of ascorbate (4 g/kg) and MnTE-2-PyP5+ (0.2 mg/kg) showed significant suppression of tumor growth relative to either MnTE-2-PyP5+ or ascorbate alone. In addition to optimal vo(Asc)ox, the compound must be also bioavailable at the site of interest. About 7-fold higher accumulation of MnTE-2-PyP5+ in tumor vs normal tissue was found to contribute largely to the anticancer effect.

Oxidative stress that results from an imbalance between the concentrations of reactive species (RS) and antioxidant defenses is associated with many pathologies. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase are among the key enzymes that maintain the low nanomolar physiological concentrations of superoxide and hydrogen peroxide. The increase in the levels of these species and their progeny could have deleterious effects. In this context, chemists have developed SOD and CAT mimics to supplement them when cells are overwhelmed with oxidative stress. However, the beneficial activity of such molecules in cells depends not only on their intrinsic catalytic activities but also on their stability in biological context, their cell penetration and their cellular localization. We have employed cellular assays to characterize several compounds that possess SOD and CAT activities and have been frequently used in cellular and animal models. We used cellular assays that address SOD and CAT activities of the compounds. Finally, we determined the effect of compounds on the suppression of the inflammation in HT29-MD2 cells challenged by lipopolysaccharide. When the assay requires penetration inside cells, the SOD mimics Mn(III) meso-tetrakis(N-(2′-n-butoxyethyl)pyridinium-2-yl)porphyrin (MnTnBuOE-2-PyP5+) and Mn(II) dichloro[(4aR,13aR,17aR,21aR)-1,2,3,4,4a,5,6,12,13,13a,14,15,16,17,17a,18,19,20,21,21a-eicosahydro-11,7-nitrilo-7Hdibenzo[b,h] [1,4, 7,10] tetraazacycloheptadecine-κN5,κN13,κN18,κN21,κN22] (Imisopasem manganese, M40403, CG4419) were found efficacious at 10 μM, while Mn(II) chloro N-(phenolato)-N,N′-bis[2-(N-methyl-imidazolyl)methyl]-ethane-1,2-diamine (Mn1) requires an incubation at 100 μM. This study thus demonstrates that MnTnBuOE-2-PyP5+, M40403 and Mn1 were efficacious in suppressing inflammatory response in HT29-MD2 cells and such action appears to be related to their ability to enter the cells and modulate reactive oxygen species (ROS) levels.

Although radiation therapy can be effective against cancer, potential damage to normal tissues limits the amount that can be safely administered. In central nervous system (CNS), radiation damage to normal tissues is presented, in part, as suppressed hippocampal neurogenesis and impaired cognitive functions. Mn porphyrin (MnP)-based redox active drugs have demonstrated differential effects on cancer and normal tissues in experimental animals that lead to protection of normal tissues and radio- and chemo-sensitization of cancers. To test the efficacy of MnPs in CNS radioprotection, we first examined the tissue levels of three different MnPs – MnTE-2-PyP5+(MnE), MnTnHex-2-PyP5+(MnHex), and MnTnBuOE-2-PyP5+(MnBuOE). Nanomolar concentrations of MnHex and MnBuOE were detected in various brain regions after daily subcutaneous administration, and MnBuOE was well tolerated at a daily dose of 3 mg/kg. Administration of MnBuOE for one week before cranial irradiation and continued for one week afterwards supported production and long-term survival of newborn neurons in the hippocampal dentate gyrus. MnP-driven S-glutathionylation in cortex and hippocampus showed differential responses to MnP administration and radiation in these two brain regions. A better understanding of how preserved hippocampal neurogenesis correlates with cognitive functions following cranial irradiation will be helpful in designing better MnP-based radioprotection strategies., Graphical abstract fx1, Highlights • Bioavailability of MnPs in individual brain regions were determined by LC-MS/MS. • CNS MnBuOE and MnHex levels were between 15 and 160 nM after daily administration. • MnBuOE administration ameliorated radiation effects on hippocampal neurogenesis. • MnBuOE preserved categories E&F Dcx+ neurons after cranial irradiation. • MnBuOE and irradiation lead to changes in protein S-glutathionylation in the CNS.

Combinations of radiotherapy (RT) and chemotherapy have shown efficacy toward brain tumors. However, therapy-induced oxidative stress can damage normal brain tissue, resulting in both progressive neurocognitive loss and diminished quality of life. We have recently shown that MnTnBuOE-2-PyP(5+) (Mn(III)meso-tetrakis(N-n-butoxyethylpyridinium -2-yl)porphyrin) rescued RT-induced white matter damage in cranially-irradiated mice. Radiotherapy is not used in isolation for treatment of brain tumors; temozolomide is the standard-of-care for adult glioblastoma, whereas cisplatin is often used for treatment of pediatric brain tumors. Therefore, we evaluated the brain radiation mitigation ability of MnTnBuOE-2-PyP(5+) after either temozolomide or cisplatin was used singly or in combination with 10 Gy RT. MnTnBuOE-2-PyP(5+) accumulated in brains at low nanomolar levels. Histological and neurobehavioral testing showed a drastic decrease (1) of axon density in the corpus callosum and (2) rotorod and running wheel performance in the RT only treatment group, respectively. MnTnBuOE-2-PyP(5+) completely rescued this phenotype in irradiated animals. In the temozolomide groups, temozolomide/ RT treatment resulted in further decreased rotorod responses over RT alone. Again, MnTnBuOE-2-PyP(5+) treatment rescued the negative effects of both temozolomide ± RT on rotorod performance. While the cisplatin-treated groups did not give similar results as the temozolomide groups, inclusion of MnTnBuOE-2-PyP(5+) did not negatively affect rotorod performance. Additionally, MnTnBuOE-2-PyP(5+) sensitized glioblastomas to either RT ± temozolomide in flank tumor models. Mice treated with both MnTnBuOE-2-PyP(5+) and radio-/chemo-therapy herein demonstrated brain radiation mitigation. MnTnBuOE-2-PyP(5+) may well serve as a normal tissue radio-/chemo-mitigator adjuvant therapy to standard brain cancer treatment regimens. Environ. Mol. Mutagen. 57:372-381, 2016. © 2016 Wiley Periodicals, Inc.

Cardiovascular complications are major side effects of many anticancer drugs. Accumulated evidence indicates that oxidative stress in mitochondria plays an important role in cardiac injury, but how mitochondrial redox mechanisms are involved in cardiac dysfunction remains unclear. Here, we demonstrate that 4-hydroxy-2-nonenal (HNE) activates the translocation of the mitochondrial apoptosis inducing factor (AIFm2) and facilitates apoptosis in heart tissue of mice and humans. Doxorubicin treatments significantly enhance cardiac levels of HNE and AIFm2. HNE adduction of AIFm2 inactivates the NADH oxidoreductase activity of AIFm2 and facilitates its translocation from mitochondria. His 174 on AIFm2 is the critical target of HNE adduction that triggers this functional switch. HNE adduction and translocation of AIFm2 from mitochondria upon Doxorubicin treatment are attenuated by superoxide dismutase mimetics. These results identify a previously unrecognized role of HNE with important consequences for mitochondrial stress signaling, heart failure, and the side effects of cancer therapy.

Cranial irradiation is a standard therapy for primary and metastatic brain tumors. A major drawback of radiotherapy (RT), however, is long-term cognitive loss that affects quality of life. Radiation-induced oxidative stress in normal brain tissue is thought to contribute to cognitive decline. We evaluated the effectiveness of a novel mimic of superoxide dismutase enzyme (SOD), MnTnBuOE-2-PyP5+(Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin), to provide long-term neuroprotection following 8 Gy of whole brain irradiation. Long-term RT damage can only be assessed by brain imaging and neurocognitive studies. C57BL/6J mice were treated with MnTnBuOE-2-PyP5+ before and after RT and evaluated three months later. At this time point, drug concentration in the brain was 25 nmol/L. Mice treated with MnTnBuOE-2-PyP5+/RT exhibited MRI evidence for myelin preservation in the corpus callosum compared with saline/RT treatment. Corpus callosum histology demonstrated a significant loss of axons in the saline/RT group that was rescued in the MnTnBuOE-2-PyP5+/RT group. In addition, the saline/RT groups exhibited deficits in motor proficiency as assessed by the rotorod test and running wheel tests. These deficits were ameliorated in groups treated with MnTnBuOE-2-PyP5+/RT. Our data demonstrate that MnTnBuOE-2-PyP5+ is neuroprotective for oxidative stress damage caused by radiation exposure. In addition, glioblastoma cells were not protected by MnTnBuOE-2-PyP5+ combination with radiation in vitro. Likewise, the combination of MnTnBuOE-2-PyP5+ with radiation inhibited tumor growth more than RT alone in flank tumors. In summary, MnTnBuOE-2-PyP5+ has dual activity as a neuroprotector and a tumor radiosensitizer. Thus, it is an attractive candidate for adjuvant therapy with RT in future studies with patients with brain cancer. Mol Cancer Ther; 14(1); 70–79. ©2014 AACR.

Peter Ratcliffe,1,2 Peppi Koivunen,3 Johanna Myllyharju,3 Jiannis Ragoussis,4 Judith VMG Bovée,5 Ines Batinic-Haberle,6 Claire Vinatier,7 Valérie Trichet,8 Florence Robriquet,9 Lisa Oliver,9 Betty Gardie9,10 1Target Discovery Institute, University of Oxford, 2The Francis Crick Institute, London, UK; 3Oulu Center for Cell-Matrix Research, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland; 4McGill University and Genome Quebec Innovation Centre, McGill University, Montreal, Canada; 5Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands; 6Department of Radiation Oncology, Duke University School of Medicine, Durham, NC, USA; 7INSERM UMR 1229, Regenerative Medicine and Skeleton-RMeS, Team STEP, University of Nantes, UFR Odontology, 8UMR 1238 INSERM, Université de Nantes, Faculté de Médecine, 9CRCINA, INSERM, Université de Nantes, Nantes, 10Ecole Pratique des Hautes Etudes, PSL Research University, Paris, France Abstract: The “Hypoxia Nantes 2016” organized its second conference dedicated to the field of hypoxia research. This conference focused on “the role of hypoxia under physiological conditions as well as in cancer” and took place in Nantes, France, in October 6–7, 2016. The main objective of this conference was to bring together a large group of scientists from different spheres of hypoxia. Recent advances were presented and discussed around different topics: genomics, physiology, musculoskeletal, stem cells, microenvironment and cancer, and oxidative stress. This review summarizes the major highlights of the meeting. Keywords: hypoxia, genomics, lipid metabolism, musculoskeletal, cancer, oxidative stress&nbsp

Aims: The susceptibility of CA1 over CA3 to damage from cerebral ischemia may be related to the differences in reactive oxygen species (ROS) production/removal between the two hippocampal subfields. We aimed to measure CA1/CA3 differences in net ROS production in real time in the first 30 min of reperfusion in pyramidal cells. We aimed to determine the underlying cause of the differential vulnerability of CA1 and CA3. Results: Real-time determinations of mitochondrial H2O2 and, independently, glutathione (GSH) redox status from roGFP-based probes in individual pyramidal cells in organotypic hippocampal cultures during oxygen–glucose deprivation (OGD)–reperfusion (RP) demonstrate a significantly more oxidizing environment during RP in CA1 than CA3 mitochondria. Protein levels (immunohistochemistry and Western blots), roGFP2-based probe measurements during controlled mitochondrial production of ROS, and thioredoxin reductase (TrxR) inhibition by auranofin are consistent with a more effective mitoch...

Doxorubicin (DOX), one of the most effective anticancer drugs, is known to generate progressive cardiac damage, which is due, in part, to DOX-induced reactive oxygen species (ROS). The elevated ROS often induce oxidative protein modifications that result in alteration of protein functions. This study demonstrates that the level of proteins adducted by 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, is significantly increased in mouse heart mitochondria following DOX treatment. A redox proteomics method involving 2D electrophoresis followed by mass spectrometry and investigation of protein data bases identified several HNE-modified mitochondria proteins, which were verified by HNE-specific immunoprecipitation in cardiac mitochondria from the DOX-treated mice. The majority of the identified proteins are related to mitochondrial energy metabolism. These include proteins in the citric acid cycle (TCA) and electron transport chain (ETC). The enzymatic activities of the HNE-adducted proteins were significantly reduced in DOX-treated mice. Consistent with the decline in the function of the HNE adducted proteins, the respiratory function of cardiac mitochondria as determined by oxygen consumption rate (OCR) was also significantly reduced after DOX treatment. The treatment with Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnP, an SOD mimic, averted the doxorubicin-induced mitochondrial dysfunctions as well as the HNE protein adductions. Together, the results demonstrate that free radical-mediated alteration of energy metabolism is an important mechanism mediating DOX-induced cardiac injury suggesting that metabolic intervention may represent a novel approach to preventing cardiac injury after chemotherapy.

With the goal to enhance the distribution of cationic Mn porphyrins within mitochondria, the lipophilic Mn(III)meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin, MnTnHex-2-PyP5+ has been synthesized and tested in several different model of diseases, where it shows remarkable efficacy at as low as 50 µg/kg single or multiple doses. Yet, in a rat lung radioprotection study, at higher 0.6–1 mg/kg doses, due to its high accumulation and micellar character, it became toxic. To avoid the toxicity, herein the pulmonary radioprotection of MnTnHex-2-PyP5+ was assessed at 50 µg/kg. Fischer rats were irradiated to their right hemithorax (28 Gy) and treated with 0.05 mg/kg/day of MnTnHex-2-PyP5+ for 2 weeks by subcutaneously-implanted osmotic pumps, starting at 2 h post-radiation. The body weights and breathing frequencies were followed for 10 weeks post-radiation, when the histopathology and immunohistochemistry were assessed. Impact of MnTnHex-2-PyP5+ on macrophage recruitment (ED-1), DNA oxidative damage (8-OHdG), TGF-β1, VEGF(A) and HIF-1α were measured. MnTnHex-2-PyP5+ significantly decreased radiation-induced lung histopathological (H&E staining) and functional damage (breathing frequencies), suppressed oxidative stress directly (8-OHdG), or indirectly, affecting TGF-β1, VEGF (A) and HIF-1α pathways. The magnitude of the therapeutic effects is similar to the effects demonstrated under same experimental conditions with 120-fold higher dose of ~5000-fold less lipophilic Mn(III)meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP5+., Graphical abstract, Highlights • Redox-active Mn porphyrin, MnTnHex-2-PyP5+, exerts impressive rat lung radioprotection at 0.05 mg/kg/day. • MnTnHex-2-PyP5+ reduces rat breathing frequences and lung tissue damage. • MnTnHex-2-PyP5+ affects redox-based signaling pathways whereby reducing inflammation (macrophage recruitment, DNA damage, HIF-1α, TGF-β1 and VEGF(A) expression). • Remarkable efficacy is likely due to its high cellular and mitochondrial accumulation.

Clear-cell renal carcinoma (ccRCC) is the most common type of renal cancer. The importance of oxidative stress in the context of this disease has been described, although there is only little information concerning the role of superoxide dismutase (SOD) enzymes. The importance of SOD in different pathological conditions promoted the development of SOD mimics (SODm). As such, manganese(III) porphyrins can mimic the natural SOD enzymes and scavenge different reactive oxygen species (ROS), thus modulating the cellular redox status. In this study, the exposure of 786-O human renal cancer cells to MnTnHex-2-PyP5+ (MnP), a very promising SODm, led to a concentration and time-dependent decrease in cell viability and in the cell proliferation indices, as well as to an increase in apoptosis. No relevant effects in terms of micronuclei formation were observed. Moreover, the exposure to MnP resulted in a concentration-dependent increase in intracellular ROS, presumably due to the generation of H2O2 by the inherent redox mechanisms of MnP, along with the limited ability of cancer cells to detoxify this species. Although the MnP treatment did not result in a reduction in the collective cell migration, a significant decrease in chemotactic migration was observed. Overall, these results suggest that MnP has a beneficial impact on reducing renal cancer cell viability and migration and warrant further studies regarding SODm-based therapeutic strategies against human renal cancer.

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 (ΔΨm). 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 (B-ALL) 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.

Cationic Mn porphyrins are among the most potent catalytic antioxidants and/or cellular redox modulators. Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin chloride (MnTE-2-PyPCl(5)) is the Mn porphyrin most studied in vivo and has successfully rescued animal models of a variety of oxidative stress-related diseases. The stability of an authentic MnTE-2-PyPCl(5) sample was investigated hereon by thermogravimetric, derivative thermogravimetric, and differential thermal analyses (TG/DTG/DTA), under dynamic air, followed by studies at selected temperatures to evaluate the decomposition path and appropriate conditions for storage and handling of these materials. All residues were analyzed by thin-layer chromatography (TLC) and UV-vis spectroscopy. Three thermal processes were observed by TG/DTG. The first event (endothermic) corresponded to dehydration, and did not alter the MnTE-2-PyPCl(5) moiety. The second event (endothermic) corresponded to the loss of EtCl (dealkylation), which was characterized by gas chromatography-mass spectrometry. The residue at 279°C had UV-vis and TLC data consistent with those of the authentic, completely dealkylated analog, MnT-2-PyPCl. The final, multi-step event corresponded to the loss of the remaining organic matter to yield Mn(3)O(4) which was characterized by IR spectroscopy. Isothermal treatment at 188°C under static air for 3h yielded a mixture of partially dealkylated MnPs and traces of the free-base, dealkylated ligand, H(2)T-2-PyP, which reveals that dealkylation is accompanied by thermal demetallation under static air conditions. Dealkylation was not observed if the sample was heated as a solid or in aqueous solution up to ∼100°C. Whereas moderate heating changes sample composition by loss of H(2)O, the dehydrated sample is indistinguishable from the original sample upon dissolution in water, which indicates that catalytic activity (on Mn basis) remains unaltered. Evidently, dealkylation at high temperature compromises sample activity.

We disclose here the studies that preceded and guided the preparation of the metal-based, redox-active therapeutic Mn(III) meso-tetrakis(N-n-butoxyethylpyridyl)porphyrin, MnTnBuOE-2-PyP5+ (BMX-001), which is currently in Phase I/II Clinical Trials at Duke University as a radioprotector of normal tissue in cancer patients. N-substituted pyridylporphyrins are ligands for Mn(III) complexes that are among the most potent superoxide dismutase mimics thus far synthesized. To advance their design, thereby improving their physical and chemical properties and bioavailability/toxicity profiles, we undertook a systematic study on placing oxygen atoms into N-alkylpyridyl chains via alkoxyalkylation reaction. For the first time we show here the unforeseen structural rearrangement that happens during the alkoxyalkylation reaction by the corresponding tosylates. Comprehensive experimental and computational approaches were employed to solve the rearrangement mechanism involved in quaternization of pyridyl nitrogens, which, instead of a single product, led to a variety of mixed N-alkoxyalkylated and N-alkylated pyridylporphyrins. The rearrangement mechanism involves the formation of an intermediate alkyl oxonium cation in a chain-length-dependent manner, which subsequently drives differential kinetics and thermodynamics of competing N-alkoxyalkylation versus in situ N-alkylation. The use of numerous alkoxyalkyl tosylates, of different length of alkyl fragments adjacent to oxygen atom, allowed us to identify the set of alkyl fragments that would result in the synthesis of a single compound of high purity and excellent therapeutic potential.

Combinations of radiotherapy (RT) and chemotherapy have shown efficacy toward brain tumors. However, therapy-induced oxidative stress can damage normal brain tissue, resulting in both progressive neurocognitive loss and diminished quality of life. We have recently shown that MnTnBuOE-2-PyP(5+) (Mn(III)meso-tetrakis(N-n-butoxyethylpyridinium -2-yl)porphyrin) rescued RT-induced white matter damage in cranially-irradiated mice. Radiotherapy is not used in isolation for treatment of brain tumors; temozolomide is the standard-of-care for adult glioblastoma, whereas cisplatin is often used for treatment of pediatric brain tumors. Therefore, we evaluated the brain radiation mitigation ability of MnTnBuOE-2-PyP(5+) after either temozolomide or cisplatin was used singly or in combination with 10 Gy RT. MnTnBuOE-2-PyP(5+) accumulated in brains at low nanomolar levels. Histological and neurobehavioral testing showed a drastic decrease (1) of axon density in the corpus callosum and (2) rotorod and running wheel performance in the RT only treatment group, respectively. MnTnBuOE-2-PyP(5+) completely rescued this phenotype in irradiated animals. In the temozolomide groups, temozolomide/ RT treatment resulted in further decreased rotorod responses over RT alone. Again, MnTnBuOE-2-PyP(5+) treatment rescued the negative effects of both temozolomide ± RT on rotorod performance. While the cisplatin-treated groups did not give similar results as the temozolomide groups, inclusion of MnTnBuOE-2-PyP(5+) did not negatively affect rotorod performance. Additionally, MnTnBuOE-2-PyP(5+) sensitized glioblastomas to either RT ± temozolomide in flank tumor models. Mice treated with both MnTnBuOE-2-PyP(5+) and radio-/chemo-therapy herein demonstrated brain radiation mitigation. MnTnBuOE-2-PyP(5+) may well serve as a normal tissue radio-/chemo-mitigator adjuvant therapy to standard brain cancer treatment regimens. Environ. Mol. Mutagen. 57:372-381, 2016. © 2016 Wiley Periodicals, Inc.

Based on aqueous redox chemistry and simple in vivo models of oxidative stress, Escherichia coli and Saccharomyces cerevisiae, the cationic Mn(III) N-substituted pyridylporphyrins (MnPs) have been identified as the most potent cellular redox modulators within the porphyrin class of drugs; their efficacy in animal models of diseases that have oxidative stress in common is based on their high ability to catalytically remove superoxide, peroxynitrite, carbonate anion radical, hypochlorite, nitric oxide, lipid peroxyl and alkoxyl radicals, thus suppressing the primary oxidative event. While doing so MnPs could couple with cellular reductants and redox-active proteins. Reactive species are widely accepted as regulators of cellular transcriptional activity: minute, nanomolar levels are essential for normal cell function, while submicromolar or micromolar levels impose oxidative stress, which is evidenced in increased inflammatory and immune responses. By removing reactive species, MnPs affect redox-based cellular transcriptional activity and consequently secondary oxidative stress, and in turn inflammatory processes. The equal ability to reduce and oxidize superoxide during the dismutation process and recently accumulated results suggest that pro-oxidative actions of MnPs may also contribute to their therapeutic effects. All our data identify the superoxide dismutase-like activity, estimated by log kcat(O2–), as a good measure for the therapeutic efficacy of MnPs. Their accumulation in mitochondria and their ability to cross the blood-brain barrier contribute to their remarkable efficacy. We summarize herein the therapeutic effects of MnPs in cancer, central nervous system injuries, diabetes, their radioprotective action and potential for imaging. Few of the most potent modulators of cellular redox-based pathways, MnTE2-PyP5+, MnTDE-2-ImP5+, MnTnHex-2-PyP5+ and MnTnBuOE-2-PyP5+, are under preclinical and clinical development.

Increased understanding of the role of mitochondria under physiological and pathological conditions parallels increased exploration of synthetic and natural compounds able to mimic MnSOD - endogenous mitochondrial antioxidant defense essential for the existence of virtually all aerobic organisms from bacteria to humans. This review describes most successful mitochondrially-targeted redox-active compounds, Mn porphyrins and MitoQ(10) in detail, and briefly addresses several other compounds that are either catalysts of O(2)(-) dismutation, or its non-catalytic scavengers, and that reportedly attenuate mitochondrial dysfunction. While not a true catalyst (SOD mimic) of O(2)(-) dismutation, MitoQ(10) oxidizes O(2)(-) to O(2) with a high rate constant. In vivo it is readily reduced to quinol, MitoQH(2), which in turn reduces ONOO(-) to NO(2), producing semiquinone radical that subsequently dismutes to MitoQ(10) and MitoQH(2), completing the "catalytic" cycle. In MitoQ(10), the redox-active unit was coupled via 10-carbon atom alkyl chain to monocationic triphenylphosphonium ion in order to reach the mitochondria. Mn porphyrin-based SOD mimics, however, were designed so that their multiple cationic charge and alkyl chains determine both their remarkable SOD potency and carry them into the mitochondria. Several animal efficacy studies such as skin carcinogenesis and UVB-mediated mtDNA damage, and subcellular distribution studies of Saccharomyces cerevisiae and mouse heart provided unambiguous evidence that Mn porphyrins mimic the site and action of MnSOD, which in turn contributes to their efficacy in numerous in vitro and in vivo models of oxidative stress. Within a class of Mn porphyrins, lipophilic analogs are particularly effective for treating central nervous system injuries where mitochondria play key role. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

The early antecedents of cerebral palsy (CP) are unknown but are suspected to be due to hypoxia-ischemia (H-I). In our rabbit model of CP, the MRI biomarker, apparent diffusion coefficient (ADC) on diffusion-weighted imaging, predicted which fetuses will develop postnatal hypertonia. Surviving H-I fetuses experience reperfusion-reoxygenation but a subpopulation manifested a continued decline of ADC during early reperfusion-reoxygenation, which possibly represented greater brain injury (RepReOx). We hypothesized that oxidative stress in reperfusion-reoxygenation is a critical trigger for postnatal hypertonia. We investigated whether RepReOx predicted postnatal neurobehavior, indicated oxidative stress, and whether targeting antioxidants at RepReOx ameliorated motor deficits, which included testing of a new superoxide dismutase mimic (MnTnHex-2-PyP). Rabbit dams, 79% gestation (E25), were subjected to 40 min uterine ischemia. Fetal brain ADC was followed during H-I, immediate reperfusion-reoxygenation, and 4–72 h after H-I. Endpoints were postnatal neurological outcome at E32, ADC at end of H-I, ADC nadir during H-I and reperfusion-reoxygenation, and area under ADC curve during the first 20 min of reperfusion-reoxygenation. Antioxidants targeting RepReOx were administered before and/or after uterine ischemia. The new MRI-ADC biomarker for RepReOx improved prediction of postnatal hypertonia. Greater superoxide production, mitochondrial injury, and oligodendroglial loss occurred in fetal brains exhibiting RepReOx than in those without. The antioxidants, MnTnHex-2-PyP and Ascorbate and Trolox combination, significantly decreased postnatal motor deficits and extent of RepReOx. The etiological link between early injury and later motor deficits can thus be investigated by MRI, and allows us to distinguish between critical oxidative stress that causes motor deficits and noncritical oxidative stress that does not.