Oxidative stress has become widely viewed as an underlying condition in a number of diseases, such as ischemia-reperfusion disorders, central nervous system disorders, cardiovascular conditions, cancer, and diabetes. Thus, natural and synthetic antioxidants have been actively sought. Superoxide dismutase is a first line of defense against oxidative stress under physiological and pathological conditions. Therefore, the development of therapeutics aimed at mimicking superoxide dismutase was a natural maneuver. Metalloporphyrins, as well as Mn cyclic polyamines, Mn salen derivatives and nitroxides were all originally developed as SOD mimics. The same thermodynamic and electrostatic properties that make them potent SOD mimics may allow them to reduce other reactive species such as peroxynitrite, peroxynitrite-derived CO 3 _ À , peroxyl radical, and less efficiently H 2 O 2 . By doing so SOD mimics can decrease both primary and secondary oxidative events, the latter arising from the inhibition of cellular transcriptional activity. To better judge the therapeutic potential and the advantage of one over the other type of compound, comparative studies of different classes of drugs in the same cellular and=or animal models are needed. We here provide a comprehensive overview of the chemical properties and some in vivo effects observed with various classes of compounds with a special emphasis on porphyrin-based compounds. Antioxid. Redox Signal. 13, 877-918.

Oxidative stress, a redox imbalance between the endogenous reactive species and antioxidant systems, is common to numerous pathological conditions such as cancer, central nervous system injuries, radiation injury, diabetes etc. Therefore, compounds able to reduce oxidative stress have been actively sought for over 3 decades. Superoxide is the major species involved in oxidative stress either in its own right or through its progeny, such as ONOO−, H2O2, ·OH, CO3·−, and ·NO2. Therefore, the very first compounds developed in the late 1970-ies were the superoxide dismutase (SOD) mimics. Thus far the most potent mimics have been the cationic meso Mn(III) N-substituted pyridylporphyrins and N,N′-disubstituted imidazolylporphyrins (MnPs), some of them with kcat(O2·−) similar to the kcat of SOD enzymes. Most frequently studied are ortho isomers MnTE-2-PyP5+, MnTnHex-2-PyP5+, and MnTDE-2-ImP5+. The ability to disproportionate O2·− parallels their ability to remove the other major oxidizing species, peroxynitrite, ONOO−. The same structural feature that gives rise to the high kcat (O2·−) and kred (ONOO−), allows MnPs to strongly impact the activation of the redox-sensitive transcription factors, HIF-1α, NF-κB, AP-1, and SP-1, and therefore modify the excessive inflammatory and immune responses. Coupling with cellular reductants and other redox-active endogenous proteins seems to be involved in the actions of Mn porphyrins. While hydrophilic analogues, such as MnTE-2-PyP5+ and MnTDE-2-ImP5+ are potent in numerous animal models of diseases, the lipophilic analogues were developed to cross blood brain barrier and target central nervous system and critical cellular compartment, mitochondria. The modification of its structure, aimed to preserve the SOD-like potency and lipophilicity, and diminish the toxicity, has presently been pursued. The pulmonary radioprotection by MnTnHex-2-PyP5+ was the first efficacy study performed successfully with non-human primates. The Phase I toxicity clinical trials were done on amyotrophic lateral sclerosis patients with N,N′-diethylimidazolium analogue, MnTDE-2-ImP5+ (AEOL10150). Its aggressive development as a wide spectrum radioprotector by Aeolus Pharmaceuticals has been supported by USA Federal government. The latest generation of compounds, bearing oxygens in pyridyl substituents is presently under aggressive development for cancer and CNS injuries at Duke University and is supported by Duke Translational Research Institute, The Wallace H. Coulter Translational Partners Grant Program, Preston Robert Tisch Brain Tumor Center at Duke, and National Institute of Allergy and Infectious Diseases. Metal center of cationic manganese porphyrins easily accepts and donates electrons as exemplified in the catalysis of O2·− dismutation. Thus such compounds may be equally good anti- and pro-oxidants; in either case the beneficial therapeutic effects may be observed. Moreover, while the in vivo effects may appear antioxidative, the mechanism of action of MnPs that produced such effects may be pro-oxidat...

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Superoxide is involved in a plethora of pathological and physiological processes via oxidative stress and/or signal transduction pathways. Superoxide dismutase (SOD) mimics have, thus, been actively sought for clinical and mechanistic purposes. Manganese(III) 5,10,15,20-tetrakis(4-benzoic acid)porphyrin (MnTBAP) is one of the most intensely explored "SOD mimics" in biology and medicine. However, we show here that this claimed SOD activity of MnTBAP in aqueous media is not corroborated by comprehensive structure-activity relationship studies for a wide set of Mn porphyrins and that MnTBAP from usual commercial sources contains different amounts of noninnocent trace impurities (Mn clusters), which inhibited xanthine oxidase and had SOD activity in their own right. In addition, the preparation and thorough characterization of a high-purity MnTBAP is presented for the first time and confirmed that pure MnTBAP has no SOD activity in aqueous medium. These findings call for an assessment of the relevance and suitability of using MnTBAP (or its impurities) as a mechanistic probe and antioxidant therapeutic; conclusions on the physiological and pathological role of superoxide derived from studies using MnTBAP of uncertain purity should be examined judiciously. An unequivocal distinction between the biological effects due to MnTBAP and that of its impurities can only be unambiguously made if a pure sample is/was used. This work also illustrates the contribution of fundamental structure-activity relationship studies not only for drug design and optimization, but also as a "watchdog" mechanism for checking/spotting eventual incongruence of drug activity in chemical and biological settings.

Mn(III) N-alkylpyridylporphyrins are among the most potent known SOD mimics and catalytic peroxynitrite scavengers, and modulators of redox-based cellular transcriptional activity. In addition to their intrinsic antioxidant capacity, bioavailability plays major role in their in vivo efficacy. While of identical antioxidant capacity, lipophilic MnTnHex-2-PyP is up to 120-fold more efficient in reducing oxidative stress injuries than hydrophilic MnTE-2-PyP. Due to limitations of analytical nature, porphyrin lipophilicity has been often estimated by thin-layer chromatographic Rf parameter, instead of the standard n-octanol/water partition coefficient, POW. Herein we used a new methodological approach to finally describe the MnP lipophilicity, by the conventional log POW means, for a series of biologically active ortho and meta isomers of Mn(III) N-alkylpyridylporphyrins. Three new porphyrins (MnTnBu-3-PyP, MnTnHex-3-PyP and MnTnHep-2-PyP) were synthesized to strengthen the conclusions. The log POW was linearly related to Rf and to the number of carbons in the alkyl chain (nC) for both isomer series; the meta isomers being 10-fold more lipophilic than the analogous ortho porphyrins. Increasing the length of the alkyl chain for 1 carbon atom increases the log POW value ~ 1 log unit with both isomers. Dramatic ~4 and ~5 orders of magnitude increase in lipophilicity of ortho isomers by extending pyridyl alkyl chains from 2 (MnTE-2-PyP, log POW = −6.25) to 6 (MnTnHex-2-PyP, log POW = −2.29) and 8 carbon atoms (MnTnOct-2-PyP, log POW = −0.77) parallels the increased efficacy in several oxidative-stress injury models, particularly those of the central nervous system where transport across the blood-brain barrier is critical. Although meta isomers are only slightly less potent SOD mimics and antioxidants than their ortho analogues, their higher lipophilicity and smaller bulkiness may lead to a higher cellular uptake and overall similar effectiveness in vivo.

MnTBAP is often referred to as an SOD mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial/ill-purified samples are able to perform O 2 •− dismutation with low-to-moderate efficacy via non-innocent Mncontaining impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in the SOD-deficient E. coli model, while MnTE-2-PyP-treated SOD-deficient E. coli grew as well as wild-type strain. This SOD-specific system indicates that MnTBAP does not act as an SOD mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate, and neutrophil infiltration and activation: these events were blocked by 0.3 mg/kg of MnTE-2-PyP and to a slightly lesser extent with 10 mg/kg of MnTBAP. Also, 3-nitrotyrosine formation, an indication of the peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. indicate that MnTBAP exert some protective actions in common with MnTE-2-PyP, which are not O 2 •− -related, and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOO − adduct with CO 2 . The log k cat (O 2 •− ) value for MnTBAP is estimated to be about 3.16, which is ~5 and ~7 orders of magnitude smaller than the SOD activity of the potent SOD mimic MnTE-2-PyP and Cu, Zn-SOD, respectively. This very low value indicates that MnTBAP is very inefficient in dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. Peroxynitrite scavenging ability of MnTBAP, however, is only ~2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the SOD-active impurities in commercial MnTBAP sample (log k red (ONOO − ) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is >1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the smaller ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP one) could arise from its impurities, which, by scav...

The different biological behavior of cationic Fe and Mn pyridylporphyrins in Escherichia coli and mouse studies prompted us to revisit and compare their chemistry. For that purpose the series of ortho and meta isomers of Fe(III) meso-tetrakis-N-alkylpyridylporphyrins, alkyl being methyl to n-octyl, were synthesized and characterized by elemental analysis, UV/vis spectroscopy, mass spectrometry, lipophilicity, protonation equilibria of axial waters, metal-centered reduction potential, E1/2 for MIIIP/MIIP redox couple (M = Fe, Mn, P=porphyrin), kcat for the catalysis of O2•− dismutation, stability towards peroxide-driven porphyrin oxidative degradation (produced in the catalysis of ascorbate oxidation by MP), ability to affect growth of SOD-deficient E. coli and toxicity to mice. Electron-deficiency of the metal site is modulated by the porphyrin ligand, which renders Fe(III) porphyrins ≥ 5 orders of magnitude more acidic than the analogous Mn(III) porphyrins, as revealed by the pKa1 of axially coordinated waters. The 5 log units difference in the acidity between the Mn and Fe sites in porphyrin translates into the predominance of tetracationic (OH)(H2O)FeP complexes relative to pentacationic (H2O)2MnP species at pH ~7.8. This is evidenced in large differences in the thermodynamic parameters - pKa of axial waters and E1/2 of MIII/MII redox couple. The presence of hydroxo ligand labilizes trans-axial water which results in higher reactivity of Fe- relative to Mn center. The differences in the catalysis of O2•− dismutation (log kcat) between Fe and Mn porphyrins is modest, 2.5-5-fold, due to predominantly outer-sphere, with partial inner-sphere character of two reaction steps. However, the rate constant for the inner-sphere H2O2-based porphyrin oxidative degradation is 18-fold larger for (OH)(H2O)FeP than for (H2O)2MnP. The in vivo consequences of the differences between the Fe- and Mn porphyrins were best demonstrated in SOD-deficient E. coli growth. Based on fairly similar log kcat(O2.− values, very similar effect on the growth of SOD-deficient E. coli was anticipated by both metalloporphyrins. Yet, while MnTE-2-PyP5+ was fully efficacious at ≥20 μM, the Fe analog, FeTE-2-PyP5+ supported SOD-deficient E. coli growth at 200-fold lower doses in the range of 0.1 to 1 μM. Moreover the pattern of SOD-deficient E. coli growth was different with Mn- and Fe porphyrins. Such results suggested different mode of action of these metalloporphyrins. Further exploration demonstrated that: (1) 0.1 μM FeTE-2-PyP5+ provided similar growth stimulation as 0.1 μM Fe salt, while 20 μM Mn salt provides no protection to E. coli; and (2) 1 μM Fe porphyrin is fully degraded by 12 hours in E. coli cytosol and growth medium; while Mn porphyrin is not. Stimulation of the aerobic growth of SOD-deficient E. coli by the Fe porphyrin is therefore due to iron acquisition. Our data suggest that in vivo, redox-driven degradation of Fe porphyrins resulting in Fe release plays a major role in their biological action. Possibly, iron reconstitutes enzym...

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 mu...