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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 or poorly purified samples are able to perform O2.- dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in a SOD-deficient Escherichia coli model, whereas MnTE-2-PyP-treated SOD-deficient E. coli grew as well as a 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 MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, 3-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O2.- 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 ONOOCO2 adduct. The log kcat (O2.-) value for MnTBAP is estimated to be about 3.16, which is approximately 5 and approximately 6 orders of magnitude smaller than the SOD activities of the potent SOD mimic MnTE-2-PyP and Cu,Zn-SOD, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only approximately 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 the 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 lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O2.-, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO-/CO3.- from O2.- pathways.

Severe pain syndromes reduce the quality of life of patients with inflammatory and neoplastic diseases, partly because reduced analgesic effectiveness with chronic opiate therapy (i.e., tolerance) leads to escalating doses and distressing side effects. Peroxynitrite-mediated nitroxidative stress in the dorsal horn of the spinal cord plays a critical role in the induction and development of antinociceptive tolerance to morphine. This provides a valid pharmacological basis for developing peroxynitrite scavengers as potent adjuncts to opiates in the management of pain. The cationic Mn(III) ortho-N-alkylpyridylporphyrins MnTE-2-PyP(5+) and MnTnHex-2-PyP(5+) are among the most potent peroxynitrite scavengers, with nearly identical scavenging rate constants (approximately 10(7) M(-1) s(-1)). Yet, MnTnHex-2-PyP(5+) is significantly more lipophilic and more bioavailable and, in turn, was 30-fold more effective in blocking the development of morphine antinociceptive tolerance than MnTE-2-PyP(5+) using the hot-plate test in a well-characterized murine model. The hydrophilic MnTE-2-PyP(5+) and the lipophilic MnTnHex-2-PyP(5+) were 10- and 300-fold, respectively, more effective in inhibiting morphine tolerance than the hydrophilic Fe(III) porphyrin FeTM-4-PyP(5+). Both Mn porphyrins decreased levels of TNF-alpha, IL-1 beta, and IL-6 to normal values. Neither of them affected acute morphine antinociceptive effects nor caused motor function impairment. Also neither was able to reverse already established morphine tolerance. We have recently shown that the anionic porphyrin Mn(III) tetrakis(4-carboxylatophenyl)porphyrin is selective in removing ONOO(-) over O(2)(-), but at approximately 2 orders of magnitude lower efficacy than MnTE-2-PyP(5+) and MnTnHex-2-PyP(5+), which in turn parallels up to 100-fold lower ability to reverse morphine tolerance. These data (1) support the role of peroxynitrite rather than superoxide as a major mechanism in blocking the development of morphine tolerance and (2) show that lipophilicity is a critical parameter in enhancing the potency of such novel peroxynitrite scavengers.

Cationic Mn porphyrins are among the most potent SOD mimics and peroxynitrite scavengers. They have been widely and successfully used in different models of oxidative stress and are either progressing towards or are in phase I of clinical trials. The most frequently used compounds are Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+) or AEOL10113), its methyl analogue (MnTM-2-PyP(5+) or AEOL10112), and Mn(III) meso-tetrakis(4-benzoic acid)porphyrin (MnTBAP). A great discrepancy between the in vivo data obtained with Calbiochem preparations and those of authentic MnTE-2-PyP(5+) and MnTM-2-PyP(5+) samples were recently observed. Surprisingly, the commercial samples were invariably of poor identity and consisted of mixtures of nearly equal contributions of non-alkylated, mono-, di-, tri- and tetraalkylated porphyrins, lacking thus the major structural entity that determines their antioxidant potency, i.e., the four positively charged orthoN-alkylpyridyl groups that afford thermodynamic tuning of the active site and electrostatic guidance of anionic superoxide and peroxynitrite species toward the metal center. The MnTE-2-PyP(5+) and MnTM-2-PyP(5+) compounds were not even the major species in the commercial samples sold as "MnTE-2-PyP" and "MnTM-2-PyP", respectively. While we have already reported the insufficient impurity of the MnTBAP samples from Alexis and other suppliers, in one more recent lot the situation is dramatic, as 25% of the sample was not MnTBAP, but metal-free ligand, H(2)TBAP. The (unintentional) use of the Mn porphyrins of low quality compromises therapeutic and/or mechanistic conclusions. Simple techniques, which include thin-layer chromatography, electrospray-mass spectrometry, UV-vis spectroscopy, and electrochemistry described here could be used routinely to check the overall quality of Mn porphyrins in order to avoid misleading conclusions and waste of valuable resources (animals, compounds, time, manpower).

We have studied the role of three Mn(III)porphyrins differing in charge, alkyl substituent length and reactivity, on LDL exposed to low fluxes of peroxynitrite (PN) in the presence of uric acid. Mn(III)porphyrins (5 microM, MnTE-2-PyP(5+), MnTnOct-2-PyP(5+), and MnTCPP(3-)) plus uric acid (300 microM) inhibited cholesteryl ester hydroperoxide formation, changes in REM as well as spared alpha- and gamma-tocopherol. MnTnOct-2-PyP(5+), the more lipophilic compound, was the most effective in protecting LDL lipids, while MnTCPP(3-) exerted the lesser protection. Mn(III)porphyrins react fast with PN ( approximately 10(5)-10(7) M(-1) s(-1)) to yield a O=Mn(IV) complex. The stoichiometry of uric acid consumption was approximately 1.7 moles per mol of PN, in agreement with reactions with both the O=Mn(IV) complex and nitrogen dioxide. A shift from an anti- to a pro-oxidant action of the Mn(III)porphyrin was observed after uric acid was significantly consumed, supporting competition reactions between LDL targets and uric acid for the O=Mn(IV) complex. Overall, the data is consistent with the catalytic reduction of PN in a cycle that involves a one electron oxidation of Mn(III) to Mn(IV) by PN followed by the reduction back to Mn(III) by uric acid. These antioxidant effects should predominate under in vivo conditions having plasma uric acid concentration range between 150 and 500 microM.