A porphyrin-based fluorescence method for zinc determination in commercial propolis extracts without sample pretreatment.

The quantification of zinc in over-the-counter drugs as commercial propolis extracts by molecular fluorescence technique using meso-tetrakis(4-carboxyphenyl)porphyrin (H2 TCPP4 ) was developed for the first time. The calibration curve is linear from 6.60 to 100 nmol L-1 of Zn2+ . The detection and quantification limits were 6.22 nmol L-1 and 19.0 nmol L-1 , respectively. The reproducibility and repeatability calculated as the percentage variation of slopes of seven calibration curves were 6.75% and 4.61%, respectively. Commercial propolis extract samples from four Brazilian states were analyzed and the results (0.329-0.797 mg/100 mL) obtained with this method are in good agreement with that obtained with the Atomic Absorption Spectroscopy (AAS) technique. The method is simple, fast, of low cost and allows the analysis of the samples without pretreatment. Moreover the major advantage is that Zn-porphyrin complex presents fluorescent characteristic promoting the selectivity and sensitivity of the method.

Comparative Study on the Efficiency of the Photodynamic Inactivation of Candida albicans Using CdTe Quantum Dots, Zn(II) Porphyrin and Their Conjugates as Photosensitizers.

The application of fluorescent II-VI semiconductor quantum dots (QDs) as active photosensitizers in photodymanic inactivation (PDI) is still being evaluated. In the present study, we prepared 3 nm size CdTe QDs coated with mercaptosuccinic acid and conjugated them electrostatically with Zn(II) meso-tetrakis (N-ethyl-2-pyridinium-2-yl) porphyrin (ZnTE-2-PyP or ZnP), thus producing QDs-ZnP conjugates. We evaluated the capability of the systems, bare QDs and conjugates, to produce reactive oxygen species (ROS) and applied them in photodynamic inactivation in cultures of Candida albicans by irradiating the QDs and testing the hypothesis of a possible combined contribution of the PDI action. Tests of in vitro cytotoxicity and phototoxicity in fibroblasts were also performed in the presence and absence of light irradiation. The overall results showed an efficient ROS production for all tested systems and a low cytotoxicity (cell viability >90%) in the absence of radiation. Fibroblasts incubated with the QDs-ZnP and subjected to irradiation showed a higher cytotoxicity (cell viability <90%) depending on QD concentration compared to the bare groups. The PDI effects of bare CdTe QD on Candida albicans demonstrated a lower reduction of the cell viability (~1 log10) compared to bare ZnP which showed a high microbicidal activity (~3 log10) when photoactivated. The QD-ZnP conjugates also showed reduced photodynamic activity against C. albicans compared to bare ZnP and we suggest that the conjugation with QDs prevents the transmembrane cellular uptake of the ZnP molecules, reducing their photoactivity.

Redox potential determines the reaction mechanism of HNO donors with Mn and Fe porphyrins: defining the better traps.

Azanone ((1)HNO, nitroxyl) is a highly reactive molecule with interesting chemical and biological properties. Like nitric oxide (NO), its main biologically related targets are oxygen, thiols, and metalloproteins, particularly heme proteins. As HNO dimerizes with a rate constant between 10(6) and 10(7) M(-1) s(-1), reactive studies are performed using donors, which are compounds that spontaneously release HNO in solution. In the present work, we studied the reaction mechanism and kinetics of two azanone donors Angelís Salt and toluene sulfohydroxamic acid (TSHA) with eight different Mn porphyrins as trapping agents. These porphyrins differ in their total peripheral charge (positively or negatively charged) and in their Mn(III)/Mn(II) reduction potential, showing for each case positive (oxidizing) and negative (reducing) values. Our results show that the reduction potential determines the azanone donor reaction mechanism. While oxidizing porphyrins accelerate decomposition of the donor, reducing porphyrins react with free HNO. Our results also shed light into the donor decomposition mechanism using ab initio methods and provide a thorough analysis of which MnP are the best candidates for azanone trapping and quantification experiments.

Rational design of superoxide dismutase (SOD) mimics: the evaluation of the therapeutic potential of new cationic Mn porphyrins with linear and cyclic substituents.

Our goal herein has been to gain further insight into the parameters which control porphyrin therapeutic potential. Mn porphyrins (MnTnOct-2-PyP(5+), MnTnHexOE-2-PyP(5+), MnTE-2-PyPhP(5+), and MnTPhE-2-PyP(5+)) that bear the same positive charge and same number of carbon atoms at meso positions of porphyrin core were explored. The carbon atoms of their meso substituents are organized to form either linear or cyclic structures of vastly different redox properties, bulkiness, and lipophilicities. These Mn porphyrins were compared to frequently studied compounds, MnTE-2-PyP(5+), MnTE-3-PyP(5+), and MnTBAP(3-). All Mn(III) porphyrins (MnPs) have metal-centered reduction potential, E1/2 for Mn(III)P/Mn(II)P redox couple, ranging from -194 to +340 mV versus NHE, log kcat(O2(•-)) from 3.16 to 7.92, and log kred(ONOO(-)) from 5.02 to 7.53. The lipophilicity, expressed as partition between n-octanol and water, log POW, was in the range -1.67 to -7.67. The therapeutic potential of MnPs was assessed via: (i) in vitro ability to prevent spontaneous lipid peroxidation in rat brain homogenate as assessed by malondialdehyde levels; (ii) in vivo O2(•-) specific assay to measure the efficacy in protecting the aerobic growth of SOD-deficient Saccharomyces cerevisiae; and (iii) aqueous solution chemistry to measure the reactivity toward major in vivo endogenous antioxidant, ascorbate. Under the conditions of lipid peroxidation assay, the transport across the cellular membranes, and in turn shape and size of molecule, played no significant role. Those MnPs of E1/2 ∼ +300 mV were the most efficacious, significantly inhibiting lipid peroxidation in 0.5-10 µM range. At up to 200 µM, MnTBAP(3-) (E1/2 = -194 mV vs NHE) failed to inhibit lipid peroxidation, while MnTE-2-PyPhP(5+) with 129 mV more positive E1/2 (-65 mV vs NHE) was fully efficacious at 50 µM. The E1/2 of Mn(III)P/Mn(II)P redox couple is proportional to the log kcat(O2(•-)), i.e., the SOD-like activity of MnPs. It is further proportional to kred(ONOO(-)) and the ability of MnPs to prevent lipid peroxidation. In turn, the inhibition of lipid peroxidation by MnPs is also proportional to their SOD-like activity. In an in vivo S. cerevisiae assay, however, while E1/2 predominates, lipophilicity significantly affects the efficacy of MnPs. MnPs of similar log POW and E1/2, that have linear alkyl or alkoxyalkyl pyridyl substituents, distribute more easily within a cell and in turn provide higher protection to S. cerevisiae in comparison to MnP with bulky cyclic substituents. The bell-shape curve, with MnTE-2-PyP(5+) exhibiting the highest ability to catalyze ascorbate oxidation, has been established and discussed. Our data support the notion that the SOD-like activity of MnPs parallels their therapeutic potential, though species other than O2(•-), such as peroxynitrite, H2O2, lipid reactive species, and cellular reductants, may be involved in their mode(s) of action(s).

Molecular recognition using ruthenium(II) porphyrin thiol complexes as probes.

In situ (1)H NMR data are reported for 106 Ru(porp)(RSH)(2) species, where porp is the dianion of ß-octaethylporphyrin (OEP), meso-tetraphenylporphyrin (TPP), and its para-substituted tetraphenyl analogues (T-p-XPP; X = OMe, Me, F, Cl, CO(2)Me, CF(3)), meso-tetrakis(3,5-dimethylphenyl)porphyrin (T-m,m'-Me(2)PP), and meso-tetramesitylporphyrin (TMP), and R = Me, Et, (n)Pr, (i)Pr, (n)Bu, (t)Bu, (n)Hex, Bn (benzyl), Ph, and p-MeOC(6)H(4). The upfield shifts in the SH resonances upon coordination of the thiol reflect changes in the porphyrin ring current and are analyzed using an empirical model that depicts quantitatively the nonbonding, electronic, and steric interactions between the thiol ligands, where steric factors dominate, and the porphyrin plane, where electronic factors dominate; such interactions are typically involved in small-molecule recognition within metalloporphyrin systems. Implications of the findings to hemethiolate proteins and surface coordination chemistry are also briefly presented.

Differential coordination demands in Fe versus Mn water-soluble cationic metalloporphyrins translate into remarkably different aqueous redox chemistry and biology.

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, E(1/2) for M(III)P/M(II)P redox couple (M = Fe, Mn, P = porphyrin), kcat for the catalysis of O2(•-) dismutation, stability toward 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 additionally evidenced in large differences in the E(1/2) values of M(III)P/M(II)P redox couples. 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. On the basis of fairly similar log kcat(O2(•-)) values, a very similar effect on the growth of SOD-deficient E. coli was anticipated by both metalloporphyrins. Yet, while (H2O)2MnTE-2-PyP(5+) was fully efficacious at ≥20 µM, the Fe analogue (OH)(H2O)FeTE-2-PyP(4+) supported SOD-deficient E. coli growth at as much as 200-fold lower doses in the range of 0.1-1 µM. Moreover the pattern of SOD-deficient E. coli growth was different with Mn and Fe porphyrins. Such results suggested a different mode of action of these metalloporphyrins. Further exploration demonstrated that (1) 0.1 µM (OH)(H2O)FeTE-2-PyP(4+) provided similar growth stimulation as the 0.1 µM Fe salt, while the 20 µM Mn salt provides no protection to E. coli; and (2) 1 µM Fe porphyrin is fully degraded by 12 h 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 enzymes bearing [4Fe-4S] clusters as active sites. Under the same experimental conditions, (OH)(H2O)FePs do not cause mouse arterial hypotension, whereas (H2O)2MnPs do, which greatly limits the application of Mn porphyrins in vivo.

Intracellular targeting and pharmacological activity of the superoxide dismutase mimics MnTE-2-PyP5+ and MnTnHex-2-PyP5+ regulated by their porphyrin ring substituents.

Manganese porphyrin-based drugs are potent mimics of the enzyme superoxide dismutase. They exert remarkable efficacy in disease models and are entering clinical trials. Two lead compounds, MnTE-2-PyP(5+) and MnTnHex-2-PyP(5+), have similar catalytic rates, but differ in their alkyl chain substituents (ethyl vs n-hexyl). Herein we demonstrate that these changes in ring substitution impact upon drug intracellular distribution and pharmacological mechanism, with MnTnHex-2-PyP(5+) superior in augmenting menadione toxicity. These findings establish that both catalytic activity and intracellular distribution determine drug action.

A Novel Strategy Based on Zn(II) Porphyrins and Silver Nanoparticles to Photoinactivate Candida albicans.

Background: Photodynamic inactivation (PDI) is an attractive alternative to treat Candida albicans infections, especially considering the spread of resistant strains. The combination of the photophysical advantages of Zn(II) porphyrins (ZnPs) and the plasmonic effect of silver nanoparticles (AgNPs) has the potential to further improve PDI. Here, we propose the novel association of polyvinylpyrrolidone (PVP) coated AgNPs with the cationic ZnPs Zn(II) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin or Zn(II) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin to photoinactivate C. albicans. Methods: AgNPs stabilized with PVP were chosen to allow for (i) overlap between the NP extinction and absorption spectra of ZnPs and (ii) favor AgNPs-ZnPs interaction; prerequisites for exploring the plasmonic effect. Optical and zeta potential (ζ) characterizations were performed, and reactive oxygen species (ROS) generation was also evaluated. Yeasts were incubated with individual ZnPs or their respective AgNPs-ZnPs systems, at various ZnP concentrations and two proportions of AgNPs, then irradiated with a blue LED. Interactions between yeasts and the systems (ZnP alone or AgNPs-ZnPs) were evaluated by fluorescence microscopy. Results: Subtle spectroscopic changes were observed for ZnPs after association with AgNPs, and the ζ analyses confirmed AgNPs-ZnPs interaction. PDI using ZnP-hexyl (0.8 µM) and ZnP-ethyl (5.0 µM) promoted a 3 and 2 log10 reduction of yeasts, respectively. On the other hand, AgNPs-ZnP-hexyl (0.2 µM) and AgNPs-ZnP-ethyl (0.6 µM) systems led to complete fungal eradication under the same PDI parameters and lower porphyrin concentrations. Increased ROS levels and enhanced interaction of yeasts with AgNPs-ZnPs were observed, when compared with ZnPs alone. Conclusion: We applied a facile synthesis of AgNPs which boosted ZnP efficiency. We hypothesize that the plasmonic effect combined with the greater interaction between cells and AgNPs-ZnPs systems resulted in an efficient and improved fungal inactivation. This study provides insight into the application of AgNPs in PDI and helps diversify our antifungal arsenal, encouraging further developments toward inactivation of resistant Candida spp.

Thiol, disulfide, and trisulfide complexes of Ru porphyrins: potential models for iron-sulfur bonds in heme proteins.

Thirty-two Ru(porp)L(2) complexes have been synthesized, where porp = the dianion of meso-tetramesitylporphyrin (TMP) or meso-tetrakis(4-methylphenyl)porphyrin (H(2)T-pMe-PP), and L = a thiol, a sulfide, a disulfide, or a trisulfide. Species studied were with RSH [R = Me, Et, (n)Pr, (i)Pr, (t)Bu, Bn (benzyl), and Ph], RSR (R = Me, Bn), RSSR (R = Me, Et, (n)Pr, Bn) and MeSS(t)Bu, and RSSSR (R = Me, Bn). All the species except two, which were the isolated Ru(T-pMe-PP)((t)BuSH)(2) and Ru(TMP)(MeSSMe)(2), were characterized in situ. The disulfide complex was characterized by X-ray analysis. (1)H NMR data for the coordinated thiols are the first reported within metalloporphyrin systems, and are especially informative because of the upfield shifts of the axial sulfur-containing ligands due to the porphyrin π-ring current effect, which is also present in the di- and trisulfide species. The disulfide in the solid state structure of Ru(TMP)(MeSSMe)(2) is η(1)(end-on) coordinated, the first example of such bonding in a nontethered, acyclic dialkyl disulfide; (1)H-(1)H EXSY NMR data in solution show that the species undergoes 1,2-S-metallotropic shifts. Stepwise formation of the bis(disulfide) complex from Ru(TMP)(MeCN)(2) in solution occurs with a cooperativity effect, resembling behavior of Fe(II)-porphyrin systems where crystal field effects dominate, but ligand trans-effects are more likely in the Ru system. The η(1)(end-on) coordination mode is also favored for the trisulfide ligand. Discussed also are the remarkable linear correlations that exist between the ring-current shielding shifts for the axial ligand C(1) protons of Ru(porp)(RS(x)R)(2) and x (the number of S atoms). The Introduction briefly reviews literature on Ru- and Fe porphyrins (including heme proteins) with sulfur-containing ligands or substrates, and relationships between our findings and this literature are discussed throughout the paper.

Photoinactivation of Yeast and Biofilm Communities of Candida albicans Mediated by ZnTnHex-2-PyP4+ Porphyrin.

Candida albicans is the main cause of superficial candidiasis. While the antifungals available are defied by biofilm formation and resistance emergence, antimicrobial photodynamic inactivation (aPDI) arises as an alternative antifungal therapy. The tetracationic metalloporphyrin Zn(II) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (ZnTnHex-2-PyP4+) has high photoefficiency and improved cellular interactions. We investigated the ZnTnHex-2-PyP4+ as a photosensitizer (PS) to photoinactivate yeasts and biofilms of C. albicans strains (ATCC 10231 and ATCC 90028) using a blue light-emitting diode. The photoinactivation of yeasts was evaluated by quantifying the colony forming units. The aPDI of ATCC 90028 biofilms was assessed by the MTT assay, propidium iodide (PI) labeling, and scanning electron microscopy. Mammalian cytotoxicity was investigated in Vero cells using MTT assay. The aPDI (4.3 J/cm2) promoted eradication of yeasts at 0.8 and 1.5 µM of PS for ATCC 10231 and ATCC 90028, respectively. At 0.8 µM and same light dose, aPDI-treated biofilms showed intense PI labeling, about 89% decrease in the cell viability, and structural alterations with reduced hyphae. No considerable toxicity was observed in mammalian cells. Our results introduce the ZnTnHex-2-PyP4+ as a promising PS to photoinactivate both yeasts and biofilms of C. albicans, stimulating studies with other Candida species and resistant isolates.

Thermal Stability Kinetics and Shelf Life Estimation of the Redox-Active Therapeutic and Mimic of Superoxide Dismutase Enzyme, Mn(III) meso-Tetrakis(N-ethylpyridinium-2-yl)porphyrin Chloride (MnTE-2-PyPCl5, BMX-010).

Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin chloride (MnTE-2-PyPCl5, BMX-010, and AEOL10113) is among the most studied superoxide dismutase (SOD) mimics and redox-active therapeutics, being currently tested as a drug candidate in a phase II clinical trial on atopic dermatitis and itch. The thermal stability of active pharmaceutical ingredients (API) is useful for estimating the expiration date and shelf life of pharmaceutical products under various storage and handling conditions. The thermal decomposition and kinetic parameters of MnTE-2-PyPCl5 were determined by thermogravimetry (TG) under nonisothermal and isothermal conditions. The first thermal degradation pathway affecting Mn-porphyrin structural integrity and, thus, activity and bioavailability was associated with loss of ethyl chloride via N-dealkylation reaction. The thermal stability kinetics of the N-dealkylation process leading to MnTE-2-PyPCl5 decomposition was investigated by using isoconversional models and artificial neural network. The new multilayer perceptron (MLP) artificial neural network approach allowed the simultaneous study of ten solid-state kinetic models and showed that MnTE-2-PyPCl5 degradation is better explained by a combination of various mechanisms, with major contributions from the contraction models R1 and R2. The calculated activation energy values from isothermal and nonisothermal data were about 90 kJ mol-1 on average and agreed with one another. According to the R1 modelling of the isothermal decomposition data, the estimated shelf life value for 10% decomposition (t 90%) of MnTE-2-PyPCl5 at 25°C was approximately 17 years, which is consistent with the high solid-state stability of the compound. These results represent the first study on the solid-state decomposition kinetics of Mn(III) 2-N-alkylpyridylporphyrins, contributing to the development of this class of redox-active therapeutics and SOD mimics and providing supporting data to protocols on purification, handling, storage, formulation, expiration date, and general use of these compounds.

Efficient photodynamic inactivation of Leishmania parasites mediated by lipophilic water-soluble Zn(II) porphyrin ZnTnHex-2-PyP4.

BACKGROUND: Photodynamic inactivation (PDI) is emerging as a promising alternative for cutaneous leishmaniasis (CL). The chemotherapy currently used presents adverse effects and cases of drug resistance have been reported. ZnTnHex-2-PyP4+ is a porphyrin with a high potential as a photosensitizer (PS) for PDI, due to its photophysical properties, structural stability, and cationic/amphiphilic character that can enhance interaction with cells. This study aimed to investigate the photodynamic effects mediated by ZnTnHex-2-PyP4+ on Leishmania parasites. METHODS: ZnTnHex-2-PyP4+ stability was evaluated using accelerated solvolysis conditions. The photodynamic action on promastigotes was assessed by (i) viability assays, (ii) mitochondrial membrane potential evaluation, and (iii) morphological analysis. The PS-promastigote interaction was studied. PDI on amastigotes and the cytotoxicity on macrophages were also analyzed. RESULTS: ZnTnHex-2-PyP4+, under submicromolar concentration, led to immediate inactivation of more than 95% of promastigotes. PDI promoted intense mitochondrial depolarization, loss of the fusiform shape, and plasma membrane wrinkling in promastigotes. Fluorescence microscopy revealed a punctate PS labeling in the parasite cytoplasm. PDI also led to reductions of ca. 64% in the number of amastigotes/macrophage and 70% in the infection index after a single treatment session. No noteworthy toxicity was observed on mammalian cells. CONCLUSIONS: ZnTnHex-2-PyP4+ is stable against demetallation and more efficient as PS than the ethyl analogue ZnTE-2-PyP4+, indicating readiness for evaluation in in vivo studies as an alternative approach to CL. GENERAL SIGNIFICANCE: This report highlighted promising photodynamic effects mediated by ZnTnHex-2-PyP4+ on Leishmania parasites, opening up perspectives for applications in CL pre-clinical assays and PDI of other microorganisms.

Robust Mn(III) N-pyridylporphyrin-based biomimetic catalysts for hydrocarbon oxidations: heterogenization on non-functionalized silica gel versus chloropropyl-functionalized silica gel.

Two classes of heterogenized biomimetic catalysts were prepared and characterized for hydrocarbon oxidations: (1) by covalent anchorage of the three Mn(iii) meso-tetrakis(2-, 3-, or 4-pyridyl)porphyrin isomers by in situ alkylation with chloropropyl-functionalized silica gel (Sil-Cl) to yield Sil-Cl/MnPY (Y = 1, 2, 3) materials, and (2) by electrostatic immobilization of the three Mn(iii) meso-tetrakis(N-methylpyridinium-2, 3, or 4-yl)porphyrin isomers (MnPY, Y = 4, 5, 6) on non-modified silica gel (SiO2) to yield SiO2/MnPY (Y = 4, 5, 6) materials. Silica gel used was of column chromatography grade and Mn porphyrin loadings were deliberately kept at a low level (0.3% w/w). These resulting materials were explored as catalysts for iodosylbenzene (PhIO) oxidation of cyclohexane, n-heptane, and adamantane to yield the corresponding alcohols and ketones; the oxidation of cyclohexanol to cyclohexanone was also investigated. The heterogenized catalysts exhibited higher efficiency and selectivity than the corresponding Mn porphyrins under homogeneous conditions. Recycling studies were consistent with low leaching/destruction of the supported Mn porphyrins. The Sil-Cl/MnPY catalysts were more efficient and more selective than SiO2/MnPY ones; alcohol selectivity may be associated with hydrophobic silica surface modification reminiscent of biological cytochrome P450 oxidations. The use of widespread, column chromatography, amorphous silica yielded Sil-Cl/MnPY or SiO2/MnPY catalysts considerably more efficient than the corresponding, previously reported materials with mesoporous Santa Barbara Amorphous No 15 (SBA-15) silica. Among the materials studied, in situ derivatization of Mn(iii) 2-N-pyridylporphyrin by covalent immobilization on Sil-Cl to yield Sil-Cl/MnP1 showed the best catalytic performance with high stability against oxidative destruction and reusability/recyclability.

Fe Porphyrin-Based SOD Mimic and Redox-Active Compound, (OH)FeTnHex-2-PyP4+, in a Rodent Ischemic Stroke (MCAO) Model: Efficacy and Pharmacokinetics as Compared to Its Mn Analogue, (H2O)MnTnHex-2-PyP5+.

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-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.

Redox-Active Drug, MnTE-2-PyP5+, Prevents and Treats Cardiac Arrhythmias Preserving Heart Contractile Function.

BACKGROUND: Cardiomyopathies remain among the leading causes of death worldwide, despite all efforts and important advances in the development of cardiovascular therapeutics, demonstrating the need for new solutions. Herein, we describe the effects of the redox-active therapeutic Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, AEOL10113, BMX-010 (MnTE-2-PyP5+), on rat heart as an entry to new strategies to circumvent cardiomyopathies. METHODS: Wistar rats weighing 250-300 g were used in both in vitro and in vivo experiments, to analyze intracellular Ca2+ dynamics, L-type Ca2+ currents, Ca2+ spark frequency, intracellular reactive oxygen species (ROS) levels, and cardiomyocyte and cardiac contractility, in control and MnTE-2-PyP5+-treated cells, hearts, or animals. Cells and hearts were treated with 20 µM MnTE-2-PyP5+ and animals with 1 mg/kg, i.p. daily. Additionally, we performed electrocardiographic and echocardiographic analysis. RESULTS: Using isolated rat cardiomyocytes, we observed that MnTE-2-PyP5+ reduced intracellular Ca2+ transient amplitude, without altering cell contractility. Whereas MnTE-2-PyP5+ did not alter basal ROS levels, it was efficient in modulating cardiomyocyte redox state under stress conditions; MnTE-2-PyP5+ reduced Ca2+ spark frequency and increased sarcoplasmic reticulum (SR) Ca2+ load. Accordingly, analysis of isolated perfused rat hearts showed that MnTE-2-PyP5+ preserves cardiac function, increases SR Ca2+ load, and reduces arrhythmia index, indicating an antiarrhythmic effect. In vivo experiments showed that MnTE-2-PyP5+ treatment increased Ca2+ transient, preserved cardiac ejection fraction, and reduced arrhythmia index and duration. MnTE-2-PyP5+ was effective both to prevent and to treat cardiac arrhythmias. CONCLUSION: MnTE-2-PyP5+ prevents and treats cardiac arrhythmias in rats. In contrast to most antiarrhythmic drugs, MnTE-2-PyP5+ preserves cardiac contractile function, arising, thus, as a prospective therapeutic for improvement of cardiac arrhythmia treatment.

Impact of electrostatics in redox modulation of oxidative stress by Mn porphyrins: protection of SOD-deficient Escherichia coli via alternative mechanism where Mn porphyrin acts as a Mn carrier.

Understanding the factors that determine the ability of Mn porphyrins to scavenge reactive species is essential for tuning their in vivo efficacy. We present herein the revised structure-activity relationships accounting for the critical importance of electrostatics in the Mn porphyrin-based redox modulation systems and show that the design of effective SOD mimics (per se) based on anionic porphyrins is greatly hindered by inappropriate electrostatics. A new strategy for the beta-octabromination of the prototypical anionic Mn porphyrins Mn(III) meso-tetrakis(p-carboxylatophenyl)porphyrin ([Mn(III)TCPP](3-) or MnTBAP(3-)) and Mn(III) meso-tetrakis(p-sulfonatophenyl)porphyrin ([Mn(III)TSPP](3-)), to yield the corresponding anionic analogues [Mn(III)Br(8)TCPP](3-) and [Mn(III)Br(8)TSPP](3-), respectively, is described along with characterization data, stability studies, and their ability to substitute for SOD in SOD-deficient Escherichia coli. Despite the Mn(III)/Mn(II) reduction potential of [Mn(III)Br(8)TCPP](3-) and [Mn(III)Br(8)TSPP](3-) being close to the SOD-enzyme optimum and nearly identical to that of the cationic Mn(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin (Mn(III)TM-2-PyP(5+)), the SOD activity of both anionic brominated porphyrins ([Mn(III)Br(8)TCPP](3-), E(1/2)=+213 mV vs NHE, log k(cat)=5.07; [Mn(III)Br(8)TSPP](3-), E(1/2)=+209 mV, log k(cat)=5.56) is considerably lower than that of Mn(III)TM-2-PyP(5+) (E(1/2)=+220 mV, log k(cat)=7.79). This illustrates the impact of electrostatic guidance of O(2)(-) toward the metal center of the mimic. With low k(cat), the [Mn(III)TCPP](3-), [Mn(III)TSPP](3-), and [Mn(III)Br(8)TCPP](3-) did not rescue SOD-deficient E. coli. The striking ability of [Mn(III)Br(8)TSPP](3-) to substitute for the SOD enzymes in the E. coli model does not correlate with its log k(cat). In fact, the protectiveness of [Mn(III)Br(8)TSPP](3-) is comparable to or better than that of the potent SOD mimic Mn(III)TM-2-PyP(5+), even though the dismutation rate constant of the anionic complex is 170-fold smaller. Analyses of the medium and E. coli cell extract revealed that the major species in the [Mn(III)Br(8)TSPP](3-) system is not the Mn complex, but the free-base porphyrin [H(2)Br(8)TSPP](4-) instead. Control experiments with extracellular MnCl(2) showed the lack of E. coli protection, indicating that "free" Mn(2+) cannot enter the cell to a significant extent. We proposed herein the alternative mechanism where a labile Mn porphyrin [Mn(III)Br(8)TSPP](3-) is not an SOD mimic per se but carries Mn into the E. coli cell.

Pharmacokinetics of the potent redox-modulating manganese porphyrin, MnTE-2-PyP(5+), in plasma and major organs of B6C3F1 mice.

Mn(III) tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP(5+), a potent catalytic superoxide and peroxynitrite scavenger, has been beneficial in several oxidative stress-related diseases thus far examined. Pharmacokinetic studies are essential for the better assessment of the therapeutic potential of MnTE-2-PyP(5+) and similar compounds, as well as for the modulation of their bioavailability and toxicity. Despite high hydrophilicity, this drug entered mitochondria after a single 10 mg/kg intraperitoneal injection at levels high enough (5.1 muM; 2.95 ng/mg protein) to protect against superoxide/peroxynitrite damage. Utilizing the same analytical approach, which involves the reduction of MnTE-2-PyP(5+) followed by the exchange of Mn(2+) with Zn(2+) and HPLC/fluorescence detection of ZnTE-2-PyP(4+), we measured levels of MnTE-2-PyP(5+) in mouse plasma, liver, kidney, lung, heart, spleen, and brain over a period of 7 days after a single intraperitoneal injection of 10 mg/kg. Two B6C3F1 female mice per time point were used. The pharmacokinetic profile in plasma and organs was complex; thus a noncompartmental approach was utilized to calculate the area under the curve, c(max), t(max), and drug elimination half-time (t(1/2)). In terms of levels of MnTE-2-PyP(5+) found, the organs can be classified into three distinct groups: (1) high levels (kidney, liver, and spleen), (2) moderate levels (lung and heart), and (3) low levels (brain). The maximal levels in plasma, kidney, spleen, lung, and heart are reached within 45 min, whereas in the case of liver a prolonged absorption phase was observed, with the maximal concentration reached at 8 h. Moreover, accumulation of the drug in brain continued beyond the time of the experiment (7 days) and is likely to be driven by the presence of negatively charged phospholipids. For tissues other than brain, a slow elimination phase (single exponential decay, t(1/2)=60 to 135 h) was observed. The calculated pharmacokinetic parameters will be used to design optimal dosing regimens in future preclinical studies utilizing this and similar compounds.

SOD-like activity of Mn(II) beta-octabromo-meso-tetrakis(N-methylpyridinium-3-yl)porphyrin equals that of the enzyme itself.

Mn porphyrins are among the most efficient SOD mimics with potency approaching that of SOD enzymes. The most potent ones, Mn(III) N-alkylpyridylporphyrins bear positive charges in a close proximity to the metal site, affording thermodynamic and kinetic facilitation for the reaction with negatively charged superoxide. The addition of electron-withdrawing bromines onto beta-pyrrolic positions dramatically improves thermodynamic facilitation for the O2*- dismutation. We have previously characterized the para isomer, Mn(II)Br(8)TM-4-PyP(4+) [Mn(II) beta-octabromo-meso-tetrakis(N-methylpyridinium-4-yl)porphyrin]. Herein we fully characterized its meta analogue, Mn(II)Br(8)TM-3-PyP(4+) with respect to UV/vis spectroscopy, electron spray mass spectrometry, electrochemistry, O2*- dismutation, metal-ligand stability, and the ability to protect SOD-deficient Escherichia coli in comparison with its para analogue. The increased electron-deficiency of the metal center stabilizes Mn in its +2 oxidation state. The metal-centered Mn(III)/Mn(II) reduction potential, E((1/2))=+468 mV vs NHE, is increased by 416 mV with respect to non-brominated analogue, Mn(III)TM-3-PyP(5+) and is only 12 mV less positive than for para isomer. Yet, the complex is significantly more stable towards the loss of metal than its para analogue. As expected, based on the structure-activity relationships, an increase in E((1/2)) results in a higher catalytic rate constant for the O2*- dismutation, log k(cat)> or =8.85; 1.5-fold increase with respect to the para isomer. The IC(50) was calculated to be < or =3.7 nM. Manipulation of the electron-deficiency of a cationic porphyrin resulted, therefore, in the highest k(cat) ever reported for a metalloporphyrin, being essentially identical to the k(cat) of superoxide dismutases (log k(cat)=8.84-9.30). The positive kinetic salt effect points to the unexpected, unique and first time recorded behavior of Mn beta-octabrominated porphyrins when compared to other Mn porphyrins studied thus far. When species of opposing charges react, the increase in ionic strength invariably results in the decreased rate constant; with brominated porphyrins the opposite was found to be true. The effect is 3.5-fold greater with meta than with para isomer, which is discussed with respect to the closer proximity of the quaternary nitrogens of the meta isomer to the metal center than that of the para isomer. The potency of Mn(II)Br(8)TM-3-PyP(4+) was corroborated by in vivo studies, where 500 nM allows SOD-deficient E. coli to grow >60% of the growth of wild type; at concentrations > or =5 microM it exhibits toxicity. Our work shows that exceptionally high k(cat) for the O2*- disproportionation can be achieved not only with an N(5)-type coordination motif, as rationalized previously for aza crown ether (cyclic polyamines) complexes, but also with a N(4)-type motif as in the Mn porphyrin case; both motifs sharing "up-down-up-down" steric arrangement.

Synthetic and mechanistic aspects of a new method for ruthenium-metalation of porphyrins and Schiff-bases.

A new method is presented for metalation of a wide range of free-base, neutral, cationic, and anionic porphyrins in refluxing dimethylformamide (DMF) using an easily prepared [Ru(DMF) 6](OTf) 3 complex, and comparisons are made with the more familiar metalation procedure using Ru 3(CO) 12. Both procedures generate Ru (II)(porp)(CO)L complexes (L = solvent); use of the Ru (III)-triflate precursor gives yields comparable to, or greater than, those obtained with the carbonyl, and generates no Ru-chlorin impurities. Mechanistic studies on the meso-tetraphenylporphyrin system reveal that the DMF furnishes the CO, which in the presence of essential water reduces the metal, and metalation likely occurs via a Ru (II)-CO species. Corresponding metalation of tetradentate Schiff-bases gives trans-[Ru (III)(Schiff- base)(DMF) 2]OTf complexes in yields of approximately 50%, a limitation being the accompanying hydrolysis of the Schiff-base through the presence of trace water.

Quality of potent Mn porphyrin-based SOD mimics and peroxynitrite scavengers for pre-clinical mechanistic/therapeutic purposes.

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).