ACORDOS SUBSTITUTIVOS COMO INSTRUMENTO DE REGULAÇÃO AMBIENTAL

Acordos substitutivos podem ser utilizados como alternativa regulatória ambiental em matéria de compensação por ato lícito e por ato ilícito. São há muito aceitos pelas normas de Direito Ambiental em ambos os casos, mas praticados pela Administração Pública apenas mediante compensação por ato lícito, ou seja, por impacto ambiental. Falta de segurança jurídica constituiu histórico obstáculo à ampla utilização deste instrumento de regulação ambiental. A consensualidade vem sendo tratada como uma nova tendência pela moderna teoria de Direito Administrativo. A regulação ambiental serve de estudo de caso para demonstração da efetividade dessa alternativa regulatória na maximização do interesse público a partir da análise de conveniência no caso concreto. Recentes alterações normativas no Direito Ambiental, promovidas pelos Decretos 9.179/17 e MP 809/17, podem aumentar a segurança jurídica para otimização e promoção dos acordos substitutivos na regulação ambiental. Podem servir também de referencial normativo para outras áreas reguladas, potencializando o interesse público pela consensualidade.  

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

Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin, (HO)MnTnHex-2-PyP (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-PyP (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 model of oxidative stress. In vivo, it does not cause arterial hypotension and is less toxic to mice. We thus evaluated FeHex 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 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

CNS bioavailability and radiation protection of normal hippocampal neurogenesis by a lipophilic Mn porphyrin-based superoxide dismutase mimic, MnTnBuOE-2-PyP

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-PyP(MnE), MnTnHex-2-PyP(MnHex), and MnTnBuOE-2-PyP(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 3mg/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

CNS bioavailability and radiation protection of normal hippocampal neurogenesis by a lipophilic Mn porphyrin-based superoxide dismutase mimic, MnTnBuOE-2-PyP

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-PyP(MnE), MnTnHex-2-PyP(MnHex), and MnTnBuOE-2-PyP(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 3mg/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

Radiation-Mediated Tumor Growth Inhibition Is Significantly Enhanced with Redox-Active Compounds That Cycle with Ascorbate

Aims: We aim here to demonstrate that radiation (RT) enhances tumor sensitization by only those Mn complexes that are redox active and cycle with ascorbate (Asc), thereby producing H2O2 and utilizing it subsequently in protein S-glutathionylation in a glutathione peroxidase (GPx)-like manner. In turn, such compounds affect cellular redox environment, described by glutathione disulfide (GSSG)/glutathione (GSH) ratio, and tumor growth. To achieve our goal, we tested several Mn complexes of different chemical and physical properties in cellular and animal flank models of 4T1 breast cancer cell. Four other cancer cell lines were used to substantiate key findings. Results: Joint administration of cationic Mn porphyrin (MnP)-based redox active compounds, MnTE-2-PyP5+ or MnTnBuOE-2-PyP5+ with RT and Asc contributes to high H2O2 production in cancer cells and tumor, which along with high MnP accumulation in cancer cells and tumor induces the largest suppression of cell viability and tumor growth, while increasing GSSG/GSH ratio and levels of total S-glutathionylated proteins. Redox-inert MnP, MnTBAP3- and two other different types of redox-active Mn complexes (EUK-8 and M40403) were neither efficacious in the cellular nor in the animal model. Such outcome is in accordance with their inability to catalyze Asc oxidation and mimic GPx. Innovation: We provided here the first evidence how structure-activity relationship between the catalytic potency and the redox properties of Mn complexes controls their ability to impact cellular redox environment and thus enhance the radiation and ascorbate-mediated tumor suppression. Conclusions: The interplay between the accumulation of cationic MnPs and their potency as catalysts for oxidation of Asc, protein cysteines, and GSH controls the magnitude of their anticancer therapeutic effects

A comprehensive evaluation of catalase-like activity of different classes of redox-active therapeutics

Because of the increased insight into the biological role of hydrogen peroxide (H2O2) under physiological and pathological conditions and the role it presumably plays in the action of natural and synthetic redox-active drugs, there is a need to accurately define the type and magnitude of reactions that may occur with this intriguing and key species of redoxome. Historically, and frequently incorrectly, the impact of catalase-like activity has been assigned to play a major role in the action of many redox-active drugs, mostly SOD mimics and peroxynitrite scavengers, and in particular MnTBAP3- and Mn salen derivatives. The advantage of one redox-active compound over another has often been assigned to the differences in catalase-like activity. Our studies provide substantial evidence that Mn(III) N-alkylpyridylporphyrins couple with H2O2 in actions other than catalase-related. Herein we have assessed the catalase-like activities of different classes of compounds: Mn porphyrins (MnPs), Fe porphyrins (FePs), Mn(III) salen (EUK-8), and Mn(II) cyclic polyamines (SOD-active M40403 and SOD-inactive M40404). Nitroxide (tempol), nitrone (NXY-059), ebselen, and MnCl2, which have not been reported as catalase mimics, were used as negative controls, while catalase enzyme was a positive control. The dismutation of H2O2 to O2 and H2O was followed via measuring oxygen evolved with a Clark oxygen electrode at 25°C. The catalase enzyme was found to have kcat(H2O2)=1.5×106 M-1 s-1. The yield of dismutation, i.e., the maximal amount of O2 evolved, was assessed also. The magnitude of the yield reflects an interplay between the kcat(H2O2) and the stability of compounds toward H2O2-driven oxidative degradation, and is thus an accurate measure of the efficacy of a catalyst. The kcat(H2O2) values for 12 cationic Mn(III) N-substituted (alkyl and alkoxyalkyl) pyridylporphyrin-based SOD mimics and Mn(III) N,N′-dialkylimidazolium porphyrin, MnTDE-2-ImP5+, ranged from 23 to 88 M-1 s-1. The analogous Fe(III) N-alkylpyridylporphyrins showed ∼10-fold higher activity than the corresponding MnPs, but the values of kcat(H2O2) are still ∼4 orders of magnitude lower than that of the enzyme. While the kcat(H2O2) values for Fe ethyl and n-octyl analogs were 803.5 and 368.4 M-1 s-1, respectively, the FePs are more prone to H2O2-driven oxidative degradation, therefore allowing for similar yields in H2O2 dismutation as analogous MnPs. The kcat(H2O2) values are dependent on the electron deficiency of the metal site as it controls the peroxide binding in the first step of the dismutation process. SOD-like activities depend on electron deficiency of the metal site also, as it controls the first step of O2•- dismutation. In turn, the kcat(O2•-) parallels the kcat(H2O2). Therefore, the electron-rich anionic non-SOD mimic MnTBAP3- has essentially very low catalase-like activity, kcat(H2O2)=5.8 M-1 s-1. The catalase-like activities of Mn(III) and Fe(III) porphyrins are at most, 0.0004 and 0.05% of the enzyme activity, respectively. The kcat(H2O2) values of 8.2 and 6.5 M-1 s-1 were determined for electron-rich Mn(II) cyclic polyamine-based compounds, M40403 and M40404, respectively. The EUK-8, with modest SOD-like activity, has only slightly higher kcat(H2O2)=13.5 M-1 s-1. The biological relevance of kcat(H2O2) of MnTE-2-PyP5+, MnTDE-2-ImP5+, MnTBAP3-, FeTE-2-PyP5+, M40403, M40404, and Mn salen was evaluated in wild-type and peroxidase/catalase-deficient E. coli

Anticancer therapeutic potential of Mn porphyrin/ascorbate system

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\u3e0 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 HBL-100 cells. Bell-shape relationship, essentially identical to vo(Asc)ox vs E1/2, was also demonstrated between MnP/Asc-controlled 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 toxicity, 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. About 7-fold higher accumulation of MnTE-2-PyP5+ in tumor vs normal tissue was found to contribute largely to the anticancer effect