manuscript doi: 10.5599/admet.2.3.123 185 ADMET & DMPK 2(3) (2014) 185-190; doi: 10.5599/admet.2.3.123 Open Access : ISSN : 1848-7718 http://www.pub.iapchem.org/ojs/index.php/admet/index Short communication Electrochemistry of Redox-active Mn Porphyrin-based SOD Mimic MnTnBuOE-2-PyP 5+ - Study of Redox Species Involved in ROS/RNS Scavenging Tin Weitner 1 *, Ines Batinic-Haberle 2 1 University of Zagreb, Faculty of Pharmacy and Biochemistry, Ante Kovačića 1, Zagreb 10000, Croatia 2 Department of Radiation Oncology, Duke University School of Medicine, Durham, NC 27710, USA *Corresponding Author: E-mail: tweitner@pharma.hr; Tel.: +385-1-6394-452; Fax: +385-1-6394-400 Received: September 09, 2014; Revised: September 10, 2014; Published: September 16, 2014 Abstract Manganese ortho tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP 5+ , is a third- generation redox-active compound currently undergoing preclinical exploration. This work is intended to complement the already extensive research of its chemical and biological properties by a simple electrochemical study. The thermodynamic parameters related to the Mn(IV) porphyrin species of MnTnBuOE-2-PyP 5+ determined in this work support its observed reactivity as an efficient scavenger of peroxynitrite. The corresponding driving forces for the possible single-electron or two-electron reductions of ONOO - have been estimated as well. Keywords metalloporphyrins, oxidative stress, superoxide, peroxynitrite, cyclic voltammetry. Introduction Therapeutic potential of metalloporphyrin-based mimics of superoxide dismutase (SOD) enzyme has been well documented [1-5]. Briefly, a true SOD mimic should be able to dismutate superoxide (i.e. oxidize or reduce O2 • - ) at a catalytic rate, kcat(O2 • - ), higher than the rate of O2 • - self-dismutation (5 × 10 5 M -1 s -1 at pH = 7). The formal reduction potential of the SOD mimic should be optimally midway between the single- electron formal reduction potentials for oxygen (E 0’ = – 0.16 V vs. NHE) and superoxide (E 0’ = + 0.89 V vs. NHE) [6-7]. Over the last 15 years, a rational approach to the design of potential SOD mimics led to the development of several manganese porphyrin complexes (MnPs) with unique electronic properties and high reactivity towards O2 • - , but also other reactive species such as peroxynitrite, ONOO - [2-5]. Indeed, a structure-activity relationship (half-wave reduction potential, E1/2, vs. log kcat(O2 • - )) has been developed and found to be valid for any other class of SOD mimics as well [1]. Recent efforts have been directed towards increasing the bioavailability and reducing the toxicity of MnPs. The original structure of a potent first-generation SOD mimic, manganese ortho tetrakis(N- ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP 5+ ) was modified via lengthening the alkyl chains of pyridyl http://www.pub.iapchem.org/ojs/index.php/admet/index mailto:tweitner@pharma.hr Weitner and Batinic-Haberle ADMET & DMPK 2(3) (2014) 185-190 186 substituents, yielding the more lipophilic compound, the manganese ortho tetrakis(N-n-hexylpyridinium-2- yl)porphyrin (MnTnHex-2-PyP 5+ ) [8]. This second-generation compound showed much higher brain and mitochondrial distribution than MnTE-2-PyP 5+ , but was also found to be toxic at higher concentrations and could have a potentially limited use [9-10]. The third-generation compound, manganese ortho tetrakis(N-n- butoxyethylpyridinium-2-yl)porphyrin (MnTnBuOE-2-PyP 5+ ), was designed via introduction of oxygen atoms into the alkyl chains on the pyridyl substituents. This compound preserved high redox-activity and lipophilicity but exhibited significantly reduced toxicity relative to MnTnHex-2-PyP 5+ [11]. Comparison of MnTE-2-PyP 5+ and MnTnBuOE-2-PyP 5+ with regards to their redox properties (E1/2 for Mn III P/Mn II P redox couple), reactivities towards superoxide, log kcat(O2 • - ), and peroxynitrite, log kred(ONOO - ), as well as lipophilicities (distribution between n-octanol and water, log POW) is given in Table 1. Table 1. Physicochemical properties of redox-active Mn porphyrin-based SOD mimics MnTE-2-PyP 5+ and MnTnBuOE-2-PyP 5+ . Data are taken from references [3-5,11-16] ; a at 25 °C; b at 37 °C. Ar N Ethyl n-Butoxyethyl E1/2 (mV vs. NHE) + 228 + 277 log kcat (O2 •- ) a 7.76 7.83 log kred (ONOO - ) b 7.53 7.54 log POW -7.79 -4.10 Comprehensive studies on bioavailability of these compounds have been performed recently as well [17- 19], in order to gain further insight into the biodistribution of MnPs, but also provide guidelines for proper dosing regimens. After assessing preliminary efficacy and toxicity data, the porphyrins were administered to mice intraperitoneally (ip) or intravenously (iv) at following doses: 10 mg/kg of MnTE-2-PyP 5+ (ip) and 2 mg/kg of MnTnBuOE-2-PyP 5+ (iv). Drug levels in plasma and different organs were followed during at least 24 hours after a single dose, thus allowing the calculation of relevant pharmacokinetic (PK) parameters via non-compartmental analysis of PK curves. These studies showed the highest MnP bioavailability in liver (maximum concentration, Cmax, values of 8.1 and 13.1 µmol per L of tissue homogenate for MnTE-2-PyP 5+ and MnTnBuOE-2-PyP 5+ , respectively) and kidney, followed by spleen, lung and heart, whereas lower levels were found in plasma and lowest in the brain. Remarkably, MnTnBuOE-2-PyP 5+ has 8-fold higher brain bioavailability (expressed as the area under PK curve, AUC, divided by dose) than the less lipophilic MnTE-2-PyP 5+ (Figure 1), which may be highly significant for its potential application as a brain tumor radio- and chemo-therapeutic [20-21]. Additionally, it has been shown that kcat(O2 • – ) parallels the ability of MnPs to reduce ONOO - [4-5,14], due to the same thermodynamic and kinetic factors facilitating the reaction of electron-deficient cationic MnPs with electron-donating anionic species, such as O2 • – and ONOO - [3-5,22]. Specifically, the removal of ONOO - can occur either through its binding to the Mn(III) site followed by its one-electron reduction to NO2 • radical (coupled to the oxidation of Mn III P to Mn IV P), or the two-electron reduction of ONOO - by the Mn II P species (coupled to the oxidation of Mn II P to Mn IV P). The latter reaction produces less toxic nitrite, NO2 - , and might in fact be dominant in vivo due to (i) the abundance of cellular reductants keeping MnPs in Mn(II) state [1,13,23-25] and (ii) a higher driving force making it thermodynamically favorable [26]. ADMET & DMPK 2(3) (2014) 185-190 Redox active Mn-porphyrin SOD mimetic doi: 10.5599/admet.2.3.123 187 Figure 1. Bioavailability of redox-active Mn porphyrin-based SOD mimics MnTE-2-PyP 5+ and MnTnBuOE-2-PyP 5+ , demonstrated as 24-hour dose-adjusted AUC values: raw AUC values have been divided by dose (10 mg/kg for MnTE-2-PyP 5+ and 2 mg/kg for MnTnBuOE-2-PyP 5+ ). The values for MnTE-2-PyP 5+ determined via iv route are taken as 100 % availability (plasma bioavailability via ip route was found to be approximately 84% of bioavailability via iv route for MnTE-2-PyP 5+ ) [18]. MnPs’ charges have been omitted from the plot for simplicity. MnTnBuOE-2-PyP 5+ represents the latest generation of redox-active compounds and is currently undergoing preclinical exploration. This work is intended to complement the already extensive research of its chemical and biological properties by a simple electrochemical study. Experimental The investigated metalloporphyrin, Mn III TnBuOE-2-PyPCl5 was synthesized according to the published procedure [11]. Stock solution was prepared by dissolution of purified and characterized solid (known molar absorbance coefficient, ε456 (MnTnBuOE-2-PyP 5+ ) = 1.78 × 10 5 M -1 cm -1 ) [11]. Cyclic voltammetry of buffered and deaerated solutions was performed as reported in a previous study [26]. All potentials are reported vs. the normal hydrogen electrode (NHE), using the known potential of MnTE-2-PyP 5+ , E1/2 = + 228 mV vs. NHE at pH 7.8, as a reference [11,27-28]. Results and Discussion Cyclic voltammograms of the aqueous solutions of Mn III Ps can show two distinct electron transfers: only one current peak pair attributed to the Mn III P/Mn II P redox couple is observed in a neutral or mildly basic medium (pH = 7.8, Figure 2), whereas in the more basic medium an additional current peak pair attributed to the Mn IV P/Mn III P redox couple appears at more positive potentials (pH = 11, Figure 2). The cathodic and anodic current peak potentials, Epc and Epa, of both redox couples shift toward more negative values upon the increased pH of the solution. The cathodic-anodic peak separation, ΔEp = |Epc - Epa| for both redox processes is larger than 59 mV even at the scan rate of 0.02 V s -1 , indicating quasi-reversible electron transfer processes. Weitner and Batinic-Haberle ADMET & DMPK 2(3) (2014) 185-190 188 Figure 2. Comparison of cyclic voltammograms of 0.5 mM aqueous solutions of MnTnBuOE-2-PyP 5+ at pH=7.8 and pH = 11, ν = 0.02 V s -1 , [NaCl] = 0.1 M,  = 25 °C. Similar behavior has been reported previously for methyl, ethyl and butyl analogues [15,26,29]. Redox couples shown relate to the Mn III P/Mn II P (right cycle) and the Mn IV P/Mn III P couples (left cycle). These results are in agreement with the previously established scheme of behavior of MnPs in aqueous solutions [26,30]. At pH = 7.8 only a single-electron reduction of Mn III P can be observed, shown by eq. (1), whereas at pH = 11 the single-electron reduction of Mn IV P is accompanied by a simultaneous dissociation of two protons, according to eq. (2). The latter electron transfer cannot be observed in a neutral medium due to a formal potential more positive than that of water (E 0’ (O2(g),H + (aq.)/H2O(l)) = 816 mV vs. SHE at pH = 7) [30-31].           III 5 II 42 2 22H O Mn TnBuOE 2 PyP e H O Mn TnBuOE 2 PyP H O (1)             IV 4 III 52 2 2O H O Mn TnBuOE 2 PyP 2H e H O Mn TnBuOE 2 PyP (2) Table 2. Calculated reduction potentials for the Mn III P/Mn II P, Mn IV P/Mn III P and Mn IV P/Mn II P redox couples of MnTnBuOE-2-PyP 5+ . Values for MnTE-2-PyP 5+ are taken from reference [26] and adjusted to the value E1/2 = + 228 mV vs. NHE for its Mn III P/Mn II P redox couple at pH=7.8. The reduction potential of the Mn IV P/Mn II P couple is calculated as: E1/2(Mn IV P/Mn II P) = [E1/2(Mn III P/Mn II P) + E1/2(Mn IV P/Mn III P)]/2. MnP E1/2 (mV vs. NHE) pH = 7.8 pH = 11 Mn III P/Mn II P Mn III P/Mn II P Mn IV P/Mn III P Mn IV P/Mn II P MnTnBuOE-2-PyP 5+ + 277 + 235 + 624 + 430 MnTE-2-PyP 5+ + 228 + 208 + 592 + 400 ADMET & DMPK 2(3) (2014) 185-190 Redox active Mn-porphyrin SOD mimetic doi: 10.5599/admet.2.3.123 189 As shown previously [11], the E1/2 of Mn III P/Mn II P redox couple of MnTnBuOE-2-PyP 5+ is close to the value of the Mn SOD enzyme itself, accounting for its high reactivity towards O2 •– (even higher than that of MnTE-2-PyP 5+ , Table 1). Thermodynamic parameters related to the Mn IV P species of MnTnBuOE-2-PyP 5+ determined in this work (Table 2) support its observed reactivity as an efficient scavenger of peroxynitrite. Estimating the shift of the E1/2 (Mn IV P/Mn III P) value for +118 mV per unit decrease of pH from pH = 11, yields the values of E1/2 (Mn IV P/Mn III P) = + 1049 mV vs. NHE at the physiological pH, according to eq. (2). The E1/2 (Mn IV P/Mn II P) at the physiological pH can then be calculated as E1/2(Mn IV P/Mn II P) = [E1/2(Mn III P/Mn II P) + E1/2(Mn IV P/Mn III P)]/2 = + 663 mV vs. NHE. Finally, the driving forces for the single-electron and two-electron reduction of ONOO - , EMF (1e - ) = 551 mV and EMF (2e - ) = 637 mV, can then be calculated as EMF = Ec - Ea, where the values of Ec are the formal potentials E 0’ (ONOO - /NO2 • ) = 1.6 V and E 0’ (ONOO - /NO2 - ) = 1.3 V [32], respectively, whereas the values of Ea are the formal potentials E1/2(Mn IV P/Mn III P) and E1/2(Mn IV P/Mn II P), respectively. Conclusions Considerable interest in the impact of oxidative stress on cellular function prompted an intensive search for natural and synthetic antioxidants. High bioavailability of MnTnBuOE-2-PyP 5+ , coupled with its exceptional physicochemical properties as a redox-active ROS/RNS scavenger, makes it a promising candidate in the ongoing cancer and radioprotection preclinical studies. 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