Pursuing the Elixir of Life: In vivo antioxidative effect of manganosalen complexes, a review

: Manganosalen complexes are coordination compounds that possess a chelating salen-type ligand, a class of bis-Schiff bases obtained by condensation of salicylaldehyde and a diamine. They may act as catalytic antioxidants mimicking both the structure and the reactivity of the native antioxidant enzymes active site. Thus, manganosalen complexes have shown to exhibit superoxide dismutase, catalase, and glutathione peroxidase activities, and they could potentially facilitate the scavenging of excess ROS, thereby restoring the redox balance in the damaged cells and organs. Initial catalytic studies compared the potency of these compounds as antioxidants in terms of rate constants of the chemical reactivity against ROS, giving catalytic values approaching and even exceeding that of the native antioxidative enzymes. Although most of these catalytic studies lack of biological relevance, subsequent in vitro studies have confirmed the efficiency of many manganosalen complexes in oxidative stress models. These synthetic catalytic scavengers, cheaper than natural antioxidants, have accordingly attracted intensive attention for the therapy of ROS-mediated injuries. The aim of this review is to focus on in vivo studies performed on manganosalen complexes and their activity on the treatment of several pathological disorders associated with oxidative damage. This disorders, ranging from the prevention of fetal malformations to the extension of lifespan, include neurodegenerative, inflammatory and cardiovascular diseases, tissue injury, and other damages related to liver, kidney or lungs.


Introduction
In 2000 a research paper by Melov et al. [1] reported the extension of lifespan of nematode worms (Caenorhabditis elegans) by treatment with different manganosalen complexes which act as synthetic scavenger compounds. This study was planned to test the theory that reactive oxidative species cause aging. In announcing this research, Melov stated that "the results are the first real indication we have that aging is a condition that can be treated through appropriate drug therapy" [2]. The research paper and this statement attracted extensive coverage in the communications media, where these findings were presented as a sort of search for the Elixir of Life. The published results indicate that the two tested compounds (named as EUK-8 and EUK-134) increased the mean lifespan of the worms by 44 percent over the control group, and treatment of prematurely aging worms resulted in normalization of their lifespan, which means a 67 percent increase. However, the chosen nematode
ROS levels are regulated in the living systems by the antioxidant enzymes, which basically include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), peroxiredoxins (PRDXs), thioredoxin 2 (TRX2) or cytochrome c oxidase (complex IV). Other non-enzymatic antioxidants such as α-tocopherol, ascorbic acid and carotenes complete the antioxidant defence grid [43]. In this review, the discussion will focus on the antioxidant enzymes that may contain manganese in their active centre: SOD, CAT and GPx ( Figure 1). The global role of these cellular antioxidant defences is the reduction of ROS to water.  Superoxide radical anion is a harmful species produced during respiration from the one-electron reduction of molecular oxygen. SOD catalyzes the dismutation of two superoxide anions to yield hydrogen peroxide and molecular oxygen [44]. SODs are classified according to their metal ion cofactor as Fe-SODs, Ni-SODs, CuZn-SODs and MnSODs [45] The SOD2, also known as manganesedependent superoxide dismutase, is located within the inner mitochondrial matrix, the main site of free radical production from the electron transport chain [46], being pivotal in the ROS release in humans. The coordination environment around the manganese ion for human SOD2 is depicted in Figure 1. The metal ion shows a trigonal bipyramidal geometry, bound to three histidine ligands (His26, His74 and His163), one aspartate (Asp159) and water or hydroxide as the fifth ligand. These amino acids and ligands constitute the inner sphere with a direct interaction with the manganese. The outer sphere residues are not shown in the figure but they are also essential for efficient dismutation and comprise His30, Tyr34, Phe77, Trp78, Trp123, Gln143, Trp161 and Glu162. The superoxide disproportionation mechanism by SOD2 involves a cyclic one-electron oxidation and reduction of the manganese ion between Mn(II)/Mn(III) oxidation states ( Figure 2). The catalysis occurs at a rate approaching the diffusion-controlling region (log KSOD ⁓ 10 9 M -1 s -1 ) [47].
Hydrogen peroxide is generated as a by-product of mitochondrial electron transport of aerobic respiration. As it has been mentioned, it is one of the products of the superoxide radical anion dismutation by SOD. Although H2O2 is less reactive than superoxide, control of H2O2 levels is also critical [48]. Catalase enzymes catalyse the decomposition of hydrogen peroxide to water and oxygen [49]. The non-heme dinuclear Mn catalase was isolated from bacteria. Figure 1 shows the core of the active site of this enzyme in Lactobacillus plantarum [50]. The dimanganese core is bridged by a single carboxylate (Glu66), which spans the cluster. Two single-atom solvent bridges (oxo, hydroxo or aquo) lie between the manganese ions and provide electronic coupling between the two manganese ions. In addition, one of the manganese ions completes its octahedral geometry with a monodentate carboxylate (Glu35), a histidine (His69) and a terminally bound water molecule. The other manganese ion, also displaying an octahedral geometry, is bound to a bidentate (chelating) glutamic acid (Glu148) and a monodentate histidine (His181). The catalase reaction proceeds via a two-electron oxidationreduction cycle during turnover ( Figure 3). Peroxidases catalyse the oxidation of a broad range of substrates by hydrogen peroxide [51]. Hydrogen peroxide is then reduced by two electrons using glutathione (GSH) as a sacrificial reductant, yielding water and glutathione disulfide (GSSG). The manganese binding site of the manganese peroxidase is also shown in Figure 1. One heme propionate, two water molecules and the side chains of Glu35, Glu39 and Asp179 have been identified as the manganese(II) ligands in the inner sphere [52], giving rise to an octahedral geometry for the metal ion. GPx also contains a selenocysteine which plays an essential role during the reaction mechanism, since it involves oxidation of the selenol by one molecule of hydrogen peroxide, starting a reaction chain that release GSSG as the by-product ( Figure 4). Peroxidases are not limited to H2O2 as the substrate but also catalyse the conversion of organic peroxide to alcohol. Thus, peroxidases can eliminate lipid hydroperoxides, another ROS which contributes to the progression of disbalanced redox homeostasis [53]. Lipid hydroperoxides can be generated very easily in neuronal membranes rich in polyunsaturated fatty acids like arachidonic acid, docosahexaenoic acid, or eicosapentaenoic acid. The peroxidase activity may be successfully be used for different industrial processes since peroxidases are capable of degrading a broad range of chemical structures as lignin or different recalcitrant pollutants like polycyclic aromatic hydrocarbons [54]. The enzymatic antioxidant defence along with other non-enzimatic antioxidants act in an effective way against free radicals and other reactive oxygen species to control their damaging effects to macromolecules and body tissues. However, overproduction of ROS leads to oxidative damage of cellular structures due to an imbalance in the oxidant-antioxidant status [55]. ROS excess may be derived from exogenous sources as mentioned above, but also by faulty regulation of cellular antioxidant defences, normally associated with aging, that can lead to accumulation of toxic levels of ROS [56]. For this situation, a strategy to recover a balance between ROS generation and removal is the use of antioxidants as therapy [57][58][59][60]. Administration of exogenous native antioxidant enzymes has not been successful for therapeutic treatment of oxidative stress because of several limitations [61]: i) short half-life of the enzymes; ii) difficulty to enter in the cells due to their high molecular weight; iii) antigenicity; iv) high-manufacturing costs. To overcome these limitations, pharmacological research has pointed at the development of low molecular weight SOD/CAT mimics [62][63][64][65]. Several supplements as α-tocopherol [66], ascorbic acid [67], carotenes [68][69] or other organic molecules [70][71][72][73] with antioxidant properties have been also used for the therapeutic treatment of oxidative stress-induced diseases.

Manganosalen complexes as catalytic antioxidants
Manganosalen complexes are coordination compounds with a chelate bis-Schiff base ligand obtained by condensation of salicylaldehyde and a diamine. The acronym salen is due to the reactants used to synthezise the ligand N,N-bis(salicylidene)ethylendiamine, obtained by condensation of salicylaldehyde (sal) and ethylenediamine (en) [74]. Although the latter ligand is strictly speaking the salen ligand, actually the class of compounds known as manganosalen complexes encompasses other bis-Schiff base ligands (containing two bisimine groups), obtained from different diamines (propylenediamine, phenylenediamine, butylendiamine, etc.) as well as the group of their derivatives with different substituents both in the phenyl rings and in the diamine spacer [20,75]. Figure 5 collects some manganosalen complexes with pharmacological relevance used in in vivo studies, corresponding to the EUK series patented by Eukarion.
This type of ligands has oxygen and nitrogen donor groups, which decrease the Mn(III)/Mn(II) redox potentials upon coordination (Eº = 1.51 V for [Mn(H2O)6] 3+ , a rather oxidizing potential), and the resulting complexes constitute suitable systems to catalyse multiple redox reactions [20,[75][76][77][78][79]. Redox potentials of manganosalen complexes may be tuned by modifying other features than these substituents. In this sense, the number of ligands or the geometry around the metal ion are factors that influence the manganese redox potential of the final complex. For example, alkoxy substituents in the phenyl rings, particularly 3-methoxy or 3-ethoxy groups (see EUK-113, EUK-134, EUK-172, EUK-178 or EUK-189 in Figure 5), lowers the redox potentials for the manganosalen complexes due to the electron-donor character of these substituents, thus facilitating to achieve higher oxidation states for the manganese ion during the enzymatic activity.  The chelating bis-Schiff base ligand forms a typical almost planar MnN2O2 core where the 5-or 6-member chelate rings confer high stability. The introduction of different auxiliary ligands (halides, carboxylates, alcohols, dicyanamides, thiocyanates, etc.) alters the geometry of the compounds [20,80], giving rise to different behaviours in catalysis. Labile auxiliary ligands or solvent molecules in the axial positions favour catalytic activity through an inner-sphere electron transfer mechanism in which a vacant can be generated in this site, where the substrate molecule can be subsequently accommodated [81]. A short two-carbon chain between the imine groups in the Schiff base ligand constricts the chelate ring once the nitrogen atom coordinates to the metal, leading to tetragonally elongated geometries [23,[81][82][83][84]. On the contrary, if the Schiff base ligand has a flexible threemembered alkyl chain between the imine groups, a better stabilization of a high-symmetry octahedral symmetry is achieved, and subsequently the generation of a coordination site gets difficult. Thus, a correlation between the factor of the tetragonal elongation and catalytic activity as enzyme mimics has been reported for manganosalen complexes [23,[81][82][83][84].
Supramolecular interactions may play different crucial roles in enhancing the activity as enzyme mimics. On the one hand, hydrogen bonding and other supramolecular contacts may induce selforganization of the complexes to afford dimeric entities [85]. Thus, these supramolecular mechanisms may allow aggregation of the complexes into dimers once the monomers cross the cell membrane. On the other hand, supramolecular interactions also play an essential role in biological processes recognition [86]. Second-sphere effects of the substituents may also modulate the redox potentials of the metal centre and the metal-ligand bond strength and guide their reactivity with superoxide anion radical [87]. Alkoxy substituents on the phenyl rings can also participate in establishing supramolecular interactions through hydrogen bonding, which added to their mentioned effect on redox potentials, could explain the high activity as enzyme mimics of alkoxy substituted manganosalen complexes [88][89][90]. The relatively planar MnN2O2 core of manganosalen complexes is somewhat similar to that of natural manganese Mn-macrocycles or Mn-porphirins, and this similarity is shown in their chemical reactivity. However, the manganese ion in manganosalen compounds is coordinated to oxygen and nitrogen atoms, which contrasts with porphyrins where the metal is coordinated to nitrogen atoms only. Oxygen and nitrogen are the most common donor atoms in biological systems, so that the structure of manganosalen complexes ( Figure 6) resembles those of various native manganoenzymes. Manganosalen compounds are easily obtained from inexpensive precursors, and they are cheaper than Mn-porphirins.
One of the most interesting properties of the manganosalen complexes is that they are cellpermeable, with better bioavailability than exogenous antioxidant enzymes [91][92]. Manganosalen complexes exhibit high SOD, catalase and peroxidase activities, which has led to their development as catalytic antioxidants [6][7][8][9][10]13,[18][19][20][21]23], a term used for all cases in which one single molecule of catalyst induces the detoxification of numerous ROS molecules.
Malfroy et al. first reported the SOD mimetic properties of manganosalen complexes [93]. Based on stopped-flow analysis combined with time-resolved UV/vis spectroscopy and global spectra analysis of superoxide decay, it has been reported that manganosalen complexes possess a SOD activity of about 2 x 10 6 M -1 s -1 [94][95], both in Hepes and in phosphate buffers. The mechanism followed by the manganosalen complexes is similar to that of the native SOD enzyme, a ping-pong mechanism where a superoxide anion radical reduces the synthetic manganese complex from Mn(III) to Mn(II), which is subsequently oxidizes back to Mn(III) by a second superoxide anion radical ( Figure 7) [10,19,23].  While the SOD activity of manganosalen complexes hardly varies with derivatization, their catalase activity is highly sensitive to the substituents on the aromatic rings or the length of the alkyl chain in the spacer between the imine groups [90], ranging from inactivity to high efficiencies in the hydrogen peroxide disproportionation [81][82][83][84]96]. The efficiency of these systems seems to be related 9 of 29 to the presence of at least one vacancy or a labile coordination position on the manganese core. Electron donor substituents on the phenyl rings of the salen moiety also increase the catalase activity of manganosalen complexes [87]. The rates at which manganosalen complexes scavenge hydrogen peroxide are similar to those reported for metalloporphyrins. For instance, compound EUK-172 (see Figure 5) is reported to have a catalase rate greater than 1 mM O2/min [96], measured by monitoring the conversion of hydrogen peroxide to oxygen using a Clark type oxygen electrode [97].
The mechanism of these synthetic mimics is proposed to involve mononuclear Mn(V)=O species [98][99] (Figure 8), although some mimics may follow a mechanism through the formation of dimeric species in solution [81]. Anyhow the mechanism is different from the native catalase enzyme shown in Figure 3.
Peroxidase mimics are able to convert H2O2 to H2O but also to scavenge other peroxides, including lipid hydroperoxides or other organic peroxides [96,100], so that they could scavenge lipid peroxides in tissues. The peroxidase activity of manganosalen complexes is related to achieve square pyramidal or tetragonally elongated octahedral geometries [81,84], facilitating substrate coordination. In this way, as previously mentioned for other catalytic activities, manganese complexes with an npropyl spacer between the imine groups show little or no activity, whereas short two-carbon chains enhance the peroxidase activity. Electronic effects contributions by the substituents on the phenyl rings tune the stability of the oxidation states and modulate the reversibility of the catalytic action. Manganosalen complexes are proposed to behave as peroxidase mimics by a mechanism quite similar to that of their catalase action, through a Mn(V)=O intermediate, which is able to oxidize an organic substrate ( Figure 9) to afford the initial catalyst complex. Manganosalen complexes often have multiple antioxidant activities at the same time, showing, for instance, both SOD and catalase functions. This reactivity against different ROS is attractive since hydrogen peroxide is a product released by the SOD activity. However, recent findings open other interesting antioxidant routes, like a cascade mechanism, where the initial complex only has SOD activity but not catalase function [10,19]. In this case, the catalase active complex is activated after radical scavenger reaction, so potential redox side effects within the cell can be avoided.

Therapeutic effects of manganosalen complexes in in vivo models
The SOD, catalase and peroxidase activities shown by manganosalen complexes have attracted attention for their use as catalytic antioxidants. Subsequent in vitro studies have confirmed their efficiency in oxidative stress models [6,8,[9][10][13][14][15][20][21]23 and other references cited therein], although the focus of this review is the evaluation of the in vivo studies with different animal models. These studies are organized below according to the pathology or the oxidative damage produced by ROS.

Neurodegenerative diseases and mental disorders
The brain is prone to oxidative stress as a result of the high levels of oxygen required, which represents 20% of oxygen uptake when brain accounts for only 2% of body weight. Neurons consume a high rate of energy (4 x 10 12 ATP/minute) meaning large amounts of oxygen to maintain neural intracellular ion homeostasis [41]. Moreover, neural membranes have high concentrations of polyunsaturated fatty acids which generate lipid hydroperoxides. Additional factors, as the presence of autooxidizable neurotransmitters, increase the sensitivity of this organ to ROS-mediated damage. Excessive ROS levels have been associated with neurodegenerative disorders like Alzheimer's Disease (AD), Parkinson's Disease (PD), amyotrophic lateral sclerosis (ALS) or Huntington's Disease [42].
As previously mentioned, EUK-8, EUK-134 and EUK-189 models were injected to SOD2 nullizygous mice to rescue them from oxidative neurodegenerative process [4]. These compounds had previously shown efficacy in a variety of oxidative stress paradigms [101][102][103]. For instance, EUK-134 had found to protect most of the vulnerable neurons from excitotoxic cell death in Sprague-Dawley rats [102]. Administration of these three manganosalen compounds extended the lifespan of the mice lacking SOD2 approximately threefold and eliminated clinical signs of the associated neurobehavioral phenotype previously described. This study also indicate that these catalytic antioxidants can cross the blood-brain barrier, particularly EUK-189, which is slightly more lipophilic than EUK-134. In a later study, using the same animal model, Melov et al. reported that neural cell death in defined regions of the frontal cortex of SOD2 null mice is a consequence of endogenous mitochondrial oxidative stress [104]. In this study, they could partially rescue neural cell death by treatment with a high dose of EUK-8. Melov et al. also reported the therapeutic effects of EUK-189 in preventing a neurodegenerative phenotype in an Ah-transgenic mouse model for Alzheimer disease [105]. This manganese complex and the cyclic analogue EUK-207 were used in a study with C57BL/6N Sim middle-age mice [106], which usually exhibit a dramatic decrease in learning and memory function between 8 and 11 months of age, associated with oxidative protein damage in the brain [107]. Treatment during a 3-month period with EUK-189 or EUK-207 resulted in an almost complete reversal of age-related learning and memory deficit, showing a complete reversal in protein oxidation and a 50% reduction in age-related increase in lipid peroxidation. EUK-207 exhibits longer plasma half-life than EUK-189, and subsequently greater biological stability. Two such molecules were also used by Baudry et al. in older mice, at a lower dose, and for longer periods of time [108], preventing age-dependent cognitive decline in the same way as previously found for middle-aged individuals. Their results indicate that the age-associated deficits in learning and memory might be originated by oxidative damage to hippocampus, amygdala, or both.
EUK-8 was the first manganosalen complex that showed efficacy for the treatment of a neurodegenerative disease using an animal model (see Table 1). Malfroy et al. reported in 1997 [109] how this synthetic catalytic scavenger reduces the severity of autoimmune encephalomyelitis in guinea pigs. Watanabe et al. [110] used the same compound to treat small bowel ischemia/reperfusion injury in Sprague-Dawley rats. They compared the protective effects of EUK-8 and a Mn(III)porphirin SOD mimic, manganese-meso-tetrakis(N-methylpyridinium-2-yl)porphyrin [111]. Both manganosalen compound and Mn-porphyrin showed similar beneficial properties by the inhibition of O2, H2O2, and NO production.
Xu et al. [112] reported that EUK-8 and EUK-134 reduced the levels of oxidative stress and prolonged survival in a mouse amyotrophic lateral sclerosis model. In a later study EUK-134 afforded best results than EUK-8 in reducing brain infarct size after middle artery occlusion in a rat model [113]. After the neuroprotective effects shown by EUK-8 and EUK-134, Doctrow et al. [86] compared the in vivo antioxidant activity of different analogues, including the two cited compounds, EUK-113, EUK-161, EUK-163, EUK-172, EUK-178 and EUK-189 in a rat stroke model. They concluded that alkoxy substituents on the phenyl rings for this series of compounds confers some advantage toward the biological protective effects of these manganosalen complexes.  [114] demonstrated the efficacy of EUK-134 and EUK-189 in protecting against paraquat-induced dopaminergic cell death in adult mice via inhibition of the activation of JNKmediated apoptosis. Paraquat is an herbicide that induces selective loss of dopaminergic neurons of the substantia nigra. These two manganosalen complexes significantly inhibited caspase-3 activation, cell death, and DNA fragmentation in in vitro paraquat exposed rat cells. EUK-189 was also employed in a mouse model of human prion disease [115], giving place to a modest 5% increase of survival compared to untreated disease controls. This beneficial effect was correlated with reductions in oxidative, especially nitrative, damage to proteins. The same manganosalen complex, EUK-189, was used by Levine et al. [116] to treat the neurobehavioral defect in ataxia-telangiectasia mice, showing that this catalytic antioxidant corrected the neurobehavioral abnormality. This effect, that was reproducible over time, and it involved a reduction in the oxidation of brain fatty acids and also a retard development of thymomas.
More recent studies were focused on the manganosalen EUK-207, due to both its ability to supress oxidative stress and its greater biological stability. Thus, Raber et al. [117] reported that this compound mitigated radiation-induced cognitive impairments without affecting cognition of shamirradiated mice. Wang et al. [22,118] used EUK-207 to treat hydrogen peroxide induced DNA damage and senescence phenotype in senescence-accelerated mouse-prone 8 mice, reducing age-related loss of both hearing and hair cells degeneration. In their study, they found that cochleae cells treated with EUK-207 displayed increased levels of FOXO3a and Nrf2, two transcription factors that have been previously shown to positively regulate cellular resistance to oxidative stress [119][120].

Inflammatory diseases
The neuroprotective effects shown by manganosalen complexes are close related to their ability to attenuate inflammation makers such as cytokines or chemokimes. High amounts of hydrogen peroxide activate a number of transcription factors as NFkB, AP-1 and Nrf2 [121], whose levels may be regulated by the administration of manganosalen complexes. The anti-inflammatory effects of these catalytic antioxidants play a beneficial role not only in neurons or in the respiratory system, but also in a wide range of pathologies related to inflammation ( Table 2).
Hill et al. [122] treated Sprague-Dawley rats with EUK-189 to mitigate the DNA damage in rat lung after exposed to 10-20.5 Gy doses of gamma rays. The catalytic antioxidant resulted effective at reducing micronucleus formation in lung fibroblasts and this could be attributed to the ability of this compound to suppress the signal that would normally turn on the inflammatory response to repair the radiation insult.
EUK-134 was used by Lawler et al. [123] as ROS scavenger in myopathy in the diaphragm of the mdx mouse model. This manganosalen complex reduced hydroperoxides, markers of oxidative stress in this muscle, attenuated both by the elevation of inflammatory cell invasion and the NF-B activity and p65 subunit protein levels in the mdx diaphragm. Muzykantov et al. [124] employed PEGliposomes loaded with EUK-134 to alleviate acute pulmonary inflammation induced by endotoxin in mice. This manganosalen compound was also used by Singh et al. [125] to mitigate zinc-and paraquat-induced toxicity in rat polymorphonuclear leukocytes.
Other manganosalen compounds were used with efficacy against inflammatory episodes is EUK-207. Hill et al. [126] reported that this synthetic model mitigated the radiation-induced lung injury in 6-to 7-week-old Fisher rats, by scavenging ROS and reducing activity of the NFkB pathway. In this study, EUK-207 also showed the ability to reduce levels of TGF-β1 expression, activated macrophages and fibrosis.
A study that deserves a more detailed analysis is the carried out by Kash et al. [127] with EUK-207 to reduce lung damage and to increase survival during 1918 influenza virus infection in mice. This pandemic, caused by the H1N1 influenza A virus, infected at about a third of the world's population of 1918, and resulted in 40-60 million deaths worldwide. This influenza and SARS-CoV-2 share some similarities in the way they may lead to a respiratory failure. Many studies demonstrated that the severe lung pathology provoked by the 1918 influenza virus infection was associated with immunopathogenic immune response with excessive inflammatory and cell death Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 11 July 2020 doi:10.20944/preprints202007.0222.v1 responses [128]. EUK-207 treatment caused a marked reduction in the severity of lung pathology and substantially reduced cell death responses at both the RNA and the protein levels. The beneficial effect of this manganosalen complex in an animal model affected by the 1918 influenza is related to its ability to reduce the most relevant cytotoxic effects of ROS, thereby limiting excess of cell death responses and allowing for increased lung repair responses. As the antioxidant effects of EUK-207 regulate the response of the host, it could be a therapeutic alternative to other organism's infections than influenza. In this sense, it would be interesting to test this hypothesis with studies in animal models of the response to combined treatments of manganosalen complexes and antiviral against the SARS-CoV-2 virus. Different previous studies showed the beneficial effects of manganosalen complexes to treat lung injuries. EUK-8 was used by Fink et al. to attenuate many of the features of LPS-induced acute lung injury in a porcine model [129][130][131], by detoxifying ROS without affecting the release of other important proinflammatory mediators like 6-keto-prostaglandin F1 alpha, thromboxane B2 or tumor necrosis factor alpha. EUK-134 was the antioxidant catalytic model chosen by Kamp et al. [132] to alleviate asbestos-and H2O2-induced damage in mice, limiting pulmonary fibrosis. Hill et al. [133][134] reported the mitigation of radiation-induced lung injury by EUK-207 in Sprague-Dawley rats. Treatment with this manganese-model decreased hydroxyproline content, 8-hydroxy-2-Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 11 July 2020 doi:10.20944/preprints202007.0222.v1 deoxyguanosine, malondialdehyde levels, and activated macrophages levels. Lung levels of the cytokine transforming growth factor-β1 also decreased. Medhora et al. [135] reported the antioxidant effect of EUK-207 to reduce pneumonitis and pulmonary fibrosis after thoracic irradiation in a rat model. The anti-inflammatory effect of other catalytic antioxidants, inspired by manganosalen complexes, but not belonging to the EUK series, has also been studied. A bioinspired manganese SOD mimic, reported by Policar et al. [136], demonstrated efficiency as anti-inflammatory agent for C57BL/6 mice with DNBS-induced colitis.

Cardiovascular diseases
Inflammation and tissue damage are closely related to different pathologies, including some cardiovascular diseases. Inflammation is common for heart disease and stroke patients. The role of excessive ROS production during haemorrhagic shock and reperfusion injury has been well documented [137]. In this way, some already cited studies of manganosalen complexes treatments for anti-inflammatory models, also showed beneficial effects for cardiovascular diseases (Table 3). For instance, the commented in vivo study of Medhora et al. [135] with EUK-207 mitigated multiple vascular injuries in irradiated lungs.  [138] used EUK-8 to reduce cardiac oxidative stress in Harlequin mutant mice and their wild-type counterparts. The results of this study showed an improvement of the left ventricular end-systolic dimensions and a fractional shortening by using this manganese complex. EUK-8 also attenuated necrotic and apoptotic cell death, prevented myocardial oxidant stress, and attenuated cardiac hypertrophy and fibrosis.
EUK-189 increased 30-day survival in irradiated mice according to a study by Whitnall et al. [139]. This manganosalen complex increase the number diverse circulating blood elements like total white blood cells, lymphocytes, eosinophils, and platelets. The same catalytic antioxidant was tested by Monsalve et al. [140] to regulate ROS homeostasis and to control the vascular endothelial cells function in mice. EUK-189 restored endothelial growth factor-A signalling in peroxisome proliferator activated receptor γ co-activator 1α (PGC-1 α), a process to be relevant in metabolic disorders where Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 11 July 2020 doi:10.20944/preprints202007.0222.v1 microvascular complications are frequent, like diabetic retinopathy. Excessive ROS appeared as key factor in the alteration of the endothelial growth factor-A signalling and in the capacity of endothelial cells to form stable interactions with other endothelial cells and with the extracellular matrix, but these alterations were partially reversed by administration of EUK-189. Recent studies have shown the efficacy of other manganosalen complexes for the treatment of different cardiovascular models. Yamada el al. [141] reported that EUK-134 prevented the force decrease and the actin modifications in pulmonary hypertension diaphragm bundles in Wistar rats. In their study they found that this manganosalen complex does not alter diaphragm contractile function in normal rats. Lindner et al. [142] evaluated the therapeutic effects of EUK-207 in mice with age-dependent atherosclerosis. Long-duration therapy (40 weeks) with EUK-207 almost completely suppressed plaque development and macrophage content in thoracic aorta of the treated mice compared with control mice. However, therapy for eight weeks did not affect the area or the macrophage content.

Skin damage
In the same way as discussed for endothelial cells, any tissue, including the skin, can suffer oxidative damage both of inflammatory and non-inflammatory origins. Several studies have focused on the protective effects of the manganosalen compounds in ROS-mediated skin damage (Table 4). Skin is exposed to solar ultraviolet irradiation, ozone, smoke and air pollution. All of them are environmental sources of ROS that induce damage to lipids, proteins and DNA, playing a role in the skin aging process [143].  [144] to delay the rejection of fully allogeneic skin transplants in mice. Mice treated with EUK-189 showed the longest skin graft survival, and along with EUK-134, exhibited the longest delays of graft rejection. The three manganosalen complexes reduced anti-donor cytotoxic responses in skin-grafted mice, and they decreased proinflammatory type 1 alloresponse while promoted anti-inflammatory type 2 alloimmunity.
Declercq et al [12] carried out a study of the protective effects of EUK-134 on the human skin of 748 healthy volunteers (18-80 years of age) over a period of 4 years. EUK-134 had been previously reported to increase cell survival in normal human keratinocytes upon exposure to ultraviolet-B, superoxide or hydrogen peroxide [145][146]. In the study with the human volunteers, EUK-134 (applied at a concentration of 0.01-0.1%) reduced the level of skin surface lipid peroxidation in UVA Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 11 July 2020 doi:10.20944/preprints202007.0222.v1 exposed skin. Noteworthy that the reduction of squalene hydroperoxide levels at the skin surface was found even when applied the antioxidant after UVA-exposure. As a consequence of this study EUK-134 is now commercially available as an antioxidant for the protection of dry or irritated skin. EUK-207 was tested in a study performed by Lazar et al. [147] as a potential mitigating drug on end points relevant to radiation dermatitis, skin wound healing, and chronic oxidative stress in rats. The EUK-207-treated mice group showed reduced radiation dermatitis severity by 30 days after irradiation and displayed significantly smaller wounds than vehicle-treated rats. Thiss manganosalen complex also reversed and normalized the gene expression pattern in irradiated skin by reducing the oxidation of proteins and nucleic acids. The same compound was used by Hill et al. [148] to mitigate the radiation-induced DNA damage and the lipid peroxidation in mice. They found that EUK-207 provided some protection against DNA damage only when was delivered before irradiation. They also demonstrated significant protecting effects on radiation-induced lipid peroxidation at one or more of the three time points after local skin irradiation.

Fetal malformations
Pregnancy is a state of oxidative stress due to high metabolic activity in the fetoplacental compartment. Regulation of ROS during gestation is a complex process, whereas excessive oxidant levels cause biomolecules damage and leads to fetal malformations as a consequence of the attack by ROS formed during the resumption of placental perfusion. On the other hand, the maintenance of a physiological level of oxidant levels is essential for governing life processes through redox signalling [149]. Two studies have been reported about the fetal protection or the reduction of pregnancy complications by manganosalen complexes.
Zhang et al. [150] studied the effect of long-term high-altitude hypoxia (a severe lack of oxygen) during gestation in sheep. Uterine arteries of pregnant sheep are affected by chronic hypoxia due to an inhibition effect of the large conductance Ca 2+ activated K + (BKCa) channel activity by increasing oxidative stress. Treatment of the pregnancy sheep with EUK-134 resulted in a mitigation of the hypoxia effects on BKCa channel currents in uterine arteries, alleviating pregnancy complications such as pre-eclampsia and fetal growth restriction.
The same manganosalen complex was used by Chen et al. [151] to protect ethanol-induced limb malformations in mice. In vivo treatment with EUK-134 resulted in diminished apical ectodermal ridge cell death as well as parallel reductions in the incidence and severity of limb defects in mouse fetuses (from 67.3% to 35.9%). The forelimb malformations were partially reversed by this manganosalen complex, including postaxial ectrodactyly, metacarpal, and ulnar deficiencies.

Adrenal and liver diseases
Since the imbalance between free radicals and antioxidants can be suffered by a variety of cells and issues, practically any organ can be affected, leading to a wide variety of pathologies. Kidney and liver function can be altered by excessive ROS and, again, manganosalen complexes appear as antioxidant therapeutic alternatives.
Kregel et al. [152] used EUK-189 to prevent age-related oxidative damage associated with environmental stress. They reported that this catalytic antioxidant blocked the activation of activator protein-1 (a redox-sensitive early response transcription factor involved in the regulation of cellular stress responses), enhanced stress tolerance in aged animals by reducing cellular oxidative stress and subsequent accrual of hepatic injury in Fischer 344 rats. Yazdanparast et al. [153] reported the amelioration of diet-induced non-alcoholic steatohepatitis in rats by EUK-8 and EUK-134. These two compounds had hepatoprotective, hypolipidemic, hypocholestorolimic and hypoglycemic effects on the in vivo model. Thus, the authors reported that EUK-8 and EUK-134 reduced the sera aminotransferases, the extent of lipid peroxidation, low density lipoprotein contents, cholesterol, and protein carbonylation. The same research group published other study about the protective effects of EUK-8, EUK-15, EUK-115, EUK-122, EUK-132 and EUK-134 against CCl4-induced damages in rats [154]. The manganosalen complexes ameliorated the effects of CCl4 by decreasing the levels of ROS, lipid and protein oxidations and lipofuscin-like pigments formation on liver and brain.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 11 July 2020 doi:10.20944/preprints202007.0222.v1 EUK-134 was used by Ghouleh et al. [155] to attenuate the vascular manifestations of sepsis in lipopolysaccharide-treated pigs. This catalytic antioxidant prevented the fall in renal blow flow, an effect associated with a decrease in nitrosative stress in the kidney supporting a renal protective effect.

Lifespan extension
Harman proposed that organisms age because they accumulate oxidative damage that comes from ROS [156]. His free radical theory of aging prompted investigations to look for therapeutic antioxidants to lifespan extension. Although this theory was supported by later studies that demonstrate that increased production of ROS shortens lifespan [157], and oxidative damage increase with age [158], other reports clearly contradict the basis of this theory [159]. The aim of this review is not to assess this controversy but to present the results of the different tests in this regard.  [160] reported that increased oxidative stress caused by deletion of the mitochondrial superoxide dismutase SOD-2 extended lifespan in the same nematode model. According to this latest study, decreased antioxidant function may extend lifespan. In vivo studies are sensitive to small changes in the environment, as Gems et al. [5] hypothesized to explain their results when they tried to reproduce the lifespan extension of the same nematode using EUK-8. Since they did not find any increase in lifespan upon treatment with this SOD mimetic, they conclude that this effect reported by Melov et al. should be very sensitive to subtle differences in the manner in which the manganosalen complex is administered. Their results raised doubts about the potential utility of EUK-8 in the therapeutic attenuation of aging. A subsequent study by Gems et al. [161] tested EUK-8 and EUK-134 again in Caenorhabditis elegans. In this study, they found that the synthetic mimetics elevated in vivo SOD activity levels (increases 5-fold) and exhibited protection when the worms were treated with superoxide generators (paraquat and plumbagin). Thus, the manganosalen complexes increased lifespan in nematodes, compared to the control study, where superoxide levels were elevated but they did not retard aging in the absence of superoxide generators. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 11 July 2020 doi:10.20944/preprints202007.0222.v1 EUK-8, EUK-134 and Caenorhabditis elegans met again in the study of Lithgow et al. [162]. They reported that these manganosalen complexes extended lifespan of the worms and conferred resistance to two types of oxidative stress-inducing agents (paraquat and thermal stress). The protective effects of EUK-8 and EUK-134 were independent of insulin/IGF-I signalling, and they did not show any detrimental repercussion on development or fertility. Definitely, in vivo evaluation of these synthetic mimetics in aging studies involves careful consideration of complex concentration, complex delivery and biotic environment.
Partridge et al. [163] treated Drosophila melanogaster, fruit fly, with EUK-8 and EUK-134, reporting the antioxidant protective effects to rescue pathologies associated with elevated oxidative stress in SOD-deficient flies or normal flies exposed to induced oxidative stress. However, the synthetic antioxidants did not extend lifespan in normal, wild type animals. In a different study, Sohal et al. [164], did not find lifespan extension administrating the EUK-8 to Musca domestica, both under normoxic and hyperoxic conditions.
In in vivo mammalian models, it has already been discussed above how different manganosalen complexes increase the longevity of genetically engineered SOD2 nullizygous mice [4], extending 3fold their lifespan. In general, the life expectancy of animal models subjected to oxidative stress or with pathologies associated with this stress, is increased with treatment with manganosalen complexes, as reviewed in previous sections. However, the use of these synthetic antioxidants in normal and healthy individuals probably does not have any advantageous effect on lifespan extension.

Conclusions
Throughout the previous sections the protective effects of manganosalen complexes to combat oxidative stress and its associated pathologies have been presented and discussed. These compounds showed efficiency to reverse different oxidative damage: neurodegenerative, inflammatory, cardiovascular, adrenal and liver diseases, skin damage and fetal malformations ( Figure 10). The increase of lifespan has been also reported for organisms exposed to oxidative stress, although far from being any Elixir of Life. Not all compounds have the same activity, as they show different lipophilicities, redox properties or steric hindrance.
Of the nearly sixty in vivo trials, only a few of them did not give beneficial effects for the desired quality. Thus, Wada et al. [165] reported that EUK-134 was ineffective in promoting the restoration of prolonged low-frequency force depression, a state which may suffer skeletal muscles under vigorous activity. In this case, the antioxidant catalyst showed a positive effect on sarcoplasmic reticulum Ca 2+ release in Wistar rats but a negative effect on myofibrillar Ca 2+ sensitivity. On the other hand, Espósito et al. [166] reported the toxicity of EUK-108 (20-100 M dose) toward Danio rerio individuals (zebrafish), particularly in terms of brain damage. Finally, Vanfleteren et al. [11] found that administration of EUK-8 to starving Escherichia coli cells surprisingly enhanced the production of ROS, resulting in a massive increase of oxidative damage.
Much is still unknown about the way these manganosalen complexes work in different pathologies. Drugs often have unrecognized effects, so that we cannot be confident that the beneficial effects of these compounds were due solely to their antioxidant activities. More research is needed, particularly because of the properties showed by this type of compounds that also vary with minor structural modifications. Evaluation in animal models continues to be necessary, since it cannot be assumed that the in vivo antioxidant activity of different analogues is similar. In the search to identify the best candidate, an orally available one would be the most suitable. Efforts should be directed towards obtaining an effective antioxidant, modulating its lipophilicity and redox potentials, and that it can be administered orally.
Despite the protective effect against ROS shown by these compounds in oxidative stress models in vitro and in vivo, their practical application in humans remains highly challenge. Clinical trials are crucial to assess the efficacy of this approach, especially after the results given by other antioxidants. Thus, although coenzyme Q10, β-carotene, α-tocopherol or other antioxidant supplements showed highly encouraging results in in vivo animal models, most of their clinical trials in humans failed to Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 11 July 2020 doi:10.20944/preprints202007.0222.v1 reproduce positive results [167][168][169]. The synthetic catalytic antioxidants approach may have a plus compared with non-enzimatic antioxidants: They may regulate ROS by mimicking the mechanism of the native enzymes. Figure 10. Some of the oxidative stress-related diseases tested in in vivo animal models with manganosalen complexes.
Finally, in vivo evaluation of pharmacological responses may be affected by multiple factors. Moreover, sometimes the results are simplified by describing them as beneficial or harmful when, turning the sentence of Paracelsus, the dose makes the medicine. ROS play physiological roles in cell signalling and in the control of gene expression, processes that could be affected by antioxidant therapies. The catalytic oxidants should be administered just in the right dose to combat excessive ROS levels.