Exploiting endobiotic metabolic pathways to target xenobiotic antioxidants to mitochondria

Mitochondrion 13 (2013) 454–463

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Exploiting endobiotic metabolic pathways to target xenobiotic antioxidants to mitochondria M. W. Anders ⁎ Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA

a r t i c l e

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Available online 1 November 2012 Keywords: Mitochondria Antioxidants Drug targeting β-oxidation Prodrugs

a b s t r a c t Oxidative stress plays a role in a range of human disease entities. Hence, strategies to target antioxidants to mitochondria are an active area of investigation. Triphenylphosphonium cation-based antioxidants and SS-peptides have been described and show significant uptake by mitochondria and effectiveness in animal models of conditions linked to oxidative stress. We tested the hypothesis that the mitochondrial β-oxidation pathway could be exploited to activate the antioxidant phenolic and methimazole prodrugs. Most compounds studied underwent mitochondrial biotransformation to release their antioxidant moieties, and some were cytoprotective in a hypoxia–reoxygenation model in rat cardiomyocytes. These results demonstrate the feasibility of exploiting mitochondrial bioactivation reactions for targeted drug delivery. © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction There is significant contemporary interest in the targeting of antioxidants to mitochondria. These efforts are based on the knowledge that mitochondria are a major cellular source of reactive oxygen species (Turrens, 2003) and that oxidative stress has been implicated in a range of disease processes, including, for example, diabetes (Lowell and Shulman, 2005), neurodegeneration (Reddy, 2008; Serviddio et al., 2011), stroke (Chen et al., 2011), cardiovascular disease (Lesnefsky et al., 2001; Tompkins et al., 2006), sepsis (Galley, 2011), in the toxicity of many chemicals (Boelsterli et al., 2006), and in the chemotherapeutic and toxic effects of some anticancer drugs (Menna et al., 2010; Verrax et al., 2011; Wang et al., 2010). This review will note briefly the range of mitochondria-targeting strategies that has been reported. Our studies on the development and testing of the use of endobiotic metabolic pathways to target phenolic and methimazole antioxidants to mitochondria will also be discussed. 2. Mitochondria-targeting strategies A range of strategies for targeting antioxidants to mitochondria has been reported. These targeting strategies exploit biophysical properties of mitochondria (high negative internal potential), unique mitochondrial expression of enzymes that catalyze the release of antioxidants from prodrugs, and targeting based on the transporter-dependent delivery of antioxidants or antioxidant prodrugs to mitochondria (Anders et al.,

⁎ Corresponding author. Tel.: +1 585 230 5163; fax: +1 585 273 2652. E-mail address: [email protected].

2006; Hoye et al., 2008; Rocha et al., 2010; Sheu et al., 2006; Smith et al., 2011). Although mitochondrial targeting has been largely focused on small molecules, the feasibility of the selective delivery of macromolecules, e.g., DNA, RNA, and macromolecular assemblies, e.g., liposomes, to mitochondria has also been reported (D'Souza et al., 2007; Ibrahim et al., 2011; Muratovska et al., 2001; Wang et al., 2012; Weissig, 2011). Finally, Bognar et al. (Bognar et al., 2006) prepared a SOD mimetic, HO-3538 (2-methyl-3-(3,5-diiodo-4-{2-[N-ethyl, N-(1-hydroxy-2,2,5, 5-tetramethyl-2,5-dihydro-1H-pyrrol-3-ylmethyl) ethyl]}oxybenzoyl) benzofurane 2HCl), whose structure is based on amiodarone. HO-3538 is targeted selectively to the mitochondrial permeability transition (MPT) and is designed to detoxify free radicals in the proximity of the MPT. HO-3538 is cytoprotective in cardiomyocyte-derived H9C2 cells and in the isolated, Langendorff-perfused rat heart.

2.1. Lipophilic cation-based compounds Lipophilic cations, e.g., tetraphenylphosphonium and rhodamine 123, which are taken up by mitochondria according to the Nernst potential, are used to quantify the mitochondrial membrane potential (Chen, 1988; Grinius et al., 1970; Lichtshtein et al., 1979). This knowledge has been exploited to design a range of mitochondria-targeted antioxidants. In general, the compounds contain a triphenylphosphonium moiety tethered to an antioxidant moiety, and the high negative internal mitochondrial potential fosters their selective uptake. The use of lipophilic cations to target antioxidants to mitochondria has been pioneered by Murphy and coworkers (for reviews, see Cochemé et al., 2007; Murphy and Smith, 2007; Smith et al., 2011) and has led to the synthesis and testing of a range of functionalities tethered to a triphenylphosphonium group.

1567-7249/$ – see front matter © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved. http://dx.doi.org/10.1016/j.mito.2012.10.015

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2.1.1. MitoQ MitoQ (10-(6′-ubiquinonyl)decyltriphenylphosphonium bromide; Fig. 1) has been the focus of many studies that validate its mitochondrial uptake and antioxidant properties (Kelso et al., 2001). In mitochondria, MitoQ is reduced to ubiquinol, which is the antioxidant species that detoxifies reactive oxygen species (ROS), and is, thereby, oxidized to the ubiquinone; the ubquinone thus formed is reduced to the ubiquinol. MitoQ (1 μM) blocks hydrogen peroxide-induced apoptosis in Jurkat cells; significantly, MitoQ concentrations up to 10 μM does not perturb mitochondrial or cellular function. The lack of toxicity of MitoQ was confirmed in studies in C57BL/6 that were given MitoQ for up to 28 weeks: no apparent effects on physiology, metabolism, and gene expression were observed (Rodriguez-Cuenca et al., 2010). In addition, for example, MitoQ also protects against decreased heart dysfunction, cell death, and mitochondrial damage in a rat model of ischemia–reperfusion injury (Adlam et al., 2005); against diabetic nephropathy in an Akita mouse model (Chacko et al., 2010); against ethanol-induced steatosis in a rat model (Chacko et al., 2011); against oxidative DNA damage, increased adiposity, hypercholesterolemia, hypertriglyceridemia, hyperglycemia, and hepatic steatosis in a mouse

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model of the metabolic syndrome (Mercer et al., 2012); and against oxidative stress, mitochondrial damage, proinflammatory cytokine release in a lipopolysaccharide-peptidoglycan rat model of sepsis (Lowes et al., 2008). 2.1.2. SkQ1 SkQ1 (10-(6′-plastoquinonyl)decyltriphenylphosphonium bromide; Fig. 1) is composed of plastoquinone linked to a decyltriphenylphosphonium cation. (SkQ1 and related compounds are often referred to as “Skulachev ions” in recognition of the contributions of Professor Vladimir Skulachev to the field.) Its antioxidant actions and mitochondria-protective effects have been investigated in a range of experimental systems (Antonenko et al., 2008; Izyumov et al., 2010); SkQ1 is more potent as an antioxidant and shows less prooxidant activity than MitoQ. SkQ1 protects against hydrogen peroxide- and ischemia-induced cardiac arrhythmias, myocardial infarction, renal ischemia, and stroke in several in vivo and ex vivo rat models (Bakeeva et al., 2008); decreases the spontaneous development of tumors in p53 −/− mice and inhibits the growth of human colon carcinoma HCT116/p53 −/− xenografts in athymic mice

Fig. 1. Structures of triphenylphosphonium cation-based mitochondria-targeted antioxidants.

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(Agapova et al., 2008); protects against age-induced cataract and retinopathies in OXYS rats and experimental uveitis and glaucoma in rabbits (Neroev et al., 2008); prolongs the lifespans of a fungus, a crustacean, an insect, and a mammal (Anisimov et al., 2008); and prevents cardiolipin peroxidation and fatty acid cycling (Skulachev et al., 2010). SkQ1 also prevents a range of senescence- and age-associated disorders (Skulachev et al., 2009). 2.1.3. MitoE2 MitoE2 (2-[2-(triphenylphosphonio)ethyl]-3,4-dihydro-2,5,7,8tetramethyl-2H-1-benzopyran-6-ol bromide; Fig. 1), a mitochondriatargeted analog of α-tocopherol, is taken up by mitochondria and reduces iron/ascorbate-induced mitochondrial damage (Smith et al., 1999) and reduces both neuronal and astrocytic cell death in pyramidal neurons incubated with mercaptosuccinate or buthionine sulfoximine (Bai and Lipski, 2010). 2.1.4. MitoPeroxidase MitoPeroxidase (2-[4-(4-triphenylphosphoniobutoxy)phenyl]-1, 2-benzisoselenazol-3(2H)-one iodide; Fig. 1) is composed of the peroxidase mimic ebselen linked to the triphenylphosphonium cation (Filipovska et al., 2005). MitoPeroxidase required activation by mitochondrial glutathione and showed antioxidant properties: it degraded phospholipid hydroperoxides, blocked lipid peroxidation, protected mitochondria from oxidative damage, and decreased oxidativestress-induced apoptosis. In contrast to other triphenylphosphonium cation-based compounds, the mitochondrial uptake of MitoPeroxidase was only slightly greater than that of ebselen, perhaps because of their covalent modification of protein thiols. 2.1.5. MitoLipoic acid MitoLipoic acid ((5-[1,2]dithiolan-3-yl-pentyl)triphenylphosphonium methanesulfonate; Fig. 1) is composed of the antioxidant lipoic acid linked to the triphenylphosphonium cation (Brown et al., 2007). MitoLipoic acid was accumulated in mitochondria and was reduced to dihydroMitoLipoic acid by thioredoxin and lipoamide dehydrogenase, but not by thioredoxin reductase. In mitochondria, MitoLipoic acid was, however, inefficiently reduced, whereas lipoic acid itself underwent significant reduction; thioredoxin reductase efficiently catalyzes the reduction of lipoic acid (Arner et al., 1996). Hence, MitoLipoic acid failed to protect mitochondria from oxidative injury. 2.1.6. revMitoLipAc revMitoLipAc ((5-((5-(1,2-dithiolan-3-yl)pentanoyl)oxy)pentyl) triphenylphosphonium bromide; Fig. 1) is composed of lipoic acid linked to the triphenylphosphonium cation with a metabolically cleavable ester functional group (Ripcke et al., 2009). The design of revMitoLipAc exploits the mitochondrial location of aldehyde dehydrogenase (ALDH2), which in addition its dehydrogenase activity also catalyzes the hydrolysis of esters (Mukerjee and Pietruszko, 1992). Hence, revMitoLipAc is taken up by mitochondria and releases lipoic acid.revMitoLipAc reduces mitochondrial ROS formation in HepG2 cells. 2.1.7. TPP-OH TPP-OH ((E)-2-(3-(3,4-dihydroxyphenyl)prop-2-enamido) ethyltriphenylphosphonium methanesulfonate; Fig. 1) (Teixeira et al., 2012). Hydroxycinnamic acids are chain-breaking antioxidants that are abundant in foods (Esteves et al., 2008). TPP-OH exploits the triphenylphosphonium cation to target the phenolic antioxidant 3,4dihydroxycinnamic acid to mitochondria; there is a 300- to 550-fold accumulation of TPP-OH in mitochondria. TPP-OH protects mouse C2C12 myoblast cells against hydrogen peroxide- and linoleic acid hydroperoxide-induced cell death and is itself not cytotoxic at concentrations below 250 μM. TPP-OH blocked FeCl2 + H2O2-ascorbateinduced malondialdehyde formation in isolated mitochondria.

2.1.8. MitoTempol MitoTempol (2,2,6,6-tetramethyl-4-[5-(triphenylphosphonio) pentoxy]piperidin-1-oxy bromide; Fig. 1, n = 2) (Trnka et al., 2008), TEMPOL-TPP (4-[(2,2,6,6-tetramethyl-piperidin-4-yloxy) butyl]triphenylphosphonium bromide-N-oxide; Fig. 1, n = 1) (Dessolin et al., 2002), MitoCP (2,2,5,5-tetramethyl-3-[[[11(triphenylphosphonio)undecyl]oxy]carbonyl]-1-pyrrolidinyloxy bromide; CAS865788-68-7; Fig. 1) (Dhanasekaran et al., 2005), and MitoTempo ((2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2oxoethyl)triphenylphosphonium chloride; Fig. 1) (Dikalova et al., 2010) are analogous compounds that feature the nitroxide moiety Tempo linked to the triphenylphosphonium cation. Both TEMPOL-TPP and Tempol itself blocked selenite-induced apoptosis, but the targeted nitroxide was not superior to the parent nitroxide. The mitochondrial uptake of TEMPOL-TPP was apparently not measured (Dessolin et al., 2002). MitoTempol is accumulated several hundredfold by energized mitochondria (Trnka et al., 2008). MitoTempol is reduced to the hydroxylamine analog by reaction with coenzyme Q pool in both isolated mitochondria and Jurkat cells. MitoCP, but not the nontargeted analog ((-)-3-carboxy-2,2,5, 5-tetramethylpyrrolidinyl-1-oxy), inhibits peroxide-induced oxidative stress, apoptosis, transferrin-dependent iron uptake; inactivation of complex I and aconitase; and restores proteosomal activity in bovine aortic endothelial cells (Dhanasekaran et al., 2005). MitoTempo reduces angiotensin II-induced mitochondrial superoxide formation, decreases cellular superoxide concentrations, reduces NADPH oxidase activity, and restores the concentrations of available NO (Dikalova et al., 2010). Significantly, mice given mitoTEMPO showed decreased hypertension when given at the beginning of angiotensin II infusion and decreased blood pressure after establishment of both angiotensin II-induced and deoxycorticosterone acetate-salt hypertension, indicating that mitochondria-targeted antioxidants may find use in the management of hypertension. 2.1.9. JD-29 JD-29 is the Salen-Mn(III) complex of o-vanillin (EUK-134) linked to two triphenylphosphonium cation groups; (Fig. 1) (Dessolin et al., 2002). The Salen-Mn(III) complex of o-vanillin is a small molecule superoxide dismutase and catalase mimic that reduces lesion sizes in a rat stroke model (Baker et al., 1998). JD-29 was prepared to target EUK-134 to mitochondria. JD-29 was not, however, more effective than the non-targeted EUK-134 against staurosporine-induced apoptosis (Dessolin et al., 2002). 2.2. Mitochondria-targeted peptides Peptides with antioxidant properties represent another mitochondriatargeting strategy (for reviews, see Rocha et al., 2010; Szeto, 2006). The peptide sequences afford resistance to hydrolysis and, thus, favorable pharmacokinetic properties. The observed cell permeability of peptides is dependent on charge and lipophilicity (Horton et al., 2008; Zhao et al., 2003). Cell-permeable peptides that deliver bioactive cargoes have also been described (Yousif et al., 2009). 2.2.1. SS-Peptides SS-31 (D-Arg-Dmt-Lys-Phe-NH2 where Dmt = 2′,6′-dihydroxytyrosine; Fig. 2) and SS-02 (Dmt-D-Arg-Phe-Lys-NH2; Fig. 2) are members of a series of tetrapeptides that were synthesized as opioid agonists (Schiller et al., 2000; Zhao et al., 2004). The structural motif of these peptides is characterized by alternating aromatic and basic amino acid residues with 2′,6′-dimethyltyrosine residue as the antioxidant moiety. The observed antioxidant activity of these peptides may be attributed to the presence of the 3,5-dimethylphenol group (Wright et al., 1997). SS-31 inhibits linoleic acid and low-density lipoprotein (LDL)

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3.2. Mitochondria-targeted prodrugs that undergo mitochondrial bioactivation

Fig. 2. Structures of the SS peptides SS-31 (D-Arg-Dmt-Lys-Phe-NH2 where Dmt = 2′,6′-dihydroxytyrosine) and SS-02 (Dmt-D-Arg-Phe-Lys-NH2).

oxidation and scavenges hydrogen peroxide. SS-31 blocks tert-butyl hydroperoxide-induced ROS formation and cell death in neuronal N2A cells. An analogous SS-peptide SS-02 (Dmt-D-Arg-Phe-Lys-NH2) undergoes approximately a 100-fold accumulation in mitochondria. Studies with a fluorescent analog of SS-02, i.e., SS-19, in Caco-2 cells demonstrated its cellular uptake and mitochondrial localization. The SS-peptides also possess favorable pharmacokinetic properties (Szeto et al., 2001). The SS-peptides have been investigated in a range of in vitro and in vivo disease models. For example, in a mouse model of cerebral ischemia, SS-31 reduces ischemia-induced glutathione depletion in the cortex and reduces infarct size (Cho et al., 2007). SS-31 protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in mice (Yang et al., 2009). In a diabetic retinopathy model of glucose-induced damage to human retinal endothelial cells, SS-31 reduced ROS production, maintained mitochondrial membrane potential, decreased cytochrome c release, and decreased caspase 3 expression (Li et al., 2011). In mice with angiotensin II-induced cardiomyopathy, SS-31 reduced cardiac hypertrophy, diastolic dysfunction, and fibrosis, but failed to lower blood pressure (Dai et al., 2011). In a rat model of renal ischemia/ reperfusion injury, SS-31 prevented mitochondrial structure damage and decreased respiration after reperfusion, accelerated recovery of ATP concentrations, decreased apoptosis and necrosis of tubular cells, and improved renal tubular dysfunction (Szeto et al., 2011).

3. Use of mitochondrial bioactivation pathways to target antioxidants to mitochondria 3.1. Enzymes expressed solely or largely in mitochondria catalyze the bioactivation of a range of xenobiotics to toxic metabolites Although these mitochondrial enzymes are chiefly involved in the biotransformation of endobiotics, many also accept xenobiotics as substrates. The bioactivation of the highly toxic fluoroacetate to fluorocitrate is catalyzed by the enzymes of the citric acid cycle (Peters et al., 1953). Mitochondrial monoamine oxidase B catalyzes the bioactivation of MPTP, which causes a Parkinsonian-like syndrome, to 1-methyl-4-phenyl-2,3-dihydropyridium (MPDP+), which then undergoes a two-electron oxidation to 1-methyl-4-phenylpyridinium (MPP+) (Wu et al., 1988). The hypoglycemic agent (methylenecyclopropyl)acetate, which is formed from hypoglycins A and B, is a substrate and inactivator of the mitochondrial medium-chain acylCoA dehydrogenase (MCAD) (Lai et al., 1993). The hepatotoxicity of the valproic acid metabolite 2-n-propyl-4-pentenoyl-CoA is associated with its bioactivation by enzymes of the mitochondrial β-oxidation pathway (Baillie, 1988; Li et al., 1991).

3.2.1. Cysteine conjugate β-lyase (β-lyase) The mammalian pyridoxal-phosphate-dependent β-lyases catalyze elimination reactions of cysteine S-conjugates and are responsible for the bioactivation of nephrotoxic, haloalkene-derived cysteine S-conjugates (Anders and Dekant, 1998; Cooper et al., 2002). At least 11 cytosolic or mitochondrial enzymes with β-lyase activity have been identified (Cooper and Pinto, 2006). Some enzymes, e.g., glutamine transaminase K and high-Mr β-lyase, catalyze both elimination and transamination reactions. Elfarra and coworkers prepared S-(6-purinyl)-L-cysteine and S-(guanine-6-yl)-L-cysteine as prodrugs of 6-mercaptopurine and 6-thioguanine, respectively (Elfarra and Hwang, 1993; Elfarra et al., 1995). S-(6-Purinyl)-L-cysteine is an effective targeting agent and achieves far higher concentrations of 6-mercaptopurine in the kidney than in the liver (Hwang and Elfarra, 1991). 3.2.2. Aldehyde dehydrogenase (ALDH2) Mitochondrial aldehyde dehydrogenase catalyzes both ester hydrolysis and dehydrogenase reactions (Mukerjee and Pietruszko, 1992). As indicated above, Ripcke et al. (2009) combined mitochondrial targeting with the triphenylphosphonium cation and the mitochondrial localization of ALDH2 to target lipoic acid to mitochondria. 3.2.3. Mitochondrial β-oxidation pathway The concept that the β-oxidation pathway could be exploited to deliver antioxidants to mitochondria stems from the studies by Sir Rudolph Peters on the bioactivation of fluoroacetate (Liébecq and Peters, 1949), which showed that the toxicity of fluoroacetate is due to its mitochondrial biotransformation to fluorocitrate. Contemporary studies with a series of ω-fluoroalkanoates showed that compounds with an even number of carbon atoms are toxic, whereas compounds with an odd number of carbon atoms are nontoxic (Buckle et al., 1949) (Fig. 3); this work recapitulates the pioneering work of Knoop on β-oxidation (Knoop, 1905). Our work on the exploitation of the β-oxidation pathway to deliver antioxidants to mitochondria was an extension of work on the mechanisms of the mitochondrial toxicity of xenobiotics. S-(1,2dichlorovinyl)-L-cysteine (DCVC), a metabolite of trichloroethene, is a mitochondrial toxin (Anders and Dekant, 1998). Parker investigated the structural requirements for DCVC-induced mitochondrial toxicity (Parker, 1965). Whereas the desamino analog of DCVC 5,6-dichloro4-thia-5-hexenoate (DCTH) was highly toxic, removal of the carboxylic acid group gave a nontoxic compound (Fig. 4). Further studies confirmed the mitochondrial toxicity of DCTH and indicated the possibility that it was biotransformed as a fatty acid (Stonard, 1973; Stonard and Parker, 1971). The proposal that DCTH undergoes mitochondrial bioactivation was supported by work that showed that 4-thiaoctanoate is biotransformed to butanethiol (Lau et al., 1988, 1989). The hypothesis that DCTH undergoes bioactivation by the β-oxidation pathway was tested by Koechel and coworkers, who showed that DCTH was nephrotoxic in dogs and that the chain lengthened and shortened analogs, 6,7-dichloro-5-thia-6-heptenoate and 4,5-dichloro-3-thia-4-pentenoate, respectively, were not nephrotoxic (Koechel et al., 1993); these findings indicated a role for β-oxidation

Fig. 3. Biotransformation of ω-fluoroalkanoates. ω-Fluoroalkanoates with an even number of carbon atoms are biotransformed to the toxic fluoroacetate, whereas ω-fluoroalkanoates with an odd number of carbon atoms are converted to nontoxic metabolites.

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Fig. 4. Mitochondrial biotransformation of ω-(phenoxy)alkanoates and ω-(phenoxy)acrylates (compounds 1c and 6a are shown as examples). FACL, fatty-acid-CoA ligase; CPT-I, carnitine palmitoyltransferase-I; CACT, carnitine-acyl carnitine translocase; CPT-II, carnitine palmitoyltransferase-II; MCAD, medium-chain acyl-CoA dehydrogenase.

in the bioactivation of DCTH. Fitzsimmons and Anders provided evidence from studies with isolated rat hepatocytes that DCTH undergoes βoxidation-dependent bioactivation (Fitzsimmons and Anders, 1993): the 6-thiaalkanoate analog of DCTH, 7,8-dichloro-6-thia-7-octenoate, was cytotoxic, whereas 6,7-dichloro-5-thia-6-heptenoate and 8,9dichloro-7-thia-8-nonenoate were not cytotoxic. Benzoic acid, which lowers cellular CoA concentrations, reduced the cytotoxicity of DCTH. The desamino 4-thiaalkanoate analogs of the nephrotoxic cysteine S-conjugates S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine and S-(pentachlorobutadienyl)-lcysteine, i.e., 6-chloro-5,5,6-trifluoro4-thiahexanoate and 5,6,7,8,8-pentachloro-4-thia-5,7-octadienoate, were also cytotoxic. Further studies showed that the nephrotoxicity and hepatotoxicity of DCTH in vivo in rats is associated with its β-oxidation-dependent bioactivation (Fitzsimmons et al., 1994). Studies with purified MCAD and enoyl-CoA hydratase showed the formation of the hemithioacetal 5,6-dichloro-4-thia-3-hydroxy5-hexenoyl-CoA, which eliminates 1,2-dichloroethenethiol to give malonyl-CoA semialdehyde (Baker-Malcolm et al., 1998; Fitzsimmons et al., 1995). Finally, Fourier-transform ion cyclotron resonance mass spectrometric studies showed the formation of α-chloroethenethiolates and thioketenes from chloroalkenederived, cytotoxic 4-thiaalkanoates (Zhang et al., 1995). These studies demonstrated the efficacy of the β-oxidation pathway to deliver toxic metabolites to mitochondria. This work was, therefore, extended to test the hypothesis that the β-oxidation pathway provides a prodrug strategy to deliver antioxidants to mitochondria. 3.2.3.1. Targeting of ω-(phenoxy)alkanoates, 3-(phenoxy)acrylates, and ω-(1-methyl-1H-imidazol-2-ylthio)alkanoates to mitochondria. Previous studies showed that 4-(phenoxy)butanoate and 6-(phenoxy) hexanoate undergo β-oxidation-dependent metabolism to give phenoxyacetate (Levey and Lewis, 1947). Similarly, 4-(chlorophenoxy) butanoate herbicides are metabolized to (chlorophenoxy)acetates (Böhme and Grunow, 1974; Van Peteghem and Heyndrickx, 1975). The metabolic fate of 3- and 5-(phenoxy)alkanoates has apparently not been reported. Hence, we tested the hypothesis that ω-(phenoxy)alkanoates, 3(phenoxy)acrylates, and ω-(1-methyl-1H-imidazol-2-ylthio)alkanoates would undergo β-oxidation and deliver antioxidant phenols and methimazole to mitochondria (Roser et al., 2010). The proposed pathway for the bioactivation of 3-(2,6-dimethylphenoxy)

propanoate 1c to deliver 2,6-dimethylphenol to mitochondria is shown in Fig. 4. Fatty-acid-CoA ligase would be expected to convert 3-(2,6-dimethylphenoxy)propanoate 1c to its CoA thioester 3-(2,6dimethylphenoxy)propanoyl-CoA, which would be transported into mitochondria by the carnitine shuttle (CPT I, CACT, CPT II). In mitochondria, the regenerated CoA thioester would serve as a substrate for the MCAD, which would form (E)-3-(phenoxy)acryloyl-CoA 6a; enoylCoA hydratase-catalyzed hydration of the acryloyl-CoA thioester 6a would give the hemiacetal 3-hydroxy-3-(2,6-dimethylphenoxy) propanoyl-CoA, which would eliminate 2,6-dimethylphenol to give malonyl-CoA semialdehyde. Accordingly a panel of ω-(phenoxy)alkanoates, 3-(phenoxy)acrylates, and ω-(1-methyl-1H-imidazol-2-ylthio)alkanoates was obtained by synthesis (Fig. 5), and their biotransformation was investigated in isolated rat liver mitochondria (Fig. 6). 2,6-Dimethylphenol and other phenolic compounds are chain-breaking antioxidants (Rigobello et al., 2004; Wright et al., 1997). The antioxidant properties of methimazole are well described (Kim et al., 2001; Nakamura et al., 2004; Ogata et al., 2005; Taylor et al., 1984). The data in Fig. 6 show that the rate of biotransformation of a series of 3-(phenoxy)propanoates is markedly sensitive to methyl-group substitution on the phenoxy moiety; the observed rates followed the order: 1a> 1b> 1c≈ 1d. 5-(Phenoxy)pentanoates (2a> 2b > 2c), which require 2 cycles of β-oxidation to deliver a phenolic metabolite, behaved similarly to the analogous 3-(phenoxy)propanoates; the observed rates followed the order: 1a> 2a, 1b≈ 2b, 1c ≈2c. Interestingly, the rate of biotransformation of 3-(2-naphthoxy)propanoate 3 was comparable to that of 5-(2,6-dimethylphenoxy)pentanoate 2a. The MCAD-dependent biotransformation of fatty acid CoA esters is considered to be more rapid than that of xenobiotic alkanoate CoA esters. To circumvent this potential block, 3-(2,6-dimethylphenoxy)acrylate 6a was prepared. This compound could, after conversion to its CoA thioester, enter the β-oxidation pathway downstream from the MCAD (Fig. 4, inset). Compound 6a proved to be an excellent substrate (Fig. 6). The 2,6-diisopropyl analog 3-(2,6diisopropylphenoxy)acrylate 6b showed no detectable biotransformation, indicating that the enoyl-CoA hydratase is intolerant of bulky substituents. Previous studies showed that 4-thiaalkanoates are substrates for the MCAD (Baker-Malcolm et al., 1998; Fitzsimmons et al., 1995; Lau et al., 1989). Given that methimazole has antioxidant properties (Kim et al.,

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Fig. 5. Structures of ω-(phenoxy)alkanoates, 3-(phenoxy)acrylates, and ω-(1-methyl-1H-imidazol-2-ylthio)alkanoates. Reproduced from Chem.-Biol. Interact., 192, M. W. Anders, Putting bioactivation reactions to work: targeting antioxidants to mitochondria, pp. 8-13, 2011, with permission from Elsevier.

2001; Nakamura et al., 2004; Ogata et al., 2005; Taylor et al., 1984), 3-(1-methyl-1H-imidazol-2-yl)propanoate 4a and 5-(1-methyl-1Himidazol-2-yl)pentanoate 4b were prepared as prodrugs of methimazole (Fig. 5). Although both compounds 4a and 4b were effective substrates, compound 4a was a superior substrate to 3-(phenoxy)propanoates 1a–d and to 3-(2,6-dimethylphenoxy) acrylate 6a (Fig. 6). 3-([2,6-Dimethylphenoxy]methylthio)propanoate 5 (Fig. 5) was prepared to test the hypothesis that interposition of a methylthio group between the 2,6-dimethylphenoxy group and the propanoate moiety would minimize the steric interaction between the substrate and the MCAD; moreover, propanoate 5 would require only 1 cycle of β-oxidation to release 2,6-dimethylphenol. Propanoate 5 was a better substrate than the analogous compounds 1c and 2c (Fig. 6). Interestingly, the β-oxidation-dependent biotransformation of propanoate 5 would be expected to yield (2,6-dimethylphenoxy)methanethiol as an intermediate; this intermediate would eliminate 2,6dimethylphenol to give thioformaldehyde, which may undergo hydrolysis to give hydrogen sulfide. Hence, this pathway may be developed to target hydrogen sulfide to mitochondria. We next investigated whether methimazole prodrugs 4a and 4b and 2,6-dimethylphenol prodrugs 1c and 6a were cytoprotective in a hypoxia-reoxygenation protocol in isolated rat cardiomyocytes (Fig. 7). Prodrugs 1c, 4a, 4b, and 6a were cytoprotective, whereas the targeted compounds methimazole and 2,6-dimethylphenol were not cytoprotective. Moreover, the cytoprotective effects of prodrugs 1c, 4a, 4b, and 6a was blocked by the carnitine palmitoyl transferase-I inhibitor etomoxir (Declercq et al., 1987). These data demonstrate that the β-oxidation pathway can be used to target and deliver antioxidants to mitochondria. This strategy offers the advantages that (1) xenobiotic alkanoates appear to be processed similarly to dietary fatty acids and (2) that the transporters and enzymes of the β-oxidation pathway are well characterized.

Issues that need to be studied in the future are (1) whether the rates of bioactivation of prodrugs are large enough to deliver therapeutic concentrations of antioxidants in vivo and (2) whether the size restrictions imposed by the MCAD and enoyl-CoA hydratase limit the range of antioxidants that can be delivered (Dai et al., 2011). 4. Preclinical toxicology studies of mitochondria-targeted antioxidants The goal of targeting antioxidants to mitochondria is to develop therapeutic agents for human disease entities. Hence, preclinical toxicology studies are an important step in evaluating their potential for therapeutic development. 4.1. MitoQ Apart from MitoQ, few data about preclinical toxicology of mitochondria-targeted antioxidants have been published. Thorough studies on the preclinical toxicology of MitoQ have been conducted (for a summary of these results, see Smith and Murphy, 2010). In mice given 500 μM MitoQ in their drinking water for 20 to 28 weeks, no discernable changes in the physical activity, oxygen consumption, food consumption, and respiratory quotient were observed (Rodriguez-Cuenca et al., 2010). In addition, no notable changes in gene expression or in indices of oxidative damage were observed. 4.2. SS-Peptides Although SS-31 has been investigated in rodent models of renal and cardiac ischemia–reperfusion injury, cardiomyopathy, and amyotrophic lateral sclerosis (Dai et al., 2011; Petri et al., 2006; Song et al., 2005; Szeto et al., 2011), rodent preclinical toxicology studies have not been published (H. H. Szeto, Personal Communication).

Fig. 6. Rates of the mitochondrial biotransformation of ω-(phenoxy)-alkanoates, 3-(phenoxy)acrylates, and ω-(1-methyl-1H-imidazol-2-ylthio)alkanoates. Compounds are identified in Fig. 5.

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Oxidative stress is considered to be a component of the pathophysiology of Parkinson's disease. Hence, MitoQ was investigated in human subjects to determine whether it would slow the progression of Parkinson's disease (Snow et al., 2010) (http://www.clinicaltrials. gov; NCT00329056; date accessed: 24 March 2012). A total of 128 subjects with previously untreated Parkinson's disease were enrolled in a double-blind study that compared placebo with two doses of MitoQ over a treatment time of 12 months. No discernible difference in the progression of Parkinson's disease between subjects given placebo and those given MitoQ was observed. Oxidative stress and mitochondrial damage are associated with the liver damage observed in chronic hepatitis C virus (HCV) infection. MitoQ was investigated to test the hypothesis that antioxidant therapy would decrease the hepatic damage seen in HCV infection (Gane et al., 2010). Thirty HCV subjects were given MitoQ (40 or 80 mg) orally or placebo for 28 days and serum alanine transaminase activities and HCV RNA levels were monitored. In both treatment groups given MitoQ, significant decreases in serum alanine transaminase activities, but no change in HCV virus load, was observed (http:// www.clinicaltrials.gov; NCT00433108; date accessed: 24 March 2012). A study to investigate whether MitoQ would reduce elevated liver enzymes due to non-alcoholic fatty liver disease was initiated but was terminated due to poor participant recruitment (http://www.clinicaltrials. gov; NCT01167088; date accessed: 24 March 2012). 5.2. SS-31 (SS-31 is identified as MTP-131 and Bendavia™ in http:// www.clinicaltrials.gov)

Fig. 7. Cytoprotective effects of ω-(1-methyl-1H-imidazol-2-ylthio)alkanoates 4a or 4b, 3-(2,6-dimethylphenoxy)propanoate 1c, and 3-(2,6-dimethylphenoxy)acrylate 6a in isolated rat cardiomyocytes. Upper panel: alkanoates 4a and 4b (1 μM), methimazole (1 μM), and etomoxir (20 μM) were incubated with cardiomyocytes in a hypoxia-reoxygenation protocol. CON, control; HR, hypoxia-reoxygenation; Eto, etomoxir. Statistical analysis (one-way ANOVA, n = 3): CON versus HR, pb .05; HR versus HR +4a, p b .05; CON versus HR +4a, NS; HR + 4a versus HR + 4a + Eto, p b .05; HR versus HR + 4b, p b .05; HR +4b versus HR + 4b + Eto, p b .05; HR versus methimazole, NS. Lower panel: alkanoates 1c and 6a (1 μM), 2,6-dimethylphenol (1 μM), and etomoxir (20 μM) were incubated with cardiomyocytes in a hypoxia–reoxygenation protocol. CON, control; HR, hypoxia–reoxygenation; Eto, etoximir. Statistical analysis (one-way ANOVA, n = 3): CON versus HR, p b .05; HR versus HR +1c, p b .05; CON versus HR +1c, p b .05; HR + 1c versus HR +1c + Eto, p b .05; HR versus HR +6a, p b .05; HR + 6a versus HR + 6a + Eto, p b .05; HR versus 2,6-dimethylphenol, NS. Reproduced from Chem.-Biol. Interact., 192, M. W. Anders, Putting bioactivation reactions to work: targeting antioxidants to mitochondria, pp. 8-13, 2011, with permission from Elsevier.

4.3. 3-(1-Methyl-1H-imidazol-2-yl)propanoate Preliminary data show that 3-(1-methyl-1H-imidazol-2-yl) propanoate given orally to mice at a dose of 1 mmol/kg is well tolerated (S. Lau, J. Sapiro, I. Rojas, and M. W. Anders, unpublished data). 5. Human clinical studies Human clinical studies are key steps in the development of mitochondria-targeted antioxidants as therapeutic agents. Such studies have been undertaken with MitoQ and SS-31. 5.1. MitoQ Given that MitoQ showed effectiveness in a range of experimental animal models of human disease entities, developmental studies with MitoQ were undertaken by Antipodean Pharmaceuticals (http:// antipodeanpharma.com), which included development of a stable formulation (Smith and Murphy, 2010).

Preclinical studies demonstrated that SS-31 ameliorates cardiomyopathy in experimental animals (Dai et al., 2011; Maack and Böhm, 2011), indicating that SS-31 may find utility in the management of hypertensive cardiovascular diseases. Accordingly, human clinical trials of SS-31 have been initiated. A study to evaluate the safety, tolerability, and pharmacokinetics of escalating single intravenous infusion doses of MTP-131 has been completed, but the study results have not been posted (http:// www.clinicaltrials.gov; NCT01115920; date accessed: 24 March 2012). Participants are currently being recruited for a phase-1 randomized, double-blind crossover trial to assess whether intravenous unfractionated heparin and Bendavia™ administered together have any significant impact on the pharmacodynamic effects of unfractionated heparin and on the pharmacokinetics of Bendavia™ (http://www.clinicaltrials.gov; NCT01513200; date accessed: 24 March 2012). A third human clinical trial to assess the effect of intravenously administered Bendavia™ on the endothelial dysfunction induced by smoking a single cigarette has been registered but is not yet open for participant registration (http://www.clinicaltrials.gov; NCT01518985; date accessed: 24 March 2012). 6. Conclusions Mitochondria-targeted triphenylphosphonium cation-based antioxidants and antioxidant peptides, i.e., SS-peptides, show significant mitochondrial uptake and effectiveness in animal models of conditions linked to oxidative stress. Moreover, recent studies show that the mitochondrial β-oxidation pathway can be exploited to deliver antioxidants to mitochondria. Although the goal of these investigations is to develop compounds that may find clinical utility, few preclinical toxicology data are available. MitoQ has been thoroughly studied and appears to be safe. Both MitoQ and the SS-peptide SS-31 are undergoing human clinical trials. If the human clinical trials show effectiveness and safety, these results will provide an impetus for further development and testing of mitochondria-targeted antioxidant drugs or prodrugs.

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