Abstract: Mitochondria are central to oxidative phosphorylation and much of metabolism, and are also involved in many aspects of cell death. Consequently, mitochondrial dysfunction contributes to a wide range of human pathologies. In many of these, excessive oxidative damage is a major factor because the mitochondrial respiratory chain is a significant source of the damaging reactive oxygen species superoxide and hydrogen peroxide. However, despite the clinical importance of mitochondrial oxidative damage, antioxidants have been of limited therapeutic success. This may be because the antioxidants are not selectively taken up by mitochondria, but instead are dispersed throughout the body. To address this unmet need, a series of mitochondria-targeted antioxidants have been developed over the past few years that are selectively concentrated within mitochondria in vivo. The accumulation of an antioxidant at the site where it is needed most has been shown to improve the outcome in a large number of animal models of diseases that involve mitochondrial oxidative damage. Mitochondria-targeted antioxidants have also been developed as pharmaceuticals and have been shown to be safe and effective in human clinical trial phase IIa studies. Therefore the mitochondria-targeted antioxidants are a new class of pharmaceuticals that can be used in a wide range of human pathologies for which current therapies are of limited efficacy. Here we survey the work that has been done to date using mitochondria-targeted antioxidants and suggest future applications.
Mitochondria are central to energy metabolism and also play crucial roles in intermediary metabolism and in many other vital functions such as iron-sulfur center assembly, thermogenesis, and heme biosynthesis (Murphy and Smith, 2000; Saraste, 1999; Szewczyk and Wojtczak, 2002; Wallace, 1999). Mitochondria are also critically involved in cell death by both the apoptotic and necrotic pathways (Kroemer et al., 1997; Lemasters et al., 1998). The wide range of ways in which mitochondria contribute to the life and death of a cell makes it unsurprising that mitochondrial dysfunction plays a role in a number of human pathologies (Balaban et al., 2005; Murphy, 2009b; Murphy and Smith, 2000). These include neurodegenerative diseases, cardiac dysfunction, inflammation, ischemia-reperfusion injury in heart attack and stroke, sepsis, types I and II diabetes, metabolic syndrome, and the success of organ transplantation (Ames et al., 1993; Beckman and Ames, 1998; Balaban et al., 2005; Finkel, 2005; Green et al., 2004). Mitochondrial function can be disrupted due to genetic defects in either mitochondrial or nuclear genomes (Wallace, 1992), as a consequence of cumulative damage over the lifetime of a subject (Beckman and Ames, 1998), or in response to acute trauma (Halestrap, 2005). Furthermore, even if the primary cause of a pathology is unrelated to mitochondria, the tendency of mitochondria to initiate apoptosis or necrosis and the requirement for ATP for a cell to recover from an insult means that mitochondrial dysfunction is a significant secondary factor in determining clinical outcome. In most cases of pathological mitochondrial dysfunction, elevated oxidative damage is thought to play a role (Ames et al., 1993; Beckman and Ames, 1998; Finkel, 2005; Green et al., 2004). This is because the mitochondrion is a major source of the reactive oxygen species superoxide within the cell that leads to oxidative damage (Balaban et al., 2005; Murphy, 2009a). As mitochondria are particularly susceptible to oxidative damage this contributes to mitochondrial dysfunction and cell death in a range of diseases (Balaban et al., 2005; Murphy, 2009a).
The importance of mitochondrial oxidative damage in clinically important situations makes preventing it a compelling therapeutic target (Murphy and Smith, 2000). Furthermore, as mitochondrial oxidative damage occurs in many disorders such a therapy would be widely applicable, making this approach attractive to the pharmaceutical industry. The obvious way to decrease oxidative damage is through the use of antioxidants, thereby reducing the levels of reactive oxygen species such as superoxide and blocking the reactions that underlie mitochondrial oxidative damage. However, the many extensive clinical trials of conventional antioxidants such as Vitamin E or Vitamin C for diseases that involve mitochondrial oxidative damage have yielded disappointing results (Bjelakovic et al., 2008; Cocheme and Murphy, 2010). One possible explanation for this may be that the antioxidants distribute widely in the body, with only a small fraction being taken up by mitochondria (Murphy and Smith, 2007). Consequently, the protective agents may not be locating where they are needed most in sufficient amounts to impact on the oxidative damage. To address this unmet need a number of mitochondria-targeted antioxidants have been developed (Murphy and Smith, 2007; Murphy and Smith, 2000; Skulachev et al., 2009; Smith et al., 1999; Szeto, 2008). These antioxidants are modified so that they are selectively concentrated within mitochondria in vivo. The accumulation of the antioxidant should then preferentially reduce mitochondrial oxidative damage and improve the clinical outcome. Ideally, mitochondria-targeted antioxidants should be pharmaceutically tractable and stable small molecules with acceptable oral bioavailability that are selectively taken up by mitochondria within organs where they block oxidative damage and then can be recycled back to the active antioxidant form (Murphy and Smith, 2007). A number of approaches have been used to develop mitochondria-targeted antioxidants and here we survey these approaches, report on results obtained so far, and discuss future applications.
Targeting Antioxidants to Mitochondria
Two general strategies have so far proved most useful in targeting small molecule antioxidants to mitochondria in vivo: conjugation to lipophilic cations (Murphy and Smith, 2007) or incorporation into mitochondria-targeted peptides (Horton et al., 2008; Szeto, 2006). These approaches are outlined below.
Conjugation of antioxidants to lipophilic cations
Lipophilic cations can pass easily through the phospholipid bilayers of the plasma membrane and the mitochondrial inner membrane because the charge of the cation is effectively distributed over a large and hydrophobic surface area, thereby lowering the activation energy for their movement across the membrane (Liberman and Skulachev, 1970; Ross et al., 2005). The ability of these cations to move through phospholipid bilayers enables their accumulation into the mitochondrial matrix simply in response to the large, negative-inside mitochondrial membrane potential (Liberman and Skulachev, 1970; Ross et al., 2005) and does not require any specific import mechanism. The Nernst equation indicates that the uptake of lipophilic cations into the mitochondrial matrix driven only by the membrane potential increases 10-fold for every 61.5 mV. As the mitochondrial membrane potential in vivo is usually ~140-160 mV, this leads to ~200-400 fold accumulation of lipophilic cations into the mitochondrial matrix (Ross et al., 2005). Uptake into cells is also driven by the plasma membrane potential (30 - 60 mV, negative inside) (Ross et al., 2005), therefore the accumulation of these compounds into mitochondria in vivo relative to the extracellular environment can be up to several thousand-fold.
The best characterized and most widely used lipophilic cation for delivery of antioxidants to mitochondria is the triphenylphosphonium (TPP) cation, which was originally used to assess the mitochondrial membrane potential (Azzone et al., 1984; Liberman and Skulachev, 1970; Liberman et al., 1969). Since then the TPP cation has been conjugated to a range of antioxidants in order to target them to mitochondria (Murphy and Smith, 2007). There are a number of advantages of using the TPP cation approach as its uptake into mitochondria is well established and it is also relatively straightforward to introduce the functionality into a compound late in the chemical synthesis scheme, typically by displacing a leaving group with triphenylphosphine (Smith et al., 2004). A wide range of antioxidants have been targeted to mitochondria by conjugation to the TPP lipophilic cation, including Vitamin E (Smith et al., 1999), ebselen (Filipovska et al., 2005), lipoic acid (Brown et al., 2007), plastoquinone (Skulachev et al., 2009), nitroxides (Trnka et al., 2008; Dhanasekaran et al., 2005), and nitrones (Murphy et al., 2003). The best characterized antioxidant targeted to mitochondria by conjugation to the TPP cation is MitoQ, which we will describe in detail as the uptake of other TPP-conjugated antioxidants is broadly similar.
MitoQ consists of a ubiquinone moiety linked to a TPP moiety by a ten-carbon alkyl chain (Kelso et al., 2001; Murphy and Smith, 2007; Smith et al., 2003). The TPP moiety on MitoQ leads to its rapid uptake across the plasma membrane, driven by the plasma membrane potential, followed by its accumulation into mitochondria within the cells that is satisfactorily described by the Nernst equation (Kelso et al., 2001; Ross et al., 2008). Within mitochondria MitoQ is continually recycled to the active ubiquinol antioxidant by respiratory complex II (Asin-Cayuela et al., 2004; James et al., 2005; James et al., 2007; Kelso et al., 2001). As MitoQ is largely found adsorbed to the mitochondrial inner membrane and its linker chain enables the ubiquinol component to penetrate deeply into the membrane core, it is an effective antioxidant against lipid peroxidation (Asin-Cayuela et al., 2004; Kelso et al., 2001). MitoQ has also been shown to protect against peroxynitrite (James et al., 2007), and the ubiquinone form may also react directly with superoxide (Maroz et al., 2009). In acting as an antioxidant the ubiquinol form of MitoQ is oxidized to the ubiquinone form, which is then rapidly re-reduced by complex II, restoring its antioxidant efficacy (James et al., 2007). MitoQ has been shown to be protective in a large number of cell models of mitochondrial oxidative stress (reviewed in Murphy and Smith, 2007).
Mitochondria-targeted peptides that incorporate antioxidants
An alternative approach to targeting antioxidants to mitochondria is through the use of small, positively charged peptides called Szeto-Schiller (SS)-peptides (Zhao et al., 2004). SS-peptides comprise four alternating aromatic/basic amino acids with a D-amino acid in the first or second position along with amidation of the C-terminus to make them more resistant to degradation (Szeto, 2006). The SS-peptides have three positive charges at physiological pH, and studies with isolated cells showed their rapid uptake through the plasma membrane and accumulation by mitochondria, where they bind to the inner membrane (Zhao et al., 2003). The uptake through the cell membrane is concentration-dependent and non-saturable, suggesting that it is due to passage directly through the membrane (Zhao et al., 2003). Peptides with a similar structure of alternating basic and aromatic amino acids are also taken up by mitochondria within cells due to the influence of the mitochondrial membrane potential on the positively charged peptide (Horton et al., 2008). However, despite their positive charge, the uptake of SS-peptides into mitochondria does not seem to occur in response to the membrane potential and the mechanism that underlies their selective uptake by mitochondria is currently unclear (Szeto, 2006; Zhao et al., 2004). Some of the SS-peptides have intrinsic antioxidant activity; for example in the SS-31 peptide, the most effective tested to date, the antioxidant activity is due to a dimethyltyrosine residue which is thought to act through its phenolic moiety (Szeto, 2006; Zhao et al., 2004) while similar peptides without this residue were not protective. These SS-peptides are protective against oxidative stress in isolated mitochondria and in cell models of disease (Zhao et al., 2004; Manczak et al., 2010; Whiteman et al., 2008).
The Uptake of Mitochondria-targeted Antioxidants by Mitochondria In Vivo
The in vitro experiments with two classes of mitochondria-targeted antioxidants, exemplified by MitoQ and SS-31, indicate that they are selectively taken up by mitochondria within cells where they decrease oxidative damage. To develop effective therapies in vivo it is necessary to determine whether these compounds can be delivered safely long-term to mitochondria within living organisms. Here we assess what is known about the modes of administration, uptake, toxicity, metabolism, and distribution of these compounds in vivo.
Lipophilic cations in vivo
The intravenous (i.v.) toxicity of MitoQ in mice is relatively low with no toxicity at ~20 mg MitoQ/kg but toxicity is evident at ~27 mg MitoQ/kg (Smith et al., 2003). To measure long-term oral toxicity young C57BL/6 mice were administered 500 µM MitoQ in their drinking water for up to 28 weeks with no evident toxicity, corresponding to a dose of ~55-80 mg MitoQ/day/kg (Rodriguez-Cuenca et al., 2010). In these experiments MitoQ did not change physical activity, O2 consumption, food consumption, lean mass, glucose or insulin levels, insulin tolerance, or bone mineral density of treated mice, but there was a decrease in the percentage of body fat and liver and plasma triglyceride content (Rodriguez-Cuenca et al., 2010). The effects of MitoQ on gene expression in the heart and liver tissue, determined using the Affymetrix GeneChip MouseGene array of 28,853 genes, was not markedly affected by MitoQ expression and the few changes seen were minor and unrelated to any particular cellular process (Rodriguez-Cuenca et al., 2010). MitoQ did not affect mitochondrial oxidative damage to the phospholipid cardiolipin (Paradies et al., 2009), the accumulation of protein carbonyls (Davies et al., 2001; Levine et al., 1994), the activity of mitochondrial respiratory complexes, mtDNA copy number, or damage to mtDNA (Santos et al., 2006). It should be noted that the demonstration that MitoQ did not decrease the levels of accumulation of oxidative damage markers in these young animals may be because the inherent oxidative damage levels were low and were not significantly different from the background levels that arise during sample processing. Nonetheless together these data suggest that the long-term oral administration of MitoQ is safe and that its effects in vivo are due to its antioxidant properties and not to other factors.
Studies of the uptake of [3H]MitoQ into tissues following i.v. or intraperitoneal (i.p.) injection into mice showed that MitoQ was very rapidly cleared from the plasma and that substantial amounts of the compound were rapidly accumulated in the heart, brain, skeletal muscle, liver, and kidney (Smith et al., 2003; Porteous et al., 2010). To determine how much orally administered MitoQ was taken up into tissues, a liquid chromatography tandem mass spectrometry assay was developed to assess MitoQ content relative to a deuterated internal standard, d3-MitoQ, by multiple reaction monitoring (Rodriguez-Cuenca et al., 2010). For mice fed 500 µM MitoQ in their drinking water for 4-6 months this led to a steady-state accumulation of MitoQ that was ~113 pmol MitoQ/g in the heart, ~20 pmol MitoQ/g in the liver, and ~2 pmol MitoQ/g in the brain. Therefore, either acute i.v. or long-term oral administration of MitoQ leads to the substantial uptake of MitoQ within critical tissues such as the liver and heart.
The extensive studies undertaken in cells and isolated mitochondria strongly suggest that any MitoQ in tissues is essentially contained within mitochondria. However, to confirm that the MitoQ taken up into the tissues is predominantly mitochondrial due to the membrane potential is technically demanding. This is because isolating mitochondria from tissues requires homogenization which leads to the loss of the membrane potential and the consequent rapid efflux of MitoQ. Even so the mitochondrial localization of tissue MitoQ can be inferred from less direct methods. Administration of the mitochondrial uncoupler dinitrophenol, which decreases the mitochondrial membrane potential decreases the uptake of related TPP compounds within tissues in vivo (Porteous et al., 2010). Another approach is to use a surrogate TPP compound containing a reactive moiety that binds covalently to protein thiols and thus labels those proteins it encounters in vivo which can then be assessed by immunostaining using specific antibodies (Lin, 2002). These experiments show that in vivo TPP compounds are only found in mitochondria to any significant extent (Porteous et al., 2010; Smith et al., 2003).
A final consideration is the metabolism and excretion of TPP compounds after their administration. MitoQ is excreted in the urine and bile as unmodified MitoQ and also with sulfation and glucuronidation of the ubiquinol ring (Li et al., 2007; Ross et al., 2008). Both the uptake and efflux of TPP compound such as MitoQ is dominated by their Nernstian distribution indicating that the TPP compound is in facile equilibrium between the extracellular fluid, cytosol and mitochondria (Ross et al., 2005). Once the level of the TPP compound in the blood decreases due to its excretion through the kidney or biliary pathways, then this equilibration process between the mitochondria and the cytosol with the extracellular fluid leads to transfer of compound to the blood and its subsequent excretion. Consequently the uptake of TPP compounds in vivo is effectively self limiting due to its rapid reversibility and equilibration with the plasma and mitochondrial membrane potentials.
Substantial amounts of MitoQ can be delivered to mitochondria within tissues such as the heart and liver following administration by either the i.v. or oral routes without toxicity. This opens up two major routes of administration that can be used in clinical situations for patients. Of particular note is that oral administration of MitoQ can continue long term (Rodriguez-Cuenca et al., 2010), and that the i.v. administration of MitoQ leads to the very rapid (<5 minutes) uptake of the compounds into tissues, opening up the possibility of acute administration of these compounds (Porteous et al., 2010).
The uptake of mitochondria-targeted peptides in vivo
SS-peptides have been administered i.v., i.p., and subcutaneously to rodents, and rats have been given daily doses of 1.5 mg/kg of SS-31 i.p. long-term (Anderson et al., 2009), but there are no reports of these compounds being administered orally (Szeto, 2006). The SS-peptides are rapidly taken up by the perfused heart (Zhao et al., 2004) and, following i.v. or i.p. injection, are taken up into tissues, including the skeletal muscle (Anderson et al., 2009). They can also pass rapidly through the blood brain barrier following i.v. injection (Szeto, 2006). The plasma half life in rats and sheep for the SS-02 peptide is relatively long (Szeto et al., 2001). However, the uptake of the SS-peptides into tissues has not been quantitated in detail and their metabolism has not yet been reported (Anderson et al., 2009; Szeto, 2008; Zhao et al., 2003; Yang et al., 2009). Therefore the SS-peptides can be delivered to tissues in vivo following i.v., i.p. or subcutaneous injection although their pharmacokinetics and metabolism have not been described in detail to date.
Protective Effects of Mitochondria-targeted Antioxidants in Animal Models of Human Diseases
The studies discussed above indicate that both long-term and acute administration of both TPP- and peptide-based mitochondria-targeted antioxidants to rodents is safe. The next step is to determine whether the accumulation of these compounds in mitochondria in vivo is protective in animal models of diseases involving mitochondrial oxidative damage.
In the first study of its protective effects, MitoQ was administered to rats in their drinking water and the hearts were then isolated and exposed to ischemia-reperfusion (i/r) injury (Adlam et al., 2005). MitoQ gave protection against heart dysfunction, tissue damage, and mitochondrial dysfunction (Adlam et al., 2005). A similar study also showed that MitoQ was protective against cardiac i/r injury (Neuzil et al., 2007). MitoQ was protective against the damage to endothelial cells in vivo associated with chronic exposure to nitroglycerin, due to protecting against oxidative damage to nitroglycerin-metabolizing enzymes within mitochondria (Esplugues et al., 2006). MitoQ was protective against an increase in blood pressure in a spontaneously hypertensive rat model in which the increase in blood pressure is thought to arise from elevated mitochondrial oxidative damage in endothelial cells (Graham et al., 2009). Administering MitoQ to rats or mice prior to induction of sepsis by endotoxin led to extensive protection against cardiac damage (Supinski et al., 2009). In the lipopolysaccharide model of sepsis, infusion of MitoQ at the same time as induction of sepsis led to significant protection against liver damage (Lowes et al., 2008). MitoQ administered by intraperitoneal injection was protective against heart damage associated with the anti-cancer compound adriamycin (Chandran et al., 2009). In a rodent model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity, MitoQ protected against substantia nigra damage, preserved locomotor activity and dopamine content as well as decreased mitochondrial markers of oxidative damage (Ghosh et al., 2010). MitoQ has also shown protection against kidney damage in a model of type I diabetes (Chacko et al., 2010) and against cocaine induced cardiac dysfunction (Vergeade et al., 2010). In addition, MitoQ has proven useful in preserving function of the isolated organ in a model of kidney preservation for transplantation (Mitchell et al., 2011). Finally, a mitochondria-targeted plastoquinone is also protective against a range of disorders in vivo (Skulachev et al., 2009), and a mitochondria-targeted nitroxide is protective in vivo against hypertension (Dikalova et al., 2010). Together these findings show that MitoQ is protective against pathological changes in a number of animal models of mitochondrial oxidative damage that are relevant to human diseases. In addition, it suggests that many other antioxidant moieties can also be targeted to mitochondria by conjugation to the TPP moiety.
A number of studies have been carried out in animal models of disease with the mitochondria-targeted SS-peptides, most often with SS-31 which has a dimethyl tyrosine as its antioxidant moiety (Szeto, 2006). SS-31 was taken up into the heart in an ex vivo reperfusion system and was protective against i/r injury (Zhao et al., 2004). The peptides SS-02 and SS-31 were also protective against cardiac i/r injury when added on reperfusion (Szeto, 2008). Intraperitoneal injection of the SS-31peptide leads to uptake into the brain and protection against damage to the substantia nigra caused by MPTP, which induces brain damage that mimics the symptoms of Parkinson’s disease (Yang et al., 2009). However, the SS-20 peptide, which does not have antioxidant ability in vitro, was also protective, which suggests that the protection in this case may not be due to its antioxidant ability (Yang et al., 2009). Intraperitoneal injection of SS-31 has protective effects against insulin resistance in the skeletal muscle in a high fat fed mouse model (Anderson et al., 2009). This is consistent with SS-31 decreasing oxidative damage protective against insulin resistance in vivo (Anderson et al., 2009). Therefore the SS-peptides can be delivered in vivo by i.p. or i.v. administration and are then protective against mitochondrial damage in a wide range of animal models.
Human Studies with Mitochondria-targeted Antioxidants
To date, only one mitochondria-targeted antioxidant, MitoQ, has been used in humans. This work has been driven by Antipodean Pharmaceuticals Inc. (http://www.antipodeanpharma.com/). To make a stable active pharmaceutical of MitoQ it was found beneficial to make MitoQ with the methanesulfonate counter-anion and to complex this with β-cyclodextrin. This material was readily formulated into tablets that passed through conventional animal toxicity. The oral bioavailability was determined to be about 10% in rats and the major metabolites in urine were glucuronides and sulfates of the reduced hydroquinone form, along with demethylated compounds. In human phase I trials MitoQ showed good pharmacokinetic behavior with oral dosing at 80 mg (1 mg/kg) resulting in a plasma maximal concentration of 33.15 ng/ml after ~1 hour. MitoQ was first assessed to see if it could slow the progression of pathology in Parkinson’s disease (Snow et al., 2010). This was the PROTECT study (registered on www.clinicaltrials.gov as NCT00329056). In this 13-center study in New Zealand and Australia 128 newly diagnosed untreated patients with Parkinson’s disease were enrolled in a double-blind study of two doses of MitoQ (40 and 80 mg per day) compared with placebo to see whether, over 12 months, MitoQ would slow the progression of Parkinson’s disease as measured by the Unified Parkinson’s Disease Rating Scale. This study showed no difference between MitoQ and placebo on any measure of Parkinson’s disease progression (Snow et al., 2010). The most probable explanation for the lack of effect is that by the time Parkinsonism is clinically evident it is too late to rescue the remaining dopaminergic neurons. While there was no therapeutic efficacy, this study demonstrated that MitoQ can be safely administered as a daily oral tablet to patients for a year. The second human trial was the CLEAR trial on chronic hepatitis C virus (HCV) patients (Gane et al., 2010) (registered on www.clinicaltrials.gov as NCT00433108). HCV patients who were unresponsive to the conventional HCV virus treatments were chosen because there is evidence for increased oxidative stress and mitochondrial damage in liver dysfunction in these cases. The effect of oral MitoQ on serum aminotransferases in HCV infected patients was assessed in a double-blind trial of 40 mg or 80 mg MitoQ, or matching placebo, for 28 days. Both treatment groups showed significant decreases in serum alanine transaminase. These data suggest that MitoQ reduces liver damage during chronic inflammation. More generally, this study is the first report of a potential clinical benefit from the use of mitochondria-targeted antioxidants in humans. Coupled with the one year’s safety data for MitoQ from the Parkinson’s disease study, this strongly suggested that MitoQ should be investigated in chronic liver diseases that involve mitochondrial oxidative damage. Consequently, a multicenter Phase IIb human trial has been initiated in the U.K. to assess the efficacy of MitoQ in non-alcoholic fatty liver disease. This is the MARVEL trial study (registered on www.clinicaltrials.gov as NCT01167088).
Conclusions and Future Challenges
Despite their central role in clinically important pathologies, mitochondria have been a neglected drug-target (Murphy, 2009b). This is in part due to the difficulty of selectively targeting molecules to the cellular organelle in vivo. The development of strategies to direct therapeutic antioxidants to mitochondria in vivo and the demonstration that this decreases pathology in a range of disorders following oral, i.v., or i.p. delivery strongly supports this approach. Furthermore, one mitochondria-targeted antioxidant, MitoQ, has been shown to be well tolerated, orally active, and safe in humans and is undergoing further phase IIb trials. The field of targeting therapeutic molecules to mitochondria is just beginning, but as mitochondrial damage contributes to so many diseases it is likely that further therapeutic compounds will be developed and applied to important human pathologies. The results to date suggest that disorders such as diabetes, metabolic syndrome, ischemia-reperfusion injury, hypertension, sepsis, and the preservation of organs for transplantation are important candidates for treatment by this approach. However it is likely that mitochondria-targeted antioxidants will also be tested in much other pathologies over the next few years.
M.P.M. and R.A.J.S. hold intellectual property in the area of the TPP class of mitochondria-targeted antioxidants and hold stock in, and act as consultants for, Antipodean Pharmaceuticals Inc.
Robin A. J. Smith, Ph.D., Department of Chemistry, University of Otago, Dunedin 9032, New Zealand. Michael P. Murphy, Ph.D., Group Leader, MRC Mitochondrial Biology Unit, Wellcome Trust-MRC Building, Hills Road, Cambridge CB2 0XY, UK.
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[Discovery Medicine; ISSN: 1539-6509; Discov Med 11(57):106-114, February 2011.]