Treatment of mitochondrial electron transport chain disorders: A review of clinical trials over the past decade

Molecular Genetics and Metabolism 99 (2010) 246–255

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Treatment of mitochondrial electron transport chain disorders: A review of clinical trials over the past decade Douglas S. Kerr * Center for Inherited Disorders of Metabolism, University Hospitals Case Medical Center, Rainbow Babies and Childrens Hospital, Department of Pediatrics, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106-6004, USA

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Article history: Received 20 October 2009 Received in revised form 20 November 2009 Accepted 20 November 2009 Available online 26 November 2009 Keywords: Controlled clinical trials Mitochondrial disorders Dichloroacetate Arginine Coenzyme Q10 Idebenone Parkinson disease Friedreich ataxia Exercise

a b s t r a c t While many treatments for mitochondrial electron transport (respiratory) chain disorders have been suggested, relatively few have undergone controlled clinical trials. This review focuses on the recent history of clinical trials of dichloroacetate (DCA), arginine, coenzyme Q10, idebenone, and exercise in both primary (congenital) disorders and secondary (degenerative) disorders. Despite prior clinical impressions that DCA had a positive effect on mitochondrial disorders, two trials of diverse subjects failed to demonstrate a clinically significant benefit, and a trial of DCA in MELAS found a major negative effect of neuropathy. Arginine also has been used to treat MELAS with promising effects, although a controlled trial is still needed for this potentially toxic agent. The anti-oxidant coenzyme Q10 is very widely used for primary mitochondrial disorders but has not yet undergone a controlled clinical trial; such a trial is now underway, as well as trials of the co-Q analogue idebenone for MELAS and LHON. Greater experience has accumulated with multi-center trials of coenzyme Q10 treatment to prevent the progression of Parkinson disease. Although initial smaller trials indicated a benefit, this has not yet been confirmed in subsequent trials with higher doses; a larger Phase III trial is now underway. Similarly, a series of trials of idebenone for Friedreich ataxia have shown some benefit in slowing the progression of cardiomyopathy, and controlled clinical trials are now underway to determine if there is significant neurological protection. Uncontrolled trials of exercise showed an increase of exercise tolerance in patients with disorders of mitochondrial DNA, but did not selectively increase the percentage of normal mtDNA; a larger partially controlled trial is now underway to evaluate this possible benefit. In summary, none of the controlled trials so far has conclusively shown a benefit of treatment with the agents tested, but some promising therapies are currently being evaluated in a controlled manner. These experiences underscore the importance of controlled clinical trials for evaluation of benefits and risks of recommended therapies. Application of such clinical trials to future more effective therapies for mitochondrial disorders will require multi-center collaboration, organization, leadership, and financial and advocacy support. Ó 2009 Elsevier Inc. All rights reserved.

Introduction Despite remarkable progress in delineating the biochemical and genetic basis of mitochondrial disorders over the past 20 years, progress in establishing effective treatment has generally been limited. Many potential interventions have been proposed, most of which have not been demonstrated objectively to be beneficial. The goal of this review is to trace the history of recent clinical trials for mitochondrial disorders in children and adults with disorders of the mitochondrial electron transport (respiratory) chain. This includes both recognized congenital ‘‘primary’’ disorders of established biochemical or genetic basis as well as acquired ‘‘secondary’’ neurodegenerative disorders associated with mito-

* Fax: +1 216 844 8900. E-mail address: [email protected]. 1096-7192/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2009.11.005

chondrial dysfunction, such as Parkinson disease and Friedreich ataxia. A partial list of the many agents that have been used or proposed to have beneficial potential is shown in Table 1. These include vitamins or cofactors involved in energy metabolism, metabolic intermediates, enzyme activators, natural products, various anti-oxidants, aerobic exercise, transplantation of cells or tissues, and gene transfer or gene selective strategies. Some of these are widely used in current practice, such as ‘‘cocktail’’ combinations including carnitine, coenzyme Q10, and various vitamins. Others have been shown to have promising effects in cultured cells or experimental animal models. The rationale and reported experience in using most of these therapies will not be covered in this review, but several excellent reviews of these alternatives have been published recently [1,2]. The clinical trials that will be reviewed include those published within the past decade which were controlled or partially con-

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trolled and clinical trials that are currently in progress. Case reports are generally not included, except in their context of prompting clinical trials. Emphasis is given to the principles of design of these trials, successive series of trials, and what might be learned from relatively small trials and larger trial networks that are applicable to future planning for controlled, collaborative clinical trials of therapy of mitochondrial diseases. These cover trials of dichloroacetate, arginine, coenzyme Q10, idebenone, and exercise in children and adults with various mitochondrial disorders, MELAS, LHON, Parkinson disease (PD), and Friedreich ataxia (FA). Dichloroacetate Dichloroacetate (DCA)1 is a naturally occurring analogue of pyruvate that inhibits phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase. This results in activation (dephosphorylation) of the pyruvate dehydrogenase complex (PDC), which is the rate limiting gateway to intra-mitochondrial oxidative metabolism of carbohydrate. Prior clinical trials had shown that DCA lowers blood lactate in acute clinical conditions associated with lactic acidemia [3], which led to use of DCA in individual cases of children with congenital lactic acidosis associated with mitochondrial dysfunction. A review of these case reports suggested potential benefit without apparent severe side effects (although peripheral neuropathy had been reported in animals and one adult), warranting a clinical trial [4]. This led to development of proposals to the National Institutes of Health to conduct such a multi-center, controlled trial of DCA for children with congenital lactic acidosis, which were not funded after several submissions. That, in turn, led to two smaller, separate simultaneous clinical trials at the University of California, San Diego (UCSD), and the University of Florida (UF). The trial at UCSD, funded by the Orphan Product Division of FDA, was an open label, long-term observational trial, because of concern about the ethics or practicality of recruiting for a placebo controlled [5]. The starting dose of DCA was initially higher (40– 50 mg/kg d) in most cases, and later lowered (to 25 mg/kg d), because of the confirmatory observation that, with time, DCA inhibits its own metabolism. Thiamine was provided routinely (100 mg/d), as this had been shown to reduce the risk of peripheral neuropathy in rats [6]. The primary outcome for this trial was to lower blood and CSF lactate. Secondary outcomes included a neurological exam inventory, subjective assessments, and DCA kinetics. Eligibility for the trial included any enzymatic or genetic defect of mitochondrial energy metabolism and blood or CSF lactate equal to or higher than 26 mg/dl. A total of 37 subjects were recruited into this trial (15 females/22 males), ranging in age from 6 months to 53 years. Of these, 11 had MELAS, six defects of complex I, five defects of complex IV, three with NARP, three with PDC deficiency, and one with Kearns–Sayre syndrome. Only 22 subjects completed 3–7 years in the trial; nine died, presumably from their underlying disease, and seven discontinued treatment. The result of the primary outcome was that there was some lowering of lactate in many cases, but the overall decrease was not significant after 12 months for either baseline blood lactate (31–17 mg/dl), blood lactate after a glucose load, or CSF lactate (47–35 mg/dl), due to the large variation amongst the subjects (Fig. A, Supplement). Some 49% of the subjects continuing in the study reported subjective improvement, 22% reported worsening, and the remainder did not change. However, in the largest subgroup, seven of the 11 MELAS cases (64%) reported improvement. 1 Abbreviations used: ETC, electron transport chain; DCA, dichloroacetate; MELAS, mitochondrial encephalopathy lactic acidosis and strokes; LHON, Leber hereditary optic neuropathy; PDC, pyruvate dehydrogenase complex; mtDNA, mitochondrial DNA; PD, Parkinson disease; FA, Friedreich ataxia; cGMP, cyclic guanosine monophosphate.

Table 1 A partial list of agents that have been used or suggested for treatment of mitochondrial disorders. Thiamine Riboflavin Creatine Pyruvate Ketogenic diet Dichloroacetate

Vitamin C Vitamin E Lipoic acid Coenzyme Q10 Idebenone Curcumin

Carnitine Acetylcarnitine Acetyluridine Exercise Transplantation Gene therapy

Four of the 37 subjects developed symptomatic peripheral neuropathy and/or ataxia during the trial. In a separate report, 12 of 25 patients with normal baseline nerve conduction showed decline within a year of being treated with DCA [7]. Eight of the nine subjects who died had been treated with DCA, mostly precipitated by infections. Although nearly half of the enrolled subjects reported improvement, overall neurological function and developmental delay were not improved by DCA treatment. The concurrent clinical trial of DCA in congenital lactic acidosis at UF was also funded by the Orphan Product Division of the FDA [8]. The design was a double blind, randomized, controlled trial. All subjects received placebo for 6 months, then either DCA or placebo for 6 months, and then DCA for at least 12 months. The dose of DCA was 25 mg/kg d throughout. All subjects received thiamine (1 mg/kg d). Thus, all subjects at some point in the trial received placebo and DCA. The primary outcome was a composite score including neuromuscular, behavioral, and quality of life assessments, called ‘‘Global Assessment of Treatment Efficacy’’ (GATE). Secondary outcomes included growth, post-prandial (standard meal, Ensure) blood lactate, and illness frequency. Eligibility criteria for this trial was similar to that at UCSD, including an enzymatic or genetic defect of mitochondrial energy metabolism and blood or CSF lactate equal or greater to 2.5 mM, but the age range was limited. Forty-three cases were enrolled (22 F and 21 M), with an age range of 0.9–19 yr. These included 11 with PDC deficiency, six with MELAS, six complex I defects, five complex IV defects, and 15 with other or mixed ETC. defects. Of these, 36 completed the first 12 months, three died, and two dropped out. The primary outcome of this UF trial was based on comparison of change in the GATE score between 6 and 12 months. There was

Fig. 1. Comparison of Global Assessment of Treatment Efficacy (GATE) ratings in subjects receiving DCA or placebo. The ratio of the proportions of concordant (better), discordant (inferior), and tied (same) pairs of ratings by each blinded evaluator and all evaluators were used to calculate the generalized odds ratio point estimates and 95% confidence intervals for each major outcome variable. Since the 95% confidence limits of all evaluators included the value of 1, no significant difference in DCA treatment vs. placebo groups was observed. (from [8], with permission of the publisher).

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no difference of the overall GATE score between those subjects receiving DCA vs placebo (Fig. 1). There was a significant improvement of the 1 h post-prandial lactate (1.2 mM for DCA vs.+0.12 for placebo), but there was no significant change of basal blood or CSF lactate. Again, DCA kinetics showed a significant increase of half-life after 6 months. An unexpected finding was an increase of urinary excretion of d-aminolevulinate and maleylacetone while receiving DCA treatment. These findings were attributed to inhibition of maleylacetoacetate isomerase (in the pathway of tyrosine catabolism). Half of the subjects were found to have evidence of peripheral neuropathy prior to receiving DCA treatment, but no worsening during DCA treatment was observed. In conclusion, DCA treatment was found to reduce post-prandial blood lactate, but did not reduce basal blood or CSF lactate or improve clinical outcome in this study population. Following these two studies, one of which indicated that MELAS patients might have greater benefit from DCA treatment, and case reports indicating that MELAS patients improved with DCA [9,10], a controlled clinical trial was organized at Columbia University in collaboration with University of Florida, supported by NIH and private grants [11]. The design of this study was a double blind, randomized, placebo controlled, crossover trial. Subjects received either DCA (25 mg/kg d) or placebo for 24 months and then were crossed over to placebo or DCA for the remaining 12 months. All subjects received a ‘‘cocktail’’ of thiamine, coenzyme Q10, carnitine, and a-lipoic acid. The timing of the crossover was also doubleblinded and was deliberately not at the midpoint to minimize presumptuous bias. The primary outcome was again the GATE composite score, including neurological and neuropsychological exams, health events, and activities of daily living. Secondary outcomes included brain lactate (determined by MRS), blood and CSF lactate, MRI, EMG, and safety measures. Eligibility criteria were

now specifically restricted to subjects with the MELAS phenotype (including stroke-like episodes) associated with the common mutation of mtDNA, A3243G, and elevation of CSF lactate P2.75 mM and MRS brain lactate >5 IU. Thirty subjects were recruited, evenly male and female, mean age 30 years (±14 years, SD). However, of these 30 subjects, only six completed the full planned 36 months, three died, and three withdrew. The outcomes of this trial were presented in a complex but illuminating graph (Fig. 2). None of the 15 subjects initially assigned to DCA treatment were able to remain on treatment for 24 months, as all stopped treatment at some point because of an adverse event. In contrast, seven of the subjects initially assigned to placebo treatment completed 24 months on treatment, but four of these discontinued treatment during the 3rd year because of an adverse event. The most common adverse event was peripheral neuropathy, associated with suggestive clinical symptoms in 19 of 22 patients treated with DCA. Nerve conduction velocity decreased within 6 months in 84% of the subjects receiving DCA. This led to termination of the study by the Data Safety Monitoring Board (DSMB), before almost half of the subjects had completed 24 months in the study. Six of the subjects receiving DCA were so incapacitated that they were not able to travel to the study site. There was no significant difference in GATE scores, the primary outcome at 3, 6, or 12 month intervals between the two treatment groups, and there were no significant differences of the secondary outcomes including blood, CSF, or brain lactate. The authors concluded that DCA treatment at the doses used is not effective and is associated with unacceptable worsening of neuropathy in the majority of the subjects. They state: ‘‘Our experience underscores the importance of randomized, controlled trials in evaluating the efficacy of new treatments for MELAS’’. Indeed, this statement can be generalized to all mitochondrial disorders. The example of

Fig. 2. Diagram of follow-up for all MELAS subjects, indicating periods of exposure to DCA or placebo, extent of study completion, and reasons for not receiving study medication or not completing the study. The top panel shows subjects assigned initially to DCA; the bottom panel shows subjects assigned initially to placebo. The timeline is shown in one year columns. Dark shaded areas represent periods of receiving DCA, light shaded areas represent periods of receiving placebo. Dotted areas represent periods of treatment interruption (without assigned study medication). +, death; AE, study medication discontinued due to adverse event (mostly neuropathy related); T, study terminated; *, drug dispensing error; w/d, withdrew consent; M, missed visit due to inability to travel. (from [11] with permission of the publisher).

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these trials of DCA treatment in mitochondrial disorders is particularly significant, in that a therapy initially considered so beneficial that it seemed unacceptable to conduct a controlled trial was eventually shown by a controlled trial to be more harmful than helpful in the specific subgroup that was believed to benefit the most from such therapy. L-Arginine

Arginine has been used acutely and chronically for treatment of MELAS with apparently promising results, but has not yet been evaluated in a well-controlled clinical trial. The rationale for using arginine is that it is a substrate for nitric oxide synthase, which produces citrulline and nitric oxide, stimulating guanosyl cyclase, and thereby dilating blood vessels and presumably reducing the neurological consequences of stroke-like episodes in MELAS. Arginine has been widely used for a variety of medical conditions across the life-span for similar reasons. It appears generally safe and can be well tolerated chronically in fairly high doses in certain urea cycle disorders. However, concerns have been raised about the safety of acute or long term use of arginine, and several deaths associated with acute administration of arginine–HCl and dosing errors have been reported [12]. The most published experience with use of arginine for MELAS was initiated from Kurume University, including collaboration with other medical centers in Japan and also Moscow. This experience has been reported in a series of publications concerning a series of short-term studies and open label long-term studies [13–15]. In the short-term studies, L-arginine–HCl was administered intravenously at a dose of 500 mg/kg, and compared with infusion of 5% glucose. In the long-term trial, L-arginine (free base) was administered orally at a dose of 150–300 mg/kg d. The primary outcome measures in the short-term studies were neurological recovery and changes in neuroimaging following admission for acute stroke-like episodes, or vasodilatation of the brachial artery, or changes of plasma amino acids, nitric oxide, and cGMP. In the long-term studies, the primary outcomes were frequency and severity of readmission for stroke-like episodes or vasodilatation of the brachial artery. The eligibility criteria included presence of the A3243G mutation at 50% or greater heteroplasmy in muscle associated with

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the MELAS phenotype. 15 or 20 subjects participated in the various acute studies and six or 15 subjects in the long-tem studies, who were ages 8–30 years and about 2/3 were male. Flow-mediated vasodilatation of brachial artery diameter responses to hyperemic flow was measured by high resolution ultrasonography [14]. This showed no significant change from before or 2 h after infusion of arginine–HCl in normal controls (Fig. B, Supplement). The MELAS subjects initially had significantly less vasodilatation response of their brachial arteries, which increased 2 h after arginine–HCl infusion, although the final diameter reached was still less than that of controls. However, after 2 years of oral arginine, the baseline responsiveness was significantly increased in the MELAS subjects and similar to that of normal controls. In the short-term studies, symptomatic and neurological improvement from acute stroke-like episodes was more rapid in the MELAS subjects who received arginine–HCl infusions vs. those who received glucose [13]. Neuroimaging of cerebral blood flow by statistical parametric mapping (SPM-SPECT) also showed improvement after arginine infusion in two of these cases [14]. There were increases of plasma levels of arginine (20-fold), citrulline (2-fold), nitric oxide (2-fold), and cGMP (5-fold) within 30 min of arginine–HCl infusion (but not after glucose) [13]. During long-term follow-up of 6 MELAS patients over 48 months, the frequency and severity of stroke-like episodes decreased 8- to 10-fold respectively during the 24 months when they received oral L-arginine, compared with the prior 24 months (Fig. 3) [15]. In summary, administration of arginine–HCl to MELAS patients with acute stroke-like episodes was associated with more rapid symptomatic improvement and increases of plasma nitric oxide and cGMP, whereas long-term administration of oral arginine (base) was associated with normalization of brachial artery vasodilatation responsiveness and, in a subset of these cases, dramatically decreased frequency and severity of stroke-like episodes. Although the short-term studies included a placebo (glucose), it is not clear that they were randomized or double blinded, and the long studies were open label. Reportedly, further clinical trials of use of arginine in MELAS are in progress or planned. Because of these promising effects, and concerns about safety of arginine therapy, it would appear timely for a well-controlled long-term clinical trial of arginine therapy in MELAS.

Fig. 3. Severity and frequency of stroke-like episodes in 6 MELAS patients before and after oral L-arginine supplementation (0.15–0.30 g/kg d). The frequency of stroke-like episodes (indicted by the number of bars) after treatment (0.09 ± 0.09) was significantly decreased compared with that before supplementation (0.78 ± 0.42) (p = 0.03). The severity score of each episode (indicted by the height of the bar), after treatment (0.17 ± 0.18) was also significantly decreased compared to that before supplementation (2.04 ± 0.34) (p = 0.03). Plasma concentrations of L-arginine during supplementation ranged from 82 to 120 lM (92 ± 17; mean ± SD). (from [15] with permission of the publisher).

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Coenzyme Q10 and Idebenone in ‘‘primary’’ mitochondrial disorders Coenzyme Q10 is the most widely used potentially therapeutic agent currently employed in medical management of patients with mitochondrial disorders, usually in combination with other agents. This is the natural form that is an intrinsic part of the electron transport chain, and coenzyme Q10 is synthesized denovo in most organisms. It includes an aromatic ring that may be in either the oxidized (quinone) or reduced (quinol) form, and is lipophilic because of its long side chain of 10 isoprene moieties (50 carbon atoms). Various coenzyme Q analogues have been employed therapeutically, particularly the synthetic form, idebenone, that has only 10 carbons in its aliphatic side chain and therefore is more water soluble (Fig. 4) [16]. Idebenone can substitute for coenzyme Q10 in vitro in the electron transport chain and has similar anti-oxidant properties. The rationale for using coenzyme Q10 (or idebenone) is: (1) it might be deficient, which rarely can be due to biosynthetic defects; (2) it could serve as part of a shuttle, along with ascorbic acid, in cases where complex III activity is inadequate; and (3) it is a good anti-oxidant and can serve as a protective scavenger for reactive oxygen species, which are thought to be commonly increased in disorders of impaired electron transport and oxidative phosphorylation. The biochemical basis, diagnosis, and treatment of defects of coenzyme Q10 biosynthesis have recently been reviewed [17]. The only case of effective bypass of complex III, reported 25 years ago [18], used menadione (coenzyme Q10 was not available) and ascorbic acid, but presumably substitution of coenzyme Q10 (or idebenone) would be safer and at least as effective, but not yet reported. So the anti-oxidant properties of these agents would be expected to be most widely effective in the diverse population of subjects with various primary or secondary mitochondrial disorders. In contrast to the first two mechanisms described above, supplemental coenzyme Q10 or idebenone would not be expected to primarily increase oxidative phosphorylation (ATP formation) in the short term, although might have a protective effect on that process in the long-run. The therapeutic efficacy of coenzyme Q10 has not yet been evaluated by a clinical trial within this general group of ‘‘primary’’ genetic-metabolic disorders of the electron transport chain or oxidative phosphorylation in which it is so commonly used. A clinical trial that is intended to address this question in children with disorders of the electron transport chain is currently underway, entitled ‘‘Phase III Trial of Coenzyme Q10 in Mitochondrial Disease’’ (www.clinicaltrials.gov, NCT00432744) [19]. This is a multi-center, collaborative trial, sponsored by the FDA Office of Orphan Product Development and the University of Florida, with collaborating centers at Cincinnati Children’s Hospital Medical Center, Sick Children’s Hospital Toronto, and Case Western Reserve University. This is a double blind, randomized, placebo controlled, crossover trial. Subjects receive either coenzyme Q10 (LiQ-NOLÒ,

provided by Tishcon Corporation) 10 mg/kg d (up to 400 mg) or placebo for 6 months, and then are crossed over to receive placebo or coenzyme Q10 for the next 6 months. All subjects also receive a ‘‘cocktail’’ containing thiamine, riboflavin, vitamin C, and carnitine. The dose selected for LiQ-NOLÒ, divided bid, was previously evaluated in a separate study of children with Down Syndrome and was found to result in an average plasma level of coenzyme Q10 of 10.7 lM, which compares very favorably with that achieved with much higher doses of other forms of coenzyme Q10 used in adult clinical trials for PD (see Coenzyme Q10 in Parkinson Disease below) [20]. The primary outcomes of this trial are standardized assessments of Gross Motor Function and quality of life. Secondary outcomes include the standardized NIMH neurological exam, Child Development Inventory, sleep questionnaire, and plasma coenzyme Q10. Eligibility is more stringent than was used in prior pediatric trials of DCA, specifically a deficiency of ETC complexes I, III, and/or IV with one ETC complex 625% of the mean or two or more complexes 635% of their means and at least three other mitochondrial enzymes P60% of their means (to assure sample quality) or a defined pathogenic mutation of mtDNA or a nuclear gene involved in the function of ETC/oxidative phosphorylation with phenotypic features. Only about half of the potential mitochondrial disorder subjects screened so far have been eligible by these criteria (unpublished results). The recruitment goal is to enroll 50 cases, ages 1–17 years. A clinical trial of idebenone for MELAS also is underway. This trial, entitled ‘‘Study of Idebenone in the Treatment of Mitochondrial Encephalopathy Lactic Acidosis & Stroke-like Episodes (MELAS)’’ (www.clinicaltrials.gov, NCT00887562) [21]. The trial is sponsored by Santhera Pharmaceuticals and Columbia University. This is a phase IIa, double blind, randomized, placebo controlled, dose-finding study. Subjects in group A receive idebenone 900 mg/d, in group B receive idebenone 2250 mg/d, and in group C receive placebo, all for one month. The primary outcome is lowering of cerebral lactate, estimated by MRS. Secondary outcomes will include venous lactate, fatigue severity scale, and quality of life. The eligibility criteria are similar to the previous trial of DCA in MELAS, including presence of the A3243G mutation and the MELAS phenotype with addition of MRS lactate >5 IU. The goal is to recruit 21 cases, ages 8–65 years. Another clinical trial entitled ‘‘Study to Assess Efficacy, Safety and Tolerability of Idebenone for Leber’s Hereditary Optic Neuropathy’’ is approaching completion (www.clinicaltrials.gov, NCT00747487) [22]. This trial also is sponsored by Santhera Pharmaceuticals in collaboration with Hopital Notre-Dame, Montreal, University of Munich, and Royal Victoria Infirmary, Newcastle Upon Tyne. It is a phase II double blind, randomized, placebo controlled study, in which subjects receive idebenone, 900 mg/d or placebo for 6 months. The primary outcome is recovery of logMAR visual acuity. Eligibility requires genetically proven LHON and impaired visual acuity in at least one eye, ages P14 years and 665 years, with estimated enrollment of 84 subjects.

Coenzyme Q10 in Parkinson Disease

Fig. 4. Chemical structures of coenzyme Q10 and idebenone. (from [16] with permission of the publisher).

Inclusion of Parkinson Disease (PD) in this review serves two purposes. First, there is evidence that mitochondrial function is altered in PD, which may be considered an example of a ‘‘secondary’’ form of mitochondrial disorder [23]. Inhibitors of ETC complex I, MPTP and rotenone, have been used to produce experimental animal models of PD, and some patients with PD have been exposed to MPTP. Lower complex I activity has been reported in cells and tissues from PD patients [24]. Complex I defects are considered a major source of reactive oxygen species. Considerable effort has been

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directed to determining whether anti-oxidants slow the degenerative course of PD [23]. Secondly, there has been far more experience with clinical trials for PD than for ‘‘primary’’ mitochondrial disorders, with larger numbers of subjects, better standardized criteria for outcomes, greater multi-center collaboration, and well organized infrastructure. While all this may not be applicable to less common ‘‘primary’’ mitochondrial disorders, there are significant lessons to be learned from this larger experience. The first major collaborative trial of coenzyme Q10 in PD was sponsored by NINDS and led by UCSD, including the University of Rochester, Cornell University, Emory University, and Oregon Health Science University [25]. This was a phase II, double blind, randomized, placebo controlled, parallel group design. Subjects received a placebo or one of three doses of coenzyme Q10 at 300, 600, or 1200 mg/d for 16 months, or until the PD clinically deteriorated such that the subject required treatment with l-DOPA. All subjects also received 1200 mg/d of vitamin E, as a lipophilic carrier. The primary outcome was change in the Unified Parkinson Disease Rating Scale (UPDRS), which includes physician assessment of the severity of disability as previous agreed upon by the Parkinson Disease Study Group. Secondary outcomes included plasma CoQ10, platelet ETC enzyme assays, safety and tolerability. The entry criteria were presence of hallmark clinical features of PD (asymmetrical resting tremor, bradykinesia, and rigidity), diagnosis of PD within the last 5 years, and no PD medication, such as L-DOPA within the last 60 days. Eighty subjects were recruited, about 2/3 male, of whom only three dropped out. The primary outcome, change in the UPDRS, was significantly less (more favorable) in the group receiving the highest dose of coenzyme Q10, 1200 mg/d than the placebo group (Fig. 5A). Those taking the lower doses had intermediate UPDRS scores, but the overall difference of those on placebo vs. coenzyme Q10 reached the predetermined threshold for considering a positive trend. The dose of coenzyme Q10 correlated with the plasma level of coenzyme Q10 (Fig. C, Supplement), which reached 4 lg/ml (approximately 4.6 lM). Platelet ETC activity did not show a significant change of complex I activity (not dependent on endogenous coenzyme Q10), but the activity of the combined assay of complexes I and III (which is dependent on endogenous coenzyme Q10) did increase significantly. Since the highest dose had the greatest beneficial effect, the next question was whether even higher doses would be tolerated and have an even greater positive effect. These observations and considerations led to a phase I, open label, pilot dose-finding trial of higher doses of coenzyme Q10, also at UCDC and supported by Enzymatic Therapy Inc. [26]. 17 subjects received successively escalating doses of coenzyme Q10 at 1200, 1800, 2400, and 3000 mg/d for two weeks each, plus all received 1200 mg of vitamin E. Eligibility criteria were the same as in the prior trial. The results showed that indeed the plasma level of coenzyme Q10 continued to increase, up to a maximum average of 7.4 lg/ml (or 8.6 lM), which was achieved at 2400 mg/d and not exceeded by the higher dose of 3000 mg/d. The higher doses were generally well tolerated and led to the conclusion that these findings set the stage for planning another phase II clinical trial at a coenzyme Q10 dose of 2400 mg/d. The follow-up trial was led by the University of Rochester and Medical University of South Carolina, with collaborative participation of the NINDS NET Parkinson Disease Investigators [27]. It was supported by NINDS and Guilford Pharmaceuticals Inc., who provided one of the therapeutic agents to be tested, GPI-1485. This was a phase II, double blind, randomized, placebo controlled, multi-center, calibrated ‘‘futility’’ trial, designed to eliminate agents that show low potential for further development, based on a prespecified threshold for change in the primary outcome. The design included three arms of subjects who for 12 months received either: (1) coenzyme Q10 2400 mg/d and placebo for GPI-1485, (2) placebo

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Fig. 5. (A) Comparison of changes (mean ± SEM) of the total Unified Parkinson’s Disease Rating Scale (UPDRS) in PD subjects treated with placebo or three different doses of coenzyme Q10. Attenuation of worsening of the total UPDRS score by the highest dose of coenzyme Q10 (1200 mg/d) also was seen in each of the three parts of the UPDRS (mental, activities of daily living, and motor). (from [25] with permission of the publisher). (B) Change from baseline of the total UPDRS over one year in PD subjects treated with placebo, coenzyme Q10 (2400 mg/d), or GPI-1485 (71 subjects in each group). This analysis excluded visits conducted after patients needed symptomatic treatment. Missing visits were imputed with worst change score for the group. Bars represent standard errors of the means at 1, 3, 6, 9, and 12 months. (from [27] with permission of the publisher).

for coenzyme Q10 and GPI 4000 mg/d, or (3) placebos for both coenzyme Q10 and GPI-1485. All subjects received vitamin E, 1200 mg/d. The primary outcome was a change in the UPDRS compared with historical controls. Secondary outcomes included a conditional comparison to an updated estimate of progression in the placebo group, UPDRS subscores, and other ADL scores. Eligibility was the same as the prior studies, with age P30 years. 213 subjects were enrolled, about 2/3 males, and randomized to the three groups. Only eight subjects failed to complete the 12 months; data from all was included in the final analysis. Remarkably, the results showed no significant difference of progression of the total UPDRS between the three groups (Fig. 5B). However, that was not the pre-specified primary outcome, and neither the coenzyme Q10 nor GPI-1485 groups exceeded the futility threshold. This paradoxical difference was attributed to a historical change in medical practice such that the investigative neurologists have tended to put their PD patients on dopaminergic therapy at an earlier time when they have had less deterioration in their

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UPDRS, resulting in an apparent improvement of the UPDRS scores in the remaining untreated PD patients. The authors note that it could not be concluded from this study that either agent is effective for treatment of PD, but this study was not designed to determine whether either agent was effective and a phase III trial would be needed for that purpose, dependent on consideration of the costs, availability of other therapeutic agents, and other ongoing clinical trials. Around the same time, an independent trial of coenzyme Q10 in PD was conducted by the German Coenzyme Q10 Group sponsored by the German Parkinson Association and led by the University of Ulm [28]. This was a randomized, double blind, placebo controlled, multi-center trial, comparing nanoparticular coenzyme Q10 at 300 mg/d vs. placebo in 131 subjects with relatively mild PD, of whom 106 completed 3 months of treatment per protocol. This lower dose of nanoparticular coenzyme Q10 produced an average plasma level of 6 uM, comparable to that achieved between about 1200–1800 mg/d with regular coenzyme Q10 [26], indicating that this formulation is more efficiently absorbed. There was a paradoxical decline (improvement) from baseline of the UPDRS in both groups, but no significant difference between the two groups. However, the number of subjects and duration of this study were not sufficient to determine if there could be a beneficial effect. To answer these uncertainties of potential efficacy in PD, a larger phase III trial is now underway, sponsored by NINDS (www.clinicaltrials.gov, NCT007407140) [29]. This trial, led by Cornell University and the University of Rochester, includes 68 study locations, and is a double blind, randomized, placebo controlled, parallel, treatment/efficacy/safety trial. Subjects will be divided into three groups who will receive for 16 months: (A) coenzyme Q10, 2400 mg/d, (B) coenzyme Q10, 1200 mg/d, or (C) a placebo. All will receive vitamin E, 1200 mg/d. The primary outcome will again be change in the UPDRS. Secondary outcomes include five other scales for independent function, cognition and quality of life. Eligibility criteria will be the same as previous trials. The recruitment goal is to enroll 600 subjects, randomized into the three groups. It is notable that each of these successive clinical trials for PD over the past 10 years has been designed using evidence from previous trials, identical or very similar criteria for eligibility and primary outcomes, and a large network of collaborative centers and familiar infrastructure.

Idebenone in Friedreich Ataxia As is the case with PD, Friedreich ataxia (FA) may be considered a secondary form of a disorder of the mitochondrial electron transport chain. Deficiency of frataxin results in inadequate formation of iron-sulfur clusters which are intrinsic to electron transport chain complexes I, II, and III, and intra-mitochondrial accumulation of iron, as frataxin is thought to serve as an iron carrier and donor for Fe–S cluster synthesis [30]. Disruption of electron transport results in over-production of ROS and impaired response to oxidative stress. The clinical consequences of frataxin deficiency include progressive neurological degeneration with gait and limb ataxia, dysarthria, areflexia, sensory neuropathy, and weakness; systemic effects may include hypertrophic cardiomyopathy and diabetes [31]. Over the past 11 years, there have been some 13 clinical trials (11 completed) of idebenone to assess its efficacy in attenuating the progression of FA [32]. Three of the published trials were randomized controlled trials, initially with relatively small numbers of subjects and low doses of idebenone (5–10 mg/kg d). These showed some benefit in slowing progression of cardiomyopathy, but no significant neurological benefit. More recently, larger doses

of idebenone (up to 55 mg/kg d) were shown in phase I open-label trials to be well tolerated and result in higher blood levels [33]. This resulted in a randomized, placebo controlled, parallel design trial, led by the National Institutes of Neurological Diseases and Strokes and supported by Santhera Pharmaceuticals with help from the Friedreich Ataxia Research Alliance [34]. Subjects received idebenone over 6 months at doses of: (A) 5 mg/kg d, (B) 15 mg/kg d, or (C) 45 mg/kg d. The primary endpoint was a change in urinary 8-hydroxy-20 -deoxyguanosine (8OH20 dG), a marker of oxidative damage of DNA. Secondary endpoints included previously standardized assessment scales, including the International Cooperative Ataxia Rating Scale (ICARS) and FA Rating Scale (FARS). Eligibility requirements were genetically confirmed FA, ages 9– 17 years; 48 subjects were recruited. The outcome was that there was no significant change in 8OH20 dG in any of the groups. However, there was an indication of a dose-dependant change of the ICARS score, and if subjects with more advanced progression of FA (non-ambulatory) were excluded from analysis, this change was significant (Fig. D, Supplement). The higher doses appeared to be generally well tolerated, although one younger subject developed neutropenia. As a result of this extensive clinical investigative experience with idebenone in FA, phase III clinical trials have been undertaken in the US and Europe. One of these trials, ‘‘Study of the Efficacy, Safety, and Tolerability of Idebenone in the Treatment of Friedreich Ataxia’’ (www.clinicaltrials.gov, NCT00537680) has now been completed [35]. This trial, led the University of California, Los Angeles and Children’s Hospital of Philadelphia, is a randomized double blind, placebo controlled, parallel design trial, comparing moderate and high dose idebenone with placebo over 6 months. The primary endpoint is the ICARS, with FARS as a secondary endpoint. The eligibility criteria are similar to the prior study [34], and the enrollment target was 70 subjects. The history of this series of studies of idebenone in FA is similar to that of coenzyme Q10 in PD, in that successive clinical trials have built on the experience of prior trials, using similar standardized outcomes, initially determining safety, tolerance, and maximal doses of the agent, with preliminary suggestions of efficacy leading to larger multi-center controlled clinical trials.

Exercise The rationale for thinking exercise may benefit individuals with mitochondrial diseases is based on observations that disorders of mitochondrial DNA are frequently associated with mitochondrial proliferation and that sustained exercise also is associated with increased mitochondrial content in muscle. Therefore, the questions are: (1) Would exercise further increase mitochondrial proliferation and work capacity in subjects with mtDNA disorders? and, if so (2) In heteroplasmic conditions, would exercise selectively stimulate replication of normal mtDNA? Investigation of these questions has been led by the University of Texas, Southwest Medical Center, Dallas, and collaborators elsewhere [36,37]. The initial studies were open label, sequential, with multi-center collaboration, supported by the Muscular Dystrophy Association and VA Merit Review program. The design of the first study was a baseline evaluation followed by 14 weeks of standardized bicycle exercise (50 sessions), and final re-evaluation. The primary outcomes selected for these studies were changes in physiological parameters (work capacity, cardiac output, and creatine phosphate replenishment, estimated by 31P MRS of the vastus lateralis muscle). Secondary outcomes included a muscle biopsy to measure mitochondrial enzymes, and estimation of the percent mutant mtDNA. Eligibility was defined as mitochondrial myopathy and exercise intolerance. 10 subjects were enrolled, ages 24–

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53 years, including 4 males, all but one of whom had defined genetic mutations of mtDNA or nuclear genes that encode mitochondrial proteins involved in oxidative phosphorylation. In the follow-up study, baseline physiological studies were performed in 30 additional subjects with mitochondrial myopathies and 32 controls. The outcome of the initial studies was that exercise does significantly increase work capacity (30%) and the rate of phosphocreatine synthesis (40%), but does not increase cardiac output. Analyses of muscle biopsies showed an increase of cytochrome oxidize (complex IV; 25%) as well as citrate synthase (49%) and succinate dehydrogenase (43%). Mutant mtDNA increased slightly (9%) [36]. In baseline studies of a larger number of subjects with mitochondrial myopathy and normal controls, it was found that the percentage of mutant mtDNA correlates indirectly with exercise tolerance [37]. Further follow-up collaborative and independent studies have confirmed these physiological benefits of exercise and safety in subjects with mtDNA deletions or point mutations, including MELAS [38–40]. One of these studies showed that total mtDNA increases after exercise in subjects with mtDNA mutations (but not normal subjects) without significantly altering the ratio of normal/mutant mtDNA [39]. Currently, these observations of benefits and safety are being followed by a larger clinical trial, ‘‘The Effects of Exercise vs. Inactivity on People with Mitochondrial Muscle Diseases’’ (www.clinicaltrials.gov, NCT00457314) [41]. This trial, also led by UTSW Medical Center and sponsored by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, is a phase II, randomized, ‘‘open label’’, uncontrolled, crossover, interventional safety/efficacy study. The design is that subject Group 1 will have exercise training for 6 months, and then no exercise training for 6 months, whereas Group 2 will have no exercise training for 6 months and then have training for 6 months. The primary outcomes will be changes in the percentage of mutant mtDNA, total amount of mtDNA, and the previous physiological measures of oxidative metabolism. Secondary measures will include physical endurance, heart function, and quality of life. Eligibility will include mitochondrial myopathy due to deletions or point mutations of mtDNA in subjects ages 18–65 years. The enrollment goal is 50 subjects, to be randomized to one of the two groups.

Discussion In summary, none of the controlled clinical trials described above have yet clearly established efficacy and safety for the interventions tested for therapy of mitochondrial diseases, including ‘‘primary’’ genetic disorders or ‘‘secondary’’ degenerative diseases associated with mitochondrial dysfunction. However, it is clear from the experience with successive trials of DCA and from trials of coenzyme Q10 and idebenone that controlled clinical trials are necessary to establish efficacy and safety, and that repetitive collaborative trials may be necessary to establish even relatively small incremental benefits. Agents which currently appear to be potentially beneficial in uncontrolled trials include arginine for MELAS (perhaps chronically) and exercise in mtDNA defects. Controlled trials for both of these interventions should be pursued, and are anticipated or underway, recognizing the difficulty of ‘‘controlling’’ (i.e.: blinding) a trial of something like exercise. Controlled trials of the already widely used anti-oxidants, coenzyme Q10 and idebenone, are now underway for children with defined metabolic or genetic defects of the mitochondrial respiratory chain and children or young adults with MELAS or LHON. Coenzyme Q10 replacement for its biosynthetic deficiency is potentially the most effective therapy yet found for mitochondrial disorders, but further detailed clinical investigation may still be needed to establish criteria for early

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diagnosis of these rare conditions and selection of optimal formulations and doses for adequate treatment to prevent its consequences. Larger multi-center collaborative controlled trials should be anticipated in evaluation of forthcoming therapies for mitochondrial disorders. With this prospect in mind, it is useful to consider some of the features of successful controlled clinical trials in general and those that have been included in this review. Some of these are quite obvious, but may be difficult to achieve, as follows: 1) Subjects: It is definitely advantageous to select a relatively homogenous group of subjects for investigation, recognizing that even subjects with the same identical genotype may show phenotypic variability, and the number of subjects recruited must be sufficient to lead to statistically significant conclusions. 2) Outcomes: Primary outcomes should be kept at a minimum, usually not more than two, be clinically relevant to the disorder being investigated, and ideally generally agreed upon within the professional community caring for such disorders. Criteria for secondary outcomes need not be so limited, but too many secondary outcomes can become an excess burden to the subjects and increase the effort and costs of the study. 3) Study design: A variety of designs may be feasible, provided they are controlled (randomized, double blinded) and compatible with assessment of the primary outcomes and the number of subjects recruited. For larger trials of therapies for slowing the progression of degenerative disorders (e.g.: PD), a parallel design is necessary. However, for studies of therapy for rare diseases with fewer subjects, a crossover design may be essential to achieve statistical significance, provided relevant short-term outcomes can be assessed reliably (e.g.: for MELAS). 4) Infrastructure: Although not addressed directly in this review, a well organized infrastructure that is experienced in the conduct of multi-center clinical trials is very important for protocol design, investigator training, recruitment, data collection and analysis, and monitoring by an external data safety monitoring board (DSMB). The DSMB can determine if adverse events are related to the blinded therapy and whether the study should be terminated due to adverse effects or very significant benefits of the therapeutic intervention. 5) Investigative team: Successful study teams depend on experienced, effective leadership, a well motivated, collaborative multi-center team, and frequent communication or meetings. Once established, such teams can continue to conduct successive or multiple trials. 6) Support: Promotion by patient advocacy groups and professional societies is critical for publicity and recruitment, since the potential benefits of therapies being evaluated may not be known or presumed to be effective without evidence. At the same time, the study protocol must be attractive to subjects and provide incentives for participation. Finally, adequate funding is essential. Collaborative clinical trials are relatively expensive compared to other forms of research, since they require a lot of time from many professionals. Under-funded studies have a high risk of failing. Multiple sources of funding for a particular trial help spread the cost. Further considerations are needed to apply this list to the investigation of primary mitochondrial disorders, including children, fewer potential subjects, and a relatively smaller group of clinical investigators than is the case for conditions like PD or FA. The larger, highly organized studies of PD and FA and other more common

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disorders may serve as role models, but cannot be directly imitated. Nevertheless, the obvious conclusion remains that controlled clinical trials are essential for advancement of therapy for primary mitochondrial disorders. Multi-center collaboration is especially needed for rare diseases, since any single center is not likely to have sufficient subjects for such a trial. To this end, it appears critical that the mitochondrial disease community establish a wider, more collaborative, clinical trial network organization. Such a group could conduct a variety of successive clinical trials that would justify investment in the necessary infrastructure. Financial support from governmental, private foundations, and industry is essential and likely to become available if the organizational infrastructure is developed. Such efforts have been initiated and are underway. To keep these efforts alive and ultimately succeed, greater collaboration will be needed in order to maintain adequate funding, to begin to attain small incremental gains, and to become prepared for required evaluation of anticipated future more effective therapies for mitochondrial disorders. Acknowledgments This review was initially prepared for a workshop on Mitochondrial Diseases presented at the International Congress of Inborn Errors of Metabolism, San Diego, CA, 8/31/09, organized by Drs. Steven Cederbaum and Salvatore DiMauro, and modified for publication. The author would like to thank his colleagues at Case Western Reserve School of Medicine, Drs. Laura Konczal, Shawn McCandless, Charles Hoppel, and Arthur Zinn for helpful comments, as well as Drs. Petra Kaufmann, Columbia University, Peter Stacpoole, University of Florida, and Michael Miles, University of Cincinnati, for specific suggestions in preparation of this review. Dr. Kerr is a co-investigator in a Phase III Trial of Coenzyme Q10 in Mitochondrial Disease supported by FDA Grant R01 FD003032-01A1 [19]. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ymgme.2009.11.005. References [1] S. DiMauro, P. Rustin, A critical approach to the therapy of mitochondrial respiratory chain and oxidative phosphorylation diseases, Biochim. Biophys. Acta 1792 (2009) 1159–1167. [2] R. Horvath, G. Gorman, P.F. Chinnery, How can we treat mitochondrial encephalomyopathies? Approaches to therapy, Neurotherapeutics 5 (2008) 558–568. [3] P.W. Stacpoole, N.V. Nagaraja, A.D. Hutson, Efficacy of dichloroacetate as a lactate-lowering drug, J. Clin. Pharmacol. 43 (2003) 683–691. [4] P.W. Stacpoole, C.L. Barnes, M.D. Hurbanis, S.L. Cannon, D.S. Kerr, Treatment of congenital lactic acidosis with dichloroacetate: a review, Arch. Pediatr. Adolesc. Med. 77 (1997) 535–541. [5] B.A. Barshop, R.K. Naviaux, K.A. McGowan, F. Levine, W.L. Nyhan, A. LoupisGeller, R.H. Haas, Chronic treatment of mitochondrial disease patients with dichloroacetate, Mol. Genet. Metab. 83 (2004) 138–149. [6] P.W. Stacpoole, H.J. Harwood Jr., D.F. Cameron, S.H. Curry, D.A. Samuelson, P.E. Cornwell, H.E. Sauberlich, Chronic toxicity of dichloroacetate: possible relation to thiamine deficiency in rats, Fundam. Appl. Toxicol. 14 (1990) 327–337. [7] L. Spruijt, R.K. Naviaux, K.A. McGowan, W.L. Nyhan, G. Sheean, R.H. Haas, B.A. Barshop, Nerve conduction changes in patients with mitochondrial diseases treated with dichloroacetate, Muscle Nerve 24 (2001) 916–924. [8] P.W. Stacpoole, D.S. Kerr, C. Barnes, S.T. Bunch, P.R. Carney, E.M. Fennell, N.M. Felitsyn, R.L. Gilmore, M. Greer, G.N. Henderson, A.D. Hutson, R.E. Neiberger, R.G. O’Brien, L.A. Perkins, R.G. Quisling, A.L. Shroads, J.J. Shuster, J.H. Silverstein, D.W. Theriaque, E. Valenstein, Controlled clinical trial of dichloroacetate for treatment of congenital lactic acidosis in children, Pediatrics 117 (2006) 1519– 1531. [9] S. Saitoh, M.Y. Momoi, T. Yamagata, Y. Mori, M. Imai, Effects of dichloroacetate in three patients with MELAS, Neurology 50 (1998) 531–534. [10] M. Mori, T. Yamagata, T. Goto, S. Saito, M.Y. Momoi, Dichloroacetate treatment for mitochondrial cytopathy: long-term effects in MELAS, Brain Dev. 26 (2004) 453–458.

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