EPI-743 Reverses the Progression of the Pediatric Mitochondrial Disease–
Genetically Defined Leigh Syndrome
Diego Martinelli, Michela Catteruccia, Fiorella Piemonte, Anna Pastore,
Giulia Tozzi, Carlo Dionisi-Vici, Giuseppe Pontrelli, Tiziana Corsetti, Susanna Livadiotti, Viktoria Kheifets, Andrew Hinman, William D. Shrader,
Martin Thoolen, Matthew B. Klein, Enrico Bertini, Guy Miller
PII: S1096-7192(12)00348-4
DOI: doi: 10.1016/j.ymgme.2012.09.007
Reference: YMGME 5391
To appear in: Molecular Genetics and Metabolism
Received date: 4 September 2012
Accepted date: 4 September 2012
Please cite this article as: Martinelli, D., Catteruccia, M., Piemonte, F., Pastore, A.,
Tozzi, G., Dionisi-Vici, C., Pontrelli, G., Corsetti, T., Livadiotti, S., Kheifets, V., Hinman, A., Shrader, W.D., Thoolen, M., Klein, M.B., Bertini, E. & Miller, G., EPI-743 Reverses the Progression of the Pediatric Mitochondrial Disease– Genetically Defined Leigh
Syndrome, Molecular Genetics and Metabolism (2012), doi: 10.1016/j.ymgme.2012.09.007
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EPI-743 Leigh Syndrome Phase 2 Manuscript: FINAL Confidential
Page 2
Genetically defined Leigh syndrome is a rare, fatal inherited neurodegenerative disorder that
predominantly affects children. No treatment is available. EPI-743 is a novel small molecule
developed for the treatment of Leigh syndrome and other inherited mitochondrial diseases. In
compassionate use cases and in an FDA Expand Access protocol, children with Leigh
syndrome treated with EPI-743 demonstrated objective signs of neurologic and
neuromuscular improvement. To confirm these initial findings, a phase 2A open label trial of
EPI-743 for children with genetically-confirmed Leigh syndrome was conducted and herein
we report the results.
A single arm clinical trial was performed in children with genetically confirmed Leigh
syndrome. Subjects were treated for six months with EPI-743 three times daily and all were
eligible for a treatment extension phase. The primary objective of the trial was to arrest
disease progression as assessed by neuromuscular and quality of life metrics. Results were
compared to the reported natural history of the disease.
Ten consecutive children, ages 1-13 years, were enrolled; they possessed seven different
genetic defects. All children exhibited reversal of disease progression regardless of genetic
determinant or disease severity. The primary endpoints– Newcastle Pediatric Mitochondrial
Disease Scale, the Gross Motor Function Measure, and PedsQL Neuromuscular Module–
demonstrated statistically significant improvement (p< 0.05). In addition, all children had an
improvement of one class on the Movement Disorder-Childhood Rating Scale. No significant

drug-related adverse events were recorded.
In comparison to the natural history of Leigh syndrome, EPI-743 improves clinical outcomes
in children with genetically confirmed Leigh syndrome.
Keywords: Leigh syndrome, mitochondrial disease, EPI-743

Subacute necrotizing encephalopathy, or Leigh syndrome, is a rare, fatal inherited
neurodegenerative disorder that predominantly affects children. Originally described in 1951
by Leigh, the histopathological findings consist of necrotizing focal bilateral lesions in the
brain stem, thalamus, and/or basal ganglia.[1] Leigh syndrome arises from DNA defects in
mitochondrial,[2] nuclear,[3-5] or X-linked genes.[6-9] The estimated incidence of Leigh
syndrome is 1:30,000.[4, 10, 11] There are no approved drugs for Leigh syndrome.
EPI-743 is a small-molecule therapeutic being developed for the treatment of
inherited respiratory chain diseases, including Leigh syndrome. EPI-743 is a parabenzoquinone
targeting repletion of reduced intracellular glutathione. [12, 13]
Given EPI-743’s favorable preclinical safety and efficacy profile, the lack of
approved therapies and the significant morbidity and mortality associated with inherited
respiratory chain diseases, the United States Food and Drug Administration granted approval
for the study of EPI-743 in an Expanded Access Protocol for children with geneticallyconfirmed
inherited respiratory chain diseases within 90 days of end of life care
(NCT01370447). Children with Leigh syndrome treated under Expanded Access have
consistently demonstrated arrest of disease progression and neurologic and neuromuscular
improvement on clinical and radiographic examination. In addition, none of these children
experienced drug-related serious adverse events. [12]
Based on these data, a phase 2A study was undertaken to further study the efficacy
and safety effects of EPI-743 in children with Leigh syndrome in a controlled trial. Herein
we report on the clinical response of 10 consecutive children treated with EPI-743 for six

months in a single-site, open-label phase 2A study.

2.1 Study Overview & Subjects
We performed a prospective single arm subject-controlled trial of EPI-743 in children
with genetically confirmed Leigh syndrome. This study was conducted at the Bambino Gesù
Children’s Hospital, Rome, Italy. Institutional Review Board approval was obtained prior to
study initiation (Vatican Ethics Committee Reference Number: EPI-2011-004) and registered
in Europe (EudraCT Number: 2012-001294-84).
Subjects were children with a genetically confirmed diagnosis of Leigh syndrome
who had a Newcastle Pediatric Mitochondrial Disease Scale (NPMDS) score on sections I
through III greater than 15– signifying at least moderately severe disease. All participants
were required to have MRI confirmation of necrotizing encephalopathy. In addition, all
children were required to discontinue the use of CoQ10 and any other antioxidant
supplements for the duration of the trial. Informed consent was obtained from the parents of
each child.
2.2 Treatment
Subjects were treated for six months with 100 mg of EPI-743 three times daily orally
or via gastrostomy tube and evaluated using disease-relevant functional, neurologic and
physiologic assessments. All adverse and serious adverse events were tracked in the trial
database. In addition, all held doses due to intolerance of EPI-743 or any other reason were

The primary endpoints of this study were the Newcastle Pediatric Mitochondrial
Disease Scale (NPMDS), the Gross Motor Function Measure (GMFM) and the PedsQL
Neuromuscular Module (PedsQL). The NPDMS is a scale developed for and validated in
children with inherited mitochondrial diseases to assess disease severity. Sections I-III of the
NPDMS assess organ-specific function and section IV is a quality of life assessment. In this
study we utilized serial NPMDS measurements to assess EPI-743 effect on disease
progression. Appropriate age-specific versions of the NPMDS were utilized: 0-24 months; 2-
11 years; and 12-18 years. The GMFM is an observational tool used to assess gross motor
function over time in children with neuromuscular disorders[14]. The GMFM has been
validated as an outcome measurement instrument in intervention trials of children with
neuromuscular disorders.[14] The PedsQL is a validated measurement of pediatric quality of
life and the neuromuscular module utilized in this study is specific to children with
neuromuscular disorders[15].
In addition, clinical response was measured using the Movement Disorder-Childhood
Rating Scale (MD-CRS), a validated instrument for assessing movement disorders and their
impact on development in children.[16, 17]
All assessments at all time points were performed by a single physician with expertise
in pediatric neurology and mitochondrial disease (DM).
2.4 Pharmacokinetic Data
Complete sample sets (T0 to T8h) for analysis of plasma concentrations following the
first dose of EPI-743 at 100 mg were obtained from 5 of the patients selected randomly.
Plasma concentrations were analyzed using a liquid chromatography-tandem mass

spectroscopy method.

2.5 Review of Natural History
Given the open label nature of this trial, treatment response was also assessed against
the natural history of Leigh syndrome. A search of the PubMed database was conducted
using the following criteria: i) case reports or series describing children with Leigh
syndrome; ii) written in English; iii) published from 2000-2012. All identified publications
were reviewed and those that included case histories of children with one of the seven
mutations evaluated in this study (see Table 1) formed the basis of the analysis. Each child’s
clinical outcome was categorized as improved, stable, progressing, or death based on the
description in the paper. When available, patient sex, age at diagnosis, and age at last followup
were recorded.
2.6 Statistical Analyses
Descriptive statistics were performed on the entire cohort of children enrolled in this
study. Changes in outcome measurement were calculated for each subject and overall mean
changes from baseline were analyzed using a Wilcoxon signed rank test. Given that different
versions of the NPMDS were used based on patient age, the Wilcoxon test was used to
determine overall significance of treatment effect across the entire population and mean
changes were calculated separately for each age group. Statistical significance was defined as
p< 0.05.

A total of 10 children with seven forms of genetically confirmed Leigh syndrome
were enrolled in this study and treated with EPI-743 for at least four months. Nine of the 10
subjects completed six months of EPI-743 treatment. The parents of one patient discontinued
treatment after 111 days of therapy due to (parent) concerns related to patient somnolence.
This symptom was deemed by the investigators to be related to the underlying disease.
The baseline patient characteristics are shown in Table 1. Mean patient age was 6.3
years (range 1-13) and six of the 10 children were male. The average baseline NPMDS score
was 45.4 (range 19.6 - 62), signifying that all children enrolled in the study had advanced
3.1 Clinical outcome
The clinical outcome results are summarized in Table 2 and Figure 1.
All children– regardless of age, genotype, and starting NPMDS score– demonstrated
arrest of disease progression and/or reversal. Mean changes in NPMDS scores for sections IIII
(organ function) and section IV (quality of life) were calculated based on patient age
group given that different versions of the measurement are used for different age groups.
Eight subjects were assessed with version for children ages 2-11. Sections I-III improved by
a mean of 7.5 ± 3.9 (p = 0.01) and for section IV (quality of life), scores improved an average
of 5.0 ± 5.3 (p = 0.02). For subject 3 (age 1) and subject 6 (age 3), scores on sections I-III
and section IV also decreased following treatment with EPI-743.
There was also significant increase in GMFM scores over the treatment period with a
mean improvement of 10.1 ± 11.1 (p = 0.006). On the MD-CRS– a validated measurement
of pediatric movement disorders and dystonia– each child had improvement of one class,

indicating significant improvement in dystonia and spasticity symptoms.[16, 17] The subject
who had treatment discontinued at 16 weeks had initially improved one level on the MDCRS
but returned to baseline following discontinuation of treatment.
Finally, there was a significant improvement (15.0 ± 20.8; p = 0.02) in quality of life
score on the PedsQL neuromuscular module.[15] This significant improvement is consistent
with the improvement observed in section IV of the NPMDS, which is focused on quality of
The patient (#8) who discontinued treatment at 16 weeks had an initial improvement
in all clinical outcome measurements, but returned to baseline levels following cessation of
EPI-743 therapy (Figure 2) on the NPMDS, GMFM and MD-CRS.
3.2 Pharmacokinetic Data
Pharmacokinetic data are summarized in Table 3. EPI-743 was rapidly absorbed (Tmax 2-4
hours) and was distributed or eliminated rapidly with an estimated half-life of 3.9 to 5.6
hours. Plasma exposure to EPI-743 appeared to be dependent on body weight of the patients.
Two patients with body weight of 7.9 and 9 kg showed Cmax and AUC 0-8 hour values of
1377 and 1167 ng/mL and 3011 and 4353 ng.h/mL, respectively, whereas the patients with
higher body weights (21-28.6 kg) had Cmax values of 144 – 215 ng/mL and AUC 0-8 values
of 771 – 894 ng.h/mL. Correcting the Cmax and AUC 0-8 for dose (mg/kg) resulted in a
substantially smaller variability (Cmax/dosemg/kg range 10.7 to 36.25 ng/mL; AUC/dosemg/kg
range 84.6 to 168.6 ng.h/mL).
3.3 Safety and Tolerability
Treatment with EPI-743 was found to be safe and well tolerated other than the one
subject who had therapy discontinued for what parents reported to be increased somnolence

Eight serious adverse events requiring hospitalization occurred in four patients during
the six-month trial. They were all due to underlying disease and not associated with drug:
three cases of upper respiratory tract infection, one case of bronchopneumonia, one case of
salmonellosis, one case of metabolic acidosis, and one case of an apneic spell. All adverse
events resolved without sequelae. Two non-serious adverse events, deemed to be possibly
drug-related, included mild and transient sleepiness and hypotonia. There were no clinical
chemistry or laboratory abnormalities attributed to the drug.
3.4 Natural History
A total of 71 publications were identified and reviewed that described clinical data on
children affected by six of the seven genetic causes of Leigh syndrome in our series. No ND
1 publications were identified. 44 of these 71 publications provided specific data on patient
clinical course.
Demographic and clinical outcome data were ascertainable on 180 children (Table 4
and Figure 3). 99.4% of children either had progressive neurologic deterioration or died.
There was report of only one child demonstrating signs of clinical improvement over time.

The natural history of Leigh syndrome was recently reviewed by Finsterer and
characterized as a pediatric progressive neurodegenerative disorder in which children rapidly
deteriorate, resulting in death by five years of age.[18] Given the absence of any approved
drugs, we undertook a therapeutic development program for this devastating medical
EPI-743 is a rationally designed, catalytic, 2-electron transfer NQO1 cofactor.[13]
The 2,3-dimethyl para-benzoquinone chemical nucleus was selected for lead optimization
based on its favorable electrochemical coupling between NADPH, NQO1, and glutathione
reductase. It possesses a redox potential of -175 mV that exists between the flavin redox
potentials of NQO1 and glutathione reductase. Final structure optimization was directed at
blood-brain-barrier penetration and oral bioavailability.
In this open label trial, 10 children with seven different genetic mutations consistent
with Leigh syndrome were enrolled at a single European site. In order to ensure that these
children all had evidence of moderate disease progression, a threshold of an NPMDS of
greater than 15 was required for enrollment. Four outcome measures were used to capture the
clinical spectrum of Leigh syndrome and response to EPI-743 treatment. These included the
mitochondrial disease-specific NPMDS, the more generalized pediatric GMFM, the MDCRS,
and the PedsQL. A statistically significant improvement was shown in all four of these
outcome measurements. Central nervous system and neuromuscular function improved
following treatment with EPI-743 as assessed by the NPMDS sections 1-3, the GMFM, and
the MD-CRS. Consistent with improvement in physician-recorded outcome measures, patient
families reported improvement in quality of life measures (section 4 of NPMDS and the PedsQL), both of which were statistically significant. Improvement was independent of
genetic determinant, age, sex, and disease severity. These results confirm the initial findings
of Leigh syndrome subjects treated with EPI-743 under FDA-approved expanded access and
physician-sponsored IND protocol in the United States and compassionate treatment in
Europe. 12
The clinical results obtained in this study were compared to an historical cohort
obtained from the published natural history of Leigh syndrome over the last 12 years. In
contrast to the frequency of the combined disease progression and mortality (179/180 =
99.4%), 100% of the EPI-743 treated subjects reversed disease progression and improved.
These results occurred across each of the major Leigh syndrome genetic determinants (Table
4 and Figure 3). While these cohorts are not directly comparable due to the fact that children
in the study were followed for only six months, the results do confirm the progressive natural
history of Leigh syndrome and the fact that there is only one documented case of
spontaneous clinical improvement over time.
In addition to efficacy parameters, both clinical and standard laboratory metrics of
drug safety were prospectively assessed. No significant drug-related adverse events were
recorded. Coupled with pharmacodynamic data, the safety data established herein
demonstrate a favorable therapeutic index. Separately, as of August 1, 2012 an estimated 125
subjects with mitochondrial disease have been treated with EPI-743 with a cumulative patient
exposure of > 42,000 days, with only one reported possibly drug-related adverse event.
Based on the collective experience of the treatment of subjects with genetically defined
mitochondrial disease and data from non-clinical safety assessment studies, a safety margin

of at least 10-fold for the 100 mg three times daily dosing has been assigned to EPI-743.[12,
19, 20]
One child in this study had EPI-743 discontinued following 16 weeks of therapy due
to parental concerns over increased somnolence. The investigators deemed this symptom
disease related. Review of this patient’s clinical data demonstrates an improvement in all
clinical outcome measurements up to the point of therapy discontinuation, with a return to
baseline (i.e. pretreatment) levels shortly after therapy cessation. These findings not only
confirm the potential EPI-743 treatment benefit, but also suggest a change in clinical signs
and symptoms that are drug-dependent.
There are several limitations to this study principally related to its open label design.
First, given the lack of placebo group it is difficult to determine the rate of disease
progression absent treatment over a six-month period. However, while there are sporadic
reports in the literature of children with Leigh syndrome demonstrating signs of clinical
improvement, the observation that all children on treatment demonstrated improvement on all
outcome variables suggests a bona fide treatment effect. In addition, the physician
performing the clinical outcome measurement assessments knew that all children were
receiving treatment which potentially biases the results. Finally, given the rarity of Leigh
syndrome and the absence of previous clinical trials in this population, there is a lack of well
instantiated outcome measurements that have been appropriately validated to assess
treatment effect.
In conclusion, EPI-743 treatment resulted in neurological and neuromuscular
improvement in genetically defined Leigh syndrome subjects independent of genetic

determinant and reverses disease progression – results that have not previously been recorded
in a progressive mitochondrial disease. While these results are promising, they must be
considered in the context of the study’s open label design. A randomized placebo controlled
trial will be conducted to confirm the efficacy and safety findings.

We thank: Mitocon, Italy for assistance in recruiting patients; Telethon Italy for grant no.
GUP09004 to establish a mitochondrial disease clinical data base; Professor Paolo Rossi for
scientific guidance; Audrey Akin for illustration and graphics; Dr. Gaia Scerbo, Dr. Viviana
Loconte, Dr. Anna Iacono for preparing the pharmaceutical formula for administration in
children; Dr. Ferdinando Ceravolo and Dr. Sara Boenzi for participating in laboratory
analysis; Dr. Roberto Torriero, Dr. Alessia Scarselli, Dr. Serena Sinibaldi, Dr. Virginia
Messia and S.ra Claudia Frillici, for clinical support at Clinical Trials Center of Bambino
Gesù Children Hospital; and the Association “La Vita è un Dono” for supporting the
fellowship of Dr. Diego Martinelli.

Figure 1
Clinical outcome measurements
Clinical outcomes prior to and following six months of EPI-743 treatment are shown. There
was significant improvement on NPMDS sections I-III (organ function) and NPMDS section
IV (quality of life) as shown in Figure 1A and Figure 1B, respectively. Figure 1C
demonstrates the overall percentage improvement in each subject following three months of
EPI-743 treatment. Figure 1D and Figure 1E demonstrate the change from baseline to three
months in neuromuscular function as measured by the GMFM (Figure 1D) and health related
quality of life as measured by the PedsQL (Figure 1E). Figure 1F demonstrates the one class
improvement in each patient on the MD-CRS.
Figure 2
Patient 8 clinical outcome
This patient was treated for 16 weeks with EPI-743 prior to therapy cessation by the child’s
parents. The results of the NPMDS (Figure 2A and 2B), the GMFM (Figure 2C), the MDCRS
(Figure 2D) demonstrate initial improvement followed by return to baseline following
cessation of therap. The PedsQL score improved following cessation of therapy (Figure 2E).
Figure 3
Analysis of Leigh syndrome natural history cohort
The natural history from published case reports of children with Leigh syndrome with
mutations evaluated in this study was categorized as improved, stable, progressed or death. Of the 180
children described in the literature, 179 either died or had progressive neurologic

• We performed a prospective open label trial of the novel therapeutic EPI-743 for
children with Leigh syndrome
• There was a statistically significant improvement in all trial outcome measurements
signifying arrest and reversal of disease progression
• There were no drug related adverse events or clinical laboratory abnormalities

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