Improved Muscle Function in Duchenne Muscular Dystrophy through L-Arginine and Metformin: An Investigator-Initiated, Open-Label, Single-Center, Proof-Of-Concept-Study

Altered neuronal nitric oxide synthase function in Duchenne muscular dystrophy leads to impaired mitochondrial function which is thought to be one cause of muscle damage in this disease. The study tested if increased intramuscular nitric oxide concentration can improve mitochondrial energy metabolism in Duchenne muscular dystrophy using a novel therapeutic approach through the combination of L-arginine with metformin. Five ambulatory, genetically confirmed Duchenne muscular dystrophy patients aged between 7–10 years were treated with L-arginine (3 x 2.5 g/d) and metformin (2 x 250 mg/d) for 16 weeks. Treatment effects were assessed using mitochondrial protein expression analysis in muscular biopsies, indirect calorimetry, Dual-Energy X-Ray Absorptiometry, quantitative thigh muscle MRI, and clinical scores of muscle performance. There were no serious side effects and no patient dropped out. Muscle biopsy results showed pre-treatment a significantly reduced mitochondrial protein expression and increased oxidative stress in Duchenne muscular dystrophy patients compared to controls. Post-treatment a significant elevation of proteins of the mitochondrial electron transport chain was observed as well as a reduction in oxidative stress. Treatment also decreased resting energy expenditure rates and energy substrate use shifted from carbohydrates to fatty acids. These changes were associated with improved clinical scores. In conclusion pharmacological stimulation of the nitric oxide pathway leads to improved mitochondria function and clinically a slowing of disease progression in Duchenne muscular dystrophy. This study shall lead to further development of this novel therapeutic approach into a real alternative for Duchenne muscular dystrophy patients. Trial Registration ClinicalTrials.gov NCT02516085


Background 2a
Scientific background and explanation of rationale 3-4 2b Theories used in deigning behavioural interventions N.A.

Participants 3a
Eligibility criteria for participants, including criteria at different levels in recruitment/sampling plan (e.g., cities, clinics, subjects) 5 3b 3c 3d Method of recruitment (e.g., referral, self-selection), including the sampling method if a systematic sampling plan was implemented Recruitment setting Settings and locations where the data were collected

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Interventions 4a Details of the interventions intended for each study condition and how and when they were actually administered, specifically including -Content: what was given? -Delivery method: how was the content given? -Unit of delivery: how were the subjects grouped during delivery? -Deliverer: who delivered the intervention? -Setting: where was the intervention delivered? -Exposure quantity and duration: how many sessions or episodes or events were intended to be delivered? How long were they intended to last? -Time span: how long was it intended to take to deliver the intervention to each unit? -Activieties to increase compliance or adherence (e.g., incentives)

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Objectives 5 Specific objectives and hypotheses 4 Outcomes 6a Clearly defined primary and secondary outcome measures 6b Methods used to collect data and any methods used to enhance the quality of measurements Information on validated instruments such as psychometric and biometric properties 6-9 6-7 Sample size 7a How sample size was determined and, when applicable, explanation of any interim analyses and stopping rules 5 Assignment Method 8a Unit of assignment N.A. 8b 8c Methods used to assign units to study conditions, including details of any restriction Inclusion of aspects employed to help minimize potential bias induced due to nonrandomization N.A. N.A.

Blinding 9
Whether or not participants, those administering the interventions, and those assessing the outcomes were blinded to study condition assignment; if so, statement regarding how the blinding was accomplished and how it was assessed.

9-10 1
Unit of Analysis 10a 10b Description of the smallest unit that is being analysed to assess intervention effects If the unit of analysis differs from the unit of assignment, the analytical method used to account for this

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Statistical methods 11a Statistical methods used to compare study groups for primary methods outcome(s), including complex methods of correlated data 9 11b Statistical methods used for additional analyses, such as a subgroup analysis and adjusted analysis.

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11c Methods for imputing missing data, if used N.A. 11d Statistical software or programs used

Participant flow
12a Flow of participants through each stage of the study: enrolment, assignment, allocation, and intervention exposure, follow-up, analysis

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Baseline equivalence Numbers analyzed 15 Data on study group equivalence at baseline and statistical methods used to control for baseline differences 9-20 16a 16b Number of participants included in each analysis for each study condition, particularly when the denominators change for different outcomes; statement of the results in absolute numbers when feasible Indication of whether the analysis strategy was "intention to treat" or, if not, description of how non-compliers were treated in the analyses.

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Outcomes and estimation 17a 17b 17c For each primary and secondary outcome, a summary of results for each estimation study condition, and the estimated effect size and a confidence interval to indicate the precision Inclusion of null and negative findings Inclusion of results from treating pre-specified causal pathways through which the intervention was intended to operate, if any N.A.

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Ancillary analyses Generalizability 21 Generalizability (external validity) of the trial findings, taking into account the study population, the characteristics of the intervention, length of follow-up, incentives, compliance rates, specific sites /settings involved in the study, and other contextual issues