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Effect of Combination l-Citrulline and Metformin Treatment on Motor Function in Patients With Duchenne Muscular Dystrophy

Key Points Question Does treatment with l-citrulline and metformin combination therapy reduce motor function decline in ambulant patients with Duchenne muscular dystrophy? Findings In this randomized clinical trial of 47 ambulant male children aged 6.5 to 10 years with Duchenne muscular dystrophy, treatment with a combination of l-citrulline and metformin therapies provided a clinically relevant but not statistically significant reduction in motor function decline, as assessed by the transfer and standing posture dimension of the Motor Function Measure scale. No indications of harm were found in the intention-to-treat population. Meaning Treatment with a combination of l-citrulline and metformin therapies may slow muscle function decline in a specific subgroup of patients with Duchenne muscular dystrophy, but additional clinical trials with greater statistical power are warranted.

L-citrulline have been tolerated without side effects (6). Therefore, in this study all actively treated patients will get a daily dose of 3x 2.5g L-citrulline, , respectively placebo (provided in single sachets which have to be dissolved in a glass of water or fruit juice) per day during 26 weeks. L-citrulline sachets have to be stored at room temperature (15-25°C).

Metformin
For the planned study a dosing of 250 mg metformin, respectively placebo, three times a day (750 mg daily) will be given during 26 weeks. In our mentioned recent pilot study we used 2 x 250 mg/d metformin and observed a very good safety profile, no serious side effects, and no drop-outs. To increase the efficacy and facilitate the dosage regimen, we want to increase the metformin dose and provide it also at a three times daily dosage (750 mg daily). A dose up to 2000 mg/d seems to be associated with a low incidence of gastrointestinal side effects in children (7) thus a dose of 750 mg/d seems to be safe and might even more activate the muscle metabolism. At the beginning of the treatment with metformin nausea, vomiting, diarrhoea and abdominal pain may occur. Usually these symptoms decrease spontaneously after a few days.
To ensure the blinding, metformin capsules produced by the hospital pharmacy of the University Hospital Basel will be used in this trial. They have to be stored at room temperature (15-25°C).

Background, reason for the study and objective of the study
Duchenne muscular dystrophy (DMD) is an X-linked recessive disease and it is the most common inherited muscle disorder. DMD is characterized by replacement of normal muscle tissue by connective and fatty tissue resulting in progressive weakness with early loss of free ambulation. At the age of 3-4 years patients first present with muscle weakness due to an irreversible, progressive loss of skeletal muscle, which confines patients to a wheelchair by about 10 years of age. Typically, death occurs around 25 years of age due to cardio-respiratory complications. Current therapeutic management is supportive (8,9).
In muscle the disease causing gene product, dystrophin, is located at the inner surface of the plasmalemma. It interacts both with a number of membrane proteins that form the dystrophinassociated glycoprotein complex (DGC) and the intracellular cytoskeletal proteins. Loss of dystrophin in DMD is associated with a loss of cytoskeletal integrity. This structural defect gives rise to a misregulation of calcium ions and activation of proteases such as calcium-dependent neutral proteases (calpains). The loss of DGC proteins also results in a severe reduction of neuronal nitric oxide (NO) synthase (nNOS) in DMD (10). In normal subjects, nNOS activity leads to intramuscular L-arginine degradation to NO. Arginase II (that competes with nNOS) degrades L-arginine to L-ornithine and is upregulated in DMD (11). NO concentrations have not been measured in DMD, but the reduced nNOS activity and increased arginase II activity in DMD suggest a lower NO concentration in DMD muscle compared to controls (Fig 1a, b). NO stimulates upregulation of nuclear genes involved in mitochondrial biogenesis, including SIRT1 and PGC-1α (12,13). In addition, NO activates AMP-activated protein kinase (AMPK), which is a critical regulator of muscular energy balance. AMPK stimulates fatty acid uptake, increases fatty acid oxidation, and mitochondrial function and biogenesis. AMPK exerts its action partially via nNOS phosphorylation (14). Thus, NO and AMPK have both been shown to increase mitochondrial function and biogenesis in a synergistic mode of action, but both also act independently.
In DMD an increased production of reactive oxygen species (ROS) has been observed, which can cause further protein and membrane damage (15). The major sources of intracellular ROS are mitochondria, implying an altered mitochondrial function in DMD. Indeed, decreased mitochondrial oxidation rates in muscle biopsies from DMD patients were reported by Sperl et al (16). In accordance to an impaired mitochondrial function a low fat utilization at an early stage of the disease has been suggested and muscle tissue is increasingly being replaced by fatty tissue (17,18). Therefore, we suggest that increasing NO concentrations seems to be promising to ameliorate the devastating effects in DMD via stimulation of mitochondrial function, improved fat utilisation, and energy production (Fig. 1c). Indeed, direct NO donors can improve prednisone effects on muscular dystrophy in the mdx mouse diaphragm (19). L-arginine treatment also improved functional abilities of mdx mice (20). In addition expression of a muscle-specific, nNOS transgene increases the endurance of mdx mice during treadmill running (21).  Individual and predicted (calculated using data from the literature (23,24)) MFM and the 2 min walking distance values of all treated patients are presented as follows:

Questionnaire, trial population
Our aim is to confirm in a double blind placebo controlled trial the observed changes of our own 16 week pilot study in a broader population being treated as postulated by current medical guidelines, including patients under steroid treatment. The precursor study performed on 5 ambulatory DMD patients that showed an improved lipid metabolism, improved functional abilities, prolonged walking distances, and slowing of the rate of progressive fatty degeneration of involved muscle observed on quantitative MRI.
We plan to perform a 26 week placebo controlled RCT (randomized controlled trial) with a total number of 40-50 ambulant DMD patients aged between 6.5 and 10 years. If L-citrulline and metformin are helpful to improve muscle function, a better symptomatic treatment and slowing of muscle degeneration in DMD could be expected, other multicenter trial will be performed.
To obtain normal values for measures of muscle metabolism and mitochondrial function blood and urine samples will be analysed in 10 -20 healthy age matched male children and 10 -20 healthy male controls.

Hypothesis
Combined both causes, the increased reduction of L-arginine by arginase II and the decreased NO production by reduced nNOS should be treated to enhance the NO concentration and thus to achieve an improvement of muscular strength, a deceleration of muscle atrophy and long-term an extension of time until loss of ambulation.

Trial design
This is a randomised placebo controlled trial. We plan to enrol 40-50 ambulant DMD patients aged between 6.5 and 10 years. All actively treated patients will get a daily dose of L-citrulline of 7

Visit 1 = Screening (Day -1)
After signing the informed consent form the inclusion and exclusion criteria are verified. If the criteria are fulfilled the patient will be enrolled in the study.
During this visit the following procedures will be performed:  muscle force of knee extension and elbow flexion using hand held dynamometry  10 m within 10 s walking test

Visit 2 = Baseline (Week 0)
During this visit the following procedures will be performed: If the patient still qualifies for the study, he/she will be randomized and receive study medication.

Visit 3 (Week 13, ±7 days)
During this visit the following procedures will be performed:  muscle force of knee extension and elbow flexion using hand held dynamometry  10 m within 10 s walking test In case of screening failure due to exclusion criteria the patient can be re-screened once for this study.

Healthy volunteers
10-20 age matched healthy boys within the same age range and 10 -20 healthy male controls will be enrolled to obtain normal values of mitochondrial and muscle metabolism. All healthy controls will be seen at two time points (screening and for blood drawing).
Healthy children (and their caregivers) will be asked to participate in our study while undergoing an elective surgery at our hospital. By this blood drawing will be done as part of the routine safety protocol while the child is already under anaesthesia. An additional amount of 4 ml of blood will be drawn for this study but no additional venous puncture will be necessary for this study. Healthy adult controls will be identified for potential recruitment using IEC/IRB approved newspaper advertisements, mailing lists, websites or database available at UKBB/USB.

Visit 1 = Screening
After signing the informed consent form the inclusion and exclusion criteria are verified. If the criteria are fulfilled the patient will be enrolled in the study.

Visit 2 = Baseline
During this visit the following procedures will be performed: • check inclusion/exclusion criteria • blood drawing

Criteria for stopping the study
The following conditions/events can lead to an early termination of the study:  withdrawal of consent

Conditions for unblinding
Unblinding is the process by which the allocation code is broken so that the investigator, clinical staff and/or the trial statistician becomes aware of the intervention for a person participating in a trial. The usual reason for unblinding is that a person participating in the study has encountered an urgent medical problem necessitating that the clinician know his/her intervention allocation.
The principal investigator assess the need for unblinding where a serious adverse event has occurred and the treatment or allocation code is required in order to enable clinical treatments to Unblinding envelopes: A series of envelopes, each labelled with the randomisation number that contain the allocation for that person. These envelopes are sealed by the central randomisation service, and are only opened if emergency unblinding is required.

Measure to minimise bias (randomising, blinding)
Patients who meet the study admission criteria will be enrolled in the study and a single subject identification number will be assigned. Patients are allocated to the two study groups, L-citrulline and metformin or placebo, in a 1:1 ratio. The assigned number will be recorded in the CRF Visit 2. Drop outs after baseline visit (randomisation) will not be replaced.
The physiotherapists who perform the tests were trained and certified in Lyon where MFM has been established and validated.
This is a double blind, placebo controlled, randomized study.

Recruitment
The study patients will be recruited among the affected children in the neurologic clinic of the UKBB as well as using the Swiss and German DMD Register. Currently approximately 120 DMD patients are registered in the Swiss Register. To find the patients the Swiss DMD Register will be searched in alphabetic, systematic order for inclusion and exclusion criteria and matching patients will be contacted and informed about the study. Furthermore, an advertisement will be placed on the website Swiss and German DMD Register after approval of the Ethics Committee and Swissmedic.
Healthy volunteers will be recruited from our hospital. Children and their caregivers having a routine blood drawing prior to an elective surgical procedure will be asked if they are willing to give an additional amount of blood for this study.

Inclusion criteria
 Molecular diagnosis of DMD

Inclusion criteria healthy volunteers  Male sex
 Aged between 6.5-10 and 18-60 years.

5.5
Exclusion criteria healthy volunteers

Safety parameter: measuring methods and time
At every study visit a clinical examination will be performed and the vital parameters blood pressure and heart rate will be measured. The assessment of blood chemistry and of the following parameters is foreseen at every visit (respectively visit 1 or 2): The intake of metformin has to be stopped in case of clinically significant changes (increase of creatinine and transaminasis >2 x ULN) If pathologic changes independent of the known muscle disease should be detected, the affected patients will be informed immediately and the possibilities of further investigation, respectively treatment of these abnormalities according to current medical knowledge will be discussed.

Follow-up observation for trial subjects with adverse reactions
Patients with adverse reactions which have occurred in the context of the study will be followed up by the investigator up to 30 days after the last visit. For sample size estimation the MFM D1 subscore was chosen. Baseline and post-treatment measurements of 5 patients are available from the pilot study. A semi-parametric approach for sample size estimation was chosen that made use of these data. For the 2 min. walking distance, a mean change from baseline to post-treatment of 10 m was expected (estimated from pilot data). For the MFM D1 subscore, a mean change from baseline to post-treatment of 6.1 % was expected (estimated from pilot data). Sample size estimation was repeated for a range of values smaller and larger than the expected mean changes. Further, the influence of two randomisation ratios for the verum/placebo groups, 1:1 and 2:1, on sample size was checked.
Each sample size, n i=1,...,40 = 11, ..., 50 for the MFM D1 subscore, was evaluated by drawing 99 times an individual data set of size n i from the pilot study data set (sampling with replacement). In each of these individual data sets, each patient was randomly assigned to a verum or a placebo group. For each patient, the post-treatment value was total sample size calculated as the sum of the baseline value and a random variate drawn from a normal distribution with mean and standard deviation estimated from the pilot data. In the case of the placebo group, the mean was set to -17.2/54 × 16 for the MFM D1 subscore (value taken from Table 2

Definition of the evaluation groups
Intention-to-treat (ITT) analysis

Trial-specific preventive measures and duties
Trial-specific preventive measures and duties are not necessary.

Final examination in case of premature withdrawal from the trial
In this case it is necessary to perform a final medical examination for the safety of the child (patient group). The following procedures will be performed:

Study conduct
The study will be conducted according to the protocol, GCP and the legal requirements as required by Swiss law.
All changes of protocol as well as a final report will be submitted to the Ethics Committee and Swissmedic.

Defining Adverse Events
An adverse event (AE) is any untoward medical occurrence in a patient during or following administration of an investigational product and which does not necessarily have a causal relationship with treatment. An AE can therefore be any unfavourable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporarily associated with the use of the trial drugs, whether or not considered related to the trial drugs.

Defining Serious Adverse Events (SAEs)
A Serious Adverse Event is defined in general as an untoward (unfavourable) event, associated with trial drug or trial procedure, which: • is fatal. Death may occur as a result of the basic disease process. Nevertheless, all deaths occurring until the last administration of the study agent must be treated as an SAE and reported as such.
• is life-threatening • requires or prolongs hospitalisation • results in persistent or significant disability or incapacity • is a congenital anomaly or a birth defect, or • may require medical or surgical intervention to prevent one of the outcomes listed above • Any other significant clinical event, not falling into any of the criteria above, but which in the opinion of the investigator requires reporting.

Defining Suspected Unexpected Serious Adverse Reactions (SUSARs)
All SAEs assigned by the local investigator as both suspected to be related to the trial drugs and unexpected are subject to expedited reporting. An event is unexpected when information is not consistent with the available product information or investigator brochure, or if they add significant information on the specificity or severity of an expected reaction.

Reporting AEs
AEs will be collected for all patients from screening until 30 days after the last dose of treatment with a protocol IMP.
Information about AEs, whether volunteered by the patient, discovered by the investigator questioning or detected through physical examination, laboratory test or other investigation will be collected and recorded in the study files.
If requested, details of collected AEs will be made available after completion of the study.

Reporting SAEs
SAEs will be collected for all patients beginning with informed consent. SAEs resulting in death or Serious Adverse Drug Reaction (fatal or life-threatening) have to be reported to the EC within 7 calendar days of the PI (or his research team) being informed of the event.

Reporting SUSARs
All SAEs assigned by the local investigator as both suspected to be related to study protocol (treatment/procedures) and unexpected (see definition in section 10.2.3) will be classified as SUSARs and will be subject to expedited reporting to concerned ethic committees (EC) and regulatory authorities (RA).
• SUSARs must be reported to the EC / RA within 7 calendar days of the PI (or his research team) being informed of the event, if they result in death or are deemed to be life-threatening.
• Any SUSARs not resulting in death or deemed to be life-threatening must be reported to the EC / RA within 15 calendar days of the PI (or his research team) being informed of the event.
In addition, the sponsor shall collect these events. The information can be aggregated in a line listing of SUSARs which has to be submitted to the EC and the RA annually by the PI, respectively by the sponsor.

Insurance
The University Children's Hospital (UKBB) will compensate the patients for damage that occurs within the framework of the clinical trial. For this purpose, the University Children's Hospital (UKBB) has taken out insurance in favour of the patients with HDI Gerling Industrieversicherung AG, Dufourstrasse 46, 8034 Zürich.
If the patient or the parents observe health problems or other damages they should contact the responsible physician (PD Dr. med. Dirk Fischer). He will undertake the necessary measures.

Evaluation of the risk-benefit ratio
Duchenne's muscular dystrophy is a serious progressive neuromuscular disease with only little symptomatic possibilities of therapy so far. Affected patients mostly die aged between 20 and 30 from a generalised muscle weakness, mostly from cardiorespiratory complications. In our opinion, the infaust prognosis justifies the broad clinical and imaging procedures in this study population.

Description of why trial subjects requiring particular protection have been included
The Duchenne's muscular dystrophy becomes manifest from toddlerhood and an early therapy (mainly glucocorticoid therapy and physiotherapy) can delay severe cardiovascular as well as respiratory complications and thus maintain the quality of life longer.
To investigate the efficacy of metformin and L-citrulline on the muscle in children with Duchenne's muscular dystrophy validly, it is necessary to include subjects requiring particular protection (children between 6.5 and 10 years). At this age changes in the muscle are identifiable, however the muscle mass is mostly maintained and functionally assessed. With the progression of the disease all patients are wheelchair bound why then the (remaining) clinical abilities can only be evaluated to a limited extent.

Other ethical aspects
The participation in this study is voluntary. If the patient/the parents do not want to participate, they will not experience any disadvantages concerning the further medical treatment. The same applies if the parents and the patient withdraw their consent at a later time point. They have this possibility anytime. A possible withdrawal of consent respectively the withdrawal from the study can occur without giving any reason. In case of withdrawal the data collected until this time point will be used and the samples (blood) collected in the context of the study will be destroyed. In case of withdrawal the patient will undergo a final visit for medical examination for his own safety.
A placebo group is necessary to demonstrate the efficacy of the used combination (L-citrulline and metformin) and to exclude a possible placebo effect.
There is a great potential that positive results of this study could lead to other large multicentre trials. Furthermore, if we can show an effect of L-citrulline and metformin on muscle function, this will be a major break through and mean a better symptomatic treatment of the fatal muscle degeneration in DMD and probably in other muscular dystrophies.
Blood drawing in healthy children will be done as part of a routine safety procedure before an elective surgery. This will be done while children are already under general anaesthesia and children will not suffer any discomfort from the venous puncture. A small additional amount of blood will be obtained for our study at the same time and no additional venous puncture will be necessary.

Quality control
To assure the quality of the study conduct and of the data a monitoring of the study will be performed by a person independent of the study (Clinical Trial Unit, University Hospital Basel).
All inclusion and exclusion criteria will be checked, if the data have been recorded correctly in the CRF, if the drug accountability is correct and if during the study SAEs have occurred.

Data protection, archiving and destruction
In this study personal patient data will be captured. This data will be anonymised and is only accessible to experts. The appropriate experts of the sponsor (or their designees) can survey the conduct of the study with monitoring or audits. In case of inspections these experts and also members of the appropriate authorities can get access to the original data. Also the responsible Ethics Committee can get access to the original data. The confidentiality of the data will be strictly protected during the whole study and when performing the mentioned controls. The name of the patient will not be published in no way in reports or publications arisen from the study.
The paper documents will be stored in a lockable room during 10 years in the archive of the UKBB in a dedicated shelf. The investigator records in the medical file that the patient participates in the study. All data which will be collected in the context of the trial will first be recorded in the medical file. At a later time point the transcription of the data in the CRF will be done. No data will be captured directly in the CRF.

PROCEDURE FOR DRUG ACCOUNTABILITY
During the visits 2 and 3 the patients will be given L-citrulline in the vials prepared by the hospital pharmacy as well as metformin capsules 250 mg each or matching placebo. The patient will be asked to return the study medication as well as the empty vials and boxes to every visit. A member of the study team counts and balances the returned tablets and vials and can check the correct intake. This will be captured in an appropriate form.

PROCEDURE TO RECORD COMPLIANCE
A qualified person of the study team will check the number of dispensed/taken medication and complete a study specific drug accountability form.

DESCRIPTION FORESEEN FOR THE TEST PRODUCT
All study medication will be labelled. The labels will contain the following information: Study number, name of the investigator, for clinical trial, lot number, expiry date, storage conditions, patient number/randomization number, keep out of reach of children.

PUBLICATION OF THE RESULTS
The results of the study will be published independent of the results. These will be published in a medical journal.