Patients

Patients were recruited into this double-blind, randomised, placebo-controlled Phase 2 study at six hospital sites in France (Marseille, Lille, Limoges, Lyon, Nantes and Paris) from December 2010 to October 2011; the study ended in November 2012 (EudraCT Number: 2010-023097-40, ClinicalTrials.gov Identifier: NCT01401257). Inclusion criteria were: age (18–65 years), CMT1A diagnosis based on clinical examination and confirmation by genotyping (duplication in 17p11.2), weakness in at least foot dorsiflexion, and a Charcot Marie Tooth Neuropathy Score (CMTNS) ≤ 20, i.e. a mild to moderate disability. Key exclusion criteria included any neurological disease other than CMT1A, the use of unauthorized concomitant treatments (including but not limited to baclofen, naltrexone, sorbitol, ascorbic acid, opioids, levothyroxine, and potentially neurotoxic drugs), history of significant hematologic, kidney or liver disease, insulin-dependent diabetes or porphyria. Women of childbearing age were excluded if they were pregnant, breastfeeding, or not using adequate contraception. The protocol was approved by the local ethics committee and regulatory authority (Agence Française de Securité Sanitaire des Produits de Santé). The study was done in accordance with the Declaration of Helsinki and Good Clinical Practice (GCP), and all patients gave written informed consent before participation.

Selection of doses

The doses tested in human were chosen based on extrapolation [24] from effective doses tested in CMT1A rats for an oral administration twice daily in accordance with the available pharmacokinetic data of the three individual compounds.

Randomisation, masking and blinding

After a screening visit, eligible patients were randomly assigned in a 1:1:1:1 ratio to receive daily for one year Placebo, Low dose (LD = 0.6 mg baclofen, 0.07 mg naltrexone and 21 mg sorbitol), Intermediate dose (ID = 1.2 mg baclofen, 0.14 mg naltrexone and 42 mg sorbitol) or High dose (HD = 6 mg baclofen, 0.7 mg naltrexone and 210 mg sorbitol) of PXT3003 (Additional file 1: Table S1). A block randomisation scheme stratified by study centre was used to ensure a balance of treatment groups within each centre. Once the eligibility of the patient was confirmed by the investigator, patients were assigned a randomisation number according to a randomisation process using an interactive web randomisation system (IWRS). Study assignments were numbered according to a material randomisation list, separate from the subject randomisation list. Each patient’s assigned medication was determined by his/her randomisation number. Blinding was ensured and both patients and investigators were unaware of the treatment allocation.

Both PXT3003 and Placebo were provided in amber glass bottles containing 100 mL of clear solution, with the same appearance and taste. The three dosages above contain a quantity of sorbitol small enough to be counterbalanced by the acetate buffer and easily masked by the banana flavouring, not allowing to differentiate them (by sight or taste) from Placebo. Study drug was taken orally, using an adaptable graduated plastic pipette for medication dispensation, twice a day (morning and evening) during 12 months.

Procedures

Potentially eligible subjects were evaluated at screening visits that included history, confirmation of CMT1A genotyping, physical examination including vital signs measurement (blood pressure, heart rate and weight), electrocardiogram (ECG), concomitant medications, recording of CMTNS and Overall Neuropathy Limitations Scale (ONLS), centralised laboratory examinations blood samples including Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), gamma-GT, total, conjugated and un-conjugated bilirubinaemia, alkaline phosphatase, creatininaemia, complete blood count (CBC) and differential count, platelet count, sodium, potassium, chloride and blood glucose. Blood pregnancy test was obtained for women of childbearing age at screening and after 12 months of treatment. If subjects met eligibility criteria, they were randomised and enrolled in the study. An independent Data Monitoring Committee (DMC) was established to review all safety data at specified intervals throughout the study. All patient data related to the study were recorded on an electronic case report form (eCRF).

Safety and tolerability

We monitored the safety of PXT3003 during the study up to 30 days after the last day of study drug administration, based on adverse events reports from patients and laboratory tests (biochemistry and haematology). All adverse events, whether or not considered by the investigator to be related to the study medication, were to be recorded in the patient’s medical records and in the eCRF. Serious adverse events were reported by the study investigators. A last follow-up visit consisting of a clinical examination, adverse events records, concomitant treatments and laboratory tests was done 30 days after the last day of treatment. The primary endpoint is the Incidence of related Adverse Events (including possibly and probably related AE) with moderate or severe intensity.

Compliance was measured after 1, 3, 6, 9 and 12 months by returned empty bottle count and volume measurement (full and opened bottles). In the present study, mean (s.d.) duration of exposure overall was 11.69 (1.53) months and was similar among the four groups, and mean (s.d.) compliance with study drug was 97.3 (9.41%). Patients with compliance below 80% were considered as low compliant.

Pharmacokinetics

Blood samples for pharmacokinetic (PK) assessment of baclofen were drawn at randomisation (at peak only, 90 minutes after the first dose intake), 1, 6 and 12 months (at trough and peak, before and after 90 minutes of drug intake) or at the end of study drug administration in the case of patient’s discontinuation. Plasma concentration of baclofen was measured using a validated high-performance liquid chromatography coupled with mass spectrometry (LC-MS/MS) in a central laboratory. Baclofen plasma concentration at trough and at peak allowed us to check the compliance of the study drug and to show the kinetic of the drug according to the 3 doses.

However, because of the very low doses of naltrexone, for most of the patients the plasmatic concentration was found below the lower limit of quantification (LLOQ) of the validated method (30 pg/mL) even at peak. At 12 months, naltrexone could be dosed at peak for only 16 patients (11 patients in the HD group, 3 in the ID group and 2 in the LD group), and at trough for only 2 patients in the HD group, while baclofen concentration could testify that the patient had correctly taken PXT3003. Sorbitol was not dosed for technical reasons as it could not be detected under the conditions of the sampling.

Electrophysiology

Nerve conduction studies were performed using standard techniques at skin temperature of 32°C. They included motor and sensory responses of median and ulnar nerves of the non-dominant upper limb.

For motor parameters (amplitude of Compound Muscle Action Potential (CMAP), Motor Conduction Velocity (MCV) and Distal Motor Latency (DML)), the median nerve was stimulated at the wrist and antecubital fossa, and responses were recorded over the abductor pollicis brevis. The ulnar nerve was stimulated at the wrist and below the elbow, and responses were recorded over the abductor digiti minimi.

For sensory parameters (amplitude of Sensory Nerve Action Potential (SNAP) and Sensitive Conduction Velocity (SCV)), we used an antidromic method and ring electrodes by stimulating the nerves at the wrist. For the median nerve, the active electrode was placed at the base of the second digit and the reference electrode between the fourth and the fifth metacarpals of the second digit. For the ulnar nerve, the active electrode was placed at the base of the fifth digit and the reference electrode between the fourth and the fifth metacarpals of the fifth digit.

Efficacy

Two composite neurological scores were used to address efficacy in this study. The Charcot-Marie-Tooth Neuropathy Score (CMTNS) was proposed and validated by Shy et al. to provide a single and reliable measure of impairment in CMT [25,26]. It is a 36-point scale based on 9 items comprising 5 of impairment (sensory symptoms, pin sensibility, vibration, strength arms and legs), 2 of activity limitations (motor symptoms arms and legs) and 2 of electrophysiology (amplitudes of ulnar CMAP and SNAP). Higher scores indicate worsening function, and the score categorises disability as mild (0–10), moderate (11–20) and severe (21–36). The Charcot-Marie-Tooth Examination Score (CMTES) is a sub-score of the CMTNS comprising only the first 7 items, excluding the 2 electrophysiological items CMAP and SNAP.

Although CMTNS is an accepted measure of CMT severity, its sensitivity to change is still debated and it was agreed that some CMTNS components are not sensitive, mainly because of floor and ceiling effects [27]. For instance, if the patient had surgery (fixation of the ankle), the ‘motor legs symptoms’ definitively scores 3, and no further decrease can be scored. The usefulness of the SNAP component is also limited as it is frequently absent in CMT1A patients who then receive the maximum score on their entry visit and thus have a low sensitivity to change. Komyathy et al. (2013) pointed out that sensory and motor symptoms items are based on subjective opinion from patients, concerning their leg and arm strength and loss of sensation in their legs, and that pin sensibility, vibration and strength are based on a neurological examination which depends on patient cooperation and examiner consistency to obtain reproducible results [28]. Finally, some measurement of CMTNS, such as SNAP or vibratory sensation, may decrease with age, so that scores from patients could increase slightly as they get older, independently of CMT. A second, modified version of the scale (CMTNS2) was therefore proposed by Murphy et al. (2011), at a time when the present study was ongoing, to attempt to reduce floor and ceiling effects and to standardise patient assessment [29]. Although CMTNS sensitivity in detecting the effects of a therapeutic intervention has not been demonstrated up to now, it is the only CMT-specific outcome measure (although not specific to CMT1A subtype), used as primary outcome in the clinical trials for ascorbic acid [16-18], and therefore remains of particular interest for inter-study comparisons.

The Overall Neuropathy Limitations Scale (ONLS) was derived and improved from the ODSS by Graham and Hughes (2006) to measure limitations in the everyday activities of the upper limbs (rated on 5 points) and the lower limbs (rated on 7 points) [30]. The total score goes from 0 (= no disability) to 12 (= maximum disability).

Although the functioning of patients with peripheral neuropathy may be influenced by other factors in addition to their physical capacity, ONLS measures the perceived ability of the patient to move and enjoy a normal life, and thus is expected to be associated with quality of life. It was initially validated in a pool of 100 patients with diverse peripheral neuropathies (mainly of dysimmune origin but including 9 CMT patients), and it showed significant relationships with measures of impairment, disability and quality of life, although it did not correlate significantly with the SF-36 Role Limitation Physical Subscale in patients with neuropathies other than Guillain-Barré Syndrome (GBS) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP). ONLS was recommended as a core disability scale for CMT1A studies [26,27]. Its reliability was further validated in studies of CMT patients [31] and it was also used in ascorbic acid therapeutic trials on CMT1A patients [16,17].

The importance of both scores to assess impairment and disability in CMT1A led us to consider them a priori as main efficacy outcomes.

All other individual efficacy outcome measurements were considered as secondary endpoints and were treated equally. They included:

i) Functional measures: 6-Minute Walk Test (6MWT) [32] to assess gait velocity by the distance walked during six minutes (in metres). 9-Hole Peg Test (9HPT) to assess finger dexterity by the time required to put nine pegs in nine holes (in seconds, non-dominant hand considered). Quantified Muscular Testing (QMT) [33] to assess muscle strength by Ankle Dorsiflexion (in Newton metres, mean of left and right side considered) and Hand Grip (in kilogrammes, non-dominant hand considered).

ii) Electrophysiological parameters: Amplitude of Compound Muscle Action Potential (CMAP, in millivolt), Motor Conduction Velocity (MCV, in metres per second) and Distal Motor Latency (DML, in millisecond) measured from the mean motor responses of the median and ulnar nerves (non-dominant side considered). Amplitude of Sensory Nerve Action Potential (SNAP, in microvolt) and Sensitive Conduction Velocity (SCV, in metres per second) measured from the mean sensory responses of the median and ulnar nerves (non-dominant side considered). The nerve sensory measures have to be interpreted with caution because of a particularly high content of missing values in SCV and of 0 values in SNAP, the latter reflecting measures below the detection threshold. Myelin state of non-degenerated axons is generally assessed by MCV, DML and SCV while CMAP and SNAP inform on the degree of axonal loss, but it must be stressed that the variability attached to these measures is high.

iii) Self-assessment-based measurements: Visual Analog Scales (VAS) for pain, fatigue and global state were assessed by the patient; Clinical Global Impression (CGI) for global improvement, illness severity and therapeutic effect was assessed by the physician.



All efficacy outcomes were assessed at randomisation and at 6 and 12 months.

Additional exploratory outcomes

A set of additional, exploratory secondary outcomes were monitored during the study.

Intra-epidermal axon density in cutaneous biopsy of the lateral malleolus.

mRNA expression of PMP22 in cutaneous biopsy of the lateral malleolus.

Plasma concentrations of a series of bio-chemical biomarkers.

Their analysis and interpretation of the results are still ongoing, and will be the subject of future publications.

Between-sites standardisation

To standardise the outcome measures, the sponsor supplied the material required for the 6MWT in the hospital ward, the required tool to perform the 9HPT and the required tool (MicroFet2 Jamar) to perform QMT. A technical document detailed the techniques and material used for these evaluations. Before the study began, all examiners attended training sessions; an additional training session was implemented at the opening of each study site; furthermore, an audit was conducted during the study on each site to confirm that instructions were correctly followed. Moreover, all analyses (pharmacokinetic and clinical laboratory evaluations) were centralised and performed in two central laboratories.

Statistical analysis

Analysis population

All the analyses were conducted on the Full Analysis Set (all randomised patients, on an intent-to-treat basis).

Baseline analysis

Demographic and clinical patient characteristics were presented for each group [34].

Safety and tolerability analysis

Safety and tolerability analyses were based on the reported treatment-emergent, adverse events (TEAEs) and other safety information (vital signs, electrocardiogram and laboratory tests). The percentage of patients with treatment-emergent adverse events after one year was descriptively compared between groups and tested by a Fisher’s Exact Test.

Efficacy analysis

The composite scores used in this study (CMTNS and ONLS) were considered as the main efficacy outcomes. The monotonicity of the dose-effect was tested through a Spearman’s rank correlation between percentage of improvement and doses (numerically coded as Placebo = 0, LD = 1, ID = 2, HD = 3). Differences between treatment groups were assessed by Analysis of Covariance (ANCOVA) on log-transformed values by adjusting for baseline values. Estimates (LS-means) were provided as mean percentage change over baseline. This analysis was performed independently on CMTNS and ONLS. The significance of the treatment effect on both CMTNS and ONLS was then assessed through the significance of the O’Brien’s OLS test [35,36] considered as the most powerful method for the statistical inference of multiple outcomes. In the context of the assessment of a combination of old drugs restrained to very low doses, we suspected that the low dose and middle dose are a priori located below the inflexion point of the expected logistic dose–response function. Consequently, the Minimum Effective Dose search was performed by closed Step-Down procedure for comparing ordered doses with a control on Helmert contrasts (SD2H algorithm, the ith dose level mean is compared to the average of all the lower dose level means, including the zero dose level) [37,38]. Based on the assumption of monotonicity of the doses on the main endpoints, the detection of a Minimum Effective Dose among groups and a linear correlation coefficient of at least R = 0.75 between baseline and final values, a Standardised Mean Difference (also known as Cohen’s d) of at least s.m.d. = 0.5 should be detected with a power of at least 80% when the sample size reaches 20 patients per group and at a one-tailed 5% significance level [39,40]. The treatment-by-centre interaction was assessed on CMTNS and ONLS and we concluded that there was no interaction. The ANCOVA analysis was repeated for the other efficacy outcomes (6MWT, 9HPT, Ankle Dorsiflexion, Grip, CMAP, MCV, DML, SNAP, SCV, VAS and CGI). Finally, we considered the patients non-deteriorated (including stabilization or improvement) after the 12-month treatment. Deterioration was evaluated by the percentage of change averaged over CMTNS and ONLS. The proportions of non-deteriorated patients in each group were compared using a logistic regression model [41].

General procedures

Statistical analyses were performed with R version 3.0.1 (http://cran.r-project.org). Data distribution and within-group variation were preliminary assessed in order to guide our methodological choices [39-41]. Missing data imputation was performed by mixed model approach considered as the most appropriate technique for intent to treat longitudinal studies [42]. Statistical tests were conducted at a 5% significance level.