A randomized placebo controlled clinical trial to evaluate the efficacy and safety of minocycline in patients with Angelman syndrome (A-MANECE study)

Angelman Syndrome (AS) is a neurodevelopmental disorder characterized by delayed development, intellectual disability, dysmorphic features as prognathism and tongue protrusion, severe speech impairment, seizures, puppet-like ataxic movement, paroxysms of laughter, and abnormal sleep patterns.

For over 20 years it was considered a rare disorder, and although the occurrence of families with affected sibs suggested a genetic aetiology, no known cause could initially be identified. In 1987 Magenis et al. [1] identified a deletion of chromosome 15q11–13 in two patients with AS and subsequent work has shown that AS can be caused by a variety of genetic mechanisms which involve this imprinted region of the genome. All of these mechanisms affect expression of the maternal ubiquitin-protein ligase E3A (UBE3A) gene in the brain. All patients carry at least one copy of paternal UBE3A, which is intact but silenced by a nuclear-localized long non-coding RNA, UBE3A antisense transcript. The absence of the protein product, an E3 ubiquitin ligase, results in the accumulation of regulatory proteins in the post-synaptic density, which is believed to cause abnormal dendritic spine morphology and density in hippocampal pyramidal neurons leading to aberrant synaptic function [2]. These alterations in spine morphology and synaptic function in neurons provides an explanation for the severe profound intellectual disability (ID), lack of speech, difficulties with motor control and planning, significant sleep difficulties, seizures, and unique behavioural features.

There are 4 known aetiologies of AS responsible for the silencing of the UBE3A gene: deletion in chromosome 15q11-q13 (70% of cases), paternal uniparental disomy (UPD; 2% of cases), imprinting defect (3% of cases), and point mutation (10% of cases) [3]. There are 2 documented deletion types classified based on the proximal breakpoint (BP)—class I (BP1-BP3) and class II (BP2- BP3). Class I deletions are bigger, with implications for greater severity in phenotype.

In recent years clearer delineation of the clinical phenotype of AS and improved diagnostic testing has led to improved recognition of the condition and the incidence of AS is now estimated to be between 1 in 10,000 and 1 in 40,000 [4] [5]. Studies of the specific cognitive and behavioural features associated with AS and of the seizure disorder have improved management of the condition and provided insight into the long term outlook for affected patients [6].

Treatment and management is symptomatic with no therapy that addresses the underlying aetiology. A multidisciplinary treatment approach is normally required, relying on appropriate therapies for the physical and neurological problems encountered in this condition, and provisions for special educational needs. Given the very specific cognitive profiles and behavioural features of AS, the treatment should be tailored individually based on the most prominent symptoms. Intensive courses of conductive therapies, similar to those carried out in children with cerebral palsy, have been attempted in AS, with some improvements reported in short-term mobility and communication [7].

Language acquisition is one of the most marked problems in AS. No single communication method works best in AS so every attempt should be made to find a communication system which works for an individual AS child.

Seizures occur in 80–95% of children with AS and usually start in childhood. Seizure types include myoclonic, atypical absence, generalized tonic–clonic, and atonic (“drop”) seizures. Many individuals exhibit multiple seizures types. Seizures usually require broad-spectrum anticonvulsant medication and often combination therapy. The treatment of the epilepsy in AS is often difficult, especially in the early years [8] [9].

Several clinical trials have produced negative results. Attempts to increase transcription from the paternal allele through the use of pro-methylation vitamin supplements did not result in any noticeable improvement. It has been postulated that levodopa/carbidopa and minocycline for its mechanism of action could play a role in therapeutics of AS, but at present evidence supporting its use in clinical practice for AS patients is lacking. Among the reasons postulated to explain these negative findings are thatsome of these treatments do not target the underlying pathophysiology of the disease or that may be the therapeutic attempts are made too late to revert the phenotypic deficits [10].

Minocycline is a semi synthetic tetracycline antibiotic. It is effective against gram-positive and negative infections. Minocycline has been shown to have antioxidant, anti-inflammatory, antiapoptotic and neuroprotective properties in animal models, making it appealing as a potential treatment for neurological disorders [11].

Minocycline has been studied in different neurological disorders, including Fragile X Syndrome (FXS), multiple sclerosis, Alzheimer’s, Parkinson’s disease, stroke, traumatic brain injuries, spinal cord injury, unipolar depression, amyotrophic lateral sclerosis [12‐14] and Angelman Syndrome [15]. In Angelman Syndrome, Grieco et al. performed an open label trial in children aged 4–12 years, which showed statistically significant improvementsfollowing 8-week treatment with minocycline in the mean raw scores of the subdomains communication and fine motor ability of the BSID-III (Bayle Scales of Infant and Toddler Development3rd edition), the subdomains auditory comprehension and total language ability of the PLS-IV (Preschool Language Scale 4th edition), the receptive communication subdomain of the VABS-II (Vineland Adaptive Behaviour Scales 2^ndEdition), and mean scores of the BSID-III self-direction subdomain and CGI scale score. The treatment emergent adverse eventsreported, considered related to minocycline treatment, included lethargy and dizziness. The authors concluded that the administration of minocycline to children with AS is safe and well tolerated, and that minocycline improved the adaptive behaviour of these children suggesting this drug may be an effective treatment for this disorder.

Prior to the publication of study results in medical literature, patients became aware of these results by other means. Expectations and a strong treatment demand emerged among parents and patients associations. This raised the need to conduct a double-blind, placebo controlled clinical trial in order to evaluate the efficacy and safety of minocycline in patients with Angelman Syndrome.

Consistently, the analysis of patients that crossed-over in the second period shows lack of differences between minocycline and placebo in the mean changes of the development index of the MP-R scale. A significant period effect has been shown; with higher magnitude of changes observed in the first treatment period regardless of the actual treatment received either minocycline or placebo, which might be explained by the expectations from study participants. This is consistent with the higher rates of impression of improvement reported by parents compared to physicians. Thus, observed changes appear mostly related to a study effect than a truly treatment effect as no significant differences between active treatment and placebo could be shown for any of the endpoints analyzed, nor even when comparing different sequences of treatment or active treatment durations. Changes observed may be explained by parent’s and clinician’s expectations, although the contribution of a multidisciplinary intervention during the trial and a potential training effect in the patients cannot be totally ruled out.

It was hypothesized that the 8-week study duration might have been not long enough for the full minocycline effect to be expressed. However, the single arm study conducted by Grieco et al. was able to show significant changes from baseline following 8 weeks treatment with minocycline in patients with AS. Our study was aimed to assess if these encouraging findings were a true treatment effect by including a placebo arm, which addresses the main limitation of the previous trial. Contrary to the previous findings, our study shows that short-term treatment with minocycline is not efficacious in the treatment of AS. The question on whether longer term treatment duration might have been needed remains formally unanswered and the lack of knowledge on the actual mechanism of action of minocycline in the treatment of AS add further uncertainty. However, based on the disappointing results observed in the subset of patients undergoing 16 weeks treatment with minocycline, it is doubted that longer treatment durations may prove to be effective.

This trial was not large enough or of sufficient long-term duration to adequately assess the safety of minocycline in the treatment of AS. However, minocycline belongs to an old class of medicinal products for which an extensive experience of use does exist; making the safety profile of minocycline reasonably well known. Overall, treatment with minocycline was safe and well tolerated.

Our trial has some limitations. The limited sample size and heterogeneity of the studied population precludes drawing firm conclusions on the generalisability of the results to the general AS population and also makes it difficult drawing conclusions in relevant subsets of patients. In addition, longer than 8-week treatment duration might have been needed to express the full potential effect of treatment, but based on previous and current findings in the cohort that followed 16-week treatment, this is very unlikely. Furthermore, despite the control for potential confounding factors, the lack of changes in the rest of educational and therapeutic measures, and the double-blind assessment, an important study effect due to expectations from participants plus a potential training effect could not be prevented. Another weakness is that drug-related side effects have the potential for unblinding both subjects and investigators; for minocycline, these include gastrointestinal, teeth greying and photosensitivity, however there were no significant differences in these effects or any other side effects between the two study groups. We had no episodes of unblinding due to SAEs and only one patient discontinued due to AEs. The addition of a placebo control we aimed to confirm if previous findings were due to a true treatment effect or rather due to a study effect. In addition, the cross-over study design allowed warranting access to active treatment to all patients, making recruitment of subjects less difficult considering that this is a population with a high demand of treatment. It is recognized that this might not be the most appropriate study design to fully explore the potential of minocycline in the treatment of a chronic neurodegenerative condition. Nevertheless, the introduction of a placebo control arm addresses one of the main drawbacks of previous studies in the field and it allows concluding that the previously observed effects cannot be regarded as a short-term benefit due to minocycline in the treatment of AS.

In conclusion, in this first randomized placebo-controlled study conducted in patients with AS minocycline failed to demonstrate any benefits in the relevant neurological areas affected in AS. These results do not warrant the use of minocycline in AS.

From January 2014 through September 2014, we conducted this randomized, double-blind, placebo controlled phase 2 trial at Hospital Universitario Puerta de Hierro Majadahonda. The protocol was approved by the local REC and the national regulatory authority (AEMPS).Written informed consent was obtained from guardians after the procedure(s) had been fully explained. The study was registered at the European Clinical Trial database (EudraCT 2013–002154-67) and with Clinical.​Trials.​gov (NCT02056665). The study was conducted according to Good Clinical Practice ICH E6 guideline.