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Methods
Study Design
This study took place at the University of South Florida and was approved by USF's Human Research Protection Program's Institutional Review Board (Pro00004716). The USF IRB issued a waiver of assent; however written informed consent was obtained from both the mother and father of each participant. Since no previous research existed showing how children with AS would respond to minocycline, statistical methods could not be employed to determine sample size. Therefore, human subject protections mandated a single arm open-label study design be implemented with no placebo control. After baseline testing (T1), 25 children with AS were prescribed minocycline for 8 weeks. Concurrent administration of medication necessary for seizure control or other comorbid conditions was allowed. The time course and dosing was determined from previous research in which children with FRX were administered minocycline. The study participants were evaluated again after 8 weeks of treatment (T2) and 8 weeks after minocycline was discontinued (T3). The objective of this study was to evaluate the tolerability of minocycline in children with AS and provide preliminary neuropsychological and electroneurophysiology data.
Recruitment
Participants of this study were recruited through the websites of Angelman parent organizations and clinicaltrials.gov. Children that met the screening criteria were pooled and participants were selected at random using a computer generated randomization schedule (SAS, Cary, NC). To minimize the chance of screen failure, parents were required to have their child's primary care provider or neurologist complete a short questionnaire attesting the child met the inclusion criteria and provide an indication of the severity of the child's disability due to Angelman syndrome.
Inclusion criteria included: 1) A molecularly confirmed diagnosis of Angelman syndrome. 2) Male or female. 3) Age between 4 to 12 years old at the time of recruitment. 4) A CGI-S score of 4 or greater indicating at least moderate severity of symptoms. 5) An acceptable surrogate capable of providing consent on the participant's behalf.
Exclusion criteria included: 1) A known allergy to minocycline or any tetracycline. 2) No molecular confirmation of the AS diagnosis. 3) Participation in another study in which a drug, vitamin or dietary manipulation was used to treat AS within 6 months preceding enrollment. 4) Severe or uncontrolled seizures or any other medical condition rendering the child medically unstable. 5) A history of cardiovascular, respiratory, liver, kidney or hematologic disease or a history of systemic lupus erythematosus.
Medical and Neuropsychological Evaluation
Each participant underwent 3 identical study visits consisting of medical evaluation and neuropsychological examination at baseline, after 8 weeks of treatment with minocycline, and 8 weeks after the drug was discontinued. A board-certified pediatric neurologist completed a detailed history and physical examination, assigned a Clinical Global Impressions – Severity (CGI-S) score and interpreted the results of laboratory testing. Blood screening included a complete blood count (CBC), as well as blood urea nitrogen (BUN), creatinine, alanine amino transferase (ALT), and aspartate aminotransferase (AST) levels. Finally, a 30-minute electroencephalogram completed the medical evaluation. Neuropsychological measures were administered during each study visit. These outcome measures included the Bayley Scales of Infant and Toddler Development 3rd Edition (BSID-III), Vineland Adaptive Behavior Scales, 2nd Edition (Vinland-II), and Preschool Language Scale, 4th Edition (PLS-IV). To ensure compliance with the dosing regimen, caregivers were asked to record the date and time each dose of minocycline was administered.
Safety and Adverse Event Monitoring
Prior to the initiation of any study procedures both parents (or the legally authorized representative) of participants were required to sign an informed consent document in person. The document detailed all of the study procedures and each of the known side effects of minocycline. During the course of the study, caregivers were asked to report any observed side effects and/or changes in behavior immediately via telephone. After 4 and 8-weeks of minocycline treatment as well as 4 and 8-weeks after the drug was discontinued caregivers were asked to complete online questionnaires to document adherence to the medication regimen and to document any observation the caregiver my have made. To assess the safety of minocycline on multiple organ systems, the aforementioned blood-screening tests were reviewed during each study visit. When an adverse event was reported, the duration, severity, relatedness and treatment status were documented (Table 1).
Minocycline Dosing
After baseline testing, each subject was prescribed minocycline according to his or her body weight (3 mg/kg/day, not exceeding 200 mg a day). The drug was dispensed in 50 mg caplets to be taken orally twice daily (BID). While the lack of speech made it difficult to discern, 3 participants taking 200 mg per day appeared to suffer from intolerable lethargy and/or dizziness (Table 1). The adverse effects resolved when the dose was reduced to 100 mg daily. The dosages used here are equivalent to those used in clinical practice for children greater than 8 years of age and have been established as tolerable and safe in similar patient populations.
Statistical Analysis
For each dependent measure, the effect of minocycline treatment was assessed using repeated measures analysis of variance (ANOVA) with P values of less than 0.05 considered significant. Post hoc Dunnett's tests were performed to isolate significant changes from baseline assessment values. A 2 × 3 mixed factor ANOVA was performed with age (≤9 or >9 years old) as a between groups measure and assessment time as a repeated measure. For all analyses, partial η (effect size) was calculated according to the guidelines of Cohen (0.01 = small effect, 0.06 = moderate effect, 0.14 = large effect).
Primary Outcome Measures
Bayley Scales of Infant and Toddler Development, 3rd Edition was administered consistent to the test manual, under the direct supervision of a board-certified neuropsychologist, and by a single pyschometrician who was blind to the purpose and phase of the study. The BSID-III is a measure of development used to assess the cognitive, language and motor abilities of children ages 1 to 42 months. The BSID-III yields scores for five developmental domains: Adaptive Behavior (self-care and self-direction), Cognitive (attention, memory, sensorimotor, and visual preference), Language (receptive and expressive language functions), Motor (fine and gross motor) and Social-Emotional (using emotional signals for self regulation and communication needs). Internal reliability of the BSID-III is high, ranging from 0.086 to 0.93 in healthy subjects. We chose to administer this test because: 1) it has been shown to be reliable and valid with high correlation coefficients for test-retest reliability in children with other neuropathologies; 2) the BSID-III is a common data element suggested by the National Institutes of Health (NINDS) for clinical research involving children with Epilepsy, stroke and other neurologic disorders; and 3) literature suggests the BSID-III is an appropriate measure to use in children, such as those with AS, that exhibit profound developmental delays. Under normal circumstances raw scores are converted to standard scores based on age-matched healthy normative data. Children in this sample exhibited raw scores that were well below age-matched peers performances, which would result in standard scores at the floor of the distribution. Past research using the BSID-II in children with AS reported raw scores. Moreover, reporting raw scores adheres to the STROBE reporting guidelines. Finally, utilizing raw scores may better reflect clinical change in functional ability that could be observed for children with profound neurocognitive deficits (e.g., increase in the number of spoken words, or initial expression of speech) that remains far below expectations for age-matched healthy peers and not reflected in standardized scores. Therefore, this study employed raw scores to provide a quantitative assessment of skills and abilities of the BSID-III domains in our analyses.
Secondary Outcome Measure
The secondary outcome measures include the Vineland Adaptive Behavior Scales 2nd Edition (VABS-II) and the Preschool Language Scale 4th Edition (PLS-IV). The VABS-II is a designated NIH common data element for assessment of adaptive skills across 4 behavioral domains: communication, daily living skills, socialization, motor ability and also assesses maladaptive behaviors. Scores are based on subjective ratings of parent's/primary care provider's perception of a child's ability to complete various behaviors/tasks. The VABS-II was designed for special needs children, including those with intellectual disabilities, autistic spectrum disorders and ADHD. The test provides normative data for individuals from birth to age 90 years old. Internal reliability for early childhood, birth through 36 months, is 0.79 to 0.95 and varied from 0.83 to 0.93 for children aged 4 to 5 years old. Inter-rater reliability of two different caregivers for the same individual aged birth to 6 years old were moderate to large, ranging from 0.61 to 0.82.. The PLS-IV is well-recognized interactive, play-based comprehensive assessment of developmental language for children aged birth to 7 years, 11 months of age. Assessment provides scores for Total Language Ability, Auditory Comprehension and Expressive Communication. Internal reliability of measures are generally high, ranging from 0.80 to 0.97. Both the Vineland-II and the PLS-IV have been used extensively in research evaluating developmental language deficits across a variety of developmental disabilities, including Angelman syndrome.
Clinical Assessment
A physical examination and EEG assessment was performed at baseline (T1), after 8 weeks of minocycline treatment (T2) and after an 8-week washout period (T3). At every time point, a board-certified pediatric neurologist utilized the clinical global impressions severity scale to rate the severity of the participant's condition, where 0 represents no symptoms and 7 the most severe symptoms. This scale provides a quantitative measure of symptom severity that allows the clinician to take into account the participant's history, symptoms, behavior and how his or her disability impacted daily living before and after treatment.
A routine 21-channel EEG study was performed utilizing a standard 10/20 system of electrode placement. 30 to 60 minute EEG recordings in the awake and, whenever possible, asleep states were obtained without sedation. Asleep EEG recordings could only be obtained from 3 participants at various time points. After the conclusion of the study, each EEG recording was de-identified, and placed in random order so that the EEG order and relation to treatment were not known. A scoring system was used to evaluate several aspects of the EEG recordings regardless of whether or not they were a part of AS specific EEG patterns. Points were assigned when a particular characteristic was observed. For example, 1 point was given if an EEG was abnormal overall. Characteristics that would be considered more abnormal were scored accordingly. For instance, when evaluating the EEG background, 1 point was assigned if primarily theta waves (mild slowing, >50%) were observed. When a mixture of theta and delta waves (moderate slowing) were observed, 2 points were assigned. Finally, when primarily delta waves (severe slowing, >50%) were recorded 3 points were assigned. Other EEG characteristics were also examined including occipital rhythm (normal-1, slow-2 and absent-3), rhythmic theta (present <50% of the time-1, present >50% of the time-2), rhythmic delta (present-3) and epileptiform abnormalities (present-1, focal-1, multifocal-1, generalized-1, seizure-2). The points were totaled resulting in a total score, ranging from 0 (most normal) to 24 (most abnormal).
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