Population and setting
This trial is conducted by Vanderbilt University Medical Center in collaboration with sponsor Disruptive Nutrition, LLC, and the onsite Angelman Syndrome clinic at the Monroe Carell Jr. Children’s Hospital at Vanderbilt is the single study site. The study is funded by the Foundation for Angelman Syndrome Therapeutics (FAST). The research team at Vanderbilt is composed of lead investigator Jessica Duis, MS, MD (pediatric geneticist, Assistant Professor, Division of Medical Genetics and Genomic Medicine, Director, Prader-Willi Syndrome Comprehensive Clinic at Vanderbilt and Director of the Angelman Syndrome Comprehensive Clinic at Vanderbilt), who is responsible for overseeing the trial, including recruitment and taking consent, the majority of clinical assessments of the subjects, and data analysis. Robert Carson, MD, PhD (pediatric neurology and epilepsy specialist, Assistant Professor of Pediatrics at Vanderbilt University), will be the lead neurologist for the study with the responsibility of electroencephalogram (EEG) data interpretation. Fenna Phibbs, MD, MPH (neurology, Associate Professor of Neurology, Vanderbilt Department of Neurology, Vanderbilt University Medical Center) will conduct the mobility tracking. Alexandra Key, MD, PhD (Research Associate Professor of Hearing & Speech Sciences and Psychiatry & Behavioral Sciences; Associate Director, IDDRC Translational Neuroimaging Core C; Director, Vanderbilt Kennedy Center Psychophysiology Lab) will be responsible for collection of the event-related potential (ERP) data and analysis. Patience Ergish, MS, RD, LDN (Clinical Dietician at Vanderbilt University), will collect patient diet information during clinic visits. LeeAnna Melton, RN, BSN, CCRP (Research Nurse Specialist III, Division of Medical Genetics at Vanderbilt University) and Lakin Householder (Clinical Trials Associate II, Vanderbilt University) are the study coordinators and point of contact for families enrolled in the trial. This Vanderbilt research team has primary responsibility for data management and analysis, with coordinated efforts from Disruptive Nutrition’s scientific staff. The Vanderbilt research team meets on a weekly basis.
Additional oversight and auditing of the trial is provided by Donna Herber, PhD, Chief Science Officer of Disruptive Nutrition, LLC with weekly conferences with members of the Vanderbilt research team. In-person meetings are planned quarterly, with data auditing semi-annually. A medical and research advisory committee has been established and consists of independent experts and key opinion leaders. The committee meets semi-annually. The Data Safety Monitoring Board is discussed in a separate section of this protocol under “Data Monitoring.”
Patients are recruited to the Nashville, Tennessee site at Vanderbilt University. Subjects with AS are recruited through the lead investigator’s practice, physician referrals, outreach through social media, and parent support groups such as the Foundation for Angelman Syndrome Therapeutics and the Angelman Syndrome Foundation. Subjects are enrolled with the following inclusion criteria: genetically confirmed diagnosis of Angelman syndrome; age 4–11 years; currently on a LGIT, KD (conventional 4:1 or 3:1, MCT, modified Atkins), or standard diet (regular diet) consistently for at least 3 months; willingness to consume the investigational formulation; and willingness to undergo protocol examinations at home and clinic visits. Subjects are excluded if they: require parenteral nutrition; have major hepatic or renal dysfunction; have a history of diabetes or have diabetes; are significantly underweight with a body mass index < 18.5; participate in other clinical intervention studies within 1 month prior to entry to the study; are unwilling to comply with the protocol requirements; or have contraindications for the use of ketogenic or low-carbohydrate diets which, in the opinion of the investigator, may influence a patient’s ability to participate in the study. Subjects are permitted to continue using all previously prescribed medications, provided the prescriptions have not changed in the 1 month prior to randomization. Parents/guardians of all eligible subjects must sign informed consent prior to study enrollment, following adequate explanation of the aims, methods, objectives, and potential hazards of the trial by the responsible investigator (see Additional file 3).
This investigation is a 16-week, randomized, crossover, double-blinded, placebo-controlled exploratory study of the safety and tolerability of a nutritional formulation containing d, l-beta-hydroxybutyrate mineral salts (sodium, calcium, magnesium) (trumacro™, Disruptive Nutrition, LLC, Durham, NC, USA) for use with a KD, a LGIT diet, and standard diet in subjects with AS. It is being conducted in accordance with the Declaration of Helsinki and is approved by the Institutional Review Board (IRB) at Vanderbilt University Medical Center (protocol number DUISJ10212017084407). See Fig. 1 (Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) Figure: schedule of enrollment, interventions, and assessments), Additional file 1 for the SPIRIT 2013 Checklist (recommended items to address in a clinical trial protocol and related documents), and Additional file 2 for the schematic of the trial design.
Members of the Vanderbilt research team, including the lead investigator and clinical trial coordinators, are responsible for obtaining informed consent (see Additional file 3). Consent is primarily sought through remote interview prior to scheduling the baseline visit in order to reduce travel burden on patients and families. On the consent form, participants will be asked if they agree to the use of their data and banked blood samples should they choose to withdraw from the trial. Participants will also be asked for permission for the research team to share relevant data with people from the institution taking part in the study as well as regulatory agencies where required. After obtaining informed consent from the parent/caregiver, subjects enter a 2-week run-in period. At the beginning of the run-in period the natural history and medical history is collected from the subject’s parent/caregiver. At baseline, a 3-day recall record of the subject’s diet, appetite, GI health, sleep, seizure, mood, and activity level are collected. While one goal of this study is to assess the effects of dietary background on the suitability of the nutritional formulation, there is no requirement for a specific number of patients per diet due to the rarity of the population. Additional study measures, including physiologic, are also collected (Fig. 1).
The study design for this nutritional intervention protocol is a randomized, double-blind, placebo-controlled, crossover study. There are two arms to the study, (1) an investigational formulation period followed by a placebo formulation period, or (2) a placebo formulation period followed by investigational formulation period. Each patient will receive both the investigational formulation and the placebo formulation, with a washout period between each arm. The randomization schedule was created by the Investigational Drug Service Pharmacy using randomization software in a 1:1 ratio with a block size of 4. Participants are enrolled in the next open slot of on the randomization table. The nutritional formulations are coded by the manufacturer lot number and, therefore, the clinical staff is blind to the actual contents of the container (active verses placebo).
Once randomized, subjects start Intervention Period 1, a 4-week period in which they receive nutritional formulation containing BHB or placebo while maintaining the diet noted at entrance into the study. Home monitoring occurs as indicated in Fig. 1. After 4 weeks of blinded intervention, subjects are evaluated in the clinic and then cease consumption of the ketone formulation or placebo for 4-week washout period. Following completion of the washout, subjects enter a second 4-week period in which they receive formulation versus placebo. They will continue the home monitoring throughout the duration of the study. Subsequently, at the completion of the second intervention, they undergo the final scheduled study visit and laboratory sampling. A washout period follows Intervention Period 2, and at approximately week 16 of the study, a follow-up call is made to the family to review symptoms and ensure the patient is not experiencing adverse events during this period. The trial schedule of enrollment, interventions, and assessments is outlined in Fig. 1 (FANS trial schedule of procedures).
Procedures and interventions
Background information regarding the subject’s natural history is collected using a REDCap© survey (Copyright 2006–2013 Vanderbilt University. All rights reserved) . Body weight and height are measured at each study visit. Serum laboratory analysis is conducted to establish a metabolic baseline: comprehensive metabolic panel, complete blood count, ketones (beta-hydroxybutyrate and acetoacetate), and lipid panel. EEG, ERP and gait analysis using the ProtoKinetics Zeno™ Walkway are performed at each study visit (Fig. 1). The utility of ERP and gait analysis to predict changes in patient function in an Angelman population has not been clearly established, but their appropriateness in similar populations has been detailed elsewhere [17, 18]. Questionnaires completed during the study include the Vineland™-3, Developmental Behavioral Checklist (Pearson Education, Inc.), which has been used previously in studies of patients with AS , and the Callier-Azusa Scale to assess areas of motor development, perceptual abilities, daily livingskills, language development, and socialization. Dietary intake, seizure frequency, sleep, and GI health are evaluated throughout the protocol through at-home monitoring. Data collected during home monitoring is recorded on tablet devices provided to the parents/caregivers upon enrollment, preloaded with necessary tracking applications. Families are provided an EarlySense® monitor with a written guidance for home sleep monitoring. External sleep monitors are important in an AS population as wearable devices are typically removed by the patient. The monitors have not been studied with patients with AS; however, their validity has been demonstrated in the general population . Urinary ketones are measured by the patient’s caregivers throughout the protocol, using Ketostix which are a validated measure of urine acetoacetate [21, 22].
Tolerability is determined at the end of each 4-week intervention period. Protocol compliance is monitored during both investigational and placebo intervention periods by recording the amount of formulation consumed. Formulation acceptability is assessed at the end of each intervention period by a parent questionnaire evaluating convenience, taste, and degree of acceptance of the nutritional formulation on a 10-point Likert scale or based on compliance with the study protocol and consumption of the formulation three times daily consistenty during the 4-week interventional period. Similar ranking systems to assess formulation acceptability have been used with other nutritional interventions in epilepsy populations [22,23,24,25].
Safety is assessed throughout the entire protocol period through (1) adverse event reporting and (2) assessment of clinical parameters collected throughout the protocol including anthropometrics, blood metabolism profile, dietary intake, seizure frequency, EEG, ERP, mobility, GI function, and sleep habits. In this population, the potential for negative effects of intervention must be closely monitored as patients are nonverbal. Additional file 4 contains details on collecting, assessing, reporting, and managing solicited and spontaneously reported adverse events.
Investigational and placebo formulations are administered with the serving size based on subject body weight. The investigational formulation provides beta-hydroxybutyrate, 2 g carbohydrate, 1 g protein, and 9 g fat, plus minerals, per 100 kcal. The placebo is matched for mineral content only. The formula is given orally as food or beverage three times per day.
Families will be encouraged to continue participation in the trial through weekly telephonic interviews. Data will be collected through three primary interfaces. Background information regarding the patient’s natural history will be collected by the clinical staff using the REDCap© survey. REDCap© is a secure web application that manages the trial database (21 CFR Part 11 and HIPAA compliant). Clinical visits will use either paper or electronic medical records. Data collected during home monitoring will be recorded using a tablet provided to the patient upon enrollment. The tablet is preloaded with the applications necessary to record all data during home monitoring. All data collected, regardless of patient completion status, will be shared with patients upon conclusion of the trial (last patient last visit) and assessment of the data. Additional file 4 contains details on data management and collection procedures.
The primary outcome of the trial is to assess the tolerability of a nutritional formulation containing BHB in patients with AS. Tolerability is demonstrated through patient compliance with the protocol as determined by the amount of formulation consumed as compared to the amount prescribed and is assessed at the end of each intervention period. Premature discontinuation of the nutritional formulation by the family is also deemed as potential intolerability. Tolerability is also assessed at the end of each intervention period through a parental questionnaire. These outcome measures were chosen as this is a vulnerable, nonverbal population with limited ability to communicate and, therefore, determining if the formulation is suitable and well tolerated is an appropriate first step.
The secondary outcomes include assessment of ketosis when consuming the nutritional formulation and safety of the nutritional formulation in patients with AS. Ketosis is evaluated daily through urine testing as well as blood analysis at the end of each intervention period. The degree and timing of ketosis may be clinically relevant to demonstrating better patient nutrition as nutritional ketosis has been related to better patient outcomes as described in the background of this manuscript. Safety is evaluated by changes in motor function, cognitive function, GI tolerance, sleep, and seizures, along with changes in height, weight, and blood metabolic panels as assessed at the end of each intervention period. In addition, adverse event reporting is monitored in real time by the study coordinator.
No studies testing ketone supplementation in patients with AS exist. Therefore, sample-size estimation is based on measures of ketosis. It is assumed from murine pre-clinical investigations, non-AS clinical studies using MCT and/or BHB, and low-carbohydrate dietary interventions, that the minimal change of the secondary outcome, degree of ketosis, from the baseline to the end of the fourth week of the investigational formulation intervention period will be 100% change from baseline (baseline for standard diet is typically < 0.5mmol/L of d-BHB in blood, or < 5 mg/dL acetoacetate in urine). The acetoacetate test used is Ketostix (Bayer Corp. Diagnostics Division, Tarrytown, NY, USA) which exhibits a sensitivity of 78% and a specificity of 96% when compared to a serum standard of 14.4 ml/dL. Considering a 25% drop-out rate, an estimate of 25 subjects need to be screened to achieve 15 completers.
Descriptive statistics, such as mean and standard deviation, are used to describe continuous variables, and frequency for the categorical variables. Means are compared using a paired two-sample t test. Statistical significance for all tests is p < 0.05. Multiple comparisons can be made including: (1) each subject to serve as their own control, comparing investigational formulation to placebo formulation, as well as to baseline, (2) each dietary background group considered in aggregate, comparing investigational formulation to placebo formulation, as well as to baseline, and (3) all subjects considered in aggregate, comparing investigational formulation to placebo formulation, as well as to baseline.
Any patient who receives the study formulation for any amount of time will be included in the intent-to-treat population for study analyses. Missing data points in this population will be addressed in discussion of any published results. Data from protocol-compliant participants is the defined population for efficacy subset analyses; any participant who received both protocol-required formulations and all required clinical assessments is considered protocol compliant. Such an efficacy subset analysis will be the primary approach for determining whether the trial endpoints are met. Those patients who complete the baseline visit, but do not start Intervention Period 1 and are withdrawn from the study, will be used for natural history reportable data only but will not be included in analyses for determining whether the outcome measures of the trial have been met.
Discontinuation/withdrawal from study participation
Participation in this study is voluntary. The parent or guardian of a participant may withdraw consent to participate in the study at any time for any reason that they deem necessary. The administration of the study formulation may be discontinued abruptly and does not require any further medical intervention and no alternate therapy will be offered.
In order to ensure the safety of the study participants, members of a Data Safety Monitoring Board (DSMB) have been appointed by the principal investigator and Vanderbilt University. The board consist of a chairperson, biostatistician, and three independent reviewers with expertise in the research field. Each member of the board meets these minimum qualifications including: (1) expertise in the field, (2) experience in the conduct of clinical trials and statistical knowledge, (3) independence from the direct management of the clinical trial, and (4) no conflict of interest. The chairperson is responsible for overseeing the meetings, developing the agenda, summarizing the meeting, and is the contact person for the DSMB. The DSMB meets three to four times per year, such schedule to be determined by the chairperson. Additional details on the charter of the DSMB can be had by contacting the Vanderbilt University Medical Center Human Research Protections Program at www.vumc.org/irb.
This study is to be conducted according to US and international standards of Good Clinical Practice (FDA Title 21 part 312 and International Conference on Harmonization guidelines), applicable government regulations and institutional research policies and procedures. This protocol and any amendments will be submitted to the Vanderbilt University IRB in agreement with local legal prescriptions, for formal approval of the study conduct. The DSMB will work in conjunction with the principal investigator and IRB to determine whether a protocol amendment warrants additional patient consent or other communication to the enrolled patients and their families. The decision of the IRB concerning the conduct of the study will be made in writing to the investigator before commencement of this study. Protection of participant confidentiality is described further in Additional file 4.
Participants may be able to claim compensation for injury caused by participation in this clinical trial. Participants who sustain injury and wish to make a claim for compensation should do so in writing in the first instance to the principal investigator, who will pass the claim to the sponsor’s insurers, via the sponsor’s office.