Research Design and Methods
The overall design for this study was an open-label, add-on pilot trial of minocycline in participants with FXS, including adolescents and young adults. Twenty subjects with FXS were enrolled and after baseline testing, they were started on an 8-week treatment course of minocycline added on to any other medications being administered at the time of enrollment. Trial length for this acute treatment period was chosen for several reasons. Firstly, the time course of FXS response to minocycline is not certain and some studies which measured aggression while using a treatment duration of less than 8 weeks, were unable to detect drug-placebo differences that did become apparent in trials of longer duration. Furthermore, shorter trial durations tend to be more vulnerable to positive expectation bias, as seen for example in the clinical trial using amantadine to treat autism [26].
Subjects
It was anticipated that minocycline may be beneficial for affected individuals of all ages and this trial included both males and females affected with FXS between 13 and 35 years of age. Adolescents and young adults were included because this is an age group that often presents with impairing problems, with aggression, agitation, and mood instability. The use of minocycline during tooth development (last half of pregnancy, infancy and childhood under the age of 13 years) may cause permanent tooth discolouration (yellow-grey-brown), so this trial did not include patients under the age of 13.
Subjects were recruited from the FXS Clinic at Surrey Place Centre or were self-referred after learning about the study from the Fragile X Research Foundation of Canada website or Newsletter. Individuals enrolled must have met at least a Clinical Global Impressions-Severity Scale (CGI-S) rating of moderate impairment (score of 4 or higher) so that the risk of exposing them to a medication was justified by a moderate level of behaviour difficulty. No changes in psychoactive medications were allowed during the 8-week treatment period. Each subject was required to be in good physical health as determined by the screening procedures described below.
Inclusion criteria included (1) diagnosis of FXS by clinical evaluation and confirmed by FMR1-DNA testing with presence of full mutation or mosaicism for the full mutation. Prior DNA test reports were accepted, when available (2) age between 13 to 35 years inclusive at the time of informed consent, (3) male or female, (4) CGI-Severity Score of at least 4, indicative of moderate or greater severity of behavioural problems, (5) a score of 9 or more on the Aberrant Behaviour Checklist - Irritability Scale (top 50th %-tile), (6) availability of parent and/or caregiver for all clinic visits and assessments, (7) English language fluency and reading level of 6th grade or greater in one caregiver (8) stable doses of psychoactive medications for at least 8 weeks prior to entry into the study, and (9) for those subjects with a seizure disorder, a stable regimen of anticonvulsants for at least four months prior to entry into the study.
Subjects were excluded from participation if they had (1) a known allergy to minocycline or tetracycline, (2) kidney disease or elevated renal function tests, (3) liver disease or elevated liver function tests, (4) neutropenia, anemia, or thrombocytopenia, (5) history of systemic lupus erythematosus or screening anti-nuclear antibody (ANA) titre of > 1:40, as minocycline may cause a lupus-like reaction, (6) individuals who did not have a parent or caregiver who was willing to participate in the clinic visits, (7) individuals who were pregnant or at risk of becoming pregnant, specifically sexually active females were excluded. (8) Presence of persistent psychotic symptoms (9) subjects with symptom severity judged to likely endanger personal safety or the safety of others or which would preclude co-operation for necessary tests. Informed written consent was obtained from either the subject or the parent prior to participation. Assent from the subject was obtained in every case in which the subject was not his own legal guardian and had sufficient cognitive ability to agree to participate. The study was approved by the Research Ethics Board at Surrey Place Centre.
Baseline Evaluation
All subjects had a baseline medical evaluation, which included detailed history, medication review, review of FXS test results, a physical exam and screening blood tests, including a CBC, BUN, creatinine, ALT, AST, ANA to assess medical health. Female patients also had a serum Beta HCG level done. The Aberrant Behaviour Checklist (ABC-C) was completed by the caregiver, along with the visual analog scale for behaviour (VAS). The Clinical Global Impression - Severity (CGI-S) scale for severity of behavioural dysfunction was completed by the PI (CP).
Minocycline Dosing
All subjects were given minocycline capsules to be taken orally on a BID basis to minimize gastric upset. In order to determine the optimum effective dose, all subjects were randomly assigned to either a low-dose group or a high-dose group. Subjects in the low-dose group received 50 mg of minocycline BID. Subjects in the high-dose group were started on 50 mg of minocycline BID but had their dose increased to 100 mg BID at the 2 week follow-up visit. These are standard doses used in general practice and were used in this trial because there is already a well established safety and side-effect profile at these doses. Any subjects who were unable to tolerate the higher dose would have been allowed to continue at the lower dose. However, none of the participants in this study required a dosage adjustment during the 8 week treatment period.
Safety, Compliance and Adverse Event Monitoring
Initial blood tests for safety monitoring included a complete blood count (CBC), blood urea nitrogen (BUN), creatinine, ALT, aspartate aminotransferase (AST), ANA, and pregnancy testing for women of childbearing potential. The CBC, ALT, AST, BUN, creatinine and ANA levels were measured again in 8 weeks. Outcome measures were conducted at baseline and at 8 weeks. A clinical visit occurred at enrolment, baseline, 2 weeks, 4 weeks, and 8 weeks. Compliance was checked at each clinic visit with a direct query for any missed doses. A medication dose tracking sheet was given to the parent or caregiver to document the date and time of every dose given. At each clinic visit a capsule count was done on each medication bottle. At each visit, subjects and families with less than an average of 90% compliance up to that date were counselled about the importance of taking the minocycline as dosed. Anyone with 80% or less compliance required discussion with the PI about continued study participation.
Assessment of Adverse Events
Caretakers were questioned regarding health problems, intercurrent illnesses and concomitant medications at each clinic visit. Any adverse event was documented regarding time it occurred, duration, severity and whether it was considered related to the minocycline or not.
At each visit the caregiver was asked to fill out a side effects questionnaire that consisted of 34 items including sedation, energy level, chest pain, nausea, vomiting, bowel and bladder problems, fever, rash, joint pains and swelling, infections and changes in teeth or skin colour, headaches or visual disturbances. These items cover all of the known side effects of minocycline.
Primary Outcome Measure
Aberrant Behaviour Checklist-Community (ABC-C)
Our main outcome measure was the ABC Irritability subtest score because previous experience with patients in this population suggested that it would be the most sensitive to the benefits of minocycline. The ABC Irritability subscale was used as the primary outcome measure and was expected to support the theory that minocycline is a specific molecular targeted treatment for FXS that will display beneficial effects on disruptive behaviour and possibly other associated features of FXS. This theory would be supported by an improvement in the irritability score after 8 weeks of minocycline treatment. The ABC is a global behaviour checklist implemented for the measurement of drug and other treatment effects in cognitively impaired individuals. It is made up of five empirically derived dimensions including irritability, lethargy/withdrawal, inappropriate speech, hyperactivity, and stereotypic behaviour based on 58 items that describe various behavioural problems. The ABC was validated on 509 (moderate to profound) cognitively impaired residents (M = 26 years old, SD = 14.5, ages 6 and up). Spearman correlation coefficients for subscales were very high, from .96 to .99, and test-retest reliability is good [27]. The 15-item Irritability Scale includes questions about aggression, self-injury, tantrums, agitation, and unstable mood on a scale of 0 to 45 with higher scores indicating greater severity. This scale has been successfully used in previous medication studies in children with autism [28] and in patients with FXS and in a controlled trial of ampakine CX516 in FXS [29]. All ABC subscales showed good reliability when used by parents and caregivers of individuals with FXS to assess behaviour in the CX516 study, and yielded intraclass correlation coefficient (ICC) values of 0.7-0.9.
Secondary Outcome Measures
Subscales of the ABC-C
Secondary outcome measures were the other subscales of the ABC-C including the Lethargy (score range 0 - 48), Hyperactivity (score range 0 - 48), Stereotypy (score range 0 -21), and Inappropriate Speech (score range 0 -12) Subscales.
Visual Analog Scale (VAS)
This methodology involves a parent-defined target behaviour and has been utilized and validated in the RUPP clinical trials particularly for the risperidone studies in autism [28, 30]. The parent or caretaker chooses a behaviour target symptom, such as tantrums or aggression. This allows the problems that are of concern to parents and the family to be targeted in the trial. The use of this methodology was originally described by Arnold et al [31] and subsequently utilized in studies of ADHD symptoms [32, 33]. In this study the family chose a target symptom at baseline and then continued with that symptom throughout the eight weeks of the study. The caregiver described the symptom including frequency, duration, time spent on it and intensity, interference in daily function or family life and other consequences. These responses were documented in a written format. We asked the caregiver to mark on a visual analogue scale (VAS), a 10 centimetre line, where the symptom lies from worst ever to no problem at all.
In the RUPP 8 week controlled trial of risperidone in autism the Parent Defined Target Symptom Scale was a valid measure of outcome and correlated (Pearson r) with the Irritability subscale of the ABC at 0.64 and with the CGI-I at 0.75 [26]. The Parent Target Symptom score demonstrated a significant difference between risperidone and placebo at weeks 4 and 8 [28]. The VAS with the Parent Target Symptom score was used for participants with FXS in the CX516 study. The visual analogue scale showed good reliability when used by parents and caregivers of individuals with FXS to assess behaviour in the CX516 study with an ICC value of 0.8.
Clinical Global Impressions Scale-Severity (CGI-S) and Improvement (CGI-I)
This scale is commonly used in drug studies because it allows the clinician to utilize the history from the parents or caretaker and incorporate it into a clinical rating first for severity and then for the clinical follow up of the patient. In the initial evaluation of each participant, we used the CGI-S to judge the severity of the symptoms requiring a rating of moderate or higher for inclusion in the study. In the follow up clinical visits we used the CGI-I. This 7- point scale is a well validated measure of clinical global impression of improvement that the clinician fills out after considering all the available new information on the participant including the parent history, the examination in clinic, reports from the school and other sources [34].
Statistical Analysis
Data were analyzed for change at 8 weeks treatment from baseline for all safety and outcome measures. A series of paired samples t-test were conducted on all outcome measures and mean group change and standard deviations were determined for all measures for which sufficient data existed from both assessments. Analysis involving the ABC-C assessed pre-post differences across the 5 separate subscales and the Total Behaviour Problems. To address the issue of multiple comparisons, Bonferroni correction procedures were used for all analyses involving the ABC-C scores [35]. More specifically, since analysis involving the ABC-C included 5 separate comparisons, a more conservative level of significance was calculated by dividing the original significance value (.05) by the number of simultaneous tests conducted (5), to yield a new significance value of .01. Distributions were within the normal range, as indicated by skewness and kurtosis across variables.
Due to the exploratory nature of the investigation, participants were randomly assigned to a low dose or high dose group to assess if there could be a dose response effect. These results were assessed using a series of repeated measures analysis of variance (ANOVA). For this analysis the independent variables were dose (high dose vs. low dose) and time (baseline vs.8 week follow-up). Consistent with previous analysis, the dependent variables included all subscales of the ABC-C, CGI, and VAS. Also, 10 participants were enrolled in the study while currently taking other types of medication. In order to assess potential drug interaction effects, a series of repeated measures ANOVA were conducted. For this analysis, the independent variables were medication group (Minocycline only group vs. Minocycline plus additional medication(s) group) and time (baseline vs. 8 week follow-up). Consistent with previous analysis the ABC-C, CGI, and VAS served as the dependent measures.