Subjects were recruited from fragile X clinics at Rush University Medical Center (RUMC) and the MIND Institute at University of California at Davis Medical Center (UCDMC). Inclusion criteria required a DNA based diagnosis of FXS, stable medication doses for at least 6 weeks before study, and ability to tolerate an intravenous catheter (IV) for 6 h for pharmacokinetic studies. Exclusion criteria included concurrent treatment with lithium, typical antipsychotics, tricyclic antidepressants, NMDA antagonists or enzyme inducing anticonvulsants, concurrent or recent initiation of cognitive behavioural therapy, significant disease in another organ system, hearing or vision impairments, psychosis, major depressive symptoms, pregnancy, drug abuse disorder, Tourette syndrome, and significant abnormalities in baseline laboratory tests. Subjects with well controlled seizures were not excluded although none of the subjects enrolled had a seizure history. Informed written consent was obtained from either the subject or the parent before participation. Assent from the subject was obtained when the subject was not his/her own legal guardian. The study was approved by the institutional review boards at RUMC and UCDMC. The sequence of subject enrolment and treatment was random, depending only on when subjects contacted the study centre and when they could be scheduled.
At the screening visit medical history, exam, vital signs, laboratory testing including routine chemistries, blood counts, thyroid functions, electrocardiogram (ECG), and a pregnancy test (females) were evaluated, and baseline CPT (Carolina Project Fragile X continuous performance test) and prepulse inhibition (PPI) outcome measures were obtained. At the treatment visit, 14–28 days after screening, an IV was inserted for blood drawing and the subject received the study medication orally. Blood for pharmacokinetic testing, blood pressure, heart rate and side effect screening was obtained at 0, 15, 30, 45, 60, 120, 180, 240, 300, and 360 min after dosing. Our side effects screening protocol involved asking both the subject and family members if the subject was having any of a structured list of symptoms relevant to potential effects of fenobam (aggression, fatigue, hyperactivity, anxiety and fidgetiness, increase in self stimulation, odd behaviour, inappropriate laughter, dizziness, vertigo, nausea, paraesthesia, and headache), and direct observation by the physician and family for the above symptoms and other behavioural changes. Subjects were all verbal and sufficiently high functioning to report side effects in response to simple questions. Post-treatment PPI was performed after the 60 min blood sampling, followed by CPT. The first subject for each gender was dosed with 50 mg fenobam, the second with 100 mg, and all subsequent subjects with 150 mg. The final dosage of 150 mg fenobam was at the middle range of individual doses in previous studies where CNS side effects were observed. Conference calls were held after each subject was dosed, to confirm absence of adverse events and justify dose escalation or maintenance for the subsequent subject of that gender.
PPI was chosen as an outcome measure to assess sensorimotor gating and inhibitory control because: (1) there is significant deficit of PPI in males and females with FXS23 24
; (2) PPI at 120 ms has excellent test-retest reliability with intraclass correlations of 0.85 for FXS and 0.88 for controls24
; (3) PPI is responsive to medication effects25 26
; (4) PPI in the mouse model of FXS can be corrected with MPEP21
; and (5) PPI represents an electrophysiological measure that is expected to be less amenable to placebo effects than other measures. The PPI protocol used for this study was a slightly modified version of the procedure previously described.24
Startle stimuli (SS) were 50 ms 105 db SPL white noise pulses and acoustic prepulses (PP) are 25 ms 75 db SPL 1 kHz tones. These trials are delivered while participants watch a silent movie to maintain compliance and interest in the procedure. Test-retest studies have demonstrated good to excellent reliability for PPI at 120 ms and 240 ms, but inadequate reliability for PPI at 60 ms. Therefore, for the current study, we eliminated the 60 ms trial type and added two trials per type, resulting in a protocol with 30 total trials (10 with SS alone, 10 with 120 ms prepulse and 10 with 240 ms prepulse). Digitised obicularis oculi electromyographic peak magnitudes recorded between 20 ms and 200 ms post startle probe onset were averaged across trials within each type. PPI was calculated as: 100 × [(mean response magnitude in the startle stimulus alone trials – mean response magnitude in the prepulse trials)/mean response magnitude in the startle stimulus alone trials]. Based on group differences obtained in our prior study and review of the literature on PPI reliability and its response to psychopharmacological intervention, we determined a priori that subjects with PPI improvement of 20% on the 120 ms trials (the most reliable trial latency) or more would be considered responders.
The Carolina Fragile X Project Continuous Performance Test (FXCPT), developed for individuals with cognitive impairment, was chosen to assess attention, impulsivity, and inhibition, as other more standard CPT measures are too difficult for individuals with FXS.27
The FXCPT showed a significant deficit in response inhibition in males with FXS, compared to mental age matched controls,28
and in a test-retest reliability study had a weighted kappa of 0.7, suggesting a good reproducibility.29
The FXCPT was administered as previously described28 29
and the number of correct hits on a target stimuli, omissions involving lack of reaction to a target (attention deficit), and commissions involving reacting to a non-target (impulsivity, poor inhibitory control) were tabulated. Individuals with FXS often hit the mouse button repeatedly and impulsively, thus achieving a perfect number of hits with low omission scores, but a poor overall performance due to a high number of commission errors.29
Thus, the commission score was the main focus of the analysis, as the more reliable marker for abnormal performance on the FXCPT.
Fenobam concentrations from plasma samples were measured with MS-MS based assays validated for human application, using positive ion liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). For comparison, plasma samples over 24 h were obtained from three healthy adult male volunteers.
For statistical analysis of PPI data, a positive response was defined as a 20% or greater improvement over baseline. One sample exact binomial 95% confidence interval was computed for the proportion of positive response. A one sample two sided exact hypothesis test was used to compare the proportion of positive response to the null proportion of 2/13 (15.4%), obtained from our prior PPI study with untreated individuals with FXS.24