Primary objectives

Secondary objectives

Randomized controlled trials: design and study samples

The two RCTs consist of 16-week multicenter randomized, double blind, placebo-controlled trials with a washout period of one week (MPH) or three weeks (FLX). Subjects are stratified into two age categories: MPH: boys aged 10–12 years, and adults aged 23–40 years. FLX: girls aged 12–14 years and adults aged 23–40 years. These two age groups are randomly assigned to either placebo or active treatment. MRI and NPA assessments will take place before treatment (baseline), during treatment (only in the MPH trial) and following the washout period (see Figure 1 for the timeline for ePOD-SSRI RCT). Baseline measurements will be compared with the results obtained at trial end, and for the ePOD-MPH RCT also during the trial. Differences in outcome measures will be compared between the two age categories (children vs. adult), in addition to healthy controls (separate study). In view of our hypothesis that the active treatment results in long lasting or even permanent changes in the developing brain, we expect no or a small change in change scores between baseline-, and post-treatment assessments, whereas in children we expect to find larger changes, as enduring changes will have taken place in the developing brain, but only transient accommodation in the developed brain. Washout periods were chosen based on chemical properties (rate of elimination based on five half–live times) and ethical considerations (time without treatment).

A total of 50 children (10–12 years of age) and 50 adult (23–40 years of age) male outpatients diagnosed with ADHD (all subtypes) and in need of pharmacological therapy will be included in ePOD-MPH RCT. A total of 40 adolescent (12–14 years of age) and 40 adult (23–40 years of age) female outpatients with moderate to severe MDD or an anxiety disorder in need of pharmacological treatment will be included in ePOD-SSRI RCT. Patients that have used medications or drugs that influence the monoamine systems before age 23 are not eligible.

Patients are recruited from clinical programs at the Child and Adolescent Psychiatry Center Triversum (Alkmaar), from the department of (Child and Adolescent) Psychiatry of the Bascule/AMC (Amsterdam), and from PsyQ mental health facility in The Hague. The diagnosis is made by an experienced psychiatrist based on the Diagnostic and Statistical Manual of Mental Disorders, (DSM-IV), Fourth Edition, [14] and confirmed by a structured interview: Diagnostic Interview Schedule for Children (National Institute of Mental Health Diagnostic Interview Schedule for Children Version IV (NIMH-DISC-IV, authorized Dutch Translation) [15], in children or in parents and the Diagnostic Interview for Adult ADHD (DIVA) [16] in adults in the RCT with MPH. For the ePOD-SSRI trial we use the Diagnostic Interview Schedule for Children in children and in adults the Composite International Diagnostic Interview (CIDI; lifetime version 2.1 authorized Dutch translation) [17]. In addition, children must have a Children’s Depression Rating scale-Revised (CDRS-R) [18] score of > 45, and a Children’s Global Assessment Scale (CGAS) [19] score < 50. In adults, a Hamilton Rating Scale for Depression (HRSD-17) [20] ≥18, a Clinical Global Impression scale (CGI) [21] > 4, and or Hamilton anxiety scale (HAM-A) > 20 [22] are required for study inclusion. Subjects must exhibit stable dysphoria/depressed mood and/or anhedonia for at least 2 weeks prior to enrollment and mood should be pervasive (defined as present most of the time in at least two of three contexts: at home, at school or with friends). In both RCTS, patients with co-morbid axis I psychiatric disorders requiring treatment with medication at study entry, with IQ lower than 80 (as measured by a subtest of the Wechsler Intelligence Scale for children-Revised (WISC-R), National Adult Reading Test (NART), authorized Dutch translation [23] and MDD patients with current risk of suicide attempt are excluded.

We chose to include only male patients in ePOD-MPH to limit subject variation and because ADHD is most prevalent in males [24]. Thus, to keep our sample as homogenously as possible and prevent inclusion problems, only male subjects are included in the ePOD-MPH study. The cut-off point of 10–12 years of age was chosen because peak prevalence of ADHD is 10 years of age [25] and also because several MRI parameters greatly change until 8–10 years of age [26], whereas the rate of increase of neuronal growth and pruning reduces after 10 years of age. The cut off point of 23 years for matured brain in the adults is chosen in line with previous studies involving a comparison between matured versus immature brain [27].

Only female subjects are included in ePOD-SSRI based on the higher prevalence of MDD and AD in this population [28]. Thus, to keep our sample as homogenously as possible and prevent inclusion problems, only female subjects are included in the ePOD-SSRI study. For the adolescent group we chose a cut-off point of 12–14 years of age because the risk of MDD and AD onset increases approximately 8 fold at this age compared to children younger than 10 years of age [29, 30].

Cohort based study: design and study sample

In the ePOD-Pharmo study, we investigate the long-term effects of age following SSRI or MPH treatment on our main outcome parameter (phMRI). Exposed subjects are stratified into two age groups: one group that has been prescribed early in life with these medications, and another group late in life. Subjects are recruited through a medical prescription database from the Pharmo Institute (Utrecht, the Netherlands). This out-patient pharmacy database is a database that contains drug dispensing data since 1986 from over 3 million residents in the Netherlands, corresponding to approximately 20% of the Dutch population. The dispensing date, prescriber, prescribed dosage regimen, and duration are known. Subjects participate in a single assessment day (cross-sectional design) with similar NPA and MRI investigations as in the ePOD RCTs, mentioned above. Subjects eligible for study participation are 23–40 years of age and presumably diagnosed with ADHD or MDD/anxiety disorder. The early exposed group contains subjects with a history of MPH (male subjects) or SSRI treatment (female subjects) before the age of 16 (thus at least 7 years ago). The late exposed group contains subjects treated between 23 and 40 years of age. The early-, and late exposed groups will be compared to an age-, and gender matched unexposed control group, consisting of medication naive subjects suffering from ADHD or MDD/anxiety disorder. Every group (six in total) will contain 25 subjects.

Clinical rating scales

For both RCTs we use a set of clinical rating scales to assess symptom severity and functioning at baseline and after treatment. For each study separately we have some additional, disorder-specific rating scales. In the ePOD-MPH RCT, an authorized Dutch translation of the Disruptive Behavior Disorders Rating Scale (DBD-RS) [31] will be used in children and in adults the ADHD-SR [32]. Clinical improvement will be rated in both RCTs by the clinician using CGAS [19] and CGI [21] scales in children, and in adults using the Global Assessment of Function [33]. In both RCTs, the Children’s Depression Inventory (CDI) [34] and the Screen for Child Anxiety Related Emotional Disorders (SCARED) [35] will be administered to children, and the Beck Depression Inventory (BDI) [36] and the Beck Anxiety Index (BAI) [37] to adults. These rating scales are also administered in the ePOD-Pharmo study.

Imaging parameters

Imaging parameters are directed towards the DAergic and 5-HTergic system. DAergic and 5-HTergic brain activity will be assessed using phMRI, which is the primary outcome measure of the ePOD project. In addition, DA connectivity will be assessed using rs-fMRI and DTI, and functional brain activity using DA-related (motor inhibition) or 5-HT-related (emotional processing) fMRI tasks. Due to time restrictions of the ePOD-SSRI scan protocol, 5-HT connectivity will only be assessed using DTI.

phMRI

Application of fMRI in combination with a pharmacological challenge (phMRI) has the potential to provide an index of changes in neurotransmitter function. With phMRI a neurotransmitter specific pharmacological challenge is given, which causes changes in neurovascular coupling and subsequent region-specific changes in brain hemodynamics. It differs from fMRI, in that the neuronal system is not activated by a motor or cognitive task, but pharmacologically. phMRI has been shown to adequately assess the DA integrity and functionality, as DA-lesioned primates showed a blunted hemodynamic response to a d-amphetamine challenge, following DA lesioning, which correlated strongly with DA transporter availability and motor function [38]. During the phMRI scan, after several minutes of baseline scanning, subjects will receive an oral dose of MPH (0.5 mg/kg with a maximum dose of 20 mg in children and 40 mg in adults). This challenge dose was chosen as it induces maximum blockade of the DAT (80% occupancy), which occurs at serum concentrations of about 8–10 ng/ml. Higher concentrations are not likely to be very effective in further blocking DAT [39]. After 90 minutes, subjects will undergo a second MRI session, and the same MRI sequences are repeated, now under the influence of MPH. The 90 minute time window was chosen, because DAT occupancy is significantly correlated with plasma concentration of MPH, which peaks between 1 and 2 hours following ingestion of MPH [40, 41]. DAT occupancy has also been shown to be relatively stable between 1 and 2 hours after ingestion of MPH [40]. Based on the literature (reduction in DAT densities in young, but not adult treated animals) [42] and experiments from our own group in d-amphetamine users with phMRI and a MPH challenge [43], we expect that treatment with MPH will induce a long-lasting changes in the brain hemodynamic phMRI response in DA rich brain areas (e.g. striatum) in children, but not adults. We expect that in adult patients MPH will be accommodated by a series of transient compensatory reactions. However, in children MPH will induce changes in the form of long-lasting developmental alterations of the system, reflecting existence of ‘neuronal imprinting’ in the human brain [44].

In the ePOD-SSRI study, an intravenous (i.v.) challenge with citalopram (5 mg in adolescents and 7.5 mg in adults) will be administered during a single scanning session. A 5-HT challenge is subject to more variability and therefore needs to enter the brain in a rapid and consistent manner over the time course of a single scan session, which requires intravenous administration [45]. Citalopram is currently the only SSRI registered for i.v. administration. When used for therapeutic purposes, intravenous citalopram is given at the same dose as the oral route of administration and it is well within the therapeutic range even for children [46]. Citalopram increases 5-HT release by inhibiting the reuptake of 5-HT by SERT. It has been used previously in phMRI studies and has been proven an adequate probe of 5-HT function [45, 47]. We have previously shown that phMRI is able to detect 5-HT neuronal imprinting effects: in young rats chronic FLX treatment resulted in an increased 5-HT reactivity as measured with phMRI, whereas in adult animals FLX it reduced 5-HT brain activity [48]. We expect an increased signal in 5-HT rich brain areas (e.g. prefrontal cortex, hippocampus and hypothalamus) after 5-HT challenge only in FLX treated adolescents when compared to pretreatment baseline scans, in line with our previous findings in rats [48].

rs-fMRI

A relatively new fMRI approach (i.e., resting-state fMRI (rs-fMRI)) allows assessment of changes in organization of whole functional networks, including DAergic and 5-HTergic networks. Rs-fMRI detects baseline brain activity related to ongoing neuronal signaling at “rest” and is performed by low-pass filtering of spontaneous blood oxygenation level-dependent (BOLD) fMRI signals [49]. A decreased functional connectivity between anterior cingulated cortex and precuneus has been found using this technique in adult ADHD patients [50]. There are a number of studies that have investigated the effects of MPH or SSRIs on this parameter [14, 51–55], which found that these drugs normalize brain activation and functional connectivity abnormalities in patients suffering from ADHD or MDD. In accordance with this literature, we expect to find age-dependent normalization of functional connectivity abnormalities.

DTI

With diffusion tensor imaging (DTI), the micro-structural organization of white matter (WM) can be visualized. By measuring the diffusion motion of water molecules, and the fact that this motion is restricted by myelin sheaths, an impression of axonal direction and integrity can be obtained [56]. Fractional anisotropy (FA) is the most commonly used readout marker in DTI and provides information about the degree of fiber organization and integrity. Any process that results in alterations in axonal architecture, such as decreased axonal outgrowth, can result in decrease in FA [57–59]. A previous DTI study in children suffering from ADHD, observed an increase, or rather normalization, of white matter volume in ADHD medicated children compared to unmedicated children [60]. In line with this, chronic treatment with MPH in pre-adolescent rats was found to increase (fold change >1.5) genes involved in striatal growth of novel axons [61]. Furthermore, in a recent study in rats we observed opposite effects of MPH on FA measures: MPH induced an increase in FA in the corpus callosum of adolescent rats, whereas a slight reduction in adult animals [62]. Therefore, we also expect to find age-related findings in the current RCT with MPH: an increase in FA in MPH treated children when compared to pre-treatment baseline scans, and no effect or a small effect in adult patients.

Considering the 5-HTergic system, we have previously shown that alterations in axonal integrity linked to the 5-HTergic system can be adequately assessed using DTI [59]. We hypothesize that chronic treatment with SSRIs leads to increased outgrowth of the 5-HT system, since 5-HT acts as a growth factor in the maturing brain [63]. Therefore we expect an increase in FA (reflecting 5-HT neuronal growth) in 5-HT rich brain areas only in FLX treated adolescents when compared to pretreatment baseline scans. Like for MPH, no effect of treatment on these scan parameters are expected in adults.

fMRI

We have selected two task-related fMRI scans either based upon their involvement of the DA system and/or the 5-HT system and the known interaction with MPH or FLX and treatment response in anxiety and depressive disorders. In view of our hypothesis we expect to find a normalized pattern of activation on these tasks in children during treatment, which will persist after the end of the trial. In contrast, the activation pattern in adult subjects will normalize during the trial and fall back to pre-treatment (hypoactivation) values after the end of the trial. The fMRI tasks consist of the following:

An emotional processing task (MPH and SSRI trials, and ePOD-Pharmo study): The BOLD response to negative emotional faces (angry and fearful faces) is measured in a block-design fMRI task [64]. Emotional responses are elicited in many different brain regions, where the amygdala seems to be a relay between visual systems en modulatory responses. Emotional processing is known to be regulated by 5-HT and to be affected in mood disorder [65]. MDD patients are believed to express a heightened responsiveness to negative emotional stimuli and a reduced detection of positive affect [66, 67], which is explained by hyperactivity of the affective neurocircuitry, including the amygdala [68]. SSRIs have been found to decrease amygdala activity in response to negative affect in both healthy subjects and MDD patients, which might (partially) explain symptom remission following antidepressant treatment in MDD [69–71].

A motor inhibition task (only MPH trial and ePOD-Pharmo study): Frontal–striatal function and its modulation by MPH will be assessed using a motor inhibition task: the go/no-go task [72]. MPH has been shown to normalize striatal hypoactivation in ADHD subjects [73]. Specifically, fronto–striatal activation during response inhibition will be measured on two versions of a go/no-go task, each with and without administration of MPH. The effects of MPH on frontal and striatal activation during response inhibition will be compared within and between groups.

Neuropsychological tests

A neuropsychological test battery (Standard Reaction Time Task, Rey Auditory Verbal Learning Task [74], Sustained Attention to Response Task (SART) [75], N-back (working memory task) [76], Maudsley Index of Delay Aversion (MIDA) [77]) will be administered, addressing reaction time, verbal memory, sustained attention, working memory and delay aversion in particular. This information can be linked to results from imaging in order to determine any links between behavioral and fMRI data and changes in the monoamine systems. We will look for correlations between altered cognitive responses and fMRI responses, phMRI responses, DTI measures and rs-fMRI response.

Actigraphy and sleep log

Restless Legs Syndrome is a chronic progressive neurological disorder that has a greater incidence in ADHD children, adolescents and adults than in the general population [78]. It is possible that RLS is co-morbid with ADHD or that they share a common DAergic deficit. Also, ADHD separately and ADHD together with RLS have been found to be associated with sleep disorders such as insomnia and a common genetic polymorphism [79–81]. In a recent study, 64% of children with ADHD were estimated to suffer from RLS judged by their nocturnal periodic limb movement [82]. It has been shown that MPH reduces total sleep time but improves sleep quality by consolidating sleep in adults [83]. However, the effect of MPH on RLS in ADHD children has never been investigated. In view of the expected inhibitory effect of MPH on DA metabolism it is important to investigate the occurrence and severity of RLS and sleep disorders in children and compare these to adults, and the effect of MPH thereupon. Sleep disorders and RLS are effective and non-invasive outcome measures to evaluate the effect of age following MPH treatment in the human brain. Therefore, we will assess RLS severity and sleep quality in the ePOD-MPH trial using questionnaires (Cambridge-Hopkins RLS questionnaire(CH-RLSq, International RLS severity scale (iRLSS), John Hopkins RLS severity scale (JH-RLS-ss), Epworth sleepiness scale (ESS) and the the Holland Sleep Diagnostic List (HSDL)) [84] and sleep log and actigraphy at three time points during the study: the week prior to the trial, during the trial, and during the washout period. Actigraphy is a non-invasive method of monitoring human rest/activity cycles. To measure gross motor activity, each patient will wear a small actigraph unit, also called an actimetry sensor, for five consecutive days. We hypothesize that due to an expected long-term reduction in DA turnover rate after early MPH treatment, there will be long lasting positive effects on RLS symptoms and sleep disorders only in children, but not adults.

Cortisol measurements

In the ePOD-SSRI study salivary cortisol levels will be determined in salivary samples taken at home on a ‘normal’ weekday in the week before baseline and washout assessment days in order to determine the cortical awakenings response (CAR) and the diurnal cortisol cycle. Samples will be collected at 5 different moments: 1) directly after waking up, 2) 30 minutes after waking up, 3) 4 hours after waking up, 4) 8 hours after waking up, and 5) and 12 hours after waking up. To determine the peak after a 5-HT challenge, one salivary sample will be collected before the MRI scan session (baseline measure) and a second sample 30 minutes after the 5-HT challenge (directly after the MRI scan) on the day of both the MRI scan sessions.

Potential confounders

The study is designed to limit several important possible confounding parameters, such as gender effects (only women are included in the FLX trial and only men in the MPH trial) and aging effect (small age range, only young adults included). A within subject approach (pre- and post-treatment measurement in every subject) is used to rule out most between subject differences in the RCTs. Because of the design of the study, we have limited power and can correct for a maximum of 2 or 3 confounders. Therefore, age (in months) and ratings of symptom severity will be taken into account as covariates. In addition in the ePOD-SSRI study, the 5-HTTLPR polymorphism will be determined. The long allele of this SERT polymorphism in the promoter region (5-HTTLPR) has an activity twice that of the short allele [85], resulting in higher densities of SERT. It is expected to be an important confounder to take into account when measuring SERT functioning. Also, significant associations between the long variant and a favorable treatment response have been repeatedly reported [86].

Power analysis

Since these trials are the first to examine 5-HT and DA functioning following FLX and MPH treatment in children and young adults using MR imaging, there is only limited and indirect data available to perform a sample size calculation. The goal of our research is to detect differences in the age-dependency effect of FLX and MPH on the outgrowth of the DA-ergic and 5-HT-ergic system if these differences are in the magnitude of a standardized effect size of 1.25. From pilot experiments in rats and studies in humans with known alterations of 5-HT and/or DA (e.g., MDMA users or d-amphetamine users) we presume that the expected differences with our methods will lead to standardized effect sizes of at least 1.25. Both current trials will have the benefit of having before and after treatment measurements data from each patient. This paired data will reduce the between subject variability. This will increase the power of our trial to detect differences between groups. A sample size of 15 patients in each treatment-by-age group (4 groups) will be sufficient to detect standardized effect size of 1.25 with a two-sided significance level of 5% and a power of 90% to demonstrate age-dependency of the effects of MPH and FLX. To account for an expected drop-out of 25%, we will include 20 patients in each group for the FLX trial. Because the expected drop-out in the MPH trial is probably higher, due to motion artifacts in MRI scanning, we will include 25 patients in each treatment-by-age group. Because of slightly higher subject variability in the ePOD-Pharmo study (age and duration of treatment) again a sample size of 25 was chosen.

Statistical analysis

To evaluate the age-dependency of the effect of MPH and FLX on the outgrowth of the DA-ergic and 5-HT-ergic system, the change in our primary outcome measures (CBF) from baseline to post-treatment will be determined for each patient (∆i). These individual changes (∆i) will be used to estimate the treatment effect in adolescents (mean ∆ in treated patients minus mean ∆ in placebo treated patients) and in adults, which will be compared, as shown also in Figure 2. All analysis will initially be conducted using the intension-to-treat principle, but for the imaging outcomes a per-protocol analysis will also be performed.

The central analysis examines whether this treatment effect is different in adolescents compared to adults (effect modification or interaction by age). This hypothesis will be formally examined using ANOVA. The model includes treatment group (2 categories), age group (2 categories), and the interaction between treatment and age to examine whether the impact of MPH and FLX treatment differs by age. Depending on the imaging modality we will use a whole brain voxel based analysis or an ROI analysis. The same approach can be used for explorative analysis on the age-dependency of the effects on secondary outcome measures such as behavioral outcome (fMRI, neuropsychological assessment) and behavioral measures, and cortisol response for the FLX trial and sleep-log actigraph for the MPH trial.