Data were collected in two separate studies that have already been described in detail in recently published papers: (a) study 1: MDMA × ethanol interaction study (Dumont et al. 2008) and (b) study 2: MDMA × THC interaction study (Dumont et al. 2010c). Studies were similar in design; relevant discrepancies between studies are described in the pertaining sections and presented in Table 1. Both studies utilized a four-way, double-blind, randomized, crossover and placebo-controlled design. Sixteen volunteers were randomly assigned to one of four treatment sequences.
Each volunteer received a capsule containing either 100 mg MDMA or placebo and a placebo-controlled intravenous ethanol clamp as described below, with a washout of 7 days between treatments. So, each participant attended four drug sessions: (1) placebo+placebo, (2) MDMA+placebo (i.e. MDMA alone), (3) ethanol+placebo (i.e. ethanol alone) and (4) MDMA+ethanol.
Each volunteer received a capsule containing either 100 mg MDMA or placebo and inhaled THC or placebo vapour as described below, with a washout of 7 days between each treatment. So, each participant attended four drug sessions: (1) placebo+placebo, (2) MDMA+placebo (i.e. MDMA alone), (3) THC+placebo (i.e. THC alone) and (4) MDMA+THC.
Participants included 16 healthy volunteers (nine men, seven women) who are regular users of ecstasy (at least eight exposures in the last 2 years) and alcohol (at least one exposure per week), 22.1 ± 2.9 (mean ± SD) years of age (range 18-29). Lifetime ecstasy exposure was on average 95 (SD = 138, range 14–431). More detailed demographic data have been reported elsewhere (Dumont et al. 2008).
Participants included 16 healthy volunteers (12 men, 4 women) who are regular users of ecstasy (at least eight exposures in the last 2 years) and THC (on average at least two exposures per week in the last year), aged 18-27. Detailed demographic data have been published elsewhere (Dumont et al. 2010c).
Participants for both studies were recruited through advertisements on the Internet and at local drug testing services. Exclusion criteria for both studies included pregnancy and (history of) psychiatric illness (assessed using the Structured Clinical Interview for DSM-IV Axis I disorders, non-patient version; First et al. 1994). Axis II disorders were excluded using the Temperament and Character Inventory (Svrakic et al. 1993), use of over-the-counter medication within 2 months prior to the study start, (history of) treatment for addiction problems, excessive smoking (>10 cigarettes/day) and orthostatic hypotension. Physical and mental health was determined by assessment of medical history, a physical and ECG examination as well as by standard haematological and chemical blood examinations, all conducted by a medical practitioner.
The time between the two studies exceeded 6 months. Four volunteers participated in both studies. These volunteers were included in the studies according to the protocol and all study procedures were trained before each study started. The studies have been approved by the local Medical Ethics Committee and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. All participants gave their written informed consent before participating in the study and were paid for their participation.
Adverse events/missing data
One participant had a mild adverse reaction (local vascular reaction) during the ethanol infusion, which led to premature interruption of the procedure, and one other participant did not refrain from drug use; both (one man, one woman) were excluded from further participation and results obtained were not included in the data analysis.
One participant did not refrain from drug use, after which further study participation was denied. Two participants experienced an adverse event that was judged to be likely related to study drug administration (one participant developed a short-lasting (55 s) heart rate increase of >180 bpm and another participant had mild hallucinations, the latter subsiding along with other drug effects). These participants were excluded from further participation; data of completed study days obtained prior to these adverse events were analysed as described.
Table 1 presents the time schedules of the events on a test day for each study that are relevant for the present paper. For a detailed description of a test day, see Dumont et al. (2008, 2010c). In both studies, participants arrived in the morning and were offered a light breakfast after a negative urine drug screen (opiates, cocaine, benzodiazepines, amphetamines, methamphetamines and delta-9-tetrahydrocannabinol) as well as a negative alcohol breath test and recording of signs and symptoms of possible health problems. EEG data were acquired at 1000 hours, 30 min prior to the first drug administration. Drug administration (MDMA or placebo capsule in study 1; MDMA or placebo capsule and THC or placebo inhalation in study 2) was scheduled at 1030 hours. The ethanol or placebo infusion in study 1 was started at 1100 hours for a duration of 3 h. The second THC or placebo inhalation in study 2 was administered at 1200 hours. In both studies, post-assessment of EEG was conducted at 1230 hours, at the time around the peak concentration of MDMA (2 h). Since subjective and cognitive effects of THC have been shown within 15–60 min after administration, the second THC inhalation was administered 30 min prior to the EEG data acquisition. Volunteers were offered lunch at 1400 hours and were sent home at 1700 hours after a medical check.
In addition to the EEG data, subjective, neuropsychological and physiological data were acquired, which have already partially been published (Dumont et al. 2008, 2009a, b, 2010a, b, c).
Drugs and dosages
MDMA (or matched placebo) was given orally as a capsule in a single dose of 100 mg. MDMA was obtained from Lipomed AG, Arlesheim, Switzerland, and encapsulated according to Good Manufacturing Practice (GMP) by the Department of Clinical Pharmacy of the Radboud University Nijmegen Medical Centre. MDMA (100 mg) orally is a relevant dose in the range of normal single recreational dosages. Previous experiments in humans used doses up to 150 mg without serious adverse events.
To standardize alcohol kinetics and dynamics, an intravenous clamping procedure has been developed, which minimizes variations in BAC that are high after oral administration (Zoethout et al. 2008). Ethanol (or glucose 5% as its placebo) was administered continuously by intravenous infusion of 10% ethanol in 5% glucose solution, aimed to maintain an ethanol blood concentration of 0.6‰ (0.6 g/L) for 3 h. The alcohol clamp was targeted at 0.6‰, by which we aimed to provide the equivalent of approximately two to three alcoholic beverages. This concentration is just above the legal limit for traffic participation in many Western countries and commonly used in social settings as it is considered to be a safe and relatively moderate level despite significant CNS effects (Zoethout et al. 2009). The infusion rate was calculated using frequent breath alcohol concentration measurements, according to a previously designed algorithm (Zoethout et al. 2008). Breath alcohol concentration was assessed using a HONAC AlcoSensor IV® Intoximeter. The process was semi-automated using a computer spreadsheet programme which uses changes in the measured breath alcohol concentrations to calculate the infusion rate that is needed to maintain the ethanol level at 0.6‰. The operator of the breath alcoholmeter and the ethanol infusion pump was unblinded for alcohol treatment, but did not communicate with the study team or the participant about the results at any stage during the trial and was not involved in the analyses. A sham procedure including a mock spreadsheet was used on ethanol–placebo occasions.
THC kinetics are highly variable in a free smoking procedure. To minimize variation due to inhalation volume and duration as well as THC content, a standardized procedure using a vaporizer, which has been shown to produce reliable and reproducible THC levels (Strougo et al. 2008), was used to administrate THC.
THC was purified according to GMP-compliant procedures (Farmalyse BV, Zaandam, the Netherlands) from the flowers of Cannabis sativa grown under Good Agricultural Practice (Bedrocan BV Medicinal Cannabis, Veendam, the Netherlands; Choi et al. 2004; Hazekamp et al. 2004). Each dose (4, 6 and 6 mg) of THC (>98% purity by HPLC/GC) was dissolved in 200 μL 100 vol.% ethanol. THC was stored in the dark at −20°C in 1-mL amber glass vials containing a Teflon screw-cap secured with Parafilm to minimize evaporation. The solvent was used as placebo.
On each study day, THC (4, 6 and 6 mg) or placebo was administered at 90-min intervals by inhalation using a Volcano® vaporizer (Storz-Bickel GmbH, Tüttlingen, Germany), a validated method of intrapulmonary THC administration (Abrams et al. 2007; Hazekamp et al. 2006; Zuurman et al. 2008). The same regimen was also used with MDMA administration where 4 mg THC was administered together with MDMA and the two subsequent doses of 6 mg of THC were administered 90 and 180 min after MDMA administration. Within 5 min before administration, THC was vaporized at a temperature of about 225°C and the vapour was stored in a polythene bag equipped with a valved mouthpiece, preventing the loss of THC between inhalations. The transparant bag was covered with a black plastic bag to prevent unblinding. The personnel responsible for drug preparation was not involved in any other part of the study. Participants were not allowed to speak, were instructed to inhale deeply and hold their breath for 10 s after each inhalation. Within 2–3 min, the bag was to be fully emptied. The inhalation procedure was practiced at screening using the solvent only. The inhalation schedule was predicted to cause THC plasma concentrations and effects corresponding to the THC contents in roughly one marijuana cigarette. The decision to proceed to the next higher THC dose was made by a physician based on adverse events and physical signs.
The EEG data were recorded using an elastic cap with 27 tin electrodes according to the 10–20 electrode international system. Data were referenced to the left mastoid with the ground electrode placed at the right mastoid. Vertical electrooculogram was recorded from electrodes attached above and below the left eye and the horizontal electrooculogram from the outer canthi of both eyes. Electrode impedance was below 5 kΩ at the start of the recording session. EEG and EOG were amplified with a bandwidth of 0.005–120 Hz. The sampling rate was 512 Hz. Participants were asked to sit quietly for 3 min with eyes closed, during which resting-state EEG was recorded.
EEG and EOG data were analyzed using the Brain Vision Analyzer software (www.brainproducts.com). EEG signals were re-referenced off-line to the average of all EEG electrodes, and the sampling rate was changed to 256 Hz with a high-pass filter of 0.04 Hz, low-pass filter of 40 Hz and a notch filter of 50 Hz. The 3-min continuous EEG data were segmented into 2-s epochs. First, a full segment baseline correction was applied. Subsequently, trials with artefacts were rejected from further analysis (absolute amplitude criterion of 120 μV and low activity criterion of 0.5 μV within a 100-ms time window for EEG data). Then, ocular artefact correction was conducted according to the Gratton et al. (1983) algorithm. Finally, EEG data were Fourier-transformed (Hanning window length of 20%) and subsequently ln-transformed. Based on previous literature (e.g. Böcker et al. 2010; Ilan et al. 2004, 2005), we selected frontal (Fz, F3, F4), central (Cz, C3, C4) and parietal (Pz, P3, P4) electrodes for the analysis.
Since alpha oscillations show large interindividual differences (Klimesch 1999) and previous drug studies reported not only a change in absolute power of specific frequency bands but also in the peak frequency of alpha oscillations (Ehlers et al. 1989), the analysis of fixed frequency bands has major limitations. Klimesch (1999) suggested adjusting the frequency windows of alpha and theta for each individual by using individual alpha peak frequency (IAF) as an anchor point. More specifically, individual frequency bandwidths for alpha and theta bands are determined as a percentage of IAF (Doppelmayr et al. 1998). Moreover, Klimesch (1999) suggested dividing alpha oscillations into three frequency bands: lower-1 alpha (i.e. 0.6*IAF–0.8*IAF), lower-2 alpha (0.8*IAF–1*IAF) and upper alpha (i.e. IAF–1.2*IAF). Similar to Klimesch (1999), in the present study, the power of individual-defined frequency bands was calculated. Individual frequency bands were calculated based on the mean IAF of each participant. First, for each participant, IAF was defined as the frequency at which alpha power was maximum within 7.5–15 Hz over the occipital electrodes (O1, Oz, O2) in the eyes closed condition. Visual inspection was conducted for peak frequencies occurring at the boundaries of the search window. Subsequently, power estimates were derived from the average for the theta (0.4*IAF–0.6*IAF), lower-1 alpha (0.6*IAF–0.8*IAF), lower-2 alpha (0.8*IAF–1*IAF) and upper alpha (1*IAF–1.2*IAF) bands (Doppelmayr et al. 1998).
Since several participants did not complete the whole experiment as described in detail above, multivariate analysis of variance with repeated measures MANOVA would limit the sample size. Therefore, mixed model analyses of variance (SPSS 16.0) were conducted to examine the effects of drug administration alone and in combination on logarithmically transformed EEG data, separately for each study (study 1: MDMA in combination with ethanol; study 2: MDMA in combination with THC). First, separately for each drug condition, IAF and theta, lower-1 alpha, lower-2 alpha and upper alpha power before drug administration were subtracted from IAF and power in the specific frequency band after drug administration, respectively. The difference value of power was then entered in the mixed model analysis of variance, separately for IAF and theta, lower-1 alpha, lower-2 alpha and upper alpha power. Participant was used as subject variable. Fixed factors were MDMA (MDMA vs. placebo), THC or ethanol (THC or ethanol vs. placebo). For theta, lower-1 alpha, lower-2 alpha and upper alpha power, the fixed factors—lead [frontal (Fz, F3, F4) vs. central (Cz, C3, C4) vs. parietal (Pz, P3, P4)] and laterality (left vs. right vs midline)—were added to the model. Power values after each drug administration and at each electrode site were treated as repeated measurements. If the interaction between MDMA × THC (or ethanol) was significant, post hoc analyses were conducted to investigate the effects of MDMA alone, THC (or ethanol) alone and combined MDMA+THC (or MDMA+ethanol) relative to placebo. The alpha level of significance was set at 0.05 two-tailed. Results are presented as mean ± SD unless indicated otherwise.
Repeated measures ANOVA was used to examine the pharmacokinetics of the drugs (see also Dumont et al. 2009a, 2010b).