Purpose: Neuroendocrinological; to investigate neuroendocrine function (specifically prolactin release after administration of d-fenfluramine) in ecstasy users, cannabis users and controls, with a focus on separating cannabis effects from ecstasy effects on neuroendocrine response.

Design: Non-experimental (retrospective), incompletely crossed between-subjects/within subjects design. Drug use (ecstasy user, cannabis user or non-drug user) served as a between-subjects factor, but with gender confounded with drug user group (data only from male cannabis users and female non-drug users). Serum prolactin concentration measured over time served as a repeated measures (within-subjects) factor. All participants received 30 mg d-fenfluramine p.o. Original study design intended to study equal numbers of ecstasy users and 2 control groups (cannabis users, non-drug users), d-fenfluramine was withdrawn from market (and presumably use in humans) during the completion of the study, so this design was not realized.

Subjects: 24 ecstasy users, 9 cannabis users and 13 non-drug user controls, probably residing in the Aachen (Germany) area. No information provided concerning recruitment; previous reports written by the same authors have recruited subjects directly through students participating in the "dance scene" and via word of mouth. Matching - Age (male ecstasy users and cannabis users, but not non-drug controls), approximately matched on cannabis use.

Criteria for Inclusion - Ecstasy Users - Regular ecstasy use for 6 months or longer, with a minimum frequency of reported use of twice a month or 25 instances in 2 years. No regular use of other legal or illegal drugs except cannabis, with regular use defined as at least once a month in past 6 months, and no heavy use of alcohol, defined as severe drunkenness 2 or more times a month. Cannabis Users - No use of ecstasy, but matched with ecstasy users on drug use, degree of cannabis use and lack of heavy alcohol use. Non-Users - No past or current use of cannabis, ecstasy or any other illicit substance, and lack of heavy alcohol use. All Groups - Absence of any major medical or psychiatric disorder (excepting substance abuse for drug user groups), ascertained through medical and psychiatric interview, and abstinence from ecstasy or cannabis for at least 7 days before study day, with compliance verified through urinary analysis. Most women underwent study during early follicular phase of menstrual cycle; 5 / 11 female ecstasy users underwent study during luteal phase, due to scheduling difficulties.

Drug Use Parameters - Ecstasy users tool an average of 79.6 +/- 96.21 tablets over a lifetime, using an average dose of 1.37 +/- 0.93 tablets per use. Average frequency of use was 2.4 +/- 1.61 times per month, and average duration of use, in months, was 25.13 +/- 18.03 months. Self-reported length of drug-free period before study day, in days was 43.46 +/- 72.24 days. The average age when ecstasy was first used = 19.4 +/- 3.3 years. Cannabis use - Cannabis was used on 15.9 +/- 12.4 days a month by ecstasy users and 20.6 +/- 12 days per month by cannabis users, at an estimated average dose (in mg) of 531.2 +/- 532.9 mg per dose for ecstasy users and 707 +/- 474.7 mg per dose for cannabis users. Duration of cannabis use, in months, was 55.8 +/- 44.5 months for ecstasy users and 24.8 +/- 8.8 months for cannabis users. Ecstasy users had last used cannabis 11.7 +/- 22.6 days before the study day and cannabis users last used cannabis 1.4 +/- 0.8 days before the study day. 15/24 ecstasy users and 6/7 cannabis users had a positive urinary screen for THC on study day. Average age of first cannabis use for ecstasy users = 16.1 +/- 2.5 years, and the average age of first cannabis use for cannabis users = 16.9 +/- 2.3 years.

Group Demographics and Matched Variables - Authors attempted to match groups (both gender and drug use) on gender, age and cannabis use, but difficulties arose due to incomplete study design. Due to small sample size, all female cannabis users (2) and all male non-drug users (3) were dropped from further analysis. Gender, as M/F Ratio - Ecstasy users, 13/11, Cannabis users, 7/2, non-drug users, 3/13. Analysis removed female cannabis users and male non-drug controls, as noted above. Education - No information provided concerning level of education attained by participants.

Measures: Prolactin - Serum prolactin concentration was assessed with a heterogeneous sandwich magnetic preparation. Serum prolactin was assessed in blood samples drawn 15 and 5 min pre-drug, and hourly for 5 h after d-fenfluramine administration.

Fenfluramine - Serum d-fenfluramine and nor-fenfluramine concentrations were assessed via HPLC. Blood was collected at 15 and 5 and 5 min pre-drug and hourly for 5 h after d-fenfluramine administration.

Analyses: Separate repeated-measures ANOVAs were conducted for female ecstasy users and non-user controls, and for male ecstasy users and cannabis users, with drug use (ecstasy use versus either cannabis user or non-drug user) as between-subjects factor and time of measurement (15 or 5 min pre-drug, hourly after drug for 5 h) as repeated measures, with p. set at 0.05. Peak and area under curve (AUC) values also calculated for all groups.

Drug Use Parameters and Serum Prolactin Values - Separate correlational analyses were performed on area under curve (AUC) and average dose per use, duration of use and frequency of use for ecstasy and for cannabis.

Results - Significant Differences: Cannabis users (all male) had significantly lower serum prolactin after administration of d-fenfluramine. (The difference in prolactin response in controls and ecstasy users (all female) was present but not significant).

Drug Use Parameters and Serum Prolactin Values - Blunted prolactin response was negatively associated with duration of ecstasy use and with high average dose per use, wherein lower duration of use and higher average dose per use was associated with lower prolactin response after d-fenfluramine. Prolactin response was positively associated with frequency of ecstasy use, with greater frequency of ecstasy use associated with less of a blunted prolactin response. Average dose of cannabis per use and frequency of cannabis use (in female sample) was positively associated with AUC for serum prolactin, with higher average dose per use and greater frequency of use associated with greater prolactin AUC after d-fenfluramine. Frequency of cannabis use was also associated with peak prolactin values, with greater frequency of use associated with higher peak prolactin values after d-fenfluramine. Estimated amount of cannabis per use and duration of regular cannabis use (in male sample) and frequency of use (in female sample) was associated with lower baseline serum prolactin concentration, wherein greater average dose per use, greater duration, and greater frequency of cannabis use were both associated with lower baseline prolactin. Yet duration of cannabis use and estimated daily dose were positively associated with peak and AUC prolactin response (or negatively associated with blunted prolactin response) with greater change in prolactin response after d-fenfluramine.

Results - No Significant Differences Found: The trend for female ecstasy users to have a weaker prolactin response after d-fenfluramine was still not significantly different from prolactin response in controls. Lifetime ecstasy use, average ecstasy dose per use and duration of ecstasy use were all not associated with change in serum prolactin after d-fenfluramine in female ecstasy users, and none of these ecstasy use parameters were associated with differences in baseline prolactin. Average ecstasy dose per use was not associated with changes in serum prolactin after d-fenfluramine or baseline prolactin in male ecstasy users. Duration of regular cannabis use was not associated with baseline prolactin or change in serum prolactin after d-fenfluramine in female ecstasy users. Frequency of cannabis use not associated with baseline prolactin level or prolactin response to d-fenfluramine in male ecstasy users or cannabis users.

Overall Effects: Males reporting ecstasy and cannabis use had a significantly less blunted prolactin response to the administration of 30 mg d-fenfluramine than did men who used cannabis alone. While prolactin response to d-fenfluramine was slightly blunted in women reporting use of both ecstasy and cannabis when compared to women reporting no drug use, the difference was not significant. In men, blunted prolactin response was associated with long duration of ecstasy use and high number of tablets taken over a lifetime, yet blunted prolactin response to d-fenfluramine was associated with low, and not high, frequency of ecstasy use in the male sample. The authors state that male ecstasy users and male cannabis users had a comparatively blunted prolactin response when compared with data for non-drug using participants reported in other studies. None of the ecstasy use parameters were associated with prolactin response to d-fenfluramine in women. Confusingly, average amount of cannabis per use and duration of cannabis use were associated with low baseline prolactin and less blunted prolactin response (higher peak and AUC values) in men (cannabis users), implying that in these cases, cannabis use was associated with a less blunted prolactin response to d-fenfluramine. Average amount of cannabis per use was associated with lower baseline prolactin and higher prolactin AUC in women (ecstasy users only). Greater frequency of cannabis use is correlated with higher prolactin AUC and peak serum values in women, but not men. Overall, more relationships were found between cannabis use and serum prolactin than with ecstasy use and serum prolactin values.

Comments: Findings in this report may be compared with findings reported in previous neuroendocrine challenge studies using fenfluramine (Gerra et al. 2000), L-tryptophan (Price et al. 1989) or the serotonin releaser mCPP (McCann et al. 1999). Unlike previous studies, this study sought to tease out effects due to use of other drugs (specifically cannabis in this case). Since cannabis use was found to have more of an effect on prolactin response than ecstasy use, findings here suggest that results from previous studies might be the result of greater use of cannabis in ecstasy users than in controls. However, it should be noted that gender and drug use were confounded in this study, since the cannabis user sample was entirely male and the non-drug user sample was entirely female. This prevented a full comparison between ecstasy users, cannabis users and no-drug using controls, so that it is possible that differences between cannabis users and non-drug users are not examined. In addition, it is possible that differences in menstrual phase in female participants may have obscured potential differences between female ecstasy users and non-drug controls, since 5 female ecstasy users were tested during the luteal phase rather than during the early follicular phase. Like other neuroendocrine challenge studies, retrospective study design also makes it difficult generalize from study results. Despite these difficulties, these findings indicate that differences in neuroendocrine response after fenfluramine challenge may not be readily interpreted as the direct result of ecstasy effects or of ecstasy-induced neurotoxicity.