The synthetic psychostimulant MDMA (±3,4-methylenedioxymethamphetamine, ecstasy) acts as an indirect serotonin, dopamine, and norepinephrine agonist and as a mechanism-based inhibitor of the cytochrome P-450 2D6 (CYP2D6). It has been suggested that women are more sensitive to MDMA effects than men but no clinical experimental studies have satisfactorily evaluated the factors contributing to such observations. There are no studies evaluating the influence of genetic polymorphism on the pharmacokinetics (CYP2D6; catechol-O-methyltransferase, COMT) and pharmacological effects of MDMA (serotonin transporter, 5-HTT; COMT). This clinical study was designed to evaluate the pharmacokinetics and physiological and subjective effects of MDMA considering gender and the genetic polymorphisms of CYP2D6, COMT, and 5-HTT. A total of 27 (12 women) healthy, recreational users of ecstasy were included (all extensive metabolizers for CYP2D6). A single oral weight-adjusted dose of MDMA was administered (1.4 mg/kg, range 75–100 mg) which was similar to recreational doses. None of the women were taking oral contraceptives and the experimental session was performed during the early follicular phase of their menstrual cycle. Principal findings show that subjects reached similar MDMA plasma concentrations, and experienced similar positive effects, irrespective of gender or CYP2D6 (not taking into consideration poor or ultra-rapid metabolizers) or COMT genotypes. However, HMMA plasma concentrations were linked to CYP2D6 genotype (higher with two functional alleles). Female subjects displayed more intense physiological (heart rate, and oral temperature) and negative effects (dizziness, sedation, depression, and psychotic symptoms). Genotypes of COMT val158met or 5-HTTLPR with high functionality (val/val or l/*) determined greater cardiovascular effects, and with low functionality (met/* or s/s) negative subjective effects (dizziness, anxiety, sedation). In conclusion, the contribution of MDMA pharmacokinetics following 1.4 mg/kg MDMA to the gender differences observed in drug effects appears to be negligible or even null. In contrast, 5-HTTLPR and COMT val158met genotypes play a major role.

Funding: This study was supported by NIDA grant 5R01BA017987-01, by grants from DIUE de la Generalitat de Catalunya (2009 SGR 718), Spanish Network on Addiction Disorders grant (FIS-RTA RD06/0001/1009), and Ministerio de Educación y Ciencia grant (MICINN FI09/00355), and the support of ISCIII-FIS-CAIBER CAI08/01/0024. R. Pardo-Lozano is a recipient of a fellowship Rio Hortega (FIS CM08/00051). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2012 Pardo-Lozano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Since the contributing factors for the observation of gender differences in MDMA induced effects have not been satisfactorily determined, and that there are no previously published reports about the influence of CYP2D6, COMT val158met, and 5-HTTLPR genotypes on its pharmacokinetics and pharmacodynamics, the present study will examine both the impact of gender and genetics on the clinical pharmacology of MDMA.

The main metabolic pathway of MDMA in humans is its O-demethylenation to 3,4-dihydroxymethamphetamine (HHMA) by the CYP2D6, followed by the O-methylation of HHMA to 4-hydroxy-3-methoxymethamphetamine (HMMA) by COMT. CYP2D6 displays a large genetic variability determined by the number of functional alleles (FA). In 70–80% of Caucasians the most prevalent phenotype of CYP2D6 is the extensive metabolizer (EM) [16] . The clinical implications derived from CYP2D6 genetic polymorphisms are a large variability of MDMA plasma concentrations and, as a consequence, a difference in drug response, adverse effects [12] , and the bioactivation of MDMA to putative neurotoxic species in humans [17] . MDMA is also a potent inhibitor of CYP2D6 through the formation of a metabolic inhibitory complex [18] – [21] . This is of relevance since MDMA can temporarily (approximately ten days) convert EM subjects to apparently poor metabolizers (PM) [22] , [23] thus increasing the risk of toxicity of other substances whose disposition is regulated by CYP2D6 [16] .

MDMA acts as an indirect serotonin (5-HT), norepinephrine (NE), and dopamine (DA) agonist. Disposition of 5-HT and DA at the synaptic cleft is modulated by the 5-HTT and the COMT enzyme, respectively [13] . The most common genetic variation of 5-HTT is the gene-linked polymorphic region (5-HTTLPR) which presents two variants: the long (l) one which results in a higher serotonin transporter mRNA transcription, and the short (s) one which causes lower transcription [14] . The COMT gene displays a functional polymorphism at codon 158 producing a valine (val) to methionine (met) substitution (Val158Met, rs4680) resulting in three genotypes (val/val, val/met, and met/met). Individuals with the met allele have a lower enzyme activity which leads to higher levels of extracellular dopamine [15] .

It has also been postulated that the genetic polymorphisms of the serotonin transporter (5-HTT), catechol-O-methyltransferase (COMT), and cytochrome P450 2D6 (CYP2D6), are implicated in MDMA pharmacology as they may modulate both its pharmacodynamics and pharmacokinetics in humans [11] , [12] . However, these genotypes have not been examined in MDMA clinical experimental pharmacology studies.

Ecstasy (±3,4-methylenedioxymethamphetamine, MDMA) is a synthetic psychostimulant derived from phenylethylamine and the third most widely consumed illegal drug in the world [1] . This increase in abuse has become a growing public health concern since MDMA can produce acute toxicity and fatal episodes [2] . In addition, its chronic consumption is associated with neurocognitive deficits and increased psychopathology prevalence [3] – [6] . Several studies suggest that women are more vulnerable to drug abuse than males [7] . In some Western countries, female users present higher rates of consumption and dependence on ecstasy than men [1] and MDMA has been shown to produce stronger effects in them [8] , [9] . Differences in drug disposition may contribute to this observation; nevertheless, the only pharmacokinetic study published to date does not have sufficient sample size to address this issue [10] .

To study gender differences with respect to pharmacokinetic (C max , AUC, T max, T 1/2 , ratio MDMA/HMMA), physiological, and subjective effect parameters (E max , AUC, T max ), ANCOVA models were used including both gender and weight-adjusted dose as independent variables. In addition, the possible effect of the COMT val158met and CYP2D6 genotypes on the plasma concentration of MDMA, MDA, HHMA, and HMA was analyzed using separate ANCOVA models for genotypes as well as gender and weight-adjusted dose as further independent variables. Concerning pharmacodynamic variables, separate ANCOVA models, including gender and weight-adjusted dose, were applied to study the role of COMT val158met and 5-HTTLPR, respectively. Due to the small sample size, instead of including the respective interaction terms in the ANCOVA models, separate nonparametric Wilcoxon tests comparing males with females were carried out for each genotype. Differences were considered to be significant if p<0.05. Statistical analyses were performed with the statistical software packages SPSS 12.0 for Windows (SPSS, Inc., 2003) and R, version 2.13.1 (The R Foundation for Statistical Computing). Data resulting from the ANCOVA model are presented with the p value and the 95% confidence interval of the mean difference between genders or genotypes.

A description of both male and female volunteers is given with means, standard deviations, and ranges (min and max values). Values of the peak plasma concentration (C max ) of MDMA and its metabolites, and the time to reach Cmax (T max ), were obtained directly from the plasma concentration-time profiles of MDMA, MDA, HHMA, and HMA. Area under the concentration-time curve values (AUC) was calculated using the trapezoidal rule. Absorption and elimination rate constant (K a and K e , respectively) were estimated by log-linear regression of initial and four terminal data points. Values from physiological measures and subjective variables were transformed to differences from baseline and their peak effect (E max ), T max and AUC were calculated from effect-time profiles. The χ 2 -square test was used to check for the Hardy-Weinberg equilibrium.

The study was approved by the local Ethics Committee (CEIC-Parc de Salut Mar), authorized by the Spanish Medicine Agency (AEMPS n°. 04–0013) of the Spanish Ministry of Health, and was conducted in accordance with the Declaration of Helsinki (Edinburgh, 2000) and good clinical practices. The study was registered at ClinicalTrials.gov (NCT01447472). Subjects gave their written informed consent before inclusion in the study.

Subjective effects were measured using a set of visual analogue scales (VAS), the Addiction Research Center Inventory (ARCI), and the Evaluation of the Subjective Effects of Substances with Abuse Potential (VESSPA) questionnaire at baseline and at 0.33*, 0.66*, 1, 1.5*, 2, 3, 4, 5, 6, 8, 10, 12, and 24 h (* only VAS) after MDMA administration. VAS. Twenty one 100-mm VAS labeled with different adjectives marked at opposite ends with “not at all” and “extremely” were administered [29] . ARCI. A Spanish validated version of a 49-item short form of ARCI was used [29] , [30] . VESSPA is a validated questionnaire measuring MDMA-induced changes in subjective variables [30] .

Readings of non-invasive systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), oral temperature (OT), pupil diameter (PD), and esophoria (ESO) were taken 15 minutes prior to drug administration, at baseline (time 0), and at 0.33, 0.66, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, and 24 hours after drug administration. SBP, DBP, HR, and OT were recorded using a Carescape™ V100 monitor (GE Healthcare. Milwaukee, WI). Pupil diameter was calculated using a pupil gauge (Haab scale). The Maddox-wing device (AM, Clement Clark, London, U.K) was used to measure the balance of extraocular muscles and quantify exophoria and esophoria (MDMA effect) [25] . For safety reasons, ECG was continuously monitored during the first 12 h with a Dash® 3000 patient monitor (GE Healthcare).

Genomic DNA was extracted from the peripheral blood leukocytes of participants using the Flexi Gene DNA kit (Qiagen Iberia, S.L., Spain). The COMT val108/158met and 5-HTTLPR genotyping was performed using polymerase chain reaction (PCR) as previously described [27] . The CYP2D6 genotypes were determined using the PHARMAchip™ DNA array (Progenika Biopharma, Derio, Spain) [28] . Subjects were split according to genotype and associated functionality (CYP2D6, carrying 1 or 2 FA; COMT val158met, carrying the val/val or met/* alleles; and 5-HTTLPR, carrying the l/* or s/s alleles) in order to have equilibrated groups for comparisons, considering genotypes and genders separately.

For determination of MDMA and metabolites, blood samples (8 mL) were collected at pre-dose, and at 0.33, 0.66, 1, 1.5, 2, 4, 6, 8, 10, 12, 25, and 48 hours after MDMA administration. Blood was collected in heparinized tubes and then centrifuged at 4°C for 10 min. The resulting four 1-mL plasma aliquots were stored at −20°C until analysis. MDMA, HMMA MDA (3,4-methylenedioxyamphetamine), and HMA (3-methoxy-4-hydroxyamphetamine) were analyzed following a previously reported method based on solid-liquid extraction and gas chromatography–mass spectrometry (GC/MS) [26] .

Doses of MDMA were chosen according to data from previous studies [25] , [21] and were within the range of the recreational doses reported for a single tablet of ecstasy. For the security of the volunteers, mainly the women, a 1.4 mg/kg of MDMA was administered (range, 75–100 mg). (R,S)-MDMA was supplied by the Spanish Ministry of Health and prepared in white, opaque, soft gelatin capsules by the Pharmacy Department of the Hospital del Mar (Barcelona, Spain).

Negative results were mandatory for participation in the session. Single doses of medication for symptomatic treatment (e.g. acetaminophen for headache) were accepted up to the week preceding the trial. An indwelling catheter was inserted into a subcutaneous vein in the forearm of the non-dominant arm to obtain blood samples. Thereafter, the subjects remained seated in a quiet room. Drug administration commenced at 8∶30 a.m. with 250 mL of water, and a light breakfast was provided 2 h later. Tobacco smoking was permitted after lunch (6 h after MDMA administration). A psychiatrist evaluated volunteers 10 hours after MDMA administration. As the experimental session lasted 25 hours, subjects were admitted to the Clinical Research Unit for the night. Adverse events and concomitant medications were recorded.

The design was open as the primary outcomes measured (pharmacokinetics) were objective. Subjects participated as outpatients, women during early follicular phase, in a session that began at 7∶30 a.m. in fasting conditions. Participants were requested to refrain from consuming any illicit drug of abuse for 2 weeks before the experimental session and were asked to follow a xanthine-free diet and alcohol-free ingestion 48 and 24 hours, respectively, prior to the beginning of each session. Before drug administration, urine samples were collected for a drug screen (opiates, cocaine metabolite, amphetamine, methamphetamine, cannabinoids, and phencyclidine) by a quick on-site test (Instant-View; ASD Inc., Poway, California, USA), and for pregnancy testing in females.

Subjects were recruited by word of mouth and gave their written informed consent before inclusion in the study. Inclusion criteria were: healthy male and female adults, the recreational use of MDMA on at least ten occasions (two in the previous year), and the EM phenotype for CYP2D6 activity determined using dextromethorphan as a selective probe drug [23] . Women had to present a regular menstrual cycle and not be taking oral contraceptives. Exclusion criteria included daily consumption >20 cigarettes and >4 standard units of ethanol in men (>2 in women), and regular ingestion of medication in the month preceding the study. Eligible subjects underwent a psychiatric interview (Psychiatric Research Interview for Substance and Mental Disorders –PRISM) [24] to exclude the presence of major psychiatric disorders, history of abuse or drug dependence (except for nicotine dependence), and psychiatric adverse reactions after MDMA consumption. To confirm health status, volunteers were interviewed by a physician and underwent a general physical examination, routine laboratory tests, urinalysis, and a 12-lead electrocardiogram (ECG). All subjects were informed about the possible adverse effects during the study and were financially compensated for any inconvenience derived from their participation.

*p<0.05, **p<0.01. Graph A corresponds to gender differences in OT, graph B corresponds to differences in OT as a function of 5-HTTLPR polymorphisms (l/l n = 11 vs. s/s n = 9). Subjects l/s (n = 7) are not represented for graph clarity, but data almost fully overlaps with the s/s trace.

MDMA produced the well-known physiological and subjective effects described in the literature [29] , [21] . It increased BP, HR, OT, PD, ESO and induced euphoria, stimulation, and well-being. None of participants required specific therapy or special care due to adverse events.

Figure 1. Plasma Concentrations of MDMA, HMMA, MDA, and HMA in both genders (mean ± standard error of the mean, SEM; for MDMA and HMMA: women n = 11 vs. men n = 15; for MDA and HMA: women n = 11 vs. men n = 12).

No major gender differences were found for most of the pharmacokinetic parameters evaluated for MDMA and its metabolites ( Table 3 and Figure 1 ). The only significant differences observed concerned MDMA plasma clearance (Cl) and the MDMA vs. HMMA AUC 0–12 h ratio. Men presented a higher Cl compared to women (p = 0.001, 95% CI: 6.13–21.08); and women displayed a higher MDMA vs. HMMA AUC 0–12 h ratio (p = 0.027, 95% CI: 0.14–2.12) than men.

Due to technical problems during analysis and/or not enough sample volume for complete analysis, the data available from pharmacokinetics and COMT genotype were for fewer subjects than for pharmacodynamics ( Tables 3, 3b , 4 , 5 ).

The subjects included were tobacco smokers (10 W, 11 M), and had previous experience using alcohol (all 27 subjects), cannabis (8 W, 14 M), cocaine (6 W, 13 M), and gammahydroxybutirate (GHB, 4 M). In Table 1 anthropometric data and the oral and weight-adjusted MDMA doses are summarized according to gender and genotypes . Weight, height, and total MDMA dose administered were higher in men than in women, but body mass index was similar in both genders. According to genotypes, few differences were observed in MDMA doses in the distribution of gender and anthropometric data among the subgroups, age, however, varied in the 5-HTTLPR genotype subgroups. Gender, genotypes, and the MDMA doses administered for each participant are specified in Table 2 . The COMT (p = 0.44) but not the 5-HTTLPR (p = 0.018) genotype fulfill the Hardy-Weinberg equilibrium.

A total of 45 Caucasian subjects [26 women (W), 19 men (M)] were recruited, but only 27 (12 W, 15 M) were included and completed the study. The reasons for the volunteers’ exclusion (n = 18) were: 12 W (10 having previous mental disorders [6 abuse of/dependence on drugs of abuse; 4 affective disorders]), 1 CYP2D6 PM phenotype, and 1 due to a positive drug screen test for cannabis and cocaine); and 3 M (2 CYP2D6 PM phenotype, and 1 with criteria of drug dependence). There were 3 withdrawals (2 W, 1 M) before the experimental session due to personal reasons.

Discussion

To our knowledge, this is the first study on the human pharmacology of MDMA that reports differences in pharmacokinetics and physiological-subjective effects, taking into account gender and genetics, after the administration of doses compatible with its recreational consumption. We have confirmed that there are marked gender differences in MDMA pharmacology: women experienced heightened physiological effects, in particular, cardiovascular ones. They also reported greater negative subjective effects (e.g. dizziness, depression/sadness, and sedation). These observations were not related to MDMA or MDA pharmacokinetics as no major differences in their metabolic disposition at the dose studied were reported between genders or studied genotypes. In the studied population of extensive metabolizers the CYP2D6 genotype was only relevant in the plasmatic concentrations of HMMA depending on the carried number of functional alleles. Furthermore, gender and COMT val158met and 5-HTTLPR polymorphisms played a major role in the physiological and subjective MDMA induced effects. Genotypes corresponding to protein high functionality (e.g. 5-HTTLPR l/* genotype or COMTval158met val/val genotype) were associated with heightened cardiovascular effects.

Pharmacokinetics The main findings are that women, when they received similar weight-adjusted doses as men (1.4 mg/kg, but at a significantly lower total dose, 80 mg vs. 95 mg), reached similar plasma concentrations of MDMA and metabolites. Our results partially differ from those of Kolbrich et al [10] where a multiethnic group of 17 subjects (6 females) received low (1.0 mg/kg) and high (1.6 mg/kg) oral MDMA doses in a double-blind, randomized, placebo-controlled study. While our results are similar to those reported by Kolbrich et al [10] at the high dose as no gender differences were observed; in contrast, gender differences were observed with the lower dose. This may be due to the fact that at lower MDMA doses the impact of CYP2D6 autoinhibition in its metabolic disposition is less relevant than at the higher ones. Secondly, discrepancies observed between our study and Kolbrich´s work could be due to differences in the power of the sample size (n = 27 vs. n = 17), genetic polymorphisms of CYP2D6, COMT, and 5-HTT (not measured in Kolbrich’s study), and ethnicity that might have influenced the activity of these proteins [31]–[33]. MDMA. Similar plasma concentrations in men and women are consistent with Kolbrich´s results at high dose [10]. Furthermore, our results in K a and Vd are also in agreement with the literature [18], [10]. From a metabolic point of view, although oral doses assayed reached the same level of CYP2D6 inhibition, men 80% and women 86% (p = 0.15) [18], the slightly higher MDMA concentrations in women are most probably related to a lower CYP2D6 baseline activity than men [22], and to a lower plasmatic MDMA Cl. According to the elimination parameters, men presented a significantly higher MDMA Cl than women, but no gender differences were observed in relation to K e and T 1/2 . Variations in Cl, although not determinant to generate gender differences in MDMA plasma concentrations, could be due to the fact that the Cl of drugs is generally higher in men than in women (25%) [34], [22], [10]. In addition, the higher baseline CYP2D6 activity in men would imply, despite the autoinhibition of the enzyme, a greater capacity to clear the drug [22]. HMMA. Our results show that HMMA formation is conditioned more by CYP2D6 polymorphism than by COMT val158met. A higher CYP2D6 enzymatic activity (carriers of 2 FA) is determinant in HMMA plasma concentrations. In contrast, the different enzymatic activity of COMT val158met genotype may partially explain the inter-individual variability in susceptibility to MDMA-induced neurotoxicity [17], [20], [35]. Finally, gender does not appear to be relevant in HMMA formation, although men tend to present greater concentrations because of the higher activities of CYP2D6 and COMT [22], [36], as reflected in the lower MDMA vs. HMMA AUC 0-12 h ratio. CYP2D6 and COMT genotypes have been recently associated with lower sodium in plasma and/or higher cortisol levels among MDMA users [37], [38]. MDA. Similar plasma concentrations in men and women are consistent with Kolbrich´s results at the 1.6 mg/kg dose [10]. Slightly higher concentrations of MDA observed in women (Table 3) could be due to the lower CYP2D6 activity that implies both a higher availability of substrate (MDMA) for N-demethylation to MDA and a simple accumulation of MDA, since the O-demethylenation pathway from MDA to HHA is also regulated by CYP2D6 [21].

Physiological Effects In the absence of pharmacokinetic differences between the genders, the most important findings in our study were that female gender and the 5-HTTLPR and the COMT val158met polymorphisms play a major role in modulating the physiological and subjective effects of MDMA. The main results show that women presented more intense effects in SBP, HR, and OT than men, while subjects with high functionality in 5-HTTLPR (l/* alleles) or COMT val158met (val/val alleles) genotypes experienced increased cardiovascular effects. The MDMA-induced cardiovascular effects are mainly due to the release of NE and 5-HT [39], [40]. Gender differences in the 5-HT neurotransmission system, which is modulated by sex steroids, could have contributed to our results. Women exhibited an enhanced serotonergic function in relation to men [15], [41]. On the other hand, men have larger reserves of 5-HT, and, therefore, more extensive disturbances in 5-HT synthesis/transmission may be required to respond to MDMA [42]. Genetics determined blood pressure (BP) and HR effects. Our results are plausible since MDMA promotes 5-HT release through the translocation of 5-HTT and, as a result, the availability of 5-HT in the synapse will be greater in the l/* carriers because they present higher expression of 5-HTT in relation to the s/s carriers. Furthermore, results of BP according to COMT val158met genotype are consistent with the literature [43], and independent of BP results regarding 5-HTTLPR genotype since the proportion of subjects with l/* and s/s alleles in val/val and met/* groups was similar. MDMA-induced effects on HR are determined by female gender and 5-HTTLPR genotype. However, gender might be more important than genetics; in fact, women showed higher HR increases than men in all four subgroups of genotypes (no differences in HR increase were observed between women carrying l/* and s/s allelic-variants, results not shown). The results on HR are clinically relevant because the mean 20 beats/min (bpm) HR increase developed into tachycardia in some cases. Furthermore, HR increase would be greater in a real context of drug consumption than that observed in our laboratory controlled study because consumers dance for hours under stressful conditions (loud music and high ambient temperature), and repeated doses may be taken per session. Globally, this would worsen observations made in the experimental setting. Naturalistic studies suggest that the HR of ecstasy users easily reaches 100 bpm. [44], [45]. Values that are comparable with those observed during physical exercise of variable intensity in healthy people [46], [47]. The modest increase observed in OT, which is well-documented, seems to be influenced by gender, although the contribution of the 5-HTTLPR polymorphism should not be completely discarded [5], [48], [30].The sexual dimorphism in the 5-HT system (previously discussed) may explain gender differences. When comparing OT values of l/l vs. s/s carriers there were differences in the AUC 0-8 h (p = 0.015) (Figure 2). The increase of OT observed between 8–12 h post-administration of MDMA could be simply physiological (circadian rhythm). In the work of Liechti et al [8] whilst no gender differences were reported in DBP, HR, and peripheral body temperature they were, however, observed in SBP where men displayed significantly higher values. Women showed a significant increase in SBP and DBP compared to placebo. Discrepant results between our study and Lietchís could be due to the fact that the volunteers of Liechti and colleagues were drug naïve, received similar or higher MDMA doses (1.35–1.8 mg/kg) than ours, and genetic polymorphisms were not considered.