Because carvedilol has been shown to prevent MDMA‐induced hyperthermia and rhabdomyolysis in rats ( Sprague et al ., 2005 ) and the cardiostimulant response to cocaine in humans ( Sofuoglu et al ., 2000a ), we evaluated the effects of carvedilol on the cardiovascular and hyperthermic response to MDMA in healthy subjects.

Statistical analysis. Values were transformed to differences from baseline. The maximal effect (E max ) values were determined for repeated measures and analysed by two‐way General Linear Models repeated‐measures anova with the two drug factors MDMA (MDMA vs. placebo) and carvedilol (carvedilol vs. placebo) using STATISTICA 6.0 software (StatSoft, Tulsa, OK, USA). Tukey's post hoc comparisons were performed based on significant main effects or interactions. Additional anovas were performed, with drug order as an additional factor, to exclude carry‐over effects. The criterion for significance was P < 0.05. A sample‐size estimation based on previous data ( Hysek et al ., 2011; 2012 ) showed that eight subjects would be needed to detect a relevant change in the primary study outcome with 80% power using a within‐subjects study design.

Pharmacokinetic analysis. The data for the plasma concentrations of MDMA and MDA were analysed using non‐compartmental methods. C max and time to C max were obtained directly from the concentration–time curves of the observed values. The area under the plasma concentration–time curve (AUC) 0–6 h was calculated using the linear trapezoidal rule. Plasma concentrations were only determined up to 6 h after MDMA administration because the aim of the study was to assess potential changes in plasma levels of MDMA during the time of the pharmacodynamic effects of MDMA.

Samples of plasma for the determination of MDMA and ±3,4‐methylenedioxyamphetamine (MDA), the active metabolite of MDMA, were collected 1 h before and 0 (just before), 0.33, 0.66, 1, 1.5, 2, 2.5, 3, 4 and 6 h after MDMA or placebo administration. The plasma concentrations of MDMA and MDA were determined using HPLC coupled to tandem MS as described previously ( Hysek et al ., 2012 ).

Plasma catecholamines. Blood samples to determine the concentrations of NA and adrenaline were taken 1 h before and 1 and 2 h after MDMA or placebo administration. All of the blood samples were collected on ice and centrifuged within 10 min at 4°C. The plasma was then stored at −20°C until analysis. The plasma levels of free catecholamines (NA and adrenaline) were determined by HPLC with an electrochemical detector as described previously ( Hysek et al ., 2011 ).

Vital signs. Vital signs were assessed repeatedly 1 h before and 0, 0.33, 0.66, 1, 1.5, 2, 2.5, 3, 4, 5 and 6 h after MDMA or placebo administration. Heart rate, systolic blood pressure and diastolic blood pressure were measured using an OMRON M7 blood pressure monitor (Omron Healthcare Europe, Hoofddorp, The Netherlands) in the dominant arm after a resting time of 5 min. Measures were taken twice per time point with an interval of 1 min, and the average was used for analysis. Core (tympanic) temperature was assessed using a GENIUS 2 ear thermometer (Tyco Healthcare Group, Watertown, NY, USA).

±MDMA hydrochloride (Lipomed AG, Arlesheim, Switzerland) was prepared as gelatine capsules (100 and 25 mg of the salt). Identical placebo (lactose) capsules were prepared. MDMA was administered in a single oral dose of 125 mg, corresponding to a dose of 1.93 ± 0.36 mg·kg −1 body weight. Carvedilol tablets (50 mg, Dilatrend, Roche Pharma AG, Basel, Switzerland) were encapsulated within opaque gelatine capsules, and identical placebo (lactose) capsules were prepared. An oral dose of carvedilol (50 mg) was used that has previously been shown to attenuate the smoked cocaine‐induced increases in heart rate and blood pressure in humans ( Sofuoglu et al ., 2000a ). At this dose, carvedilol is expected to inhibit both α 1 ‐ and β‐adrenoceptors ( Tham et al ., 1995 ; Sofuoglu et al ., 2000a ). Carvedilol or placebo was administered 1 h before MDMA or placebo administration so that the maximal plasma concentration (C max ) of carvedilol was reached ( Morgan, 1994 ) shortly before the C max of MDMA occurred. Oral medication administration was supervised by study personnel.

Sixteen healthy subjects (eight men, eight women) with a mean (SD) age of 24.2 (2.2) years and a mean body weight of 67 (13) kg were recruited from the university campus. The allocation to treatment order was performed by drawing from blocks of eight different balanced drug treatment sequences by two pharmacists not involved in the study. Each code was stored in a sealed envelope until the termination of the study. Data from all 16 subjects were available for the final analysis. The exclusion criteria included the following: (i) age <18 or >45 years; (ii) pregnancy determined by a urine test before each test session; (iii) body mass index <18.5 kg·m −2 or >25 kg·m −2 ; (iv) personal or family (first‐degree relative) history of psychiatric disorder [determined by the structured clinical interview for Axis I and Axis II disorders according to the Diagnostic and Statistical Manual of Mental Disorders , 4th edition ( Wittchen et al ., 1997 ) supplemented by the SCL‐90‐R Symptom Checklist ( Derogatis et al ., 1976 ; Schmitz et al ., 2000 )]; (v) regular use of medications; (vi) chronic or acute physical illness assessed by physical examination, electrocardiogram, standard haematology and chemical blood analyses; (vii) smoking more than seven cigarettes per day; (viii) a lifetime history of using illicit drugs more than five times, with the exception of cannabis; (ix) illicit drug use within the last 2 months; and (x) illicit drug use during the study, determined by urine tests conducted before the test sessions using TRIAGE 8 (Biosite, San Diego, CA, USA). The subjects were asked to abstain from excessive alcohol consumption between test sessions and limit alcohol use to one glass on the day before each test session. All of the subjects were non‐smokers. All of the subjects, with the exception of one, had previously used cannabis. Four subjects reported using illicit drugs, in which three subjects had tried amphetamine once and one had tried ecstasy once and amphetamine three times. All of the subjects were phenotyped for cytochrome P450 (CYP) 2D6 activity using dextromethorphan as the probe drug. Nine extensive, six intermediate and one poor CYP2D6 metabolizer were identified in the study. The female subjects were investigated during the follicular phase (day 2–14) of their menstrual cycle when the reactivity to amphetamines is expected to be similar to men ( White et al ., 2002 ). All of the subjects provided their written informed consent before participating in the study, and they were paid for their participation.

The subjects completed a screening visit, four test sessions and an end‐of‐study visit. The test sessions were conducted in a quiet hospital research ward with no more than two research subjects present per session. The mean (SD) room temperature was 23.3°C (0.7°C). At the beginning of each test session, an indwelling i.v. catheter was placed in the antecubital vein for blood sampling. Carvedilol (50 mg) or placebo was administered at 8 h 00 min. MDMA (125 mg) or placebo was administered at 9 h 00 min. A standardized lunch was served at 12 h 00 min, and the subjects were sent home at 15 h 00 min.

We used a double‐blind, double‐dummy placebo‐controlled, randomized, crossover study design with four experiential conditions (placebo‐placebo, carvedilol‐placebo, placebo‐MDMA and carvedilol‐MDMA) in a balanced order. The washout periods between the sessions were at least 10 days. The study was conducted at the University Hospital of Basel in accordance with the Declaration of Helsinki and International Conference on Harmonization Guidelines on Good Clinical Practice and approved by the Ethics Committee of the Canton of Basel, Switzerland, and Swiss Agency for Therapeutic Products (Swissmedic). The use of MDMA in healthy subjects was authorized by the Swiss Federal Office of Public Health. The study was registered at ClinicalTrials.gov (NCT01270672). The reduction in the MDMA‐induced increase in blood pressure by carvedilol was the predefined primary outcome of this clinical trial.

Pharmacokinetics (A) and pharmacokinetic–pharmacodynamic relationship (B). Carvedilol non‐significantly increased the exposure to MDMA and MDA (A). The values are expressed as mean ± SEM in 16 subjects. Carvedilol was administered at t =−1 h. MDMA was administered at t = 0 h. MDMA effects on systolic blood pressure plotted against MDMA plasma concentration (B). The values are expressed as means of the changes from baseline in 16 subjects. The time of sampling is noted next to each point in min or h after MDMA administration. Carvedilol produced a downward and rightward shift of the concentration‐blood pressure response curve of MDMA (B).

The decrease in the cardiovascular and thermogenic response to MDMA after carvedilol pretreatment was not attributable to a pharmacokinetic interaction between carvedilol and MDMA. Carvedilol did not affect the C max or AUC 0–6 h of MDMA or MDA ( Table 2 and Figure 4A ). The effect of MDMA on blood pressure in relation to the plasma concentration of MDMA is illustrated by the hysteresis curves in Figure 4B . Carvedilol produced a pronounced downward shift in the E max of the systolic pressure response to MDMA and a rightward shift in the C max of MDMA in the concentration‐effect curve ( Figure 4B ). The pharmacokinetic parameters of MDMA did not depend on CYP2D6 phenotype or the dextromethorphan : dextrorphan ratio in our small study sample.

MDMA increased the total adverse effect score on the List of Complaints, both 3 and 24 h after drug administration compared with placebo ( Table 1 ). Carvedilol had no effect on the MDMA‐induced increase in the total score. However, fewer subjects reported palpitations and hot flushes after carvedilol and MDMA co‐treatment ( n = 2 and n = 2, respectively) compared with MDMA treatment alone ( n = 6 and n = 5, respectively). Frequent adverse effects of MDMA and carvedilol‐MDMA were thirst ( n = 10 and n = 11, respectively), lack of appetite ( n = 9 and n = 7, respectively), sweating ( n = 8 and n = 7, respectively), restlessness ( n = 7 and n = 5, respectively) and bruxism ( n = 7 and n = 7, respectively). No severe adverse effects were reported.

Time course of subjective drug effects on Visual Analogue Scale ratings. MDMA increased scores on all scales. Carvedilol did not affect any of the MDMA‐induced increases in Visual Analogue Scale ratings. Carvedilol was administered at t =−1 h. MDMA was administered at t = 0 h. The values are expressed as mean ± SEM percentage of maximal values in 16 subjects.

Effects of carvedilol and MDMA on circulating catecholamines. Carvedilol alone increased the plasma levels of noradrenaline (A) compared with placebo. MDMA alone produced a similar non‐significant increase in noradrenaline. Co‐administration of carvedilol and MDMA increased the concentrations of circulating noradrenaline (A) and adrenaline (B) compared with placebo. The values are expressed as mean ± SEM changes from baseline in 16 subjects.

Physiological effects of carvedilol and MDMA. Carvedilol reduced MDMA‐induced elevations in systolic (A) and diastolic (B) blood pressure, heart rate (C) and body temperature (D). Carvedilol was administered at t =−1 h. MDMA was administered at t = 0 h. The values are expressed as mean ± SEM changes from baseline in 16 subjects.

MDMA significantly increased blood pressure, heart rate and body temperature compared with placebo ( Table 1 and Figure 1 ). Carvedilol significantly inhibited the MDMA‐induced increases in blood pressure, heart rate and body temperature ( Table 1 and Figure 1 ). Carvedilol alone also moderately lowered blood pressure and heart rate compared with placebo. The effect of carvedilol on the pressure and hyperthermic response to MDMA was more pronounced than the effect of carvedilol alone compared with placebo, corroborated by the significant carvedilol × MDMA interaction in the two‐way anova. Carvedilol alone increased the plasma concentration of NA compared with placebo. MDMA also tended to increase circulating NA compared with placebo, but the effect was not significant. The co‐administration of carvedilol and MDMA significantly increased both circulating adrenaline and NA ( Table 1 and Figure 2 ).

Discussion

The α 1 ‐ and β 1,2,3 ‐adrenoceptor antagonist carvedilol reduced the cardiostimulant and hyperthermic response to MDMA in healthy subjects. Carvedilol similarly reduced MDMA‐induced hyperthermia in rats (Sprague et al., 2004a; 2005). Additional studies in rats and mice showed that the transient and early hypothermic effect of MDMA are enhanced by blocking α 1 ‐receptors (Bexis and Docherty, 2008), whereas the late hyperthermic response to MDMA is blunted by blocking β 3 ‐receptors (Sprague et al., 2004a; Bexis and Docherty, 2008). Moreover, α 1 ‐receptors mediate peripheral vasoconstriction and heat dissipation, which are impaired by MDMA (Pedersen and Blessing, 2001). Administration of β 1,2 ‐receptor antagonists had no effect on the thermogenic response to MDMA in rats (Sprague et al., 2005) or humans (Hysek et al., 2010). These data suggest a role for both α 1 ‐ and β 3 ‐receptors in MDMA‐induced hyperthermia. Carvedilol should be considered for the treatment of hyperthermia associated with ecstasy use because it effectively reduced MDMA‐induced hyperthermia in both animals and humans and reversed established hyperthermia in rats (Sprague et al., 2005).

In addition to adrenoceptors, other sites have been implicated in stimulant‐induced hyperthermia. MDMA primarily induces the release of 5‐HT, NA and dopamine through their respective presynaptic monoamine transporters (Rudnick and Wall, 1992; Rothman et al., 2001; Verrico et al., 2007). MDMA binds to α 2 ‐adrenoceptors, 5‐HT 2A ‐receptors, H 1 ‐histamine and trace amine‐1 receptors (Battaglia et al., 1988; Bunzow et al., 2001). The 5‐HT 2A ‐receptor antagonist ketanserin inhibited the thermogenic effects of MDMA in rats (Shioda et al., 2008), mice (Di Cara et al., 2011) and humans (Liechti et al., 2000). In both mice and humans, ketanserin administered alone lowered body temperature compared with vehicle and placebo, respectively (Liechti et al., 2000; Di Cara et al., 2011). Thus, no interactive effect of ketanserin and MDMA on body temperature was observed, in contrast to carvedilol and MDMA in the present study. Furthermore, ketanserin has α 1 ‐adrenoceptor‐blocking properties (Brogden and Sorkin, 1990), and its ability to reduce MDMA‐associated hyperthermia may be explained, at least partially, by α 1 ‐receptor antagonism. A recent study showed that mice that lack trace amine‐1 receptors did not exhibit the early hypothermic response to MDMA, indicating a role for this receptor in the early hypothermic effects of MDMA (Di Cara et al., 2011). D 1 ‐ and D 2 ‐dopamine receptors, α 2 ‐adrenoceptors and 5‐HT 1 ‐receptors do not appear to be involved in the effects of MDMA on body temperature, demonstrated by preclinical (Docherty and Green, 2010; Di Cara et al., 2011) and clinical (Liechti and Vollenweider, 2000; Hysek et al., 2010; 2012) studies.

Recreational users of ecstasy report subjective increases in body temperature, sweating and hot flushes (Parrott et al., 2008). Hot flushes and sweating were also reported after administration of MDMA in the present and in previous studies (Liechti et al., 2001; Freedman et al., 2005). Carvedilol did not reduce the number of subjects who reported MDMA‐induced subjective sweating but reduced the number of subjects reporting flushes. Interestingly, in another laboratory study, MDMA did not influence the perceptions of warmth and cold but delayed the onset of sweating at a warm ambient temperature along with an MDMA‐induced increase in body temperature (Freedman et al., 2005).

Carvedilol also reduced the cardiostimulant response to MDMA, including blood pressure and heart rate. The α‐ and β‐blockers carvedilol and labetalol have similarly been shown to inhibit the blood pressure response to cocaine in humans (Boehrer et al., 1993; Sofuoglu et al., 2000a,b). Blockade of β‐receptors alone did not reduce the pressure response to cocaine (Ramoska and Sacchetti, 1985) or MDMA (Hysek et al., 2010) in humans and enhanced cocaine‐induced coronary vasoconstriction (Lange et al., 1990). In rats, the blockade of α 1 ‐receptors inhibited both the pressure response and vasoconstriction in isolated vessels in response to cocaine (Mo et al., 1999). The data indicate that dual α,β‐blockers, but not selective β‐blockers, should be used in the treatment of psychostimulant‐associated hypertension and myocardial ischaemia. The data indicate that carvedilol could be useful in the treatment of both psychostimulant‐induced hypertension and hyperthermia.

Circulating catecholamine levels were increased by both MDMA and carvedilol. Plasma adrenaline is mainly derived from the adrenals, whereas plasma NA stems largely from transmitters released by sympathetic nerves and the escape of NA into the circulation (Esler et al., 1990; Eisenhofer et al., 1995). Circulating NA is therefore considered an indicator of sympathetic system activation. We observed a marked increase in plasma NA concentrations after carvedilol administration. This compensatory sympathoadrenal response with enhanced levels of catecholamines has previously been documented after α 1 ‐ or α‐ and β‐adrenoceptor blockade (Omvik et al., 1992; Mazzeo et al., 2001). The MDMA‐induced increase in circulating NA in the present study did not reach statistical significance compared with previous work (Dumont et al., 2009; Hysek et al., 2011; 2012). It is possible that the peak effect was missed because we took only two samples. The catecholamine response was enhanced when MDMA was administered following carvedilol. A similar potentiation of the exercise‐induced increases in plasma catecholamines has been shown following blockade of α 1 ‐adrenoceptors or α‐ and β‐adrenoceptors (Berlin et al., 1993).

Preclinical and clinical studies suggest that NA contributes to the mediation of the subjective effects of MDMA and other psychostimulants (Sofuoglu and Sewell, 2009; Hysek et al., 2011; Newton, 2011). For example, MDMA is more potent in releasing NA than 5‐HT or dopamine from monoamine‐preloaded human embryonic kidney cells transfected with the corresponding human monoamine transporters (Verrico et al., 2007). Additionally, doses of stimulants that produce amphetamine‐type subjective effects in humans correlated with their potency to release NA (Rothman et al., 2001). Furthermore, the NA transporter inhibitor reboxetine attenuated the cardiovascular and subjective response to MDMA in humans, indicating a role for MDMA‐induced transporter‐mediated NA release in the psychostimulant effects of MDMA (Hysek et al., 2011). Similarly, atomoxetine attenuated the effects of amphetamine in humans (Sofuoglu et al., 2009). Clonidine, which blocks the vesicular release of NA, did not affect the psychological effects of MDMA in humans (Hysek et al., 2012). Although these data suggest a role for transporter‐mediated NA release in the psychotropic effects of psychostimulants, how and which postsynaptic adrenoceptors are involved are still unclear. Carvedilol did not alter the subjective effects of MDMA in the present study. Similar to our results, carvedilol and labetalol did not affect the subjective responses to cocaine in humans at doses of cocaine that effectively inhibited the cardiostimulant effects of the drug (Sofuoglu et al., 2000a,b). The available clinical data do not support a critical role for α 1 ‐ and β 1,2,3 ‐receptors in the subjective effects of psychostimulants. Alternatively, the carvedilol concentrations in humans may not have been high enough to produce sufficient adrenoceptor occupancy in the brain. Carvedilol is lipophilic and enters the brain (Elsinga et al., 2005). However, carvedilol is a substrate of the efflux transporter P‐glycoprotein in the blood‐brain barrier (Elsinga et al., 2005; Bachmakov et al., 2006), and P‐glycoprotein activity is known to limit brain exposure to carvedilol (Elsinga et al., 2005).

Preclinical studies indicate that α 1 ‐receptors are involved in the mechanism of action of psychostimulants, including MDMA. For example, pretreatment with the α 1 ‐receptor antagonist prazosin inhibited locomotor stimulation induced by cocaine (Wellman et al., 2002), amphetamine (Vanderschuren et al., 2003) and MDMA (Fantegrossi et al., 2004; Selken and Nichols, 2007) in rats and mice. Additionally, α 1 ‐receptor activation in the ventral tegmental area contributed to the amphetamine‐induced release of dopamine in the nucleus accumbens (Pan et al., 1996). Injection of prazosin directly into the ventral tegmental area also blocked the locomotor response to MDMA in rats (Selken and Nichols, 2007). Furthermore, administration of prazosin in the rat prefrontal cortex also blocked amphetamine‐induced dopamine release in the nucleus accumbens and hyperactivity (Forget et al., 2011). Finally, α 1 ‐adrenoceptor knockout mice do not show increased amphetamine‐induced dopamine release in the nucleus accumbens (Auclair et al., 2002) or behavioural sensitization to amphetamine or cocaine (Drouin et al., 2002). In contrast to α 1 ‐antagonism, the β‐blocker propranolol enhanced both cocaine‐induced locomotion and the cocaine‐induced increase in dopamine in the nucleus accumbens (Harris et al., 1996). Altogether, the preclinical studies indicate that α 1 ‐adrenoceptors, but not β‐receptors, play a role in the hyperlocomotion and dopaminergic neurochemical response to psychostimulants. However, the role of adrenoceptors in the reinforcing effects of psychostimulants is unclear. For example, prazosin reduced the self‐administration of cocaine (Wee et al., 2008) and nicotine (Forget et al., 2011) in rats. In contrast, prazosin had no effect on cocaine self‐administration in rhesus monkeys (Woolverton, 1987). The β‐blocker propranolol also inhibited cocaine self‐administration in rats (Harris et al., 1996). Carvedilol lowered the number of cocaine self‐administrations in humans at a low but not high dose (Sofuoglu et al., 2000a). At low doses, carvedilol preferentially blocks β‐receptors (Tham et al., 1995; Sofuoglu et al., 2000a) and active metabolites of carvedilol may contribute to the β‐ but not the α‐adrenoceptor blocking effects of the drug (Spahn‐Langguth and Schloos, 1996). The antagonism of α 1 ‐adrenoceptors by carvedilol may not have been sufficient in the brain to attenuate the subjective effects of MDMA and we cannot exclude a role for these receptors. The efficacy of carvedilol to reduce cocaine use or abstinence in addicted patients is currently being investigated in ongoing clinical trials [(Sofuoglu and Sewell, 2009) clinicaltrials.gov identifier: NCT00566969 and NCT01171183]. Further trials have investigated the effects of selective α 1 ‐blockers on the acute response to MDMA (NCT01386177) and cocaine (NCT01062945) and abstinence from cocaine use (NCT00880997).

Pharmacokinetic interactions between carvedilol and MDMA need to be considered in the interpretation of the present findings, because both drugs are metabolized by CYP2D6 (Graff et al., 2001; O'Mathuna et al., 2008). We therefore assessed the potential effects of carvedilol on the pharmacokinetics of MDMA. We found that carvedilol non‐significantly increased the plasma exposure to MDMA or MDA. Thus, the reduced haemodynamic and thermogenic effects of MDMA after carvedilol pretreatment did not result from lower plasma levels of MDMA or MDA. We did not assess the plasma concentrations of carvedilol. MDMA inhibits CYP2D6 (O'Mathuna et al., 2008). CYP2D6 inhibition has been shown to increase the exposure to carvedilol but not its pharmacodynamic or adverse effects in humans (Graff et al., 2001).

Our laboratory study has a few limitations. The study design is limited by the use of single doses. We did not use a dose–response study because we did not want to expose the subjects to more than two doses of MDMA in a within‐subject design. However, moderate to highly effective doses of both drugs were selected. The primary goal of the study was to investigate the role of adrenoceptors in the mechanism of action of MDMA in humans. Therefore, the study provides only indirect support for the use of carvedilol in the treatment of stimulant toxicity, in which carvedilol would be administered following the ingestion of ecstasy or other stimulants. Furthermore, the MDMA‐induced increase in body temperature in our study was moderate, and we do not know whether carvedilol would also be effective in cases of severe hyperthermia following ecstasy use. Finally, thyroid function may modulate the thermogenic effects of MDMA (Martin et al., 2007; Sprague et al., 2007) and thyroid function parameters were not assessed in this study.