MDMA

In vivo effects of 3,4-methylenedioxymethamphetamine (MDMA) and its deuterated form in rodents: Drug discrimination and thermoregulation.

This rat study (n=41) compared the hyperthermic side effects of MDMA ( and a deuterium-substituted analog d2-MDMA in rats and found that d2-MDMA produced increases in body temperature that were shorter-lasting and of lower magnitude compared to equivalent doses of MDMA.

Authors

Berquist, M. D.
Fantegrossi, W. E.
Kristensen, J. L.

Published

March 1, 2020

Drug and Alcohol Dependence

individual Study

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Abstract

Background: Recent clinical studies support the use of 3,4-methylenedioxymethamphetamine (MDMA) as an adjunct treatment for post-traumatic stress disorder (PTSD). Despite these promising findings, MDMA administration in controlled settings can increase blood pressure, heart rate, and body temperature. Previous studies indicate that O-demethylated metabolites of MDMA contribute to its adverse effects. As such, limiting the conversion of MDMA to reactive metabolites may mitigate some of its adverse effects and potentially improve its safety profile for therapeutic use.Methods: We compared the interoceptive and hyperthermic effects of a deuterium-substituted form of MDMA (d2-MDMA) to MDMA using rodent drug discrimination and biotelemetry procedures, respectively.Results: Compared to MDMA, d2-MDMA produced full substitution for a 1.5 mg/kg MDMA training stimulus with equal potency and effectiveness in the drug discrimination experiment. In addition, d2-MDMA produced increases in body temperature that were shorter-lasting and of lower magnitude compared to equivalent doses of MDMA. Last, d2-MDMA and MDMA were equally effective in reversing the hypothermic effects of the selective 5-HT2A/2C antagonist ketanserin.Conclusion: These findings indicate that deuterium substitution of hydrogen at the methylenedioxy ring moiety does not impact MDMA’s interoceptive effects, and compared to MDMA, d2-MDMA has less potential for producing hyperthermic effects and likely has similar pharmacodynamic properties. Given that d2-MDMA produces less adverse effects than MDMA, but retains similar desirable effects that are thought to relate to the effective treatment of PTSD, additional investigations into its effects on cardiovascular functioning and pharmacokinetic properties are warranted.

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Research Summary of 'In vivo effects of 3,4-methylenedioxymethamphetamine (MDMA) and its deuterated form in rodents: Drug discrimination and thermoregulation.'

Introduction

Berquist and colleagues situate this work in the context of renewed clinical interest in 3,4-methylenedioxymethamphetamine (MDMA) as an adjunct pharmacotherapy for post-traumatic stress disorder (PTSD). Earlier clinical and preclinical studies have reported persistent symptom reductions after MDMA-assisted therapy and generally acceptable tolerability, but acute adverse effects—most notably increases in blood pressure, heart rate and body temperature—remain a concern, particularly in patients with comorbid cardiovascular or renal disease. Prior laboratory work implicates O-demethylated MDMA metabolites (HHMA and HHA) in some acute and long-term adverse outcomes; these metabolites can be oxidised to reactive species that form neurotoxic adducts and have cardiovascular actions in rodents.

Methods

Analyses included calculation of ED50 values from linear portions of dose–effect curves (normalized 0–100%), percent drug-lever selection, and response rates. Repeated-measures and mixed-model ANOVAs were used to compare dose–effect curves and time courses; Dunnett's tests compared response rates to saline and post hoc Tukey or Šídák tests followed significant omnibus effects. Temperature endpoints from biotelemetry included maximal core temperature and duration above 39 °C (defined as hyperthermic). Statistical significance was set at p < .05.

Results

In the ketanserin pretreatment paradigm, both MDMA and d2-MDMA reversed ketanserin-induced hypothermia across the tested dose range. Time-course analyses showed significant time × treatment interactions and main effects of time for comparisons at each dose level; at the 120-min time point a two-factor ANOVA revealed a main effect of MDMA/dose (F[2,12] = 6.22, p = .01) but no main effect of treatment (MDMA versus d2-MDMA) and no interaction, indicating both compounds were similarly effective at restoring temperature over the session.

Discussion

The investigators caution that larger doses of d2-MDMA might produce equal or greater hyperthermia, so a simple potency difference cannot be fully excluded. They also report that both compounds equivalently reversed ketanserin-induced hypothermia, which the authors take as evidence that deuterium substitution does not substantially alter MDMA's serotonergic pharmacodynamics relevant to thermoregulation. Limitations and next steps acknowledged include the need for direct pharmacodynamic assays (for example, in vitro transporter/receptor binding), pharmacokinetic investigations of parent compound and metabolite formation to confirm reduced O-demethylation, and assessment of cardiovascular effects. The discussion also notes translational considerations: the 1.5 mg/kg MDMA training dose has some correspondence to human dosing ranges used in therapeutic research, supporting the relevance of the discrimination findings.

Conclusion

The authors conclude that d2-MDMA elicits discriminative stimulus effects indistinguishable from MDMA while producing hyperthermic effects of lower magnitude and shorter duration. They suggest that deuterium substitution may improve tolerability and safety by altering metabolic fate without changing desirable pharmacodynamic actions, and recommend further studies of cardiovascular outcomes and pharmacokinetics to evaluate d2-MDMA as a potential alternative to MDMA for therapeutic use.

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INTRODUCTION

3,4-Methylenedioxymethamphetamine (MDMA) has regained clinical attention as an adjunct pharmacotherapy to treat posttraumatic stress disorder (PTSD). The U.S. Food and Drug Administration (FDA) granted Breakthrough Therapy designation to MDMA for treating PTSD on. Indeed, volunteers in phase II clinical trials have experienced persistent reductions in PTSD symptoms following MDMA-assisted therapy sessions, and clinical use of MDMA is considered well tolerated. MDMA is expected to be an FDA-approved medication for PTSD by 2021. Despite the promising findings from these preclinical and clinical studies, a subset of volunteers in clinical trials with MDMA report anxiety, jaw clenching, headaches, and increased sensitivity to cold after MDMA administration. In addition, participants show acute reactions of increased blood pressure, heart rate, and body temperature after MDMA administration in controlled settings, which may have a dampening effect on MDMA's therapeutic utility. Although risks from MDMA use in clinical settings should be distinguished from recreational use (reviewed in, the effects of MDMA on cardiovascular functioning and thermoregulation may be problematic in clients who have comorbid issues, such as cardiovascular disease, renal disease, or psychiatric disorders. Indeed, prolonged traumatic stress increases risk for developing hypertension and cardiovascular disease (reviewed in, perhaps highlighting PTSD patients as particularly susceptible to these adverse effects of MDMA. Moreover, future treatment strategies with MDMA may include more frequent and prolonged administrations (e.g., in treatment-resistant individuals). Many studies show that chronic MDMA exposure is associated with persistent neurochemical and behavioral alterations in experimental animals (e.g.,and humans. There are concerns of MDMA use in clinical settings (e.g.,, and efforts to reduce MDMA's acute adverse effects and risks for developing toxicity, while retaining its desirable therapeutic effects, are of critical importance for maximizing MDMA as a pharmacotherapeutic tool for PTSD and other disorders. Previous studies show that MDMA's ring-opened phase I metabolites, 3,4-dihydroxymethamphetamine (HHMA) and 3,4-dihydroxyamphetamine (HHA; a metabolite of 3,4-methylenedioxyamphetamine) contribute to its acute and long-term adverse effects (e.g.,. In particular, HHMA and HHA can readily oxidize into reactive ortho-quinones that conjugate with glutathione and N-acetylcysteine to form neurotoxic adducts. In addition, subcutaneous administration of HHMA and HHA in rats produces increases in heart rate and blood pressure, which suggests that these metabolites may account for some acute adverse cardiovascular effects after MDMA administration. Deuterium substitution is one strategy to reduce the rate of MDMA metabolism into these phase I metabolites. We have synthesized a deuterated form of MDMA (d2-MDMA) that includes deuterium substitution of hydrogen at the methylenedioxy ring moiety of MDMA (Fig.). Deuterium-carbon bonds are more stable and resistant to cleavage from cytochrome P450 (CYP) enzymes than hydrogencarbon bonds (reviewed in. As such, we predict that d2-MDMA produces smaller concentrations of HHMA and HHA by limiting the rate of O-demethylation of MDMA by CYP enzymes, and, as a result, d2-MDMA may produce less adverse effects following acute administration. In addition to potentially reducing acute adverse effects of MDMA, it is important to confirm that d2-MDMA retains its desirable therapeutic effects.suggest that MDMA's complex releasing effects at monoamine transporters (e.g.,and on neurohormones mediate its desirable subjective effects in clinical studies. Drug discrimination procedures are used in preclinical studies to characterize the subjective effects of psychoactive substances, and it is well known that MDMA has discriminative stimulus effects which rats can be trained to detect (e.g.,. Therefore, we used a two-lever drug discrimination procedure in rats trained to discriminate 1.5 mg/kg MDMA from saline to characterize the discriminative stimulus effects of d2-MDMA. In addition, because MDMA is known to increase body temperature in rodents (e.g.,and in humans in controlled settings, we assessed the hyperthermic effects of d2-MDMA in the present study using two experiments conducted in mice. In the first experiment, core temperature dose-effect curves were generated for d2-MDMA and MDMA using implanted biotelemetry probes. It is also known that MDMA administration attenuates the hypothermic effects of the 5-HT 2A/2C antagonist ketanserin in mice, which suggests a role for 5-HT 2 receptors in mouse thermoregulation. As such, to determine if d2-MDMA has a similar capacity to MDMA in reversing ketanserin-elicited hypothermia and thus provide supportive proof-ofconcept that deuterium substitution does not alter MDMA's pharmacodynamic effects (i.e., effects that are mediated by serotonin 5-HT 2A/2C receptor activation), a separate experiment was conducted in which groups of mice were pretreated with saline or ketanserin and then administered MDMA or d2-MDMA.

DRUGS AND CHEMICALS

Racemic deuterated MDMA hydrochloride was prepared by Sebastian Leth-Petersen (Copenhagen, Denmark) from commercially available CD 2 Cl 2 and 3,4-dihydroxybenzaldehyde, following the procedures outlined in WO2008016677A2 with minor modifications (see Supplemental File 1 for details and H 1 -NMR and C 13 -NMR spectra). Racemic MDMA hydrochloride and ketanserin hydrochloride were provided by the National Institute on Drug Abuse (NIDA) and as a generous gift from Gantt P. Galloway, Ph.D. (Addiction & Pharmacology Research Laboratory, California Pacific Medical Center Research Institute, San Francisco, CA.) All doses are expressed as weight of the salt, were dissolved in 0.9 % physiological saline, and were delivered via intraperitoneal injection (ip) in a 10 ml/kg injection volume (mice) or a 1 ml/kg injection volume (rats).

ANIMALS

Eight male Sprague-Dawley rats (Charles River Laboratories Inc., Wilmington, MA) weighing 422-532 g (16 weeks old) at the start of the experiment were pair housed in polycarbonate cagesin corncob bedding in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility. The animal colony room was maintained at 22 ± 2 °C and 45-50 % humidity with lights set to a 10:14 h light-dark cycle (lights on at 0600 h). Each cage contained a plastic tunnel for enrichment. Rats were placed under a food-restricted diet such that their body weights were reduced to 85-90 % of their free-feeding weights. Rats were fed once daily at least 10 min after their experimental training sessions and were given ad libitum access to tap water while in their home cages. Thirty-three male NIH Swiss mice (Charles River Laboratories Inc., Wilmington, MA) were housed three per cage in polycarbonate cages (15.24 × 25.40 × 12.70 cm) in corncob bedding. Colony rooms were maintained at 22 ± 2 °C and 45-50 % humidity, with lights set to a 12:12-h lightdark cycle (lights on at 0600 h). Mice were given ad libitum feeding of standard rodent chow and tap water prior to any handling. All mice were drug naïve (with the exception of surgical anesthetics for biotelemetry experiment) before testing. All experiments were conducted during the animals' light cycle. All experimental protocols were conducted in accordance with the Guide for the Care and Use of Laboratory Animalsand approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences.

APPARATUS

Eight computer-operated standard rat operant conditioning chambers (ENV-001; MED Associates Inc., St. Albans, VT, USA) were equipped with two retractable levers, a food pellet dispenser, a 28 V overhead house light, a clicker, and a tone generator. Each chamber was housed within a light-and sound-attenuating cabinet equipped with a fan for ventilation and noise masking. Experimental events were programmed and controlled using Version IV Med-PC software (MED Associates Inc., St. Albans, VT, USA). Reinforcers were 45 mg Dustless Precision Pellets (F0021, BioServ, Flemington, NJ, USA). 1 Supplementary material can be found by accessing the online version of this paper atd=DwICaQ&c=27AKQ-AFTMvLXtgZ7shZqsfSXu-Fwzpqk4BoASshREk& r=nFMlJyJ07nZmJgkJiibtOQTNZYoNXYUNgIvrE82fBGk& m=KVQ9tizWjQUC0Td5OoNrAY7WjUz81xDIT2WkqP1xUpg&s=-57-KH9-ZQtrgmHmQId-qjun1IWaEflkcbh-ZJijsVc&e= and by entering doi.

TRAINING PROCEDURES

Rats were trained using previously developed procedures (e.g., Berquist and Baker, 2017). Briefly, rats were trained to lever press for food pellets under a fixed ratio (FR) schedule of reinforcement that gradually increased from FR 1 to FR 20. Each lever press was paired with an audible click from the clicker, and delivery of a pellet was paired with a 1 s tone from the tone generator. Following acquisition of the lever press response, rats underwent eight errorless training sessions to establish stimulus control on each lever. Fifteen minutes prior to an errorless training session, rats were injected with 0.9 % physiological saline (ip; S) or the training drug of 1.5 mg/kg MDMA (ip; D) and placed into a darkened chamber for a total of eight 20 min sessions in the following order: S, S, S, D, D, D, S, D. The 15 min pre-session injection interval and 1.5 mg/kg MDMA training dose were chosen from previous drug discrimination studies with MDMA in rats. The house light illuminated and the levers extended into the operant chamber after the 15 min period. The lever (i.e., the right or left lever) assigned to each injection condition was counterbalanced among animals across the eight operant chambers. Following errorless training, discrimination training proceeded in a pseudorandomized order with no more than three consecutive drug or saline sessions. During discrimination training, both levers extended into the operant chamber, but only responses on the lever paired with the injection for that session resulted in the delivery of a food pellet. Correct responses on the drug or saline lever were reinforced under a FR 20 resetting schedule in which the response requirement reset to 0 if a rat switched to the other lever before completing the FR 20. Rats were considered ready for substitution testing once they achieved 4 out of 5 consecutive discrimination training sessions wherein the percentage of responses during the first FR and the percentage of responses for the entire 20 min session were ≥80 % on the injection-appropriate lever.

STIMULUS SUBSTITUTION TESTS

Stimulus substitution test sessions were similar to discrimination training sessions except no food pellets were delivered and the sessions ended upon completion of the first FR 20 or 5 min, whichever occurred first. Rats were required to complete at least one S and one D discrimination training session wherein they met criteria (described above) in between substitution test sessions. The order of substitution tests was as follows: saline, training drug (1.5 mg/kg MDMA; ip), 0.1-3 mg/kg MDMA (ip), and 0.1-3 mg/kg d2-MDMA (ip). Doses of test compounds were assessed in a counterbalanced order among rats.

THERMOREGULATION EXPERIMENTS 2.4.1. SURGICAL PROCEDURES

All surgical procedures were similar to those previously reported. Immediately after surgical implantation of biotelemetry probes, the twelve mice (weighing 28-33 g) were singly-housed in polycarbonate cages in corncob bedding and were given a small cotton nestlet. Animals had ad libitum access to rodent chow and water for the duration of the biotelemetry experiment. Mice were housed in an experimental testing room maintained at 20-22 °C and 40-45 % humidity with lights set to a 12:12 h light-dark cycle (lights on at 0600) for 7 days after surgery to recover and acclimate to the testing room.

BIOTELEMETRY PROCEDURES

Twelve mice were randomly assigned to d2-MDMA (n = 6) or MDMA (n = 6) treatment groups. The assignment of mice to radiotelemetry receiver pads was counterbalanced across treatment conditions. Receiver pads were calibrated to record body core temperature (in °C) on one channel and locomotor activity (horizontal counts) on the other. On experimental day 1, mice were injected with 10 mg/kg (ip) of d2-MDMA or MDMA and core temperature and locomotor activity was recorded for 24 h. Mice received ascending doses of MDMA or d2-MDMA (10, 32, 56 mg/kg; ip) and each injection was spaced four days apart. That is, mice were injected with 10, 32, and 56 mg/kg of their associated treatments on experimental days 1, 5, and 9, respectively.

KETANSERIN PRETREATMENT EXPERIMENT

Twenty-one mice were used for the ketanserin pretreatment experiment. Briefly, mice were randomly assigned to 5.6 mg/kg ketanserin + saline (n = 3); 5.6 mg/kg ketanserin + 10, 32, or 56 mg/kg d2-MDMA (n = 3 per d2-MDMA dose); or, 5.6 mg/kg ketanserin + 10, 32, or 56 mg/kg MDMA (n = 3 per MDMA dose) treatment conditions. Mice were initially housed three per cage in their colony room, but were transferred to a separate experimental room and were individually housed in new cage for at least one hour prior to the experiment. Rectal temperature was recorded with a commercially available rodent thermometer (Physitemp; Model BAT-12; 0.1 °C resolution) equipped with a mouse rectal probe (Model RET-3; 1.91 cm long; 0.17 cm ball tip diameter). Prior to each rectal temperature measurement, the probe was wiped clean with a 70 % isopropyl alcohol swab and lubricated with a bacteriostatic surgical lubricant (Surgilube). The rectal probe was inserted until a relatively stable temperature reading was acquired (< 5 s). A baseline rectal temperature was acquired 15 min prior to administration of 5.6 mg/kg ketanserin (ip). The 5.6 mg/kg dose of ketanserin and the 15 min pretreatment time were chosen from a previous study. Then, 15 min after the ketanserin injection, mice were administered saline, 10-56 mg/kg d2-MDMA, or 10-56 mg/kg MDMA according to their treatment assignment. A rectal temperature measurement was taken just before an injection and every 15 min after an injection for a total duration of 120 min.

DATA ANALYSIS

Acquisition of drug stimulus discrimination was quantified as the number of discrimination training sessions required for each subject to have met criteria necessary to begin stimulus substitution testing. Acquisition data are presented as mean (with 95 % confidence interval and range). Stimulus substitution test results are expressed as mean ( ± SE) percent MDMA-lever selection for each dose tested. Percent MDMA-lever selection was determined as the number of responses emitted on drug lever as a percentage of the total number of responses emitted on both levers. Full substitution was defined as ≥80 % drug lever selection. Response rate was calculated as the average number of responses per second emitted during the session. Response rate data were analyzed using separate Dunnett's multiple comparison tests for MDMA and d2-MDMA to compare drug-lever selection values after drug administration to saline administration. In addition, a two-factor repeated-measures ANOVA was used to compare the dose-effect curves of MDMA to d2-MDMA. Linear regression analyses were performed for each subject (data normalized 0-100 %) to determine effective dose (ED 50 ) values for MDMA and d2-MDMA dose-effect curves. Average ED 50 values were compared using 95 % confidence intervals. Data from biotelemetry and ketanserin pretreatment experiments are presented as means ( ± SE) in degrees Celsius or horizontal counts. For the biotelemetry experiment, dose-effect curves of maximal core temperature, duration (in minutes) of core temperature exceeding 39 °C (considered "hyperthermic" in this experiment), and total horizontal counts were generated. Temperature and locomotor dose-effect curves were analyzed using two-factor analysis of variance (ANOVA) procedures. The Gessier-Greenhouse adjustment was applied to all two-factor ANOVAs with a within-subjects factor, where necessary. Tukey's or Šídák multiple comparisons tests were performed following statistically significant main effects (or simple main effects were assessed using Tukey's or Šídák tests in the case of a statistically significant interaction). Creation of graphs and statistical analysis were achieved using GraphPad Prism (version 8.0; La Jolla, CA, USA). Statistical significance was declared at p < .05 for all analyses.

DISCRIMINATIVE STIMULUS EFFECTS OF D2-MDMA IN RATS

Two rats were removed from the study because one died due to factors unrelated to the experiment and the other failed to show reliable discrimination performance. Rats (n = 6) required an average of 25.5 (95 % CI: 13.63, 37.37; range = 17-44) discrimination training sessions to meet criteria for substitution testing. MDMA and d2-MDMA produced full substitution in rats trained to discriminate 1.5 mg/kg MDMA from saline (Fig.; left panel). Administration of TD, 1.78 and 3 mg/kg MDMA and d2-MDMA produced full substitution and there were no differences in percent MDMA-lever selection between MDMA and d2-MDMA at any dose. Linear regression analysis on the linear portion of each dose-effect curve revealed no difference in ED 50 values between MDMA (ED 50 = 0.88; 95 % CI: 0.27, 1.49) and d2-MDMA (ED 50 = 0.95; 95 % CI: 0.43, 1.47). MDMA and d2-MDMA did not substantially alter response rate (Fig.; right panel). Indeed, a Dunnett's multiple comparisons tests revealed that response rate after administration of 3 mg/kg MDMA differed from saline (p < .05), and a separate Dunnett's test revealed no differences in response rate after administration of any dose of d2-MDMA. In addition, a two-way ANOVA failed to reveal a statistically significant main effect of drug treatment (i.e., d2-MDMA vs. MDMA) (F[1, 5] = 4.90, p > .05), a main effect of dose= 2.72, p > .05), or an interaction between drug treatment and dose= 2.85, p = .05).

D2-MDMA AND MDMA EFFECTS ON BODY CORE TEMPERATURE FOLLOWING ASCENDING DOSE ADMINISTRATION IN MICE

The temperature data from one mouse in the MDMA treatment group was lost during the experiment (due to faulty probe), so its temperature data is excluded from all graphs and data analysis; however, the locomotor data from this mouse was retained. Administration of d2-MDMA and MDMA increased body core temperature within the dose range of 10-56 mg/kg (Fig.); however, this effect was more pronounced in MDMA-treated mice compared to d2-MDMA-treated mice. Indeed, a two-factor mixed-measures ANOVA on maximal core temperature (Fig.) revealed a statistically significant main effect of dose.39] = 47.09, p < .0001) and an interaction between treatment and dose (F[2, 18] = 5.57, p = .01), but no main effect of treatment (i.e., d2-MDMA vs. MDMA) (F[1, 9] = 4.44, p > .06). MDMA-treated mice showed a significantly higher maximal core temperature after administration of 56 mg/kg compared to mice injected with 56 mg/kg d2-MDMA (Fig.). In addition, a two-way ANOVA on duration of core temperature exceeding 39 °C (Fig.) revealed a statistically significant main effect of treatment (F[1, 9] = 6.62, p = .03), a main effect of dose.18] = 6.39, p = .01), and an interaction between treatment and dose (F[2, 18] = 4.13, p = .03). Posthoc multiple comparisons tests failed to reveal the statistically significant comparisons. MDMA-treated mice showed a significantly longer duration of core temperature exceeding 39 °C after administration of 56 mg/kg compared to mice injected with 56 mg/kg d2-MDMA (Fig.). No differences were observed between treatment groups in locomotor activity (Fig.), however, there was a statistically significant main effect of dose in this measure.81] = 103.7, p < .0001) with 32 and 56 mg/kg producing significantly greater locomotor activity than 10 mg/kg.

CAPACITY OF D2-MDMA AND MDMA TO ATTENUATE KETANSERIN-INDUCED HYPOTHERMIA IN MICE

Administration of MDMA and d2-MDMA within the dose range of 10-56 mg/kg reversed the hypothermic effects of 5.6 mg/kg ketanserin (ip) (Fig.). Separate two-factor ANOVAs on time courseactivity curves with the ketanserin experiments revealed time-dependent increases in rectal temperature following administration of MDMA or d2-MDMA compared to saline-treated mice (Fig.): for the 5.6 mg/kg ketanserin +10 mg/kg MDMA and d2-MDMA comparison there was a statistically significant interaction between session time and treatment (F[20, 60] = 3.49, p < .0001) and a main effect of session time.391] = 62.28, p < .0001), but no main effect of treatment; for the 5.6 mg/kg ketanserin + 32 mg/kg MDMA and d2-MDMA comparison there was a statistically significant interaction between treatment and session time= 11.43, p < .0001), a main effect of session time.66] = 41.09, p < .0001, and a main effect of treatment (F[2, 6] = 18.69, p = .0026); and, for the 5.6 mg/kg ketanserin + 56 mg/kg MDMA and d2-MDMA comparison there was a statistically significant interaction between treatment and session time= 29.07, p < .0001), a main effect of session time.04] = 54.66, p < .0001), and a main effect of treatment (F [2, 6] = 36.62, p = .0004). In addition, a two-factor ANOVA on rectal temperature measured at 120 min of the experimental session revealed a main effect of dose (F[2, 12] = 6.22, p = .01), but no effect of treatment and no interaction between treatment and dose (Fig.).

DISCUSSION

This is the first report to characterize the interoceptive and hyperthermic effects of d2-MDMA in rodents. Rats in this study met discrimination training criteria within 17-44 sessions, which is consistent with a previous drug discrimination study that used a 1.5 mg/kg MDMA training dose and similar training criteria (e.g.,. Clinical studies with MDMA report that volunteers receive MDMA amounts of 25-150 mg, which, for a 70 kg adult, is equivalent to approximate doses of 0.35-2.14 mg/kg. In addition, humans can discriminate 1.5 mg/kg MDMA using drug discrimination procedures in controlled experimental settings. Together, these findings suggest that the 1.5 mg/kg MDMA training dose used in this study and previous studies has some translational value in regard to the treatment of PTSD. Deuterium-substitution of hydrogen at the methylenedioxy ring moiety of MDMA did not alter its interoceptive effects, as evidenced by an almost identical stimulus substitution doseeffect curve. Indeed, there were no differences in the ED 50 values and maximal percent MDMA-lever selection values between d2-MDMA and MDMA. Moreover, 3 mg/kg MDMA reduced response rate compared to saline, whereas 3 mg/kg of d2-MDMA did not reduce response rate, which may suggest that d2-MDMA has lower potential to elicit direct effects that interfere with discrimination performance. However, the response rate results must be interpreted with caution as an ANOVA failed to detect a difference between d2-MDMA and MDMA in response rate. Nevertheless, both MDMA and d2-MDMA produced full substitution after administration of 1.5-3.0 mg/kg without producing a complete suppression of responding, which suggests that d2-MDMA may retain the desirable subjective effects that are thought to relate to the effective treatment of PTSD. We used previously-developed biotelemetry procedures (e.g.,to simultaneously assess the thermoregulatory and locomotor effects of MDMA and d2-MDMA in mice. Both MDMA and d2-MDMA impacted body core temperature and locomotor activity within the dose range of 10-56 mg/kg, which is consistent with previous experiments using similar doses of MDMA, species, and procedures. We found that d2-MDMA produced increases in core temperatures that were shorter-lasting and of lower magnitude compared to equivalent doses of MDMA. It is possible that d2-MDMA could produce increases in body core temperature equal to (or greater than) those produced by MDMA observed in this study if larger doses of d2-MDMA were tested, which may suggest a simple potency difference between MDMA and d2-MDMA instead of a difference in maximal effectiveness. Nevertheless, the equal potency of MDMA and d2-MDMA in terms of interoceptive effects, coupled with the reduced potency (and perhaps effectiveness) of d2-MDMA in terms of thermoregulatory effects, likely suggests an improved therapeutic window for d2-MDMA. We report that d2-MDMA is equally effective to MDMA in reversing the hypothermic effects of the selective 5-HT 2A/2C antagonist ketanserin. These findings suggest that deuterium substitution at the methylenedioxy ring does not alter the pharmacodynamic properties of MDMAat least at the serotonin transporter and/or serotonin receptors. This is an important observation because MDMA's pharmacodynamic actions (and biobehavioral effects that follow) likely relate to the effective treatment of PTSD. Additional and more direct studies that assess the pharmacodynamic actions of d2-MDMA (e.g., in vitro binding at monoamine transporters) and provide comparisons between d2-MDMA and MDMA with respect to metabolite formation (e.g., pharmacokinetic analysis of parent compounds and metabolites) are warranted. MDMA is also known to stimulate the release of hormones (e.g., oxytocin, cortisol) that may contribute to its therapeutic profile (e.g., reviewed in. Given that MDMA produces a complex array of neurochemical and behavioral effects, future research will likely uncover the specific mechanisms that contribute to MDMA's therapeutic potential (e.g., oxytocin-related prosocial effects, effects on synaptic plasticity), and perhaps tailor treatments to subpopulations of individuals at risk for developing PTSD (e.g., those who may be more vulnerable to PTSD based on variation in the expression of the oxytocin receptor gene;. In any case, deuterium substitution appears as a viable strategy for improving the tolerability and safety of pharmacotherapeutics by precisely modulating the metabolic fate of the parent compound. As such, toward the goal of improving MDMA for clinical use, we believe that this relatively modest modification of MDMA's chemical structure can mitigate some of the adverse effects associated with MDMA use while retaining desirable therapeutic effects, and thus represent as a suitable alternative to MDMA for treating psychiatric problems.

CONCLUSIONS

In summary, d2-MDMA produced hyperthermic effects that were of lower magnitude and shorter duration than MDMA, and elicited discriminative stimulus properties that are indistinguishable from equivalent doses of MDMA. Together, these findings support the idea that deuterium substitution may improve the safety and tolerability of a compound without altering its desirable pharmacodynamic actions. Based on these promising findings, future studies focused on the cardiovascular effects and pharmacokinetic properties of d2-MDMA are warranted.

CONTRIBUTORS

MB and WF participated in research design. SLP and JLK participated in d2-MDMA synthesis. MB conducted experiments and performed data analysis. MB, WF, SLP, and JLK wrote the manuscript. All authors approved the final manuscript.

ROLE OF FUNDING SOURCE

Funding for this work was provided in part by the NIH National Institute on Drug Abuse (T32 DA022981 and GM110702), and DEA/ FDA contract HHSF223201610079C.