Pharmacokinetic and pharmacodynamic analysis of d-amphetamine in an attention task in rodents.

Amphetamine is a common therapeutic agent for alleviating the core symptoms associated with attention-deficit hyperactivity disorder (ADHD) in children and adults. The current study used a translational model of attention, the five-choice serial reaction time (5-CSRT) procedure with rats, to examine the time-course effects of d-amphetamine. Effects of different dosages of d-amphetamine were related to drug-plasma concentrations, fashioned after comprehensive pharmacokinetic/pharmacodynamic assessments that have been employed in clinical investigations. We sought to determine whether acute drug-plasma concentrations that enhance performance in the 5-CSRT procedure are similar to those found to be therapeutic in patients diagnosed with ADHD. Results from the pharmacokinetic/pharmacodynamic assessment indicate that d-amphetamine plasma concentrations associated with improved performance on the 5-CSRT procedure overlap with those that have been reported to be therapeutic in clinical trials. The current findings suggest that the 5-CSRT procedure may be a useful preclinical model for predicting the utility of novel ADHD therapeutics and their effective concentrations.

Acquisition of MDMA self-administration: pharmacokinetic factors and MDMA-induced serotonin release.

The current study aimed to elucidate the role of pharmacokinetic (PK) parameters and neurotransmitter efflux in explaining variability in (±) 3, 4-methylenedioxymethamphetamine (MDMA) self-administration in rats. PK profiles of MDMA and its major metabolites were determined after the administration of 1.0 mg/kg MDMA (iv) prior to, and following, the acquisition of MDMA self-administration. Synaptic levels of 5-hydroxytryptamine (5HT) and dopamine (DA) in the nucleus accumbens were measured following administration of MDMA (1.0 and 3.0 mg/kg, iv) using in vivo microdialysis and compared for rats that acquired or failed to acquire MDMA self-administration. Effects of the 5HT neurotoxin, 5,7 dihydroxytryptamine (5, 7-DHT), on the acquisition of MDMA and cocaine self-administration were also determined. In keeping with previous findings, approximately 50% of rats failed to meet a criterion for acquisition of MDMA self-administration. The PK profiles of MDMA and its metabolites did not differ between rats that acquired or failed to acquire MDMA self-administration. MDMA produced more overflow of 5HT than DA. The MDMA-induced 5HT overflow was lower in rats that acquired MDMA self-administration compared with those that did not acquire self-administration. In contrast, MDMA-induced DA overflow was comparable for the two groups. Prior 5,7-DHT lesions reduced tissue levels of 5HT and markedly increased the percentage of rats that acquired MDMA self-administration and also decreased the latency to acquisition of cocaine self-administration. These data suggest that 5HT limits the initial sensitivity to the positively reinforcing effects of MDMA and delays the acquisition of reliable self-administration.

Methylphenidate and impulsivity: a comparison of effects of methylphenidate enantiomers on delay discounting in rats.

RATIONALE: Current formulations of methylphenidate (MPH) used in treatment of attention-deficit/hyperactivity disorder (ADHD) result in significantly different bioavailability of MPH enantiomers. Daytrana®, a dl-MPH transdermal patch system, produces higher levels of l-MPH than when dl-MPH is administered orally (e.g., Ritalin®). One potential limitation of increased l-MPH was indicated in a preclinical study showing l-MPH may attenuate effects of d-MPH. OBJECTIVES: The objective of the study was to investigate the interactive effects of MPH enantiomers by (1) assessing drug effects via a preclinical model of "impulsivity" and (2) performing a quantitative dose equivalence analysis of MPH enantiomer interactions. METHODS: Sprague-Dawley rats were trained to emit either of two responses, one producing an immediate food pellet, the other producing four pellets delivered at increasing delays (0, 8, and 32 s). The percent selection of the larger food amount was graphed as a function of delay with the area under the curve (AUC) assessed. Increases in AUC are consistent with decreases in "impulsivity" (i.e., selection of the smaller, immediate over the larger, delayed reinforcer). RESULTS: Systemic administration of dl-MPH and d-MPH dose-dependently increased AUC, while l-MPH, morphine, and pentobarbital did not alter AUC. An analysis based upon dose equivalence indicated that dl-MPH produced additive effects that were not different from that predicted from effects of the enantiomers administered alone. CONCLUSIONS: The present results indicate pharmacologically selective effects in that only drugs prescribed for the treatment of ADHD symptoms decreased a measure of "impulsivity" and that l-MPH likely does not attenuate or enhance the effects of d-MPH in the current delay-discounting task.

Behavioral effects and pharmacokinetics of (±)-3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) after intragastric administration to baboons.

(±)-3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") is a popular drug of abuse. We aimed to characterize the behavioral effects of intragastric MDMA in a species closely related to humans and to relate behavioral effects to plasma MDMA and metabolite concentrations. Single doses of MDMA (0.32-7.8 mg/kg) were administered via an intragastric catheter to adult male baboons (N = 4). Effects of MDMA on food-maintained responding were assessed over a 20-hour period, whereas untrained behaviors and fine-motor coordination were characterized every 30 minutes until 3 hours postadministration. Levels of MDMA and metabolites in plasma were measured in the same animals (n = 3) after dosing on a separate occasion. MDMA decreased food-maintained responding over the 20-hour period, and systematic behavioral observations revealed increased frequency of bruxism as the dose of MDMA was increased. Drug blood level determinations showed no MDMA after the lower doses of MDMA tested (0.32-1.0 mg/kg) and modest levels after higher MDMA doses (3.2-7.8 mg/kg). High levels of 3,4-dihydroxymethamphetamine (HHMA) were detected after all doses of MDMA, suggesting extensive first-pass metabolism of MDMA in the baboon. The present results demonstrate that MDMA administered via an intragastric catheter produced behavioral effects that have also been reported in humans. Similar to humans, blood levels of MDMA after oral administration may not be predictive of the behavioral effects of MDMA. Metabolites, particularly HHMA, may play a significant role in the behavioral effects of MDMA.

Single oral doses of (±) 3,4-methylenedioxymethamphetamine ('Ecstasy') produce lasting serotonergic deficits in non-human primates: relationship to plasma drug and metabolite concentrations.

Repeated doses of the popular recreational drug methylenedioxymethamphetamine (MDMA, 'Ecstasy') are known to produce neurotoxic effects on brain serotonin (5-HT) neurons but it is widely believed that typical single oral doses of MDMA are free of neurotoxic risk. Experimental and therapeutic trials with MDMA in humans are underway. The mechanisms by which MDMA produces neurotoxic effects are not understood but drug metabolites have been implicated. The aim of the present study was to assess the neurotoxic potential of a range of clinically relevant single oral doses of MDMA in a non-human primate species that metabolizes MDMA in a manner similar to humans, the squirrel monkey. A secondary objective was to explore the relationship between plasma MDMA and metabolite concentrations and lasting serotonergic deficits. Single oral doses of MDMA produced lasting dose-related serotonergic neurochemical deficits in the brains of squirrel monkeys. Notably, even the lowest dose of MDMA tested (5.7 mg/kg, estimated to be equivalent to 1.6 mg/kg in humans) produced significant effects in some brain regions. Plasma levels of MDMA engendered by neurotoxic doses of MDMA were on the order of those found in humans. Serotonergic neurochemical markers were inversely correlated with plasma concentrations of MDMA, but not with those of its major metabolites, 3,4-dihydroxymethamphetamine and 4-hydroxy-3-methoxymethamphetamine. These results suggest that single oral doses of MDMA in the range of those used by humans pose a neurotoxic risk and implicate the parent compound (MDMA), rather than one of its metabolites, in MDMA-induced 5-HT neural injury.

Studies of (±)-3,4-methylenedioxymethamphetamine (MDMA) metabolism and disposition in rats and mice: relationship to neuroprotection and neurotoxicity profile.

The neurotoxicity of (±)-3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy") is influenced by temperature and varies according to species. The mechanisms underlying these two features of MDMA neurotoxicity are unknown, but differences in MDMA metabolism have recently been implicated in both. The present study was designed to 1) assess the effect of hypothermia on MDMA metabolism, 2) determine whether the neuroprotective effect of hypothermia is related to inhibition of MDMA metabolism, and 3) determine if different neurotoxicity profiles in mice and rats are related to differences in MDMA metabolism and/or disposition in the two species. Rats and mice received single neurotoxic oral doses of MDMA at 25°C and 4°C, and body temperature, pharmacokinetic parameters, and serotonergic and dopaminergic neuronal markers were measured. Hypothermia did not alter MDMA metabolism in rats and only modestly inhibited MDMA metabolism in mice; however, it afforded complete neuroprotection in both species. Rats and mice metabolized MDMA in a similar pattern, with 3,4-methylenedioxyamphetamine being the major metabolite, followed by 4-hydroxy-3-methoxymethamphetamine and 3,4-dihydroxymethamphetamine, respectively. Differences between MDMA pharmacokinetics in rats and mice, including faster elimination in mice, did not account for the different profile of MDMA neurotoxicity in the two species. Taken together, the results of these studies indicate that inhibition of MDMA metabolism is not responsible for the neuroprotective effect of hypothermia in rodents, and that different neurotoxicity profiles in rats and mice are not readily explained by differences in MDMA metabolism or disposition.

Identifying the serotonin transporter signal in Western blot studies of the neurotoxic potential of MDMA and related drugs.

A number of published studies have questioned the serotonin neurotoxic potential of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") and related drugs (fenfluramine, p-chloroamphetamine) based upon results from Western blot studies using a custom synthesized serotonin transporter (SERT) antibody that found no reduction in the abundance of a 50kDa protein after substituted amphetamine treatment. The purpose of this study was to collect Western blot data using the same SERT antibody used in those studies, but with positive and negative controls to identify the SERT protein signal. A 63-68 kDa band that had the regional distribution expected of rat brain SERT, was decreased by 5,7-DHT, and was absent in SERT KO animals was identified as the SERT protein. Significant, lasting decreases in the abundance of the 63-68 kDa band were evident in the rat brain after treatment with MDMA and related drugs (FEN, PCA). Thus, when the band corresponding to the SERT protein is identified in Western blots through the use of positive and negative controls, reduced abundance of the SERT protein can be readily demonstrated after substituted amphetamine treatment. These data provide further evidence of lasting loss of the SERT protein after exposure to MDMA and other substituted amphetamines.

Altered pain responses in abstinent (±)3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") users.

RATIONALE: (±)3,4-Methylenedioxymethamphetamine (MDMA) is a popular recreational drug that has potential to damage brain serotonin (5-HT) neurons in humans. Brain 5-HT neurons play a role in pain modulation, yet little is known about long-term effects of MDMA on pain function. Notably, MDMA users have been shown to have altered sleep, a phenomenon that can lead to altered pain modulation. OBJECTIVES: This study sought to assess pain processing in MDMA users using objective methods, and explore potential relationships between pain processing and sleep indices. METHODS: Forty-two abstinent MDMA users and 43 age-matched controls participated in a 5-day inpatient study. Outcome measures included standardized measures of pain, sleep polysomnograms, and power spectral measures of the sleep EEG. When differences in psychophysiological measures of pain were found, the relationship between pain and sleep measures was explored. RESULTS: MDMA users demonstrated lower pressure pain thresholds, increased cold pain ratings, increased pain ratings during testing of diffuse noxious inhibitory control, and decreased Stage 2 sleep. Numerous significant relationships between sleep and pain measures were identified, but differences in sleep between the two groups were not found to mediate altered pain perception in MDMA users. CONCLUSIONS: Abstinent MDMA users have altered pain perception and sleep architecture. Although pain and sleep outcomes were related, differences in sleep architecture in MDMA users did not mediate altered pain responses. It remains to be determined whether alterations in pain perception in MDMA users are secondary to neurotoxicity of 5-HT-mediated pain pathways or alterations in other brain processes that modulate pain perception.

Synthesis and neurotoxicity profile of 2,4,5-trihydroxymethamphetamine and its 6-(N-acetylcystein-S-yl) conjugate.

The purpose of the present study was to determine if trihydroxymethamphetamine (THMA), a metabolite of methylenedioxymethamphetamine (MDMA, "ecstasy"), or its thioether conjugate, 6-(N-acetylcystein-S-yl)-2,4,5-trihydroxymethamphetamine (6-NAC-THMA), play a role in the lasting effects of MDMA on brain serotonin (5-HT) neurons. To this end, novel high-yield syntheses of THMA and 6-NAC-THMA were developed. Lasting effects of both compounds on brain serotonin (5-HT) neuronal markers were then examined. A single intraventricular injection of THMA produced a significant lasting depletion of regional rat brain 5-HT and 5-hydroxyindoleacetic acid (5-HIAA), consistent with previous reports that THMA harbors 5-HT neurotoxic potential. The lasting effect of THMA on brain 5-HT markers was blocked by the 5-HT uptake inhibitor fluoxetine, indicating that persistent effects of THMA on 5-HT markers, like those of MDMA, are dependent on intact 5-HT transporter function. Efforts to identify THMA in the brains of animals treated with a high, neurotoxic dose (80 mg/kg) of MDMA were unsuccessful. Inability to identify THMA in the brains of these animals was not related to the unstable nature of the THMA molecule because exogenous THMA administered intracerebroventricularly could be readily detected in the rat brain for several hours. The thioether conjugate of THMA, 6-NAC-THMA, led to no detectable lasting alterations of cortical 5-HT or 5-HIAA levels, indicating that it lacks significant 5-HT neurotoxic activity. The present results cast doubt on the role of either THMA or 6-NAC-THMA in the lasting serotonergic effects of MDMA. The possibility remains that different conjugated forms of THMA or oxidized cyclic forms (e.g., the indole of THMA) play a role in MDMA-induced 5-HT neurotoxicity in vivo.

Metabolism and disposition of 3,4-methylenedioxymethamphetamine ("ecstasy") in baboons after oral administration: comparison with humans reveals marked differences.

The baboon is potentially an attractive animal for modeling 3,4-methylenedioxymethamphetamine (MDMA) effects in humans. Baboons self-administer MDMA, are susceptible to MDMA neurotoxicity, and are suitable for positron emission tomography, the method most often used to probe for MDMA neurotoxicity in humans. Because pharmacokinetic equivalence is a key feature of a good predictive animal model, we compared the pharmacokinetics of MDMA in baboons and humans. Baboons were trained to orally consume MDMA. Then, pharmacokinetic profiles of MDMA and its major metabolites were determined after various oral MDMA doses using the same analytical method recently used to perform similar studies in humans. Results indicate that MDMA pharmacokinetics after oral ingestion differ markedly between baboons and humans. Baboons had little or no MDMA in their plasma but had high plasma concentrations of 3,4-dihydroxymethamphetamine (HHMA), pointing to much more extensive first-pass metabolism of MDMA in baboons than in humans. Other less prominent differences included less O-methylation of HHMA to 4-hydroxy-3-methoxymethamphetamine, greater N-demethylation of MDMA to 3,4-methylenedioxyamphetamine, and a shorter half-life of HHMA in the baboon. To our knowledge, this is the first study to characterize MDMA metabolism and disposition in the baboon. Differences in MDMA pharmacokinetics between baboons and humans suggest that the baboon may not be ideal for modeling human MDMA exposure. However, the unusually rapid conversion of MDMA to HHMA in the baboon may render this animal uniquely useful for clarifying the relative role of the parent compound (MDMA) versus metabolites (particularly HHMA) in the biological actions of MDMA.

Inhibition of 3,4-methylenedioxymethamphetamine metabolism leads to marked decrease in 3,4-dihydroxymethamphetamine formation but no change in serotonin neurotoxicity: implications for mechanisms of neurotoxicity.

3,4-Methylenedioxymethamphetamine (MDMA)'s O-demethylenated metabolite, 3,4-dihydroxymethamphetamine (HHMA), has been hypothesized to serve as a precursor for the formation of toxic catechol-thioether metabolites (e.g., 5-N-acetylcystein-S-yl-HHMA) that mediate MDMA neurotoxicity. To further test this hypothesis, HHMA formation was blocked with dextromethorphan (DXM), which competitively inhibits cytochrome P450 enzyme-mediated O-demethylenation of MDMA to HHMA. In particular, rats were randomly assigned to one of four treatment groups (n = 9-12 per group): (1) Saline/MDMA; (2) DXM/MDMA; (3) DXM/Saline; (4) Saline/Saline. During drug exposure, time-concentration profiles of MDMA and its metabolites were determined, along with body temperature. One week later, brain serotonin (5-HT) neuronal markers were measured in the same animals. DXM did not significantly alter core temperature in MDMA-treated animals. A large (greater than 70%) decrease in HHMA formation had no effect on the magnitude of MDMA neurotoxicity. These results cast doubt on the role of HHMA-derived catechol-thioether metabolites in the mechanism of MDMA neurotoxicity.

Dopamine is not essential for the development of methamphetamine-induced neurotoxicity.

It is widely believed that dopamine (DA) mediates methamphetamine (METH)-induced toxicity to brain dopaminergic neurons, because drugs that interfere with DA neurotransmission decrease toxicity, whereas drugs that increase DA neurotransmission enhance toxicity. However, temperature effects of drugs that have been used to manipulate brain DA neurotransmission confound interpretation of the data. Here we show that the recently reported ability of L-dihydroxyphenylalanine to reverse the protective effect of alpha-methyl-para-tyrosine on METH-induced DA neurotoxicity is also confounded by drug effects on body temperature. Further, we show that mice genetically engineered to be deficient in brain DA develop METH neurotoxicity, as long as the thermic effects of METH are preserved. In addition, we demonstrate that mice genetically engineered to have unilateral brain DA deficits develop METH-induced dopaminergic deficits that are of comparable magnitude on both sides of the brain. Taken together, these findings demonstrate that DA is not essential for the development of METH-induced dopaminergic neurotoxicity and suggest that mechanisms independent of DA warrant more intense investigation.

Sleep apnea in young abstinent recreational MDMA ("ecstasy") consumers.

BACKGROUND: Methylenedioxymethamphetamine (MDMA, "ecstasy") is a popular recreational drug of abuse and a selective brain serotonin neurotoxin. Functional consequences of MDMA neurotoxicity have defied ready characterization. Obstructive sleep apnea (OSA) is a common form of sleep-disordered breathing in which brain serotonin dysfunction may play a role. The present study sought to determine whether abstinent recreational MDMA users have an increased prevalence of OSA. METHODS: We studied 71 medically healthy recreational MDMA users and 62 control subjects using all-night sleep polysomnography in a controlled inpatient research setting. Rates of apneas, hypopneas, and apnea hypopnea indices were compared in the 2 groups, controlling for body mass index, age, race, and gender. RESULTS: Recreational MDMA users who had been drug free for at least 2 weeks had significantly increased rates of obstructive sleep apnea and hypopnea compared with controls. The odds ratio (95% confidence interval) for sleep apnea (mild, moderate, and severe combined) in MDMA users during non-REM sleep was 8.5 (2.4-30.4), which was greater than that associated with obesity [6.9 (1.7-28.2)]. Severity of OSA was significantly related to lifetime MDMA exposure. CONCLUSIONS: These findings suggest that prior recreational methylenedioxymethamphetamine use increases the risk for obstructive sleep apnea and lend support to the notion that brain serotonin neuronal dysfunction plays a role in the pathophysiology of sleep apnea.

Sleep deprivation differentially impairs cognitive performance in abstinent methylenedioxymethamphetamine ("Ecstasy") users.

Methylenedioxymethamphetamine (MDMA; "Ecstasy") is a popular recreational drug and brain serotonin (5-HT) neurotoxin. Neuroimaging data indicate that some human MDMA users develop persistent deficits in brain 5-HT neuronal markers. Although the consequences of MDMA-induced 5-HT neurotoxicity are not fully understood, abstinent MDMA users have been found to have subtle cognitive deficits and altered sleep architecture. The present study sought to test the hypothesis that sleep disturbance plays a role in cognitive deficits in MDMA users. Nineteen abstinent MDMA users and 21 control subjects participated in a 5 d inpatient study in a clinical research unit. Baseline sleep quality was measured using the Pittsburgh Sleep Quality Inventory. Cognitive performance was tested three times daily using a computerized cognitive battery. On the third day of admission, subjects began a 40 h sleep deprivation period and continued cognitive testing using the same daily schedule. At baseline, MDMA users performed less accurately than controls on a task of working memory and more impulsively on four of the seven computerized tests. During sleep deprivation, MDMA users, but not controls, became increasingly impulsive, performing more rapidly at the expense of accuracy on tasks of working and short-term memory. Tests of mediation implicated baseline sleep disturbance in the cognitive decline seen during sleep deprivation. These findings are the first to demonstrate that memory problems in MDMA users may be related, at least in part, to sleep disturbance and suggest that cognitive deficits in MDMA users may become more prominent in situations associated with sleep deprivation.

Further studies on the role of metabolites in (+/-)-3,4-methylenedioxymethamphetamine-induced serotonergic neurotoxicity.

The mechanism by which the recreational drug (+/-)-3,4-methylenedioxymethamphetamine (MDMA) destroys brain serotonin (5-HT) axon terminals is not understood. Recent studies have implicated MDMA metabolites, but their precise role remains unclear. To further evaluate the relative importance of metabolites versus the parent compound in neurotoxicity, we explored the relationship between pharmacokinetic parameters of MDMA, 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymethamphetamine (HHMA), and 4-hydroxy-3-methoxymethamphetamine (HMMA) and indexes of serotonergic neurotoxicity in the same animals. We also further evaluated the neurotoxic potential of 5-(N-acetylcystein-S-yl)-HHMA (5-NAC-HHMA), an MDMA metabolite recently implicated in 5-HT neurotoxicity. Lasting serotonergic deficits correlated strongly with pharmacokinetic parameters of MDMA (C(max) and area under the concentration-time curve), more weakly with those of MDA, and not at all with those of HHMA or HMMA (total amounts of the free analytes obtained after conjugate cleavage). HHMA and HMMA could not be detected in the brains of animals with high brain MDMA concentrations and high plasma HHMA and HMMA concentrations, suggesting that HHMA and HMMA do not readily penetrate the blood-brain barrier (either in their free form or as sulfate or glucuronic conjugates) and that little or no MDMA is metabolized to HHMA or HMMA in the brain. Repeated intraparenchymal administration of 5-NAC-HHMA did not produce significant lasting serotonergic deficits in the rat brain. Taken together, these results indicate that MDMA and, possibly, MDA are more important determinants of brain 5-HT neurotoxicity in the rat than HHMA and HMMA and bring into question the role of metabolites (including 5-NAC-HHMA) in MDMA neurotoxicity.

Direct comparison of (+/-) 3,4-methylenedioxymethamphetamine ("ecstasy") disposition and metabolism in squirrel monkeys and humans.

The present study compared the disposition and metabolism of the recreational drug (+/-) 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") in squirrel monkeys and humans because the squirrel monkey has been extensively studied for MDMA neurotoxicity. A newly developed liquid chromatography-mass spectrometric procedure for simultaneous measurement of MDMA, 3,4-dihydroxymethamphetamine, 4-hydroxy-3-methoxymethamphetamine, and 3,4-methylenedioxyamphetamine was employed. In both humans and squirrel monkeys, a within-subject design permitted testing of different doses in the same subjects. Humans and squirrel monkeys were found to metabolize MDMA in similar, but not identical, pathways and proportions. In particular, amounts of 3,4-dihydroxymethamphetamine (after conjugate cleavage) and 3,4-methylenedioxyamphetamine were similar in the 2 species, but formation of 4-hydroxy-3-methoxymethamphetamine was greater in squirrel monkeys than in humans. Both species demonstrated nonlinear MDMA pharmacokinetics at comparable plasma MDMA concentrations (125-150 ng/mL and above). The elimination half-life of MDMA was considerably shorter in squirrel monkeys than in humans (2-3 versus 6-9 hours). In both species, there was substantial individual variability. These results suggest that the squirrel monkey may be a useful model for predicting outcomes of MDMA exposure in humans, although this will also depend on the degree to which MDMA pharmacodynamics in the squirrel monkey parallels that in humans.

Hydrolysis of 3,4-methylenedioxymethamphetamine (MDMA) metabolite conjugates in human, squirrel monkey, and rat plasma.

Characterizing the formation of metabolites of 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") in different species (rat, squirrel monkey, and human) may provide insight into mechanisms of MDMA neurotoxicity. Two prominent MDMA metabolites, 3,4-dihydroxymethamphetamine (HHMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA), are conjugated with glucuronic or sulfuric acid, but reference standards are not available; therefore, quantification is only possible after conjugate cleavage. Different concentrations of HHMA and HMMA were obtained in human, squirrel monkey, and rat plasma specimens when acid or enzymatic cleavage was performed. Our data document that these differences are due to species-specific influences on conjugate cleavage. Acidic hydrolysis should be used for analyzing free HHMA and HMMA in human or squirrel monkey plasma, while enzymatic hydrolysis with glucuronidase or sulfatase maximizes recovery of free HHMA and HMMA in rat plasma. Optimization of cleavage conditions showed that sulfate conjugates were more readily cleaved by acid hydrolysis and glucuronides by glucuronidase.

Simultaneous liquid chromatographic-electrospray ionization mass spectrometric quantification of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and its metabolites 3,4-dihydroxymethamphetamine, 4-hydroxy-3-methoxymethamphetamine and 3,4-methylenedioxyamphetamine in squirrel monkey and human plasma after acidic conjugate cleavage.

3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a psychoactive drug with abuse liability and neurotoxic potential. Specimen preparation of a recently presented LC-MS assay with electrospray ionization for quantifying MDMA and its main metabolites in squirrel monkey plasma was modified to include acidic hydrolysis to obtain total 3,4-dihydroxymethamphetamine and 4-hydroxy-3-methoxy-methamphetamine. Method re-validation for squirrel monkey plasma and full validation for human plasma showed selectivity for all analytes. Recoveries were > or = 71.0%. Changed specimen preparation or matrix did not affect accuracy or precision. No instability was observed after repeated freezing or in processed samples. Plasma MDMA and metabolites quantification, derived pharmacokinetic and toxicokinetic data and neurotoxicity research will benefit from this validated method.

Liquid chromatographic-electrospray ionization mass spectrometric assay for simultaneous determination of 3,4-methylenedioxymethamphetamine and its metabolites 3,4-methylenedioxyamphetamine, 3,4-dihydroxymethamphetamine, and 4-hydroxy-3-methoxymethamphetamine in rat brain.

3,4-Methylenedioxymethamphetamine (MDMA) is a psychoactive drug with abuse liability and neurotoxic potential. Mechanisms by which MDMA produces behavioral and neurotoxic effects have yet to be elucidated. By measuring concentrations of MDMA and its metabolites in relevant brain sites, it may be possible to gain insight into mechanisms underlying MDMA actions. For this purpose, an LC-MS assay with electrospray ionization was developed after homogenization of rat brain and enzymatic conjugate cleavage. The method was successfully validated with respect to selectivity, linearity, accuracy, precision, recovery, and matrix effect and its use should help to delineate the neurotoxic mechanism of action of MDMA.

Positron emission tomographic studies of brain dopamine and serotonin transporters in abstinent (+/-)3,4-methylenedioxymethamphetamine ("ecstasy") users: relationship to cognitive performance.

BACKGROUND: (+/-)3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") is a recreational drug and brain serotonin (5-HT) neurotoxin. Under certain conditions, MDMA can also damage brain dopamine (DA) neurons, at least in rodents. Human MDMA users have been found to have reduced brain 5-HT transporter (SERT) density and cognitive deficits, although it is not known whether these are related. This study sought to determine whether MDMA users who take closely spaced sequential doses, which engender high plasma MDMA concentrations, develop DA transporter (DAT) deficits, in addition to SERT deficits, and whether there is a relationship between transporter binding and cognitive performance. MATERIALS AND METHODS: Sixteen abstinent MDMA users with a history of using sequential MDMA doses (two or more doses over a 3- to 12-h period) and 16 age-, gender-, and education-matched controls participated. Subjects underwent positron emission tomography with the DAT and SERT radioligands, [11C]WIN 35,428 and [11C]DASB, respectively. Subjects also underwent formal neuropsychiatric testing. RESULTS: MDMA users had reductions in SERT binding in multiple brain regions but no reductions in striatal DAT binding. Memory performance in the aggregate subject population was correlated with SERT binding in the dorsolateral prefrontal cortex, orbitofrontal cortex, and parietal cortex, brain regions implicated in memory function. Prior exposure to MDMA significantly diminished the strength of this relationship. CONCLUSIONS: Use of sequential MDMA doses is associated with lasting decreases in brain SERT, but not DAT. Memory performance is associated with SERT binding in brain regions involved in memory function. Prior MDMA exposure appears to disrupt this relationship. These data are the first to directly relate memory performance to brain SERT density.