Novel Multi-Antigen Orf-Virus-Derived Vaccine Elicits Protective Anti-SARS-CoV-2 Response in Monovalent and Bivalent Formats.

Prime-2-CoV_Beta is a novel Orf virus (ORFV)-based COVID-19 vaccine candidate expressing both the nucleocapsid and spike proteins of SARS-CoV-2 with the receptor-binding domain (RBD) of the Beta strain. This candidate was shown to be safe and immunogenic in a first-in-human Phase I clinical trial. With the shift in the immune landscape toward the Omicron variant and the widespread vaccine- and/or infection-derived immunity, further pre-clinical research was needed to characterize Prime-2-CoV. Here, we quantified the humoral and cellular response to Prime-2-CoV_Beta in pre-immunized mice and compared the protective efficacy of mono- and bivalent variant-based Prime-2-CoV vaccine candidates in hamsters. Prime-2-CoV_Beta induced robust humoral and cellular immune responses in naïve animals but did not further boost antibody titers in the tested setting when given as repeat booster at short interval. We furthermore showed that Prime-2-CoV_Beta-based mono- and bivalent immunization strategies produced comparable immunogenicity and protection from infection. Our results highlight the potential of the Orf virus as a vaccine platform against SARS-CoV-2 and potentially other infectious viruses.

The ups and downs of caloric restriction and fasting: from molecular effects to clinical application.

Age-associated diseases are rising to pandemic proportions, exposing the need for efficient and low-cost methods to tackle these maladies at symptomatic, behavioral, metabolic, and physiological levels. While nutrition and health are closely intertwined, our limited understanding of how diet precisely influences disease often precludes the medical use of specific dietary interventions. Caloric restriction (CR) has approached clinical application as a powerful, yet simple, dietary modulation that extends both life- and healthspan in model organisms and ameliorates various diseases. However, due to psychological and social-behavioral limitations, CR may be challenging to implement into real life. Thus, CR-mimicking interventions have been developed, including intermittent fasting, time-restricted eating, and macronutrient modulation. Nonetheless, possible side effects of CR and alternatives thereof must be carefully considered. We summarize key concepts and differences in these dietary interventions in humans, discuss their molecular effects, and shed light on advantages and disadvantages.

Structural and functional characterization of the intracellular filament-forming nitrite oxidoreductase multiprotein complex.

Nitrate is an abundant nutrient and electron acceptor throughout Earth's biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein's electron transport chain, and elucidate the electron transfer pathways within the complex.

Assessing turbine passage effects on internal fish injury and delayed mortality using X-ray imaging.

Knowledge on the extent and mechanisms of fish damage caused by hydropower facilities is important for the conservation of fish populations. Herein, we assessed the effects of hydropower turbine passage on internal fish injuries using X-ray technology. A total of 902 specimens from seven native European fish species were screened for 36 types of internal injuries and 86 external injuries evaluated with a previously published protocol. The applied systematic visual evaluation of X-ray images successfully detected skeletal injuries, swim bladder anomalies, emphysema, free intraperitoneal gas and hemorrhages. Injuries related to handling and to impacts of different parts of the hydropower structure could be clearly distinguished applying multivariate statistics and the data often explained delayed mortality within 96 h after turbine passage. The internal injuries could clearly be assigned to specific physical impacts resulting from turbine passage such as swim bladder rupture due to abrupt pressure change or fractures of skeletal parts due to blade-strike, fluid shear or severe turbulence. Generally, internal injuries were rarely depicted by external evaluation. For example, 29% of individuals with vertebral fractures did not present externally visible signs of severe injury. A combination of the external and internal injury evaluation allows quantifying and comparing fish injuries across sites, and can help to identify the technologies and operational procedures which minimize harm to fish in the context of assessing hydropower-related fish injuries as well as in assessing fish welfare.

Effects of multiple stressors on the distribution of fish communities in 203 headwater streams of Rhine, Elbe and Danube.

Fishes in European rivers are threatened by manifold stressors such as structural degradation, water pollution, overexploitation, land-use changes in the catchment, invasive species and global processes including climate change. Identifying main stressors in a stream/river system is of utterly importance for efficiently utilizing the scarce funds for conservation measures in order to achieve the best possible outcome. Within 203 headwater streams of Rhine, Elbe and Danube, we quantified the relative influence of different environmental stressors (water chemistry, food availability (macroinvertebrates), terrestrial predators) and anthropogenic stressors (land use, structural modification of streams) on fish assemblages at different spatial scales based on multivariate biota-environment models. In our analyses, the predictor variables percentage of impoundments, crop farming (especially erosion-prone crops such as maize) and ground sealing in the catchments, the number of wastewater treatment plants and biogas plants in the catchments as well as structural modifications of river banks were most often identified as stressors influencing fish community composition. However, the effects of the stressors varied between the investigated survey-area scales (two different catchments sizes and riparian strips) and regionally (entire study area, major drainage systems, river catchments, stream sizes, geographical subregions). In most cases, fish community composition was simultaneously affected by multiple stressors, underpinning the need for a more holistic and ecosystem-based approach in freshwater conservation and restoration.

Toll-like receptor 4 deficiency or inhibition does not modulate survival and neurofunctional outcome in a murine model of cardiac arrest and resuscitation.

BACKGROUND: Patients experiencing cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) often die or suffer from severe neurological impairment. Post resuscitation syndrome is characterized by a systemic inflammatory response. Toll-like receptor 4 (TLR4) is a major mediator of inflammation and TLR4 has been implicated in the pathogenesis of post-resuscitation encephalopathy. The aim of this study was to evaluate whether TLR4 deficiency or inhibition can modulate survival and neurofunctional outcome after CA/CPR. METHODS: Following intubation and central venous cannulation, CA was induced in wild type (C57Bl/6J, n = 38), TLR4 deficient (TLR4-/-, n = 37) and TLR4 antibody treated mice (5mg/kg MTS510, n = 15) by high potassium. After 10min, CPR was performed using a modified sewing machine until return of spontaneous circulation (ROSC). Cytokines and cerebral TNFalpha levels were measured 8h after CA/CPR. Survival, early neurological recovery, locomotion, spatial learning and memory were assessed over a period of 28 days. RESULTS: Following CA/CPR, all mice exhibited ROSC and 31.5% of wild type mice survived until day 28. Compared to wild type mice, neither TLR4-/- nor MTS510 treated wild type mice had statistically significant altered survival following CA/CPR (51.3 and 26.7%, P = 0.104 and P = 0.423 vs. WT, respectively). Antibody-treated but not TLR4-/- mice had higher IL-1ß and IL-6 levels and TLR4-/- mice had higher IL-10 and cerebral TNFalpha levels. No differences existed between mice of all groups in early neurological recovery, locomotion, spatial learning ability or remembrance. CONCLUSION: Therapeutic strategies targeting TLR4 may not be suitable for the reduction of mortality or neurofunctional impairment after CA/CPR.

Capsid protein structure, self-assembly, and processing reveal morphogenesis of the marine virophage mavirus.

Virophages have the unique property of parasitizing giant viruses within unicellular hosts. Little is understood about how they form infectious virions in this tripartite interplay. We provide mechanistic insights into assembly and maturation of mavirus, a marine virophage, by combining structural and stability studies on capsomers, virus-like particles (VLPs), and native virions. We found that the mavirus protease processes the double jelly-roll (DJR) major capsid protein (MCP) at multiple C-terminal sites and that these sites are conserved among virophages. Mavirus MCP assembled in Escherichia coli in the absence and presence of penton protein, forming VLPs with defined size and shape. While quantifying VLPs in E. coli lysates, we found that full-length rather than processed MCP is the competent state for capsid assembly. Full-length MCP was thermally more labile than truncated MCP, and crystal structures of both states indicate that full-length MCP has an expanded DJR core. Thus, we propose that the MCP C-terminal domain serves as a scaffolding domain by adding strain on MCP to confer assembly competence. Mavirus protease processed MCP more efficiently after capsid assembly, which provides a regulation mechanism for timing capsid maturation. By analogy to Sputnik and adenovirus, we propose that MCP processing renders mavirus particles infection competent by loosening interactions between genome and capsid shell and destabilizing pentons for genome release into host cells. The high structural similarity of mavirus and Sputnik capsid proteins together with conservation of protease and MCP processing suggest that assembly and maturation mechanisms described here are universal for virophages.

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.

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.

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.

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.