Sex Differences in Plasma Lysophosphatidic Acid Species in Patients with Alcohol and Cocaine Use Disorders.

Preclinical evidence suggests a main role of lysophosphatidic acid (LPA) signaling in drug addiction. Recently, we reported alterations in the plasma concentrations of LPA species in patients with alcohol use disorder (AUD). As there are sex differences in drug addiction, the main aim of the present study was to investigate whether relevant LPA species (16:0-LPA, 18:0-LPA, 18:1-LPA, 18:2-LPA and 20:4-LPA) were associated with sex and/or substance use disorder (SUD). This exploratory study was conducted in 214 abstinent patients with lifetime SUD, and 91 healthy control subjects. The SUD group was divided according to the diagnosis of AUD and/or cocaine use disorder (CUD). Participants were clinically assessed, and plasma samples were collected to determine LPA species and total LPA. We found that LPA concentrations were significantly affected by sex, and women showed higher concentrations than men. In addition, there were significantly lower 16:0-LPA, 18:2-LPA and total LPA concentrations in patients with SUD than in controls. Namely, patients with CUD and AUD + CUD showed lower LPA concentrations than controls or patients with AUD. In conclusion, our data suggest that LPA species could be potential biomarkers for SUD in women and men, which could contribute to a better stratification of these patients in treatment programs.

Determination of up to twenty carboxylic acid containing compounds in clinically relevant matrices by o-benzylhydroxylamine derivatization and liquid chromatography-tandem mass spectrometry.

Carboxylic acid containing compounds (R-COOH) are involved in a large number of biological processes and they are relevant for several pathological processes such as neurodegeneration or cancer. Comprehensive methodologies for the quantitative determination of R-COOH in biological samples are required. In this study we have developed a LC-MS/MS method for the quantification of 20 endogenous R-COOH belonging to different pathways such as kynurenine metabolism, serotoninergic pathway, glycolysis, tricarboxylic acid cycle, dopaminergic pathway, short chain fatty acids and glycine metabolism. The approach included derivatization with o-benzylhydroxylamine (reaction time 1 h), liquid-liquid extraction with ethyl acetate and LC-MS/MS detection (run time 10 min). The method was optimized and validated in 5 different matrices (urine, plasma, saliva, brain and liver) following two different approaches: (i) using surrogate matrices and (ii) using actual human samples by standard additions. A suitable linearity was obtained in the endogenous range of the analytes. Adequate intra and inter-assay accuracies (80-120%) and intra- and inter-assay precisions (<20%) were achieved for almost all analytes in all studied matrices. The method was applied in several scenarios to confirm (i) human urinary changes produced in glycolysis after exercise, (ii) metabolic changes produced in rat brain and plasma by methamphetamine administration and (iii) metabolic alterations in human plasma caused by vitamin B6 deficiency. Additionally, the application of the method allowed for establishing previously unreported alterations in R-COOH metabolites under these conditions. Due to the comprehensive analyte and matrix coverage and the wide applicability of the developed methodology, it can be considered as a suitable tool for the study of R-COOH status in health and disease by targeted metabolomics.

Sex-Specific Effects of Synbiotic Exposure in Mice on Addictive-Like Behavioral Alterations Induced by Chronic Alcohol Intake Are Associated With Changes in Specific Gut Bacterial Taxa and Brain Tryptophan Metabolism.

Chronic alcohol intake has been shown to disrupt gut microbiota homeostasis, but whether microbiota modulation could prevent behavioral alterations associated with chronic alcohol intake remains unknown. We investigated the effects of synbiotic dietary supplementation on the development of alcohol-related addictive behavior in female and male mice and evaluated whether these effects were associated with changes in bacterial species abundance, short-chain fatty acids, tryptophan metabolism, and neurotransmitter levels in the prefrontal cortex and hippocampus. Chronic intermittent exposure to alcohol during 20 days induced escalation of intake in both female and male mice. Following alcohol deprivation, relapse-like behavior was observed in both sexes, but anxiogenic and cognitive deficits were present only in females. Synbiotic treatment reduced escalation and relapse to alcohol intake in females and males. In addition, the anxiogenic-like state and cognitive deficits observed in females following alcohol deprivation were abolished in mice exposed to synbiotic. Alcohol-induced differential alterations in microbial diversity and abundance in both sexes. In females, synbiotic exposure abrogated the alterations provoked by alcohol in Prevotellaceae UCG-001 and Ruminococcaceae UCG-014 abundance. In males, synbiotic exposure restored the changes induced by alcohol in Akkermansia and Muribaculum uncultured bacterium abundance. Following alcohol withdrawal, tryptophan metabolites, noradrenaline, dopamine, and γ-aminobutyric acid concentrations in the prefrontal cortex and the hippocampus were correlated with bacterial abundance and behavioral alterations in a sex-dependent manner. These results suggested that a dietary intervention with a synbiotic to reduce gut dysbiosis during chronic alcohol intake may impact differently the gut-brain-axis in females and males.

Mobile Device-assisted Dietary Ecological Momentary Assessments for the Evaluation of the Adherence to the Mediterranean Diet in a Continuous Manner.

Mobile device-assisted dietary ecological momentary assessments (EMAs) have emerged as a new tool allowing the evaluation of dietary intake in real time, in a real-world setting and in a continuous manner. They have the potential to minimize recall bias, participant, and investigator burden, and decrease economic and time investment while maximizing ecological validity. We developed a set of EMAs aimed at evaluating continuous adherence to the MedDiet. Four multiple-choice EMAs are sent daily in a randomized manner from a total of eight questions. The EMAs enquire about the consumption of 11 key food groups of the Mediterranean diet in the last 24-48 h in a semi-quantitative way. EMAs capture the daily frequency of consumption of fruits, vegetables, and extra virgin olive oil on different days of the week. Additionally, EMAs capture the weekly frequency of consumption of whole grain products, sugary drinks, nuts, legumes, sweets, fish and seafood, and red and processed meats. A designed scoring system behind the EMAs extracts the percentage of adherence to the MedDiet recommendations and calculates a quality index of the diet every week. Individualized reports are sent periodically to the volunteers highlighting the strengths and weaknesses of their diet. EMAs are also expected to have a behavioral effect, reinforcing the choice of Mediterranean foods.

Prevention of cognitive decline in subjective cognitive decline APOE ε4 carriers after EGCG and a multimodal intervention (PENSA): Study design.

INTRODUCTION: Subjects exhibiting subjective cognitive decline (SCD) are at an increased risk for mild cognitive impairment and dementia. Given the delay between risk exposure and disease onset, SCD individuals are increasingly considered a good target population for cost-effective lifestyle-based Alzheimer's disease prevention trials. METHODS: The PENSA study is a randomized, double-blind, controlled clinical trial that aims to evaluate the efficacy of a personalized multimodal intervention in lifestyle (diet counseling, physical activity, cognitive training, and social engagement) combined with the use of epigallocatechin gallate (EGCG) over 12 months, in slowing down cognitive decline and improving brain connectivity. The study population includes 200 individuals meeting SCD criteria and carrying the apolipoprotein E ε4 allele, who will be randomized into four treatment arms (multimodal intervention + EGCG/placebo, or lifestyle recommendations + EGCG/placebo). The primary efficacy outcome is change in the composite score for cognitive performance measured with the Alzheimer's Disease Cooperative Study Preclinical Alzheimer Cognitive Composite (ADCS-PACC-like) adding to the original version the Interference score from the Stroop Color and Word Test and the Five Digit Test. Secondary efficacy outcomes are (1) change in functional magnetic resonance imaging (fMRI) and structural neuronal connectivity (structural MRI) and (2) the safety assessment of the EGCG compound. This study is framed within the WW-FINGERS consortium. DISCUSSION: The use of new technologies (i.e., mobile ecological momentary assessments [EMAs], activity tracker) in the PENSA study allows the collection of continuous data on lifestyle behaviors (diet and physical activity) and mood, enabling a personalized design as well as an intensive follow-up of participants. These data will be used to give feedback to participants about their own performance along the intervention, promoting their involvement and adherence. The results of the study may aid researchers on the design of future clinical trials involving preventive lifestyle multicomponent interventions.

Effects of COVID-19 Home Confinement on Mental Health in Individuals with Increased Risk of Alzheimer's Disease.

We explored the impact of the Spanish COVID-19 strict home confinement on mental health and cognition in non-infected subjects (N = 16, 60-80 years) diagnosed with subjective cognitive decline and APOEɛ3/ɛ4 carriers. Mental health was monitored for 2 months on a daily, weekly, or monthly basis, and compared to pre-confinement values. Emotional distress, anxiety, and depression scores increased to pathological threshold values during and after confinement. Those with lower mood during confinement experienced a decline in their mood after confinement. Cognition did not change. These preliminary results suggest that mental health consequences of corona measures in preclinical stages of Alzheimer's disease should be further evaluated.

Potential association of plasma lysophosphatidic acid (LPA) species with cognitive impairment in abstinent alcohol use disorders outpatients.

Lysophosphatidic acid (LPA) species are bioactive lipids participating in neurodevelopmental processes. The aim was to investigate whether the relevant species of LPA were associated with clinical features of alcohol addiction. A total of 55 abstinent alcohol use disorder (AUD) patients were compared with 34 age/sex/body mass index-matched controls. Concentrations of total LPA and 16:0-LPA, 18:0-LPA, 18:1-LPA, 18:2-LPA and 20:4-LPA species were quantified and correlated with neuroplasticity-associated growth factors including brain derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1) and IGF-2, and neurotrophin-3 (NT-3). AUD patients showed dysexecutive syndrome (22.4%) and memory impairment (32.6%). Total LPA, 16:0-LPA, 18:0-LPA and 18:1-LPA concentrations, were decreased in the AUD group compared to control group. Total LPA, 16:0-LPA, 18:2-LPA and 20:4-LPA concentrations were decreased in men compared to women. Frontal lobe functions correlated with plasma LPA species. Alcohol-cognitive impairments could be related with the deregulation of the LPA species, especially in 16:0-LPA, 18:1-LPA and 20:4-LPA. Concentrations of BDNF correlated with total LPA, 18:2-LPA and 20:4-LPA species. The relation between LPA species and BDNF is interesting in plasticity and neurogenesis functions, their involvement in AUD might serve as a biomarker of cognitive impairment.

Bioavailability of Epigallocatechin Gallate Administered With Different Nutritional Strategies in Healthy Volunteers.

The flavanol epigallocatechin gallate (EGCG) is being tested for the treatment of several diseases in humans. However, its bioavailability and pharmacokinetic profile needs a better understanding to enable its use in clinical trials. There is no consensus on the most appropriate concentration of EGCG in the body to obtain the maximum therapeutic effects. Therefore, the aim of this study is to analyze the bioavailability of EGCG orally administered alone or with different food supplements after overnight fasting in order to determine its optimal conditions (high concentrations in blood and the lowest interindividual variations) to be used as a pharmacological tool in human trials. Ten healthy volunteers (5 men and 5 women) aged 25 to 35 years were recruited prospectively. Three series of clinical experiments with a washout period of seven days among each were performed: 1) Teavigo® (EGCG extract) alone, 2) Teavigo® with a standard breakfast, and 3) FontUp® (Teavigo® commercially prepared with fats, carbohydrates, proteins, vitamins, and minerals). Blood samples were collected at 0, 30, 60, 90, 120, 180, 240, and 360 min after EGCG intake. Free EGCG in plasma was measured using a liquid chromatography and mass spectrometry UPLC-ESI-MS/MS analytical method. The pharmacokinetic variables analyzed statistically were area under the curve (AUC0-360), Cmax, Cav, Cmin, T1/2, and Tmax,. EGCG (Teavigo®) alone was the group with higher AUC0-360, Cmax, and Cav both in men (3.86 ± 4.11 µg/mL/kg/6 h; 5.95 ng/mL/kg; 2.96 ng/mL/kg) and women (3.33 ± 1.08 µg/mL/kg/6 h; 6.66 ng/mL/kg; 3.66 ng/mL). Moreover, FontUp® was the group with the highest value of T1/2 both in men (192 ± 66 min) and women (133 ± 28 min). Teavigo® intake after fasting overnight revealed the highest concentration of EGCG in plasma according to its pharmacokinetic profile, indicating that this is an excellent alternative of administration if the experimental design requires good absorption in the gastrointestinal tract. Moreover, EGCG taken along with food supplements (FontUp®) improved the stability of the molecule in the body, being the best choice if the experimental design wants to reduce interindividual variation.

Soy Isoflavone Extract Does Not Increase the Intoxicating Effects of Acute Alcohol Ingestion in Human Volunteers.

Soy beans contain isoflavones, including daidzein and genistein, with biological activities related to therapeutic effects in reducing osteoporosis, decreasing adverse menopausal manifestations, providing protection from cardiovascular diseases, and reducing hormone-dependent cancers and age-related cognitive-decline. Daidzein has been described as inhibiting the aldehyde-dehydrogenase-2 enzyme (ALDH2), and reducing alcohol use in clinical pilot studies. Our aim was to evaluate the possible interactions between a soy extract product and alcohol in a crossover, single blind, randomized study. Ten healthy male volunteers participated in two experimental sessions: one with a single dose of alcohol (0.5 g/kg, Vodka Absolut, Sweden), and the other with four capsules of a soy extract product (Super-Absorbable Soy Isoflavones, Life-Extension, United States) and, 2 h later, the same dose of alcohol. Results showed no differences in vital signs except a slightly higher significative reduction in diastolic blood pressure at 2, 3, 4, and 8 h after administration with alcohol alone in comparison with soy extract+alcohol. Ethanol-induced subjective and adverse effects were similar for both conditions with the exception of headache (higher at 8 h after alcohol alone). Our results demonstrate that a single dose of a soy isoflavone extract did not influence alcohol pharmacokinetics and pharmacological effects and did not induce any disulfiram-reaction symptoms. Soy extract and alcohol did not interact and can be administered safely.

Cannabinoid type-1 receptor blockade restores neurological phenotypes in two models for Down syndrome.

Intellectual disability is the most limiting hallmark of Down syndrome, for which there is no gold-standard clinical treatment yet. The endocannabinoid system is a widespread neuromodulatory system involved in multiple functions including learning and memory processes. Alterations of this system contribute to the pathogenesis of several neurological and neurodevelopmental disorders. However, the involvement of the endocannabinoid system in the pathogenesis of Down syndrome has not been explored before. We used the best-characterized preclinical model of Down syndrome, the segmentally trisomic Ts65Dn model. In male Ts65Dn mice, cannabinoid type-1 receptor (CB1R) expression was enhanced and its function increased in hippocampal excitatory terminals. Knockdown of CB1R in the hippocampus of male Ts65Dn mice restored hippocampal-dependent memory. Concomitant with this result, pharmacological inhibition of CB1R restored memory deficits, hippocampal synaptic plasticity and adult neurogenesis in the subgranular zone of the dentate gyrus. Notably, the blockade of CB1R also normalized hippocampal-dependent memory in female Ts65Dn mice. To further investigate the mechanisms involved, we used a second transgenic mouse model overexpressing a single gene candidate for Down syndrome cognitive phenotypes, the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A). CB1R pharmacological blockade similarly improved cognitive performance, synaptic plasticity and neurogenesis in transgenic male Dyrk1A mice. Our results identify CB1R as a novel druggable target potentially relevant for the improvement of cognitive deficits associated with Down syndrome.

Improving liquid chromatography-tandem mass spectrometry determination of polycarboxylic acids in human urine by chemical derivatization. Comparison of o-benzyl hydroxylamine and 2-picolyl amine.

Due to its high sensitivity and specificity, liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) could be considered as the gold-standard in targeted metabolomics. Although LC-MS/MS allows for the direct detection of a large number of molecules, the proper quantification of highly polar compounds such as poly-carboxylic acids in complex matrices like urine is still a challenge. Chemical derivatization offers a suitable way to improve chromatographic behavior and sensitivity for these compounds. Several derivatizing agents have been proposed for the LC-MS/MS determination of carboxylic acids but studies dealing with their comparison in challenging scenarios are scarce. Here we present the evaluation of two different derivatization agents; o-benzylhydroxyl amine (oBHA) and 2-picolyl amine (2-PA); for the quantification of the (poly)-carboxylic acids belonging to the tricarboxylic acid cycle in urine. The suitability of both derivatizating agents was compared by validation of the two approaches. Derivatization with oBHA showed important advantages against 2-PA derivatization such as (i) providing better sensitivity, (ii) more stable derivatives and (iii) allowing for the proper validation of a larger number of analytes. Moreover, while 2-PA derivatization failed in the determination of the target analytes in some stored urine samples, oBHA derivatization successfully allowed for their appropriate determination in the same samples. A comparison between the concentrations obtained using oBHA derivatization and those provided by an external laboratory using UV and GC-MS detection revealed a satisfactory agreement between both results. Additionally, the concentrations obtained by the oBHA method for a set of 38 urines are in agreement with those previously reported in the literature. As a conclusion, our results show that the use of oBHA is preferred against 2-PA for the detection and quantification of (poly)-carboxylic acids in urine.

The effect of tea consumption on the steroid profile.

Green tea (GT), along with its flavonol epigallocatechin-3-gallate (EGCG), has shown to inhibit the UGT2B17 isoenzyme, which is highly involved in the glucuronidation of testosterone (T) and its metabolites. Since the steroid profile (SP) is composed of urinary concentrations of T and related metabolites excreted in both the free and the glucuronide fractions, GT consumption could alter the SP, leading to misunderstanding in doping controls. The aim of the present work was to study the effect of GT consumption on the SP. This study was performed with 29 male volunteers, which could be classified in 2 arms depending on their T/E values (0.12 ± 0.02, n = 12; 1.64 ± 0.90, n = 17). The clinical protocol was designed to evaluate the effect of GT administration on the SP biomarkers. Participants were asked to consume GT with a high content of EGCG for 7 days (5 GT beverages along the whole day for days 1-6 and 9 GT beverages on day 7, corresponding to 520 and 936 mg/day of EGCG, respectively). Urine samples were collected before and during GT consumption at different time periods. The SP was measured using gas chromatography-mass spectrometry. The excretion rates of the SP metabolites did not change after GT consumption. Moreover, the individual evaluation of the subject's steroidal biological passport resulted in normal sequences. The results obtained show that GT consumption does not distort the establishment of normal ranges of SP parameters. Therefore, GT consumption does not need to be considered a confounding factor in the SP evaluation.

Pharmacokinetic Comparison of Soy Isoflavone Extracts in Human Plasma.

The soy isoflavones daidzein and genistein produce several biological activities related to health benefits. A number of isoflavone extracts are commercially available, but there is little information concerning the specific isoflavone content of these products or differences in their bioavailability and pharmacokinetics. This study describes the development and validation of an analytical method to detect and quantify daidzein, genistein, and equol in human plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method was applied in a crossover, randomized, bioavailability study. Twelve healthy volunteers were administered the same total isoflavones dose from two isoflavone supplement preparations (Super-Absorbable Soy Isoflavones (Life Extension, USA) and Fitoladius (Merck, Spain)). The pharmacokinetic parameters (AUC0-24/dose and Cmax/dose) of the isoflavones from the two preparations differed significantly. Such differences in bioavailability and kinetics may have relevant effects on the health benefits derived from their intake.

3,4-methylenedioxymethamphetamine induces gene expression changes in rats related to serotonergic and dopaminergic systems, but not to neurotoxicity.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is an amphetamine derivative widely abused by young adults. Although many studies have reported that relatively high doses of MDMA deplete serotonin (5-HT) content and decrease the availability of serotonin transporters (5-HTT), limited evidence is available as to the adaptive mechanisms taking place in gene expression levels in the brain following a dosing regimen of MDMA comparable to human consumption. In order to further clarify this issue, we used quantitative PCR to study the long-term changes induced by acute administration of MDMA (5 mg/kg × 3) in the expression of genes related to serotonergic and dopaminergic systems, as well as those related to cellular toxicity in the cortex, hippocampus, striatum, and brain stem of rats. Seven days after MDMA administration, we found a significantly lower expression of the 5-HTT (Slc6a4) and the vesicular monoamine transporter (Slc18a2) genes in the brain stem area. In the hippocampus, monoamine oxidase B (Maob) and tryptophan hydroxylase 2 (Tph2) gene expressions were increased. In the striatum, tyrosine hydroxylase (Th) expression was decreased, and a lower expression of α-synuclein (Snca) was observed in the cortex. In contrast, no significant changes were observed in the genes considered to be biomarkers of toxicity including the glial fibrillary acidic protein (Gfap) and the heat-shock 70 kD protein 1A (Hspa1a) in any of the structures assayed. These results suggest that MDMA promotes adaptive changes in genes related to serotonergic and dopaminergic functionality, but not in genes related to neurotoxicity.

Dose-dependent metabolic disposition of hydroxytyrosol and formation of mercapturates in rats.

Hydroxytyrosol (HT), one of the major polyphenols present in olive oil, is known to possess a high antioxidant capacity. The aim of the present study was to investigate dose dependent (0, 1, 10 and 100 mg/kg) alterations in the metabolism of HT in rats since it has been reported that metabolites may contribute to biological effects. Special attention was paid to the activation of the semiquinone-quinone oxidative cycle and the formation of adducts with potential deleterious effects. Thus, we developed a novel analytical methodology to monitor the in vivo formation of the HT mercapturate, N-acetyl-5-S-cysteinyl-hydroxytyrosol in urine samples. Biomarkers of hepatic and renal toxicity were evaluated within the dose range tested. Following HT administration, dose-dependent effects were observed for the recovery of all the metabolites studied. At the lowest dose of 1 mg/kg, the glucuronidation pathway was the most relevant (25-30%), with lower recoveries for sulfation (14%), while at the highest dose of 100 mg/kg, sulfation was the most prevalent (75%). In addition, we report for the first time the formation of the mercapturate conjugate of HT in a dose-dependent manner. The biochemical data did not reveal significant toxic effects of HT at any of the doses studied. An increase in the GSH/GSSG ratio at the highest dose was observed indicating that the products of HT autoxidation are counteracted by glutathione, resulting in their detoxification. These results indicate that the metabolic disposition of HT is highly dependent on the dose ingested.

Contribution of cytochrome P450 and ABCB1 genetic variability on methadone pharmacokinetics, dose requirements, and response.

Although the efficacy of methadone maintenance treatment (MMT) in opioid dependence disorder has been well established, the influence of methadone pharmacokinetics in dose requirement and clinical outcome remains controversial. The aim of this study is to analyze methadone dosage in responder and nonresponder patients considering pharmacogenetic and pharmacokinetic factors that may contribute to dosage adequacy. Opioid dependence patients (meeting Diagnostic and Statistical Manual of Mental Disorders, [4(th) Edition] criteria) from a MMT community program were recruited. Patients were clinically assessed and blood samples were obtained to determine plasma concentrations of (R,S)-, (R) and (S)-methadone and to study allelic variants of genes encoding CYP3A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, and P-glycoprotein. Responders and nonresponders were defined by illicit opioid consumption detected in random urinalysis. The final sample consisted in 105 opioid dependent patients of Caucasian origin. Responder patients received higher doses of methadone and have been included into treatment for a longer period. No differences were found in terms of genotype frequencies between groups. Only CYP2D6 metabolizing phenotype differences were found in outcome status, methadone dose requirements, and plasma concentrations, being higher in the ultrarapid metabolizers. No other differences were found between phenotype and responder status, methadone dose requirements, neither in methadone plasma concentrations. Pharmacokinetic factors could explain some but not all differences in MMT outcome and methadone dose requirements.

Neurotoxic thioether adducts of 3,4-methylenedioxymethamphetamine identified in human urine after ecstasy ingestion.

3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a widely misused synthetic amphetamine derivative and a serotonergic neurotoxicant in animal models and possibly humans. The underlying mechanism of neurotoxicity involves the formation of reactive oxygen species although their source remains unclear. It has been postulated that MDMA-induced neurotoxicity is mediated via the formation of bioreactive metabolites. In particular, the primary catechol metabolites, 3,4-dihydroxymethamphetamine (HHMA) and 3,4-dihydroxyamphetamine (HHA), subsequently cause the formation of glutathione and N-acetylcysteine conjugates, which retain the ability to redox cycle and are serotonergic neurotoxicants in rats. Although the presence of such metabolites has been recently demonstrated in rat brain microdialysate, their formation in humans has not been reported. The present study describes the detection of 5-(N-acetylcystein-S-yl)-3,4-dihydroxymethamphetamine (N-Ac-5-Cys-HHMA) and 5-(N-acetylcystein-S-yl)-3,4-dihydroxyamphetamine (N-Ac-5-Cys-HHA) in human urine of 15 recreational users of MDMA (1.5 mg/kg) in a controlled setting. The results reveal that in the first 4 h after MDMA ingestion approximately 0.002% of the administered dose was recovered as thioether adducts. Genetic polymorphisms in CYP2D6 and catechol-O-methyltransferase expression, the combination of which are major determinants of steady-state levels of HHMA and 4-hydroxy-3-methoxyamphetamine, probably explain the interindividual variability seen in the recovery of N-Ac-5-Cys-HHMA and N-Ac-5-Cys-HHA. In summary, the formation of neurotoxic thioether adducts of MDMA has been demonstrated for the first time in humans. The findings lend weight to the hypothesis that the bioactivation of MDMA to neurotoxic metabolites is a relevant pathway to neurotoxicity in humans.

Discriminative stimulus effects of 3,4-methylenedioxymethamphetamine and its enantiomers in mice: pharmacokinetic considerations.

3,4-Methylenedioxymethamphetamine (MDMA) is a drug of abuse with mixed stimulant- and hallucinogen-like effects. The aims of the present studies were to establish discrimination of S(+)-MDMA, R(-)-MDMA, or their combination as racemic MDMA in separate groups of mice to assess cross-substitution tests among all three compounds, to determine the time courses of the training doses, to assess pharmacokinetic variables after single injections and after cumulative dosing, and to define the metabolic dispositions of MDMA enantiomers and their metabolites. All three forms of MDMA served as discriminative stimuli, and with the exception of R(-)-MDMA in mice trained to discriminate the racemate, compounds substituted for one another. The onset of interoceptive effects for S(+)-MDMA and racemic MDMA were faster than for R(-)-MDMA, and the duration of discriminative stimulus effects was shortest for R(-)-MDMA. S(+)-MDMA and its metabolites were found in higher concentrations than R(-)-MDMA and its metabolites after a bolus dose of racemic MDMA. The N-dealkylation pathway is favored in mouse plasma with MDA as the main metabolite formed. Cumulative doses of MDMA lead to higher plasma concentrations compared with an equivalent single dose. 3,4-Methylenedioxyamphetamine (MDA) concentrations are lower after the cumulative dose compared with the single dose, which, coupled with the nonlinearity observed in MDMA pharmacokinetics after increased doses of racemic MDMA, suggests autoinhibition (or saturation) of MDMA metabolism in mice. In total, these studies suggest that the discriminative stimulus effects of racemic MDMA are perhaps driven by accumulation of S(+)-MDMA and S(+)-MDA in the mouse.

Serotonergic neurotoxic thioether metabolites of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"): synthesis, isolation, and characterization of diastereoisomers.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a synthetic recreational drug of abuse that produces long-term toxicity associated with the degeneration of serotonergic nerve terminals. In various animal models, direct administration of MDMA into the brain fails to reproduce the serotonergic neurotoxicity, implying a requirement for the systemic metabolism and bioactivation of MDMA. Catechol-thioether metabolites of MDMA, formed via oxidation of 3,4-dihydroxymethamphetamine and 3,4-dihydroxyamphetamine (HHMA and HHA) and subsequent conjugation with glutathione (GSH), are selective serotonergic neurotoxicants when administered directly into brain. Moreover, following systemic administration of MDMA, the thioether adducts are present in rat brain dialysate. MDMA contains a stereogenic center and is consumed as a racemate. Interestingly, different pharmacological properties have been attributed to the two enantiomers, (S)-MDMA being the most active in the central nervous system and responsible for the entactogenic effects, and most likely also for the neurodegeneration. The present study focused on the synthesis and stereochemical analysis of the neurotoxic MDMA thioether metabolites, 5-(glutathion-S-yl)-HHMA, 5-(N-acetylcystein-S-yl)-HHMA, 2,5-bis-(glutathion-S-yl)-HHMA, and 2,5-bis-(N-acetylcystein-S-yl)-HHMA. Both enzymatic and electrochemical syntheses were explored, and methodologies for analytical and semipreparative diastereoisomeric separation of MDMA thioether conjugates by HPLC-CEAS and HPLC-UV, respectively, were developed. Synthesis, diastereoisomeric separation, and unequivocal identification of the thioether conjugates of MDMA provide the chemical tools necessary for appropriate toxicological and metabolic studies on MDMA metabolites contributing to its neurotoxicity.

Stereochemical analysis of 3,4-methylenedioxymethamphetamine and its main metabolites in human samples including the catechol-type metabolite (3,4-dihydroxymethamphetamine).

3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") is a designer drug commonly misused in large segments of young populations. MDMA is usually formulated in tablets of its racemate (1:1 mixture of its enantiomers) in doses ranging from 50 to 200 mg. MDMA has an enantioselective metabolism, the (S)-enantiomer being metabolized faster than the (R)-enantiomer. Different pharmacologic properties have been attributed to each enantiomer. The carbon responsible for MDMA chirality is preserved along its metabolic disposition. An analytical method has been developed to determine MDMA enantiomers and those from its major metabolites, 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymeth-amphetamine (HHMA), and 4-hydroxy-3-methoxymethamphet-amine (HMMA). It has been applied to the analysis of plasma and urine samples from healthy recreational users of MDMA who participated voluntarily in a clinical trial and received 100 mg (R,S)-MDMA. HCl orally. (R)/(S) ratios both in plasma (0-48 h) and urine (0-72 h) for MDMA and MDA were >1 and <1, respectively. Ratios corresponding to HHMA and HMMA, close to unity, deviate from theoretical expectations and are most likely explained by the ability of MDMA to autoinhibit its own metabolism. The short elimination half-life of (S)-MDMA (4.8 h) is consistent with the subjective effects and psychomotor performance reported in subjects exposed to MDMA, whereas the much longer half-life of the (R)-enantiomer (14.8 h) correlates with mood and cognitive effects experienced on the next days after MDMA use.