High Incidence of Signs of Neuropathy and Self-Reported Substance Use Disorder for Nitrous Oxide in Patients Intoxicated with Nitrous Oxide.

INTRODUCTION: The number of patients with excessive nitrous oxide (N2O) use and neurological disorders has been rising, indicating an addictive potential of N2O. We studied the incidence of self-reported substance use disorder (SUD)-related symptoms, signs of neuropathy, and the patterns of use in N2O-intoxicated patients. METHODS: The Dutch Poisons Information Center (DPIC) provides information by telephone on the management of intoxications to healthcare professionals. Retrospective data on signs of neuropathy and patterns of use were collected for all N2O intoxications reported to the DPIC in 2021 and 2022. Frequent and heavy use were self-reported as "often/frequent/weekly use" and as "use of tanks or >50 balloons/session," respectively. From this cohort, we included patients with excessive N2O use or signs of neuropathy in a prospective observational cohort study. Online surveys were sent 1 week, 1 month, and 3 months after DPIC consultation. The survey included the drug use disorder questionnaire (validated to measure self-reported substance abuse [SA] and substance dependence [SD] based on Diagnostic and Statistical Manual of Mental Disorders [DSM]-IV-TR criteria) and questions on patterns of use and signs of neuropathy. DSM-IV-TR criteria were translated to DSM-V criteria to score for mild, moderate, or severe SUD, with 2-3, 4-5, or ≥6 symptoms, respectively. RESULTS: We included 101 N2O-intoxicated patients in the retrospective study. Of these, 41% showed signs of neuropathy (N = 41), 53% used N2O tanks to fill balloons (N = 53), 71% used them frequently (N = 72), and 76% used them heavily (N = 77). We included 75 patients in the prospective study and 10 (13%) completed the first survey. All 10 patients fulfilled the criteria for SA and SD (DSM-IV-TR, median number of questions answered "yes" = 10/12), all used N2O tanks to fill balloons, and 90% (N = 9) experienced signs of neuropathy. After 1 and 3 months, 6/7 and 1/1 patients, respectively, continued to fulfill SA and SD criteria. Translating to DSM-V criteria, 1/10 patients fulfilled the criteria for (self-reported) mild SUD, 1/10 patients for moderate SUD, and 8/10 patients for severe SUD, 1 week after consultation. CONCLUSION: The high proportion of N2O-intoxicated patients reporting frequent and heavy use of N2O indicates an addictive potential of N2O. Although follow-up rate was low, all patients fulfilled self-reported SA, SD (DSM-IV-TR), and SUD (DSM-V) criteria for N2O. Somatic healthcare professionals treating patients with N2O intoxications should be aware of possible addictive behavior in patients. The screening, brief intervention, and referral to treatment approach should be considered to treat patients with self-reported SUD symptoms.

3-Methylmethcathinone (3-MMC) Poisonings: Acute Clinical Toxicity and Time Trend Between 2013 and 2021 in the Netherlands.

STUDY OBJECTIVE: The synthetic cathinone 3-methylmethcathinone (3-MMC, or metaphedrone) has recently gained popularity. We studied the numbers of 3-MMC poisonings over time and the clinical effects following poisonings with 3-MMC. METHODS: We performed a retrospective study on the numbers of self-reported 3-MMC poisonings to the Dutch Poisons Information Center (DPIC) from 2013 to June 2021. For poisonings reporting 3-MMC only, the symptoms were extracted and the Poisoning Severity Score (PSS) was determined. From 2016 to June 2019, a prospective cohort study on poisonings reporting only 3-MMC was performed, in which details on the clinical courses were collected through telephone interviews. RESULTS: From 2013 to June 2021, the DPIC was consulted on 184 3-MMC poisonings. The number of poisonings increased from 1 in 2013 to 70 in the first half of 2021. In 84 poisonings with only 3-MMC (46%), sympathomimetic symptoms were commonly reported, including tachycardia (n=29, 35%), hypertension (n=17, 20%), and agitation (n=16, 19%). The initial PSS was usually minor (n=37, 44%) to moderate (n=39, 46%). Five patients (6%) experienced severe effects, including 3 patients experienced severe hypertension (systolic blood pressure >180 mmHg; n=3) and nonfatal cardiac arrest (n=1). Sympathomimetic symptoms (n=8) were also reported in the prospective cohort study. The percentage of moderate poisonings increased (n=6, 75%), and 1 (13%) severe poisoning was observed. Analytical confirmation of 3-MMC exposure was performed in 2 cases. CONCLUSION: The number of 3-MMC poisonings reported to the DPIC has increased over time. Most poisonings with 3-MMC resulted in moderate toxicity and involved sympathomimetic effects, while severe effects were observed in 5 cases.

Significant toxicity following an increase in poisonings with designer benzodiazepines in the Netherlands between 2010 and 2020.

BACKGROUND: Designer benzodiazepines (DBs) are an emerging class of new psychoactive substances. While structurally derived from pharmaceutical benzodiazepines, their toxicological profile is less clear. We investigated time trends in the rate of DB poisonings and their clinical toxicity. METHODS: A retrospective observational study was performed on the incidence rate of DB poisonings, relative to all recreational drug poisonings reported to the Dutch Poisons Information Center (DPIC) from 2010 to 2020. Time-trend analysis was performed using Poisson regression. A prospective cohort study was performed on toxicity of DBs, including the Poisoning Severity Score, from January 2016-June 2019. Data was collected through telephone interviews. RESULTS: Between 2010 and 2020, the DPIC was consulted on 142 DB exposures. The incidence rate of DB exposures increased from 0.1% to 4.3%, with a year effect estimate of 1.35 (95% CI [1.14;1.54]). Twenty different DBs were reported, mostly etizolam (33%), clonazolam (17%), and flunitrazolam (8%). During consultation (often shortly after exposure), poisoning was graded moderate-severe in 29% of cases (n = 146). In the prospective cohort sample with follow-up (n = 22), 86% of cases (n = 19) showed a moderate-severe poisoning. The severity of poisoning did not differ between mono- and mixed intoxications. Frequently reported symptoms in the prospective cohort sample included drowsiness (86%), confusion (59%), and agitation (55%). Coma was observed in seven cases (32%) and respiratory depression requiring mechanical ventilation in five cases (23%). CONCLUSION: The rate of DB poisonings reported to the DPIC strongly increased from 2010 to 2020, indicating increased (ab)use of DBs. Most DB exposures resulted in moderate-severe toxicity with neurological effects.

Pregabalin poisoning: Evaluation of dose-toxicity relationship.

CONTEXT: Pregabalin poisoning is mostly benign, although coma and convulsions occasionally occur. AIM: To determine the dose-toxicity relationship of pregabalin. METHODS: Dose-toxicity data of isolated pregabalin poisonings were collected from (1) a prospective study performed by the Dutch Poisons Information Centre (4 April 2014 to 4 October 2016) and from (2) case reports and case series reported in literature. Poisonings were graded using the Poisoning Severity Score (PSS) and the relationship between dose (mg kg-1 ) and PSS was evaluated. RESULTS: In our study (n = 21 patients), the most commonly observed symptoms were drowsiness (62%), confusion (29%) and apathy (24%). PSS was none in three (14%), minor in 15 (71%), and moderate in three patients (14%). Most case series also reported a PSS of none to minor in the majority of poisonings (69-100%). For 34 individual patients (21 from our study and 13 from literature), detailed data on dose and clinical course were available to examine the dose-toxicity relationship. The median dose was significantly lower in the PSS none-minor group ("benign") (8.6 mg kg-1 , interquartile range (IQ25-75) 5.0-17.6 mg kg-1 ) than in the PSS moderate-severe group ("significant toxicity") (46.7 mg kg-1 , IQ25-75 21.3-64.3 mg kg-1 ); estimate of the median difference = 27.3 mg kg-1 (95% confidence interval (CI): 10-48.6). CONCLUSIONS: In general, higher pregabalin doses result in more severe poisonings. Below 20 mg kg-1 the majority of patients (83%) only suffer from mild poisoning. However, large interindividual differences exist in pregabalin-induced toxicity. Therefore, pre-hospital triage should not only include pregabalin dose, but also underlying illnesses, co-exposures and reported symptoms.

Combining ecstasy and ethanol: higher risk for toxicity? A review.

Ecstasy use is commonly combined with ethanol consumption. While combination drug use in general is correlated with a higher risk for toxicity, the risk of the specific combination of ecstasy (3,4-methylenedioxymethamphetamine (MDMA)) and ethanol is largely unknown. Therefore, we have reviewed the literature on changes in MDMA pharmacokinetics and pharmacodynamics due to concurrent ethanol exposure in human, animal and in vitro studies. MDMA pharmacokinetics appear unaffected: the MDMA blood concentration after concurrent exposure to MDMA and ethanol was comparable to lone MDMA exposure in multiple human placebo-controlled studies. In contrast, MDMA pharmacodynamics were affected: locomotor activity increased and body temperature decreased after concurrent exposure to MDMA and ethanol compared to lone MDMA exposure. Importantly, these additional ethanol effects were consistently observed in multiple animal studies. Additional ethanol effects have also been reported on other pharmacodynamic aspects, but are inconclusive due to a low number of studies or due to inconsistent findings. These investigated pharmacodynamic aspects include monoamine brain concentrations, neurological (psychomotor function, memory, anxiety, reinforcing properties), cardiovascular, liver and endocrine effects. Although only a single or a few studies were available investigating these aspects, most studies indicated an aggravation of MDMA-induced effects upon concurrent ethanol exposure. In summary, concurrent ethanol exposure appears to increase the risk for MDMA toxicity. Increased toxicity is due to an aggravation of MDMA pharmacodynamics, while MDMA pharmacokinetics is largely unaffected. Although a significant attenuation of the MDMA-induced increase of body temperature was observed in animal studies, its relevance for human exposure remains unclear.

The Clinical Toxicology of 4-Bromo-2,5-dimethoxyphenethylamine (2C-B): The Severity of Poisoning After Exposure to Low to Moderate and High Doses.

STUDY OBJECTIVE: We studied the severity of poisoning after exposure to low to moderate and high doses of 4-bromo-2,5-dimethoxyphenethylamine (2C-B). METHODS: Patients for whom the Dutch Poisons Information Centre was consulted for 2C-B exposure from 2016 to 2018 were included in a prospective cohort study. Data were collected through telephone interviews with the physician or patient. Patients were categorized according to the reported 2C-B dose: low to moderate (up to 20 mg), high (greater than 20 mg), or unknown. Presence of 2C-B was analyzed in leftover drug and biological samples with liquid/gas chromatography-mass spectrometry. The severity of poisoning was graded with the Poisoning Severity Score. RESULTS: We included 59 patients, of whom 32 could be followed up. Low to moderate 2C-B doses were reported by 9 patients (28%), high doses by 17 (53%), and unknown doses by 6 (19%). Poisoning was moderate in the majority of patients in both the low- to moderate-dose and high-dose groups. Frequently reported symptoms included mydriasis, agitation or aggression, hallucinations, confusion, anxiety, hypertension, and tachycardia. The presence of 2C-B was confirmed in 5 patients in urine (n=3) or drug samples (n=4). CONCLUSION: In this study, most 2C-B poisonings resulted in moderate toxicity even at high reported doses up to 192 mg. No severe cases were observed. The clinical course was usually short-lived (up to 24 hours) and typically involved hallucinations in addition to mild somatic effects.

Hyperthermia exacerbates the acute effects of psychoactive substances on neuronal activity measured using microelectrode arrays (MEAs) in rat primary cortical cultures in vitro.

Hyperthermia is a well-known, potentially life-threatening, side effect of stimulant psychoactive substances that worsens the neurological outcome of hospitalized patients. However, current in vitro methods to assess the hazard of psychoactive substances do not account for hyperthermia. Therefore, this study determined the potency of five psychoactive substances (cocaine, MDMA (3,4-methylenedioxymethamphetamine), methamphetamine, 3-MMC (3-methylmethcathinone) and TFMPP (3-trifluoromethylphenylpiperazine)) to affect neuronal activity at physiological and hyperthermic conditions. Neuronal activity of rat cortical cultures grown on microelectrode arrays (MEAs) was recorded at 37 °C before exposure. Following 30 min and 4.5 h drug exposure (1-1000 µM) at 37 °C or 41 °C, neuronal activity was measured at either 37 °C or 41 °C. Without drug exposure, hyperthermia induced a modest decrease in neuronal activity. Following acute (30 min) exposure at 37 °C, all drugs concentration-dependently inhibited neuronal activity. Increasing the temperature to 41 °C significantly exacerbated the reduction of neuronal activity ~ 2-fold for all drugs compared to 37 °C. Prolonged (4.5 h) exposure at 41 °C decreased neuronal activity comparable to 37 °C. Neuronal activity (partly) recovered following drug exposure at both temperatures, although recovery from exposure at 41 °C was less pronounced for most drugs. None of the exposure conditions affected viability. Since acute exposure at hyperthermic conditions exacerbates the decrease in neuronal activity induced by psychoactive substances, effects of hyperthermia should be included in future hazard assessment of illicit drugs and new psychoactive substances (NPS).

Novel Phenethylamines and Their Potential Interactions With Prescription Drugs: A Systematic Critical Review.

BACKGROUND: The novel phenethylamines 4-fluoroamphetamine (4-FA) and 2,5-dimethoxy-4-bromophenethylamine (2C-B) fall in the top 10 most used new psychoactive substances (NPSs) among high-risk substance users. Various phenethylamines and NPS are also highly used in populations with mental disorders, depression, or attention deficit hyperactivity disorder (ADHD). Moreover, NPS use is highly prevalent among men and women with risky sexual behavior. Considering these specific populations and their frequent concurrent use of drugs, such as antidepressants, ADHD medication, and antiretrovirals, reports on potential interactions between these drugs, and phenethylamines 4-FA and 2C-B, were reviewed. METHODS: The authors performed a systematic literature review on 4-FA and 2C-B interactions with antidepressants (citalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, duloxetine, bupropion, venlafaxine, phenelzine, moclobemide, and tranylcypromine), ADHD medications (atomoxetine, dexamphetamine, methylphenidate, and modafinil), and antiretrovirals. RESULTS: Limited literature exists on the pharmacokinetics and drug-drug interactions of 2C-B and 4-FA. Only one case report indicated a possible interaction between 4-FA and ADHD medication. Although pharmacokinetic interactions between 4-FA and prescription drugs remain speculative, their pharmacodynamic points toward interactions between 4-FA and ADHD medication and antidepressants. The pharmacokinetic and pharmacodynamic profile of 2C-B also points toward such interactions, between 2C-B and prescription drugs such as antidepressants and ADHD medication. CONCLUSIONS: A drug-drug (phenethylamine-prescription drug) interaction potential is anticipated, mainly involving monoamine oxidases for 2C-B and 4-FA, with monoamine transporters being more specific to 4-FA.

Hazard Characterization of Synthetic Cathinones Using Viability, Monoamine Reuptake, and Neuronal Activity Assays.

Synthetic cathinones are the second largest class of new psychoactive substances (NPS) on the drug market. Despite the large number of different cathinones and their abundant use, hazard characterization is mainly limited to their potential to inhibit monoamine transporters. To expand the current hazard characterization, we first investigated the acute effects of several synthetic cathinones [4-methylethcathinone (4-MEC), 3-methylmethcathinone (3-MMC), 4-MMC, methylone, pentedrone, α-pyrrolidinovalerophenone (α-PVP), and 3,4-methylenedioxypyrovalerone (MDPV)] on human dopamine, norepinephrine, and serotonin reuptake transporters (hDAT, hNET, and hSERT), which were stably transfected in human embryonic kidney (HEK) 293 cells. Next, we examined effects on spontaneous neuronal activity in rat primary cortical cultures grown on microelectrode arrays (MEAs) as an integrated endpoint for neurotoxicity. Changes in neuronal activity were assessed after acute (30 min) and prolonged (4.5 h) exposure. Moreover, we investigated whether neuronal activity recovered after washout of the exposure (24 h after the start of the 5 h exposure). Low micromolar concentrations of synthetic cathinones inhibited monoamine uptake via hDAT and hNET, while higher cathinone concentrations were needed to inhibit uptake via hSERT. Comparable high concentrations were needed to inhibit spontaneous neuronal activity during acute (30 min) and prolonged (4.5 h) exposure. Notably, while the inhibition of neuronal activity was reversible at low concentrations, only partial recovery was seen following high, but non-cytotoxic, concentrations of synthetic cathinones. Synthetic cathinones with either a pyrrolidine moiety or long alkyl-tail carbon chain more potently inhibit monoamine uptake via hDAT and neuronal activity. Monoamine uptake via hNET was most potently inhibited by synthetic cathinones with a pyrrolidine moiety. The combination of integrated measurements (MEA recordings of neuronal activity) with single target assays (monoamine reuptake transporter inhibition) indicates inhibition of hDAT and hNET as the primary mode of action of these synthetic cathinones. Changes in neuronal activity, indicative for additional mechanisms, were observed at higher concentrations.

Synthetic Cathinones and Their Potential Interactions with Prescription Drugs.

PURPOSE: Substance use disorder often coexists with other psychiatric disorders, resulting in the simultaneous use of recreational and prescription drugs. The authors aimed to identify potential pharmacokinetic and pharmacodynamic interactions between new psychoactive substances of the cathinone class and specific prescription drugs. METHODS: The authors performed a systematic literature review on interactions between synthetic cathinones (mephedrone, methylone, methylenedioxypyrovalerone, and alpha-pyrrolidinopentiophenone) and antidepressants (citalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, and venlafaxine), attention deficit hyperactivity disorder (ADHD) medications (atomoxetine, dexamphetamine, methylphenidate, modafinil) or HIV medications. RESULTS: Although no pharmacokinetic interactions have been reported in previous literatures, such interactions are likely to occur. Metabolic pathways of cathinones, antidepressants, and ADHD medications have been shown to overlap, including metabolism via cytochrome P450 enzymes and their inhibition. Consistent with this finding, interactions of bupropion (a cathinone) with antidepressants and ADHD medications have been found to increase their serum concentrations and half-lives. Additionally, limited pharmacodynamic interactions have been reported. However, as cathinones, antidepressants, and ADHD medications have been reported to increase the extracellular monoamine concentration by affecting reuptake transporters, interactions among these compounds are likely. Presumably, even higher monoamine concentrations could be observed when cathinones are combined with prescription drugs with a similar mode of action, as has been reported in animals exposed to duloxetine and bupropion. HIV medications have a different mode of action; thus, they have been reported to be less likely to have pharmacodynamic interactions with cathinones. CONCLUSIONS: Clinicians should be aware of possible interactions between synthetic cathinones and prescription drugs, which may increase the risk of drug toxicity or reduce the therapeutic efficacy of the drugs. Qualitative drug screening for cathinones using mass spectrometry methods may aid the early detection of these agents.

Cardiotoxicity screening of illicit drugs and new psychoactive substances (NPS) in human iPSC-derived cardiomyocytes using microelectrode array (MEA) recordings.

The use of recreational drugs, including new psychoactive substances (NPS), is paralleled by emergency department visits of drug users with severe cardiotoxicity. Drug-induced cardiotoxicity can be the (secondary) result of increased norepinephrine blood concentrations, but data on potential drug-induced direct effects on cardiomyocyte function are scarce. The presence of hundreds of NPS therefore calls for efficient screening models to assess direct cardiotoxicity. We investigated effects of four reference compounds (3-30 nM dofetilide, nifedipine and isoproterenol, and 1-10 µM mexiletine) and six recreational drugs (0.01-100 µM cocaine, 0.01-1000 µM amphetamine, MDMA, 4-fluoroamphetamine, α-PVP and MDPV) on cardiomyocyte function (beat rate, spike amplitude and field potential duration (FPD ≈ QT interval in ECGs)), using Pluricyte® human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes cultured on ready-to-use CardioPlate™ multi-well microelectrode arrays (mwMEAs). Moreover, the effects of exposure to recreational drugs on cell viability were assessed. Effects of reference compounds were in accordance with the literature, indicating the presence of hERG potassium (dofetilide), sodium (mexiletine) and calcium (nifedipine) channels and α-adrenergic receptors (isoproterenol). All recreational drugs decreased the spike amplitude at 10-100 µM. All amphetamine-type stimulants and α-PVP decreased the beat rate at 300 µM, while cocaine and MDPV did so at 10 µM and 30 µM, respectively. All drugs increased the FPD, however at varying concentrations. MDMA, MDPV and amphetamine affected cardiomyocyte function at concentrations relevant for human exposure, while other drugs affected cardiomyocyte function only at higher concentrations (≥ 10 µM). Cell viability was only mildly affected at concentrations well above the lowest concentrations affecting cardiomyocyte function. We demonstrate that MEA recordings of hiPSC-derived cardiomyocytes enable screening for acute, direct effects on cardiomyocyte function. Our data further indicate that tachycardia in patients exposed to recreational drugs is likely due to indirect drug effects, while prolonged repolarization periods (prolonged QTc interval) could (partly) result from direct drug effects on cardiomyocyte function.

A quarter of admitted poisoned patients have a mild poisoning and require no treatment: An observational study.

BACKGROUND: Poisoned patients are frequently admitted following Emergency Department (ED) presentation, while the necessity of such admissions is hardly investigated. We determined the proportion and characteristics of poisoned patients who were admitted, but in retrospect had an uneventful admission. METHODS: For this observational cohort study, all patients presented to the ED of a Dutch University Hospital with various poisonings during a 1.5-year period (January 2015-July 2016) were included. The uneventfulness of admissions, defined as patients with a low Poisoning Severity Score (PSS) who received no treatment, was determined in retrospect. RESULTS: We included 417 patients who visited the ED for poisoning. 247 Patients were admitted: 30% to a general ward, 58% to a MCU, and 12% to the ICU. The poisoning severity scores of the admitted patients were none to mild in 38%, moderate to severe in 59%, and fatal in 2%. Upon admission, 60% of the patients received treatment. In retrospect, 77% of the admitted patients had a moderate, severe or fatal poisoning and/or required treatment. However, 23% of the admitted patients had a mild poisoning and required no treatment. This group involved younger patients (median age of 23 versus 42 years) and a higher proportion of patients reporting exposure to only one substance (65% versus 51%). CONCLUSIONS: The majority of poisoned patients presented to the ED was admitted, while in retrospect, a quarter of these admissions were uneventful. Predictive parameters should be sought to identify patients who can be sent home safely.

Changes in neuronal activity in rat primary cortical cultures induced by illicit drugs and new psychoactive substances (NPS) following prolonged exposure and washout to mimic human exposure scenarios.

The use of new psychoactive substances (NPS) is increasing despite associated health risks and limited pharmacological and toxicological knowledge. Information is available mainly for acute effects on specific targets like monoamine transporters and receptors. Recently, we have shown the ability of several NPS and illicit drugs to modulate neuronal activity during acute exposure. While these acute measurements provide valuable information regarding the potency and possible structure-activity relationships, an exposure scenario more representative of human exposure would increase insight and aid translation to the human situation. Therefore, we investigated the effects on neuronal activity after acute (30 min) and prolonged (5 h) exposure to amphetamine-type stimulants, cathinones, hallucinogens, piperazines and cocaine using rat primary cortical cultures grown on multi-well microelectrode arrays. To investigate the reversibility of effects, activity was also measured after a washout period of 19 h. During acute exposure, all compounds concentration-dependently decreased neuronal activity. Compared to acute exposure, prolonged exposure did not further decrease neuronal activity. Following washout, effects of 3 out of 11 drugs (methamphetamine, cocaine, and benzylpiperazine) were fully reversible, whereas effects induced by MDMA, PMMA and α-PVP were partially reversible. Neuronal activity did not recover after 19 h washout following exposure to the highest concentration of MDPV, 2C-B, 25B-NBOMe, and TFMPP. On the contrary, exposure to low concentrations of methylone, and to some extent of 2C-B, increased neuronal activity after the washout period. Hazard characterization of emerging NPS should include at least an acute exposure to determine a potency rank order. Supplementing the (acute and prolonged) exposure scenario with a washout period allows investigation of the reversibility of effects. The possibility of a neuronal network to regain activity after drug exposure appears independent of drug class or IC50 values for acute and prolonged exposure. Even though neuronal activity (partly) recovers after washout following exposure to most drugs, it is perturbing that complete recovery of neuronal activity is observed only for a minority of the tested drugs.

Differential effects of psychoactive substances on human wildtype and polymorphic T356M dopamine transporters (DAT).

Many psychoactive substances affect the human dopamine (DA) reuptake transporter (hDAT). Polymorphisms in the encoding gene could affect the functionality of the transporter and consequently alter effects of psychotropic and recreational drugs. Recently, a T356 M single nucleotide polymorphism in the human SLC6A3 gene was described, which resulted in functional impairments of DA uptake. Therefore, we investigated the effects of 10 psychoactive substances (0.01-1000 µM)) on DA uptake in human embryonic kidney (HEK) 293 cells transiently overexpressing wildtype (WT) or T356 M hDAT. Our data shows that T356 M hDAT has a 3 times lower Vmax and a 3 times higher Km compared to WT hDAT. Additionally, all psychoactive substances inhibited DA uptake by T356 M and WT hDAT. The DA reuptake inhibitors (methylphenidate, cocaine, and bupropion) inhibited DA uptake by WT hDAT most potently, followed by amphetamine-type stimulants [4-fluoroamphetamine (4-FA), amphetamine and MDMA], selective serotonin reuptake inhibitors (SSRI; fluoxetine and citalopram) and arylcyclohexylamines [methoxetamine (MXE) and ketamine]. Compared to DA uptake by WT hDAT, bupropion, methylphenidate, cocaine, and MXE less potently inhibited DA uptake by T356 M hDAT, while citalopram more potently inhibited uptake. The differences in IC50 values between T356 M and WT hDAT were considerable (3-45 fold). As such, the presence of this polymorphism could affect treatment efficiency with these substances as well as susceptibly for toxicity and addiction for individuals carrying this polymorphism.

Neurotoxicity screening of new psychoactive substances (NPS): Effects on neuronal activity in rat cortical cultures using microelectrode arrays (MEA).

While the prevalence and the use of new psychoactive substances (NPS) is steadily increasing, data on pharmacological, toxicological and clinical effects is limited. Considering the large number of NPS available, there is a clear need for efficient in vitro screening techniques that capture multiple mechanisms of action. Neuronal cultures grown on multi-well microelectrode arrays (mwMEAs) have previously proven suitable for neurotoxicity screening of chemicals, pharmaceuticals and (illicit) drugs. We therefore used rat primary cortical cultures grown on mwMEA plates to investigate the effects of eight NPS (PMMA, α-PVP, methylone, MDPV, 2C-B, 25B-NBOMe, BZP and TFMPP) and two 'classic' illicit drugs (cocaine, methamphetamine) on spontaneous neuronal activity. All tested drugs rapidly and concentration-dependently decreased the weighted mean firing rate (wMFR) and the weighted mean burst rate (wMBR) during a 30 min acute exposure. Of the 'classic' drugs, cocaine most potently inhibited the wMFR (IC50 9.8 µM), whereas methamphetamine and the structurally-related NPS PMMA were much less potent (IC50 100 µM and IC50 112 µM, respectively). Of the cathinones, MDPV and α-PVP showed comparable IC50 values (29 µM and 21 µM, respectively), although methylone was 10-fold less potent (IC50 235 µM). Comparable 10-fold differences in potency were also observed between the hallucinogenic phenethylamines 2C-B (IC50 27 µM) and 25B-NBOMe (IC50 2.4 µM), and between the piperazine derivatives BZP (IC50 161 µM) and TFMPP (IC50 19 µM). All drugs also inhibited the wMBR and concentration-response curves for wMBR and wMFR were comparable. For most drugs, IC50 values are close to the estimated human brain concentrations following recreational doses of these drugs, highlighting the importance of this efficient in vitro screening approach for classification and prioritization of emerging NPS. Moreover, the wide range of IC50 values observed for these and previously tested drugs of abuse, both within and between different classes of NPS, indicates that additional investigation of structure-activity relationships could aid future risk assessment of emerging NPS.

Fatalities, Cerebral Hemorrhage, and Severe Cardiovascular Toxicity After Exposure to the New Psychoactive Substance 4-Fluoroamphetamine: A Prospective Cohort Study.

STUDY OBJECTIVE: We study adverse health effects after use of the new psychoactive substance 4-fluoroamphetamine. METHODS: All patients who reported 4-fluoroamphetamine exposure and for whom the Dutch Poisons Information Center was consulted by their physician in 2016 were included in a prospective cohort study. The clinical course was investigated through telephone interviews with the physician and/or patient, using standardized questionnaires. 4-Fluoroamphetamine was analyzed in remaining drug material and biological samples with liquid and gas chromatography-mass spectrometry techniques. RESULTS: We included 45 patients, and follow-up with the physician and/or patient was performed in 33 cases. All patients experienced adverse effects after 4-fluoroamphetamine use. Severe toxicity was reported in 8 patients. In 5 of these patients, 4-fluoroamphetamine exposure was confirmed in biological specimens. Severe toxicity that was reported included 2 fatalities, 4 patients with cerebral hemorrhage (1 fatal), 2 patients with inverted Takotsubo's cardiomyopathy, 1 patient with myocardial infarction, 1 patient with acute heart failure, and an overall high prevalence of pronounced hypertension and tachycardia. CONCLUSION: Since the introduction of 4-fluoroamphetamine to the Dutch drug market in 2007, its use continues to increase, possibly because users perceive it as "ecstasy light" and thus relatively safe. However, the proportion of patients with severe toxicity after 4-fluoroamphetamine use is relatively large in our study population. Therefore, users should be warned about the risks of 4-fluoroamphetamine.

Effect fingerprinting of new psychoactive substances (NPS): What can we learn from in vitro data?

The use of new psychoactive substances (NPS) is increasing and currently >600 NPS have been reported. However, limited information on neuropharmacological and toxicological effects of NPS is available, hampering risk characterization. We reviewed the literature on the in vitro neuronal modes of action to obtain effect fingerprints of different classes of illicit drugs and NPS. The most frequently reported NPS were selected for review: cathinones (MDPV, α-PVP, mephedrone, 4-MEC, pentedrone, methylone), cannabinoids (JWH-018), (hallucinogenic) phenethylamines (4-fluoroamphetamine, benzofurans (5-APB, 6-APB), 2C-B, NBOMes (25B-NBOMe, 25C-NBOMe, 25I-NBOMe)), arylcyclohexylamines (methoxetamine) and piperazine derivatives (mCPP, TFMPP, BZP). Our effect fingerprints highlight the main modes of action for the different NPS studied, including inhibition and/or reversal of monoamine reuptake transporters (cathinones and non-hallucinogenic phenethylamines), activation of 5-HT2receptors (hallucinogenic phenethylamines and piperazines), activation of cannabinoid receptors (cannabinoids) and inhibition of NDMA receptors (arylcyclohexylamines). Importantly, we identified additional targets by relating reported effect concentrations to the estimated human brain concentrations during recreational use. These additional targets include dopamine receptors, α- and ß-adrenergic receptors, GABAAreceptors and acetylcholine receptors, which may all contribute to the observed clinical symptoms following exposure. Additional data is needed as the number of NPS continues to increase. Also, the effect fingerprints we have obtained are still incomplete and suffer from a large variation in the reported effects and effect sizes. Dedicated in vitro screening batteries will aid in complementing specific effect fingerprints of NPS. These fingerprints can be implemented in the risk assessments of NPS that are necessary for eventual control measures to reduce Public Health risks.

Measuring inhibition of monoamine reuptake transporters by new psychoactive substances (NPS) in real-time using a high-throughput, fluorescence-based assay.

The prevalence and use of new psychoactive substances (NPS) is increasing and currently over 600 NPS exist. Many illicit drugs and NPS increase brain monoamine levels by inhibition and/or reversal of monoamine reuptake transporters (DAT, NET and SERT). This is often investigated using labor-intensive, radiometric endpoint measurements. We investigated the applicability of a novel and innovative assay that is based on a fluorescent monoamine mimicking substrate. DAT, NET or SERT-expressing human embryonic kidney (HEK293) cells were exposed to common drugs (cocaine, dl-amphetamine or MDMA), NPS (4-fluoroamphetamine, PMMA, α-PVP, 5-APB, 2C-B, 25B-NBOMe, 25I-NBOMe or methoxetamine) or the antidepressant fluoxetine. We demonstrate that this fluorescent microplate reader-based assay detects inhibition of different transporters by various drugs and discriminates between drugs. Most IC50 values were in line with previous results from radiometric assays and within estimated human brain concentrations. However, phenethylamines showed higher IC50 values on hSERT, possibly due to experimental differences. Compared to radiometric assays, this high-throughput fluorescent assay is uncomplicated, can measure at physiological conditions, requires no specific facilities and allows for kinetic measurements, enabling detection of transient effects. This assay is therefore a good alternative for radiometric assays to investigate effects of illicit drugs and NPS on monoamine reuptake transporters.