TYPE Clinical Trial PUBLISHED 17 February 2023 DOI 10.3389/fphar.2023.1122861 Pharmacological effects of OPEN ACCESS methylone and MDMA in humans EDITED BY Kabirullah Lutfy, Western University of Health Sciences, Lourdes Poyatos 1,2, Clara Pérez-Mañá 1,2*, Olga Hladun 1,2, United States Melani Núñez-Montero 1,2, Georgina de la Rosa 1,2, Soraya Martín 1, REVIEWED BY Ana Maria Barriocanal 1, Lydia Carabias 3, Benjamin Kelmendi 4, Frederick E. Williams, University of Toledo, United States Omayema Taoussi 5, Francesco Paolo Busardò 5, Michael H. Baumann, National Institute on Drug Abuse (NIH), Francina Fonseca 6,7, Marta Torrens 7,8, Simona Pichini 9, United States Magí Farré 1,2*† and Esther Papaseit 1,2† *CORRESPONDENCE 1 Clara Pérez-Mañá, Department of Clinical Pharmacology, Hospital Universitari Germans Trias i Pujol and Institut de Recerca cperezm.mn.ics@gencat.cat Germans Trias i Pujol (HUGTiP-IGTP), Badalona, Spain, 2Department of Pharmacology, Therapeutics and Magí Farré, Toxicology, Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallés, Spain, 3Department of mfarre.germanstrias@gencat.cat Pharmacy, Hospital Universitari Germans Trias i Pujol (HUGTiP), Badalona, Spain, 4Department of Psychiatry, Yale University School of Medicine, New Haven, CT, United States, 5Department of † These authors share the last authorship Excellence-Biomedical Sciences and Public Health, Università Politecnica delle Marche, Ancona, Italy, 6 Department of Medicine and Life Sciences (MELIS), Universitat Pompeu Fabra, Barcelona, Spain, SPECIALTY SECTION 7 Addiction Program, Institut de Neuropsiquiatria i Adiccions (INAD), Barcelona, Spain, 8Department of This article was submitted to Psychiatry and Forensic Medicine, Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallés, Spain, Neuropharmacology, 9 National Centre on Addiction and Doping, Istituto Superiore di Sanità, Rome, Italy a section of the journal Frontiers in Pharmacology RECEIVED 13 December 2022 ACCEPTED 06 February 2023 PUBLISHED 17 February 2023 Methylone is one of the most common synthetic cathinones popularized as a CITATION substitute for 3,4-methylenedioxymethamphetamine (MDMA, midomafetamine) Poyatos L, Pérez-Mañá C, Hladun O, owing to its similar effects among users. Both psychostimulants exhibit similar Núñez-Montero M, de la Rosa G, Martín S, chemistry (i.e., methylone is a β-keto analog of MDMA) and mechanisms of action. Barriocanal AM, Carabias L, Kelmendi B, Taoussi O, Busardò FP, Fonseca F, Currently, the pharmacology of methylone remains scarcely explored in humans. Torrens M, Pichini S, Farré M and Herein, we aimed to evaluate the acute pharmacological effects of methylone and Papaseit E (2023), Pharmacological its abuse potential in humans when compared with that of MDMA following oral effects of methylone and MDMA in humans. administration under controlled conditions. Seventeen participants of both sexes Front. Pharmacol. 14:1122861. (14 males, 3 females) with a previous history of psychostimulant use completed a doi: 10.3389/fphar.2023.1122861 randomized, double-blind, placebo-controlled, crossover clinical trial. COPYRIGHT Participants received a single oral dose of 200 mg of methylone, 100 mg of © 2023 Poyatos, Pérez-Mañá, Hladun, MDMA, and a placebo. The variables included physiological effects (blood Núñez-Montero, de la Rosa, Martín, Barriocanal, Carabias, Kelmendi, Taoussi, pressure, heart rate, oral temperature, pupil diameter), subjective effects using Busardò, Fonseca, Torrens, Pichini, Farré visual analog scales (VAS), the short form of the Addiction Research Center and Papaseit. This is an open-access Inventory (ARCI), the Evaluation of Subjective Effects of Substances with Abuse article distributed under the terms of the Creative Commons Attribution License Potential questionnaire (VESSPA-SSE), and the Sensitivity to Drug Reinforcement (CC BY). The use, distribution or Questionnaire (SDRQ), and psychomotor performance (Maddox wing, reproduction in other forums is psychomotor vigilance task). We observed that methylone could significantly permitted, provided the original author(s) and the copyright owner(s) are credited increase blood pressure and heart rate and induce pleasurable effects, such as and that the original publication in this stimulation, euphoria, wellbeing, enhanced empathy, and altered perception. journal is cited, in accordance with Methylone exhibited an effect profile similar to MDMA, with a faster overall accepted academic practice. No use, distribution or reproduction is permitted onset and earlier disappearance of subjective effects. These results suggest which does not comply with these terms. that abuse potential of methylone is comparable to that of MDMA in humans. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT05488171; Identifier: NCT05488171. Frontiers in Pharmacology 01 frontiersin.org Poyatos et al. 10.3389/fphar.2023.1122861 KEYWORDS methylone, 3,4-methylenedioxymethcathinone, MDMA, 3,4- methylenedioxymethamphetamine, new psychoactive substances, synthetic cathinones, bath salts, psychostimulants 1 Introduction and altered perception (Poyatos et al., 2021). Overall, methylone exhibits a prototypical psychostimulant and empathogenic MDMA profile. Methylone (3,4-methylenedioxy-N-methylcathinone), also known Like other synthetic cathinones, recreational users often consume as MDMC, βk-MDMA, or M1, is a synthetic cathinone that emerged in small doses after the initial large dose to induce prolonged effects; this 2004 as a new psychoactive substance (NPS), although first synthesized pattern of use increases the risk of intoxication or overdose (Liakoni in 1996 and planned for therapeutic use as an antidepressant and et al., 2017; Gomila et al., 2022) and, in the worst case scenario, death potential treatment for the Parkinson’s disease (Bossong et al., 2005). (Boulanger-Gobeil et al., 2012; Cawrse et al., 2012; Pearson et al., 2012; NPSs appear in the market as legal alternatives to common drugs of Warrick et al., 2012; Carbone et al., 2013; McIntyre et al., 2013; Barrios abuse, benefiting from their status as non-controlled substances by the et al., 2016; Shimomura et al., 2016; deRoux and Dunn, 2017). Common International Drug Control Conventions. Users can acquire these adverse effects associated with intoxication include anxiety, agitation, substances via the Internet, advertised as bath salts, plant fertilizers, palpitations, sweating, tremors, seizures, hyperthermia, and vomiting or incense. Methylone has been popularized as an alternative to MDMA (Karila et al., 2016; Liakoni et al., 2017). (3,4-methylenedioxymethamphetamine, midomafetamine), which is As previously mentioned, methylone was first designed as a chemically similar, given that methylone is considered its β-keto potential treatment for depression and Parkinson’s disease. analog, differing only by an oxygen atom added at the benzylic Recently, this therapeutic approach has resurfaced as a proposal position (Bossong et al., 2005). to use methylone to treat post-traumatic stress disorder (PTSD), Similar to MDMA, methylone is a substrate for the high-affinity anxiety, and depression (Warner-Schmidt et al., 2023). In an monoamine transporters–i.e., the dopamine transporter (DAT), observational study, methylone exhibited promising results, norepinephrine transporter (NET), and serotonin (5-HT) improving the condition of almost all patients with PTSD transporter (SERT) (Simmler et al., 2011; Sogawa et al., 2011; (Kelmendi et al., 2022). Methylone could follow the path of Baumann et al., 2012; Eshleman et al., 2013; Simmler et al., 2013; MDMA, which has demonstrated its efficacy and safety as a Elmore et al., 2017; Luethi et al., 2019). In vitro studies from rat brain potential treatment in combination with psychotherapy for severe tissue demonstrate that methylone has a similar mechanism of PTSD, anxiety, and related psychiatric disorders (Jerome et al., 2020; action as MDMA but is less potent at all three transporters Wolfson et al., 2020; Mitchell et al., 2021). To exploit the potential of (Baumann et al., 2012). Like other transporter substrates, methylone as a therapeutic tool, it is essential to comprehensively methylone evokes the release of intraneuronal monoamine clarify its underlying human pharmacology. neurotransmitters by reversing the normal direction of To date, the abuse potential of methylone has mainly been transporter flux, thereby increasing extracellular concentrations of explored in self-administration studies in animal models (Watterson dopamine, norepinephrine, and 5-HT in the brain (Schindler et al., et al., 2012; Creehan et al., 2015; Vandewater et al., 2015; Nguyen 2016; Elmore et al., 2017). Heightened levels of serotonin and et al., 2017; Javadi-Paydar et al., 2018), demonstrating reinforcing dopamine potentially contribute to the rewarding properties of properties, less potent than those of other synthetic cathinones, such methylone (Schindler et al., 2016; Elmore et al., 2017). as mephedrone, MDPV (3,4-methylenedioxypyrovalerone), and α- Although the popularity of methylone peaked from 2011 to PVP (α-pyrrolidinopentiophenone) (Schindler et al., 2016; Javadi- 2015, according to seizure reports (NFLIS, 2016), it remains one of Paydar et al., 2018). Nevertheless, data regarding its abuse potential the most commonly used synthetic cathinones, along with in humans remain scarce, and most available data on the human mephedrone. In some recent studies, methylone has been pharmacology of methylone mainly comprises user reports and the detected along with other illicit drugs in different matrixes such above-mentioned observational study (Poyatos et al., 2021). To date, as oral fluid (Axelsson et al., 2022), wastewater (Brett et al., 2022) no controlled studies have explored these effects in humans. (Australian Criminal Intelligence Commission, 2020), urine In the present study, we aimed to evaluate the abuse potential (Gomila et al., 2022), hair (Palamar et al., 2016; Palamar et al., and acute pharmacological effects of methylone compared with 2017; Salomone et al., 2017), and nails (Busardò et al., 2020). These those of MDMA and placebo after oral administration under data suggest that the pattern of methylone consumption mainly controlled conditions. focuses on weekend and nightlife scenarios. Methylone is typically administered orally as tablets or pills and less frequently via intranasal (insufflation), sublingual, intravenous, and rectal 2 Methods routes. According to users, moderate oral doses of methylone range between 100 and 200 mg, with <100 mg considered a low dose and 2.1 Participants >200 mg a high dose. After a common dose of methylone, acute effects appear within 15–60 min and last 3–5 h, with maximum effects Eighteen healthy participants (15 males, 3 females) were recruited experienced 60–90 min post-ingestion (World Health Organization, by word of mouth, but only 17 (14 males, 3 females) completed the 2014). According to the only published human study evaluating the study (see 3.1 Participants). Before enrollment, participants underwent acute effects of methylone in natural conditions, it has been reported that a medical examination, including an electrocardiogram (ECG) and methylone mainly induces euphoria, stimulation, increased sociability, blood and urine tests. In addition, the participants received training Frontiers in Pharmacology 02 frontiersin.org Poyatos et al. 10.3389/fphar.2023.1122861 regarding procedures, questionnaires, and psychomotor tests to be used at 7:45 a.m. on the day of the session after fasting overnight and during the experimental sessions. All subjects reported previous remained at the facility for approximately 11 h. Upon arrival, recreational drug use, such as methylone or other synthetic participants provided a urine sample to perform a drug urine test cathinones, amphetamines, and/or MDMA, at least twice in the last (Drug-Screen Multi 10TD Test [Multi-Line], Nal Von Minden, year and six times in their lifetime. Subjects were excluded if they had a Germany) to detect the presence of drugs of abuse current or recent (3 months prior to inclusion) organic illness or major (amphetamine, barbiturate, benzodiazepine, cocaine, MDMA, surgery or a history of mental disorders, including substance use methamphetamine, morphine, methadone, tricyclic disorder (except for nicotine), according to the Diagnostic and antidepressants, and tetrahydrocannabinol). Participants were Statistical Manual of Mental Disorders-5. requested to abstain from pre-session use of illicit drugs (1 week), alcohol (48 h), and caffeine or xanthines (24 h) (Papaseit et al., 2016; Poyatos et al., 2021). 2.2 Study design To establish levels of methylone, MDMA and their metabolites, blood and oral fluid samples were collected at baseline and 0.25, 0.5, This study was designed as a randomized, double-blind, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, and 24 h after administration. Urine placebo-controlled, crossover clinical trial to compare the samples were collected at various time points throughout the session pharmacological effects of methylone with those of MDMA and until 24 h (0–4 h, 4–8 h, 8–12 h, 12–24 h). Data on methylone and a placebo. The subjects participated in three experimental sessions, MDMA concentrations from this study are not presented, although separated by a 1-week wash-out period. In each session, participants the pharmacokinetics of methylone at doses ranging from 50 to received either one of the following three oral medications: 200 mg 200 mg from pilot studies have been published (see study design) methylone, 100 mg MDMA, and a placebo. The administered dose (Poyatos et al., 2022a). of methylone was selected after various dose-finding pilot studies evaluating doses of 50 (n = 3), 100 (n = 6), 150 (n = 5), and 200 mg (n = 7, four subjects received two different doses of methylone and 2.5 Physiological effects placebo; three subjects received methylone, MDMA and placebo and were included in the definitive study). Methylone and MDMA were Non-invasive systolic and diastolic blood pressure, heart rate, evaluated and proved to be well-tolerated (Poyatos et al., 2022a). and oral temperature were repeatedly recorded at baseline After the pilot studies, we selected the 200 mg dose of methylone (−30 and −15 min) and 0.25, 0.50, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, that evoked similar subjective effects to the 100 mg dose of MDMA and 24 h after administration using a vital signs monitor (Philips (e.g., high or liking feelings), which is considered a common dose Sure Signs VM4 monitors, Phillips, Amsterdam, Netherlands). Pupil used in previous pharmacological studies. The subjects received diameter was measured using a Haab pupil gauge as a reference compensation for possible inconveniences owing to their under constant light conditions. Electrocardiogram was participation. Ethical approval was obtained from the local continuously monitored during the sessions for safety reasons. Human Research Ethics Committee (CEI Hospital Germans Trias i Pujol, code PI-19–082). This study was conducted in compliance with Spanish legislation regarding clinical research and the 2.6 Subjective effects Declaration of Helsinki. This study has been registered at ClinicalTrials.gov (NCT05488171). Subjective effects were assessed using visual analog scales (VAS), the short form of the Addiction Research Center Inventory (ARCI), the Evaluation of Subjective Effects of Substances with Abuse 2.3 Drugs Potential questionnaire (VESSPA-SSE), the Sensitivity to Drug Reinforcement Questionnaire (SDRQ), and a pharmacological Methylone hydrochloride and MDMA hydrochloride were identification class questionnaire. acquired from the LGC Standards (Teddington, United Kingdom). VAS allowed participants to rate several adjectives from “not at The Pharmacy Department of the Hospital Universitari Germans Trias all” (0 mm) to “extremely” (100 mm) according to their sensations. i Pujol was responsible for manufacturing and dispensing identical, This instrument contained 31 items, including intensity (any effect), white, and opaque soft gelatin capsules of methylone, MDMA, or stimulated, high, good effects, bad effects, liking, changes in placebo (maltodextrin). The methylone capsules contained 50 mg of distances, changes in colors, changes in shapes, changes in lights, methylone, and MDMA capsules contained 100 mg of MDMA. During hallucinations (seeing lights or spots), hallucinations (seeing things, each session, participants received five capsules, combining capsules animals, insects, or people), changes in hearing, hallucinations with active substances and a placebo to reach the methylone or (hearing sounds or voices), drowsiness, concentration, dizziness, MDMA dose. confusion, different or changed body feeling, unreal body feeling, different surroundings, unreal surroundings, open, trust, feeling close to others, I want to be with other people, I want to hug 2.4 Procedures someone, sexual desire, and sexual arousal (Papaseit et al., 2016; Kuypers et al., 2018; Poyatos et al., 2021). One day prior to any experimental session, participants were The ARCI 49-item short form is a validated inventory developed tested for coronavirus disease 2019 (COVID-19) by performing a to evaluate the subjective effects of various substances, following five PCR test. Participants arrived at the Clinical Research Unit (UPIC) subscales: pentobarbital-chlorpromazine-alcohol group (PCAG) Frontiers in Pharmacology 03 frontiersin.org Poyatos et al. 10.3389/fphar.2023.1122861 measures sedation, morphine-benzedrine group (MBG) measures blood pressure. This method resulted in a sample size of at least euphoria, lysergic acid diethylamide (LSD) measures dysphoria and 10 participants, which was increased to 17 to improve statistical somatic symptoms, benzedrine (BG) measures intellectual efficiency power. and energy, and amphetamine (A) measures amphetamine-like For statistical analysis, values of physiological and subjective effects (Lamas et al., 1994; Papaseit et al., 2016; Poyatos et al., 2021). effects and psychomotor performance were baseline-adjusted. The standardized VESSPA-SSE questionnaire was used to The maximum effects from baseline (0 h) to 6 h (Emax) and the evaluate the subjective effects of stimulant drugs, such as time needed to achieve maximum effects (Tmax) were MDMA. This questionnaire is divided into six subscales that determined, and the area under the curve (AUC 0–6h) was assess sedation (S), psychosomatic anxiety (ANX), changes in calculated using the trapezoidal rule. One-way repeated perception (CP), pleasure and sociability (SOC), activity and measures analysis of variance (ANOVA), with treatment as a energy (ACT), and psychotic symptoms (PS) (Poudevida et al., factor, assessed differences among the three groups considering 2003; Papaseit et al., 2016; Poyatos et al., 2021). Emax and AUC values. If significant differences were detected In addition, participants completed the SDRQ (Kuypers et al., between treatments, post hoc Tukey’s multiple comparison test 2018), rating “How pleasant was the substance” (drug liking) and was performed. For Tmax values, differences were assessed using “How much you wanted to use it in that moment” (drug wanting) on the non-parametric Friedman test. A Wilcoxon signed-rank test a scale of 1–5. was performed for variables with significant differences in the In the pharmacological identification class questionnaire, previous test, with the p-value for multiple comparisons adjusted participants were required to select which pharmacological class using the Bonferroni test. better described the administered substance. The options included The time course (0–10 h) of effects was compared using a two- placebo, benzodiazepines (such as diazepam), alcohol, stimulants way repeated measures ANOVA with time and treatment as factors. (such as amphetamine), designer drugs (such as ecstasy), cocaine, When significant differences between treatments or in the hallucinogens (such as LSD), cannabinoids (such as cannabis), treatment × time interaction were detected, a post hoc Tukey’s ketamine (special K), GHB (gamma-hydroxybutyric acid; liquid multiple comparison test was performed to assess differences ecstasy), and others (Papaseit et al., 2016). between treatments at each time point. VAS (except sexual desire and sexual arousal) were performed at Statistical tests were performed using PASW Statistics, version baseline and 0.25, 0.50, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, and 24 h, but 18 (SPSS Inc., Chicago, IL, United States ). A p-value <0.05 indicated scales regarding intensity (any effect), stimulated, high, good effects, a statistically significant difference. bad effects, and liking were also performed at 2.5 h. SDRQ and VAS regarding sexual desire and arousal were performed at baseline and 1 and 10 h. ARCI and VESSPA-SSE were performed at baseline and 3 Results 1, 2, 3, 4, 6, 8, and 10 h. The pharmacological class identification questionnaire was performed at 8 h. Subjects were evaluated for 3.1 Participants psychiatric symptoms using the Young Mania Rating Scale at baseline, 0.5, 1, 4, 6, and 24 h after administration. Considering the participants who completed the study, 17 subjects had a mean age of 23 years (range 21–25 years) and a mean weight of 70.8 kg (range 52.1–84.1 kg), presenting a 2.7 Psychomotor performance mean body mass index of 23.2 kg/m2 (range 17.6–31.3 kg/m2). The participants reported previous use of MDMA (100%), Psychomotor performance was evaluated using a specific poppers (100%), cannabis (94%), mushrooms (71%), computerized psychomotor vigilance task (PVT) and a Maddox amphetamines (41%), cocaine (47%), LSD (18%), ketamine wing device. The PVT test was performed using software (PC-PVT (18%), sedatives/hypnotics (18%), nitrous oxide (12%), opioids 2.0) that quantifies the simple reaction time to a numeric stimulus (6%), dimethyltryptamine (DMT) (6%), and cathinones (6%). All (Reifman et al., 2018). The Maddox wing measures heterophoria due subjects tested negative on performing urine drug tests, to extraocular muscle imbalance and quantifies exophoria as an conducted using urine samples collected prior to the study indicator of extraocular musculature relaxation and esophoria; this session. has been previously reported in other studies evaluating the One subject was excluded after completing two experimental pharmacological effects of psychostimulants (Papaseit et al., sessions owing to undesirable effects, mainly anxiety and 2016). The PVT test was conducted at baseline and 1 and 2 h, uneasiness, attributed to the treatment administered in the while Maddox wing assessments were performed at baseline and second session. Treatment unblinding was performed after 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, and 24 h. excluding the subject, revealing that placebo and MDMA were administered during the first and second treatment sessions, respectively. Side effects that dissipated 1.5 h after appearance 2.8 Statistical analysis were attributed to MDMA, with no therapeutic intervention required to mitigate these effects. The sample size was calculated following the methodology of Except for the one excluded subject, all participants completed bioequivalence studies considering an alpha risk of 0.05, a power of their experimental sessions without reporting any serious adverse 80%, and a variability of 30% with a difference of at least 40% effects, including psychiatric symptoms, psychotic episodes, or between methylone or MDMA and placebo in systolic and diastolic hallucinations. Frontiers in Pharmacology 04 frontiersin.org Poyatos et al. 10.3389/fphar.2023.1122861 TABLE 1 Summary of results (n = 17; mean ± standard deviation; median, range) on physiological measures, subjective effects, and psychomotor performance with statistically significant differences between the three administered conditions (methylone 200 mg, MDMA 100 mg and placebo). Methylone MDMA Placebo ANOVA Tukey/Wilcoxon F/X2 p Physiological effects Systolic blood pressure Emax (mmHg) 37.35 ± 10.47 31.88 ± 15.28 −8.97 ± 7.64 89.589 <0.001 B,C Tmax (h) 0.75 (0.5–1.5) 1 (0.5–1.5) 4 (0.5–6) 17.844 <0.001 B,C Diastolic blood pressure Emax (mmHg) 12.29 ± 14.31 13.26 ± 13.02 0.47 ± 10.02 5.375 0.010 b,c Tmax (h) 1 (0.5–6) 1 (0.75–6) 1 (0.25–6) 0.677 0.713 NS Heart rate Emax (bpm) 29.38 ± 18.16 21.53 ± 22.17 0.97 ± 12.54 13.778 <0.001 B,C Tmax (h) 0.75 (0.5–3) 1 (0.25–6) 3 (0.25–6) 8.848 0.012 B Pupil diameter Emax (mm) 2.50 ± 0.66 3.10 ± 1.05 −0.15 ± 0.34 95.149 <0.001 B,C Tmax (h) 0.75 (0.5–1) 1 (0.5–2) 0 (0–4) 14.323 0.001 A Visual Analog Scales (VAS) Intensity Emax (mm) 45.41 ± 23.56 52.76 ± 30.07 1.12 ± 3.66 30.439 <0.001 B,C Tmax (h) 0.75 (0.5–1) 1 (0–2) 0 (0–1) 24.862 <0.001 a,B,C Stimulated Emax (mm) 48.65 ± 25.47 50.94 ± 32.87 1.29 ± 3.93 25.805 <0.001 B,C Tmax (h) 0.75 (0.5–1) 1 (0–2) 0 (0–1) 25.000 <0.001 a,B,C High Emax (mm) 54.65 ± 29.74 58.71 ± 29.78 1.06 ± 4.12 38.121 <0.001 B,C Tmax (h) 0.75 (0.5–1) 1 (0–2) 0 (0–1) 27.692 <0.001 a,B,C Good effects Emax (mm) 54.35 ± 30.00 57.53 ± 31.01 1.12 ± 4.36 34.635 <0.001 B,C Tmax (h) 0.75 (0.5–1) 1 (0–2) 0 (0–1) 26.226 <0.001 a,B,C Bad effects Emax (mm) 3.82 ± 7.61 11.06 ± 21.72 0 4.204 0.024 c Tmax (h) 0 (0–1.5) 0 (0–2) 0 (0–0) 9.920 0.007 NS Liking Emax (mm) 57.59 ± 31.38 57.18 ± 33.72 1.88 ± 7.51 30.962 <0.001 B,C Tmax (h) 0.75 (0.5–1) 1 (0–2.5) 0 (0–1) 25.803 <0.001 a,B,C Changes in distances Emax (mm) 7.29 ± 13.81 24.35 ± 34.45 0 5.515 0.009 C Tmax (h) 0 (0–1) 1 (0–6) 0 (0–0) 12.050 0.002 c Changes in lights Emax (mm) 11.06 ± 18.28 18.41 ± 25.25 0.53 ± 2.18 4.869 0.014 c Tmax (h) 0 (0–1) 0 (0–1.5) 0 (0–1.5) 8.486 0.014 NS Focused Emax (mm) 19.65 ± 18.05 15.06 ± 17.18 1.24 ± 5.09 8.500 0.001 B,c Tmax (h) 0.75 (0–1.5) 1 (0–4) 0 (0–1) 15.709 <0.001 B,c Dizziness Emax (mm) 10.12 ± 12.36 26.71 ± 26.23 0 11.580 <0.001 a,C Tmax (h) 0.5 (0–1.5) 0.75 (0–2) 0 (0–0) 16.128 <0.001 B,C Confusion Emax (mm) 10.71 ± 21.58 17.29 ± 22.83 0 6.165 0.005 C Tmax (h) 0 (0–1) 0.5 (0–2) 0 (0–0) 13.818 0.001 c Different body feeling Emax (mm) 32.65 ± 29.47 34.35 ± 34.15 1.18 ± 4.60 10.725 <0.001 B,C Tmax (h) 0.75 (0–1.5) 1 (0–2) 0 (0–1) 14.913 0.001 B,c Different surroundings Emax (mm) 10.35 ± 16.93 16.71 ± 26.37 0 4.311 0.022 c Tmax (h) 0 (0–1) 0 (0–1.5) 0 (0–0) 10.294 0.006 b,c (Continued on following page) Frontiers in Pharmacology 05 frontiersin.org Poyatos et al. 10.3389/fphar.2023.1122861 TABLE 1 (Continued) Summary of results (n = 17; mean ± standard deviation; median, range) on physiological measures, subjective effects, and psychomotor performance with statistically significant differences between the three administered conditions (methylone 200 mg, MDMA 100 mg and placebo). Methylone MDMA Placebo ANOVA Tukey/Wilcoxon F/X2 p Open Emax (mm) 44.82 ± 36.34 38.71 ± 31.45 1.53 ± 6.31 16.175 <0.001 B,C Tmax (h) 0.75 (0–1.5) 1.5 (0–2) 0 (0–1) 21.541 <0.001 B,C Trust Emax (mm) 43.71 ± 35.51 37.59 ± 31.27 1.53 ± 6.31 15.340 <0.001 B,C Tmax (h) 0.75 (0–1) 1 (0–3) 0 (0–1) 22.836 <0.001 a,B,C Feeling close to others Emax (mm) 41.06 ± 35.88 35.53 ± 32.48 1.53 ± 6.31 13.123 <0.001 B,C Tmax (h) 0.75 (0–1.5) 1 (0–3) 0 (0–1) 21.849 <0.001 a,B,C I want to be with other people Emax (mm) 46.41 ± 38.37 46.00 ± 35.10 1.53 ± 6.31 18.333 <0.001 B,C Tmax (h) 0.75 (0–1.5) 1 (0–2) 0 (0–1) 21.055 <0.001 B,C I want to hug someone Emax (mm) 34.71 ± 38.89 38.41 ± 35.05 1.06 ± 4.37 12.162 <0.001 B,C Tmax (h) 0.75 (0–1.5) 1 (0–2) 0 (0–1) 15.647 <0.001 B,C Sensitivity to Drug Reinforcement Questionnaire (SDRQ) How pleasant was the substance Emax (score) 2.53 ± 1.37 2.29 ± 1.45 0.24 ± 0.75 22.501 <0.001 B,C Tmax (h) 1 (0–1) 1 (0–1) 0 (0–1) 22.933 <0.001 B,C How much you wanted to use it in that moment Emax (score) 2.29 ± 1.36 1.71 ± 1.57 0.18 ± 0.73 15.221 <0.001 B,C Tmax (h) 1 (0–1) 1 (0–1) 0 (0–1) 21.875 <0.001 B,c Addiction Research Center Inventory (ARCI) ARCI PCAG Emax (score) 0.47 ± 4.11 3.35 ± 3.28 0.12 ± 0.99 9.797 <0.001 A,C Tmax (h) 1 (1–4) 2 (0–4) 0 (0–1) 21.390 <0.001 B,C ARCI MBG Emax (score) 7.76 ± 4.93 7.29 ± 5.03 0.47 ± 1.70 21.788 <0.001 B,C Tmax (h) 1 (0–1) 1 (0–3) 0 (0–1) 24.875 <0.001 B,C ARCI LSD Emax (score) 1.88 ± 2.29 2.18 ± 2.16 −0.35 ± 0.61 11.837 <0.001 B,C Tmax (h) 1 (0–2) 1 (0–2) 0 (0–1) 19.395 <0.001 B,C ARCI BG Emax (score) 2.71 ± 2.42 1.06 ± 2.77 0.53 ± 1.28 7.042 0.003 a,B Tmax (h) 1 (1–3) 1 (0–4) 0 (0–1) 23.216 <0.001 B,C ARCI A Emax (score) 4.24 ± 2.25 4.18 ± 2.72 0.47 ± 1.07 22.899 <0.001 B,C Tmax (h) 1 (1–1) 1 (0–2) 0 (0–1) 21.814 <0.001 B,C Subjective Effects of Substances with Abuse Potential (VESSPA-SSE) VESSPA S Emax (score) 0.52 ± 0.62 1.03 ± 0.98 0.08 ± 0.17 9.987 <0.001 C Tmax (h) 1 (0–6) 1 (0–3) 0 (0–4) 7.538 0.023 NS VESSPA ANX Emax (score) 1.66 ± 0.96 1.59 ± 0.98 0.13 ± 0.39 28.771 <0.001 B,C Tmax (h) 1 (1–3) 1 (0–3) 0 (0–3) 18.429 <0.001 b,C VESSPA CP Emax (score) 0.07 ± 0.17 0.30 ± 0.45 0 5.785 0.007 a,C Tmax (h) 0 (0–1) 0 (0–1) 0 (0–0) 10.667 0.005 c VESSPA SOC Emax (score) 1.90 ± 1.64 1.81 ± 1.42 0.15 ± 0.53 13.473 <0.001 B,C Tmax (h) 1 (0–1) 1 (0–3) 0 (0–1) 22.545 <0.001 B,C VESSPA ACT Emax (score) 1.82 ± 1.22 1.56 ± 1.35 0.10 ± 0.40 15.926 <0.001 B,C Tmax (h) 1 (0–2) 1 (0–3) 0 (0–1) 24.400 <0.001 B,C (Continued on following page) Frontiers in Pharmacology 06 frontiersin.org Poyatos et al. 10.3389/fphar.2023.1122861 TABLE 1 (Continued) Summary of results (n = 17; mean ± standard deviation; median, range) on physiological measures, subjective effects, and psychomotor performance with statistically significant differences between the three administered conditions (methylone 200 mg, MDMA 100 mg and placebo). Methylone MDMA Placebo ANOVA Tukey/Wilcoxon F/X2 p VESSPA PS Emax (score) 0.28 ± 0.39 0.33 ± 0.48 0.03 ± 0.12 5.094 0.012 b,c Tmax (h) 1 (0–1) 1 (0–2) 0 (0–1) 13.550 0.001 B,c Psychomotor performance Mean reaction time Emax (ms) −14.63 ± 31.09 36.40 ± 58.70 22.53 ± 39.48 6.818 0.003 A,b Tmax (h) 2 (1–2) 1 (1–2) 2 (1–2) 2.533 0.282 NS Maddox wing Emax (diopter) −0.97 ± 1.91 −1.69 ± 2.05 0.13 ± 0.43 7.070 0.003 C Tmax (h) 0.75 (0–1.5) 1 (0–2) 0 (0–6) 9.088 0.011 a Abbreviations: ARCI PCAG (sedation), MBG (euphoria), LSD (dysphoria), BG (intellectual efficiency), and A (amphetamine-like effects), VESSPA-SSE S (sedation), ANX (psychosomatic anxiety), CP (changes in perception), SOC (pleasure and sociability), ACT (activity and energy), and PS (psychotic symptoms), ms (milliseconds). Emax is maximum effects from baseline to 6 h, expressed as mean ± standard deviation. Tmax is the time needed to reach maximum effects, expressed as median (range). Differences among Emax were calculated with a one-way ANOVA (degrees of freedom 2 and 32 for all variables) and post hoc Tukey’s multiple comparisons test. Differences among Tmax were calculated with Friedman’s test and Wilcoxon signed rank test adjusted for multiple comparisons. Statistical differences in Emax between conditions are indicated as: “a” (p < 0.05) or “A” (p < 0.01) for methylone vs. MDMA, “b” (p < 0.05) or “B” (p < 0.01) for methylone vs. placebo, “c” (p < 0.05) or “C” (p < 0.01) for MDMA vs. placebo. Statistical differences in Tmax between conditions are indicated as: “a” (p < 0.016) or “A” (p < 0.003) for methylone vs. MDMA, “b” (p < 0.016) or “B” (p < 0.003) for methylone vs. placebo, “c” (p < 0.016) or “C” (p < 0.003) for MDMA vs. placebo. FIGURE 1 Time course (n = 17; mean ± standard error) of physiological effects and psychomotor performance following oral administration of 200 mg of methylone (○), 100 mg of MDMA (Δ) and placebo (□). Significant differences between methylone and MDMA are represented with *p < 0.05 and **p < 0.01. Significant differences from placebo are represented with filled symbols (p < 0.05). 3.2 Pharmacological effects performance for which the statistical analysis revealed significant differences between the three examined treatments. The results of Table 1 summarizes the results (Emax and Tmax) of AUC0–6h and time-course with significant differences between physiological effects, subjective effects, and psychomotor conditions are presented in Supplementary Table S1. Frontiers in Pharmacology 07 frontiersin.org Poyatos et al. 10.3389/fphar.2023.1122861 FIGURE 2 Time course (n = 17; mean ± standard error) of subjective effects following oral administration of 200 mg of methylone (○), 100 mg of MDMA (Δ) and placebo (□). Significant differences between methylone and MDMA are represented with *p < 0.05 and **p < 0.01. Significant differences from placebo are represented with filled symbols (p < 0.05). 3.2.1 Physiological effects The maximum effects of methylone and MDMA on cardiovascular Compared with the placebo, 200 mg methylone and 100 mg parameters and pupil diameter differed significantly from those of MDMA could significantly increase systolic blood pressure, diastolic placebo; however, these differences were not observed between blood pressure, heart rate, and pupil diameter (see Table 1; Figure 1). active treatments. Frontiers in Pharmacology 08 frontiersin.org Poyatos et al. 10.3389/fphar.2023.1122861 Regarding systolic blood pressure and heart rate, methylone particularly in ANX (psychosomatic anxiety), SOC (pleasure and administration afforded higher and earlier maximum effects than sociability), and ACT (activity and energy). Methylone produced MDMA administration, although these differences were not higher maximum effects represented by the VESSPA ANX, SOC, statistically significant. However, we noted significant differences and ACT, whereas MDMA produced higher effects sensitive to the between methylone and MDMA considering certain time-course VESSPA S (sedation), CP, and PS subscales. The VESSPA CP was points of systolic blood pressure, heart rate, pupil diameter, AUC, the only subscale with significant differences in the maximum effects and time to reach maximum effects (Tmax) of pupil diameter, with and AUC between active treatments. Comparing the time courses of methylone demonstrating lower AUC and earlier time to reach methylone and MDMA, time points between 1 and 3 h differed maximum effects than MDMA. significantly in VESSPA S, ANX, CP, and SOC. Methylone produced a sustained increase in heart rate, which Regarding SDRQ, methylone was higher rated in the “How differed significantly from placebo until 10 h post-administration, pleasant was the substance” and “How much you wanted to use it in returning to baseline after 24 h. Changes in diastolic blood pressure that moment” questions than MDMA and placebo, considering were markedly similar between methylone and MDMA, considering maximum effects and AUCs; however, differences among active maximum effects, time to achieve maximum effects, and differences substances were not statistically significant. in the time course of effects. Considering the pharmacological class identification Except at one time-course point (2 h), there were no significant questionnaire, 16 (94.1%) participants identified methylone as a differences in any temperature parameter between active treatments. designer drug, similar to MDMA, whereas one (5.9%) participant identified it as a stimulant, similar to amphetamine. After receiving 3.2.2 Subjective effects MDMA, 15 (88.2%) participants correctly identified their treatment Compared with the placebo, methylone and MDMA induced as a designer drug (MDMA), whereas one (5.9%) subject classified it significant changes in subjective effects measured using VAS and as a stimulant (amphetamine) and another (5.9%) as a placebo. The questionnaires (ARCI, VESSPA, and SDRQ) (Table 1; Figure 2). placebo was correctly identified by 16 (94.1%) participants, with The significant changes in VAS were mainly related to only one subject classifying it as a stimulant (amphetamine). stimulation and wellbeing (VAS “stimulation”, “high”, “good effects”, “liking”), altered perception (“changes in distances”, 3.2.3 Psychomotor performance “changes in lights”, “different body feeling”, “different In the PVT test, methylone was the only treatment that surroundings”), and empathy (“open”, “trust”, “feeling close to significantly improved the reaction time by reducing the mean others”, “I want to be with other people”, “I want to hug time needed to react to a numeric stimulus (see Table 1). someone”). In general, methylone-induced subjective effects Conversely, MDMA did not impact the reaction time when appeared at 0.5 h and normalized at 2.5–3 h post-administration. compared with the placebo. Considering the time course of The subjective effects of MDMA appeared later (0.75 h), returning effects, methylone could significantly decrease the reaction time to baseline at 4 h. when compared with MDMA and placebo at 1 and 2 h post- Comparing both active substances, maximum effects and AUCs administration. of most subjective effects measured with VAS did not differ After methylone and MDMA administration, the participants significantly; however, participants who received MDMA experienced an inward deviation of the eyes (esophoria) in the reported significantly more ‘dizziness’. Notably, methylone- Maddox wing device (see Table 1). Notably, MDMA produced induced maximum effects appeared significantly earlier (Tmax = higher, but statistically insignificant, levels of esophoria than 0.75 h), considering effects related to stimulation, wellbeing, and methylone, considering their maximum effects and AUC. empathy, than those induced by MDMA (Tmax = 1 h). Regarding However, methylone administration could induce maximum the time course of effects, we noted significant differences within the effects significantly earlier than MDMA administration. During first 2.5 h in several scales related to stimulation, wellbeing, altered the time course of effects, significant differences were detected perception, and empathy between both active treatments, with between the two active treatments at 2 and 3 h. higher effects of methylone typically documented at earlier time points (0.5–0.75 h), whereas those of MDMA noted at later time points (2–2.5 h) (see Supplementary Table S1). 4 Discussion Methylone and MDMA demonstrated significant changes in all subscales of the ARCI questionnaire, especially MBG (euphoria) and To the best of our knowledge, this is the first experimental, A (amphetamine effects). Methylone generated higher maximum controlled study to evaluate the acute pharmacological effects of scores on the MBG, BG (intellectual efficiency), and A subscales, methylone in humans. Our main finding was that oral doses of whereas MDMA generated higher scores for PCAG and LSD 200 mg methylone could induce notable cardiovascular and (dysphoria). However, only PCAG (sedation) and BG pleasurable effects, including stimulation, euphoria, wellbeing, (intellectual efficiency) revealed significant differences in the increased empathy, and altered perception. Methylone exhibited maximum effects between both treatments. Comparing the time an effect profile comparable to that of MDMA, with a faster onset courses of methylone and MDMA, we noted significant differences and earlier disappearance of subjective effects than MDMA. at certain time points between 1 and 4 h for ARCI PCAG, MBG, BG, Typically, the abuse potential of methylone follows the trend of and A. other cathinones, such as natural cathinone and mephedrone Compared with placebo, methylone and MDMA demonstrated (Poyatos et al., 2022b). MDMA exhibited effects similar to those significant differences in the VESSPA questionnaire in all subscales, previously reported in other published human studies (Camí et al., Frontiers in Pharmacology 09 frontiersin.org Poyatos et al. 10.3389/fphar.2023.1122861 2000; Kirkpatrick et al., 2014; Papaseit et al., 2016; Kuypers et al., of subjective effects induced by the former, a particularity that 2017; Holze et al., 2020; Studerus et al., 2021). more closely resembles mephedrone (Papaseit et al., 2016). Our results corroborate those of an observational study in which Considering the data from the pilot studies, methylone displayed the effects of methylone were evaluated under natural conditions fast pharmacokinetics at oral doses ranging from 50 to 200 mg; (Poyatos et al., 2021). In the previous observational study, eight specifically, 200 mg methylone achieved maximum plasma subjects self-administered an oral dose of methylone (100–300 mg, concentrations (Cmax) of 604 ng/mL at 2 h (Tmax), with an mean dose of 187 mg), which were self-selected according to their elimination half-life (t1/2) of 6.4 h (Poyatos et al., 2022a). These preferences and previous experiences. Under the same setting, six results suggest that the kinetics of methylone are faster than those of subjects were selected and self-administered oral MDMA MDMA (Tmax of 2–2.5h, t1/2 of 7.7–12 h) (Desrosiers et al., 2013; (75–100 mg, mean dose of 87 mg). In that study, methylone Hysek et al., 2014; Papaseit et al., 2016) but less rapid than those of exhibited a prototypical stimulant profile and empathogenic mephedrone (Tmax of 1.25, t1/2 of 2.15) (Papaseit et al., 2016). The effects frequently attributed to social drugs, such as MDMA. maximum pharmacological effects of methylone appeared earlier Moreover, methylone was shown to induce a high and sustained than the maximum concentrations, but the short elimination half- increase in the heart rate, also observed under controlled conditions. life could explain the early dissipation of most subjective effects. This Regarding subjective effects, methylone induced milder effects than fact can also be observed in other studies evaluating human MDMA in the earlier discussed naturalistic study. According to our pharmacology of other cathinones such as mephedrone (Papaseit present results, participants reported higher subjective effects after et al., 2016). In that study, mephedrone and MDMA produced methylone administration than those documented in the previous earlier maximum effects compared to their Tmax for blood study. This discrepancy could be attributed to the wider experience concentration. Effects for mephedrone peaked at 0.75h, while its with psychostimulant use among volunteers in the naturalistic study concentrations peaked at 1.25 h. In case of MDMA, effects peaked at or the distinct settings in which the studies were conducted. In 0.75–1.25 h whereas maximum concentrations were achieved at 2 h. addition, the maximum subjective effects of methylone appeared Our results, along with those from previous studies, confirm that earlier under controlled conditions. pharmacological effects do not need maximum concentrations of Furthermore, comparing our results with other synthetic the substance in blood to reach peak effects. Moreover, the cathinones, such as mephedrone, considered one of the most concentrations of its metabolite 4-hydroxy-3-methoxy- popular cathinones for recreational use, can be interesting. The N-methylcathinone (HMMC) were analyzed, revealing a plasma human pharmacology of mephedrone was evaluated in a clinical Cmax of 42.1 ng/mL, Tmax of 1.5 h, and t1/2 of 6.3 h on trial including 12 subjects well-experienced in psychostimulant use, administering 200 mg methylone. However, the psychoactive who received one oral dose of 200 mg mephedrone compared with effects might not be attributed to HMMC concentrations despite 100 mg MDMA and placebo. Methylone and mephedrone appear to its affinity for monoamine transporters (Elmore et al., 2017; Luethi share similarities in their physiological effects, given that both could et al., 2019), given the poor brain penetration capacity of produce higher maximum effects on heart rate and a lower increase hydroxylated metabolites (Centazzo et al., 2021). Data regarding in pupil diameter than MDMA. Overall, both cathinones affected the linearity of methylone remain controversial in animals, with vital signs with an early onset and similar intensity. Like methylone, evidence of non-linear behavior after subcutaneous administration mephedrone exhibits an MDMA-like profile with desirable in rats (Elmore et al., 2017) and linear pharmacokinetics after oral subjective effects, comprising stimulation, a sensation of administration (López-Arnau et al., 2013). However, the wellbeing, altered perception, and increased sociability (Papaseit pharmacokinetics of methylone seem linear in humans (Poyatos et al., 2016). et al., 2022a), contrary to MDMA’s, which has been widely described Moreover, the effects of methylone are consistent with the as non-linear (De La Torre et al., 2000). The pharmacokinetic data euphorigenic and stimulant effects of khat, a naturally occurring are consistent with the reduced potency of methylone at monoamine cathinone (Brenneisen et al., 1990; Widler et al., 1994). transporters when compared to MDMA. Additionally, methylone Interestingly, methylone displayed characteristics that may not penetrate the brain as effectively as MDMA, due to the differed from those of MDMA. Unlike MDMA and more polar β-keto group. Regarding methylone’s capacity to mephedrone, methylone could induce a sustained and penetrate into the brain, previous studies confirmed that significant increase in heart rate when compared with the methylone cross the blood-brain barrier with a brain-to-plasma placebo, which persisted for 10 h despite the normalization of ratio of 1.42 (López-Arnau et al., 2013), 4.54 (Štefková et al., 2017) or blood pressure. Moreover, methylone improved psychomotor more than 3 (range 3–12) (Centazzo et al., 2021). Studies for MDMA performance by reducing reaction time. This improvement has found brain concentrations 5- to 10-fold higher than those in plasma also been documented with amphetamine administration (Silber (Mueller et al., 2009). Methylone seems to need higher blood et al., 2006), although there is no clear evidence for MDMA concentrations to produce comparable pharmacological effects (Camí et al., 2000; Farré et al., 2004; Ramaekers and Kuypers, with MDMA. 2006; Kuypers et al., 2007). Combined with alcohol, mephedrone The limitations of the present study should be noted. The could reduce the reaction time when compared with that of statistical analysis lacked the power to detect significant alcohol alone by mitigating the sedative effects of alcohol. differences between both active drugs owing to the sample size, However, mephedrone alone produced markedly similar despite increasing the size with respect to that initially calculated. In effects to the placebo on reaction time (De Sousa Fernandes addition, although this study included participants of both sexes, the Perna et al., 2016). However, the main difference between small number of females was insufficient to explore sex differences methylone and MDMA is the earlier onset and disappearance in the acute effects of methylone. As only one dose of methylone was Frontiers in Pharmacology 10 frontiersin.org Poyatos et al. 10.3389/fphar.2023.1122861 examined, our findings could not be extrapolated to higher doses or analyses and generated the figures. LP, MF, and EP wrote the first for establishing a dose-response relationship. draft; all the authors revised, and edited the manuscript. Given its short-lived subjective effects, methylone seems suitable for redosing to extend its duration of action. This postulation is reinforced by high scores in the SDRQ questionnaire regarding the Funding desire to repeat the dose, which was performed 1 h post- administration. At that time point, the subjective effects of This work was partially supported by Instituto de Salud Carlos methylone were already decreasing in most participants, while III (ISCIII, Fondo de Investigación en Salud [FIS]-Fondo Europeo those of MDMA were at their highest level. This phenomenon is de Desarrollo Regional [FEDER] under grant numbers PI17/ also exhibited by mephedrone, a drug often readministered owing to 01962 and PI20/00879, and a predoctoral PFIS fellowship under its brief effects and rapid pharmacokinetics (Papaseit et al., 2016). grant number FI18/00179); Plataformas ISCIII de apoyo a la I + D + I en Biomedicina y Ciencias de la Salud under grant number PT20/ 00018; and ISCIII-Redes de Investigación Cooperativa Orientadas a 5 Conclusion Resultados en Salud (RICORS)-Red de Investigación en Atención Primaria de Adicciones (RIAPAd) under grant numbers RD21/ To the best of our knowledge, the present study is the first 0009/0001 and RD21/0009/0004 funded by Instituto de Salud Carlos controlled study to assess the pharmacological effects of methylone III (ISCIII), and by the European Union NextGenerationEU, in humans. Methylone enhanced cardiovascular parameters and Mecanismo para la Recuperación y la Resiliencia (MRR). It is subjective effects, characterized by stimulation, euphoria, increased also partially supported by the project “Implementation of the sociability, and altered perception. This profile was similar to the identification and study of the effects of NPS: Development of a prototypical effects associated with MDMA, which differed in onset multicenter research to enhance the database of the National drug and duration. Overall, these results suggest that the abuse potential Addiction Observatory and the Early Warning System” and by the of methylone is similar to that of MDMA in humans, although its project “National Early Warning System on Drugs” (SNAP)” shorter subjective effects with faster onset may lead to a redosing following the Italian Department of Antidrug policies. pattern of use. Conflict of interest Data availability statement The authors declare that the research was conducted in the The original contributions presented in the study are included in absence of any commercial or financial relationships that could be the article/Supplementary Material, further inquiries can be directed construed as a potential conflict of interest. to the corresponding authors. Publisher’s note Ethics statement All claims expressed in this article are solely those of the authors and The studies involving human participants were reviewed and do not necessarily represent those of their affiliated organizations, or approved by Germans Trias i Pujol Human Research Ethics those of the publisher, the editors and the reviewers. Any product that Committee (code PI-19-082). The patients/participants provided may be evaluated in this article, or claim that may be made by its their written informed consent to participate in this study. manufacturer, is not guaranteed or endorsed by the publisher. Author contributions Supplementary material MF, EP, LP, and CP-M conceptualized the study design. MF, EP, The Supplementary Material for this article can be found online CP-M, SM, LP, OH, MN-M, GdR, AB, and LC collected the data; SM at: https://www.frontiersin.org/articles/10.3389/fphar.2023.1122861/ and LP organized the database. MF, EP, and LP performed statistical full#supplementary-material References Australian Criminal Intelligence Commission (2020). National wastewater drug Barrios, L., Grison-Hernando, H., Boels, D., Bouquie, R., Monteil-Ganiere, C., and monitoring program report 10. 1–86. Available at: https://www.acic.gov.au/sites/ Clement, R. (2016). Death following ingestion of methylone. Int. J. Leg. 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