ORIGINAL RESEARCH published: 28 January 2020 doi: 10.3389/fphar.2019.01588 Mephedrone and Alcohol Interactions in Humans Esther Papaseit 1,2†, Clara Pérez-Mañá 1,2*†, Elizabeth B. de Sousa Fernandes Perna 3†, Eulalia Olesti 4,5†, Julian Mateus 4, Kim PC Kuypers 3, Eef L. Theunissen 3, Francina Fonseca 2,6, Marta Torrens 2,6, Jan G. Ramaekers 3‡, Rafael de la Torre 4,5,7‡ and Magí Farré 1,2‡ 1 Department of Clinical Pharmacology, Hospital Universitari Germans Trias i Pujol (IGTP), Badalona, Spain, 2 Department of Pharmacology, Therapeutics and Toxicology and Department of Psychiatry and Forensic Medicine, Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès, Spain, 3 Department of Neuropsychology and Psychopharmacology, Faculty of Psychology and Neuroscience, Maastricht University, Maastricht, Netherlands, 4 Integrative Pharmacology and Systems Edited by: Neuroscience Research Group, Neurosciences Research Program, IMIM-Hospital del Mar Medical Research Institute, Parc Aviv M. Weinstein, de Salut Mar, Barcelona, Spain, 5 Department of Health and Life Sciences, Universitat Pompeu Fabra (CEXS-UPF), Ariel University, Israel Barcelona, Spain, 6 Institut de Neuropsiquiatria i Adiccions, Addiction Unit and IMIM, Barcelona, Spain, 7 CIBER de Reviewed by: Fisiopatología de la Obesidad y Nutrición (CB06/03), CIBEROBN, Santiago de Compostela, Spain Matthias E. Liechti, University Hospital of Basel, Switzerland Mephedrone (4-MMC, mephedrone) is a synthetic cathinone derivative included in the Elena Escubedo Rafa, class of new psychoactive substances. It is commonly used simultaneously with alcohol University of Barcelona, (ethanol). The aim of the present study was to evaluate the interactions on subjective, Spain Matt Kirkpatrick, cardiovascular and hormone effects and pharmacokinetics between mephedrone and University of Southern California, alcohol in humans. Eleven male volunteers participated as outpatients in four experimental United States sessions in a double-blind, randomized, cross-over, and placebo-controlled clinical trial. *Correspondence: Clara Pérez-Mañá Participants received a single oral dose of 200 mg of mephedrone plus 0.8 g/kg of alcohol cperezm.mn.ics@gencat.cat (combination condition); 200 mg of mephedrone plus placebo alcohol (mephedrone † These authors share first authorship condition); placebo mephedrone plus 0.8 g/kg of ethanol (alcohol condition); and placebo ‡ These authors share senior mephedrone plus placebo alcohol (placebo condition). Outcome variables included authorship physiological (blood pressure, heart rate, temperature, and pupil diameter), psychomotor (Maddox wing), subjective (visual analogue scales, Addiction Research Specialty section: This article was submitted to Center Inventory 49 item short form, and Valoración de los Efectos Subjetivos de Neuropharmacology, Sustancias con Potencial de Abuso questionnaire), and pharmacokinetic parameters a section of the journal Frontiers in Pharmacology (mephedrone and ethanol concentrations). The study was registered in ClinicalTrials.gov, Received: 18 August 2019 number NCT02294266. The mephedrone and alcohol combination produced an increase Accepted: 09 December 2019 in the cardiovascular effects of mephedrone and induced a more intense feeling of Published: 28 January 2020 euphoria and well-being in comparison to the two drugs alone. Mephedrone Citation: reduced the sedative effects produced by alcohol. These results are similar to those Papaseit E, Pérez-Mañá C, de Sousa Fernandes Perna EB, obtained when other psychostimulants such as amphetamines and 3,4- Olesti E, Mateus J, Kuypers KPC, methylenedioxymethamphetamine are combined simultaneously with alcohol. The Theunissen EL, Fonseca F, Torrens M, Ramaekers JG, de la Torre R and abuse liability of mephedrone combined with alcohol is greater than that induced by Farré M (2020) Mephedrone and mephedrone alone. Alcohol Interactions in Humans. Front. Pharmacol. 10:1588. Keywords: 4-methylmethcathinone (mephedrone), new psychoactive substance, alcohol (ethanol), interaction, doi: 10.3389/fphar.2019.01588 pharmacologic effects, pharmacokinetics Frontiers in Pharmacology | www.frontiersin.org 1 January 2020 | Volume 10 | Article 1588 Papaseit et al. Mephedrone and Alcohol INTRODUCTION In addition, mephedrone is linked to an intensive and repetitive administration pattern in which other drugs are During the previous decade, numerous non-conventionally listed concomitantly consumed (Schifano et al., 2011; Deluca et al., psychoactive compounds, or new psychoactive substances 2012). In these social scenes (e.g., nightclubs, music festivals, rave (NPS), have emerged on the illicit drug market to replace parties), the use of mephedrone and alcohol is the most common controlled ones (EMCDDA, 2016). Ease of availability two-drug combination reported among NPS recreational users combined with relatively low prices, and high purity compared (EMCDDA, 2010; Carhart-Harris et al., 2011; Winstock et al., to classical street drugs, plus popularization via Internet-driven 2011). Under these conditions, users often report combining social media, have contributed notably to their increasing mephedrone with alcohol to either heighten its effects or presence on the drug scene. Of the 670 NPS detected by the ameliorate the come-down, a particularly unpleasant European Union’s early warning system, synthetic cathinones experience following mephedrone consumption (Newcombe, make up the second largest group (ACMD, 2010; Papaseit et al., 2009; Carhart-Harris et al., 2011; O’Neill and McElrath, 2012). 2014; EMCDDA, 2019). With respect to acute mephedrone intoxication cases, it is Mephedrone (4-methylmethcathinone, 4MMC, drone, M-CAT, notable that in 18.2% of cases, alcohol was also present White Magic, meow meow), also known as “bath salt,” “plant (EMCDDA, 2016). Indeed, this concomitant use of feeder,” and/or “legal high,” has emerged as a prototypical synthetic mephedrone and alcohol in humans has led to several acute cathinone surpassing the popularity of other NPS (Vardakou et al., toxicities (McGaw and Kankam, 2010) and fatalities (Maskell 2011; Bretteville-Jensen et al., 2013). It is a beta-keto amphetamine et al., 2011; Regan et al., 2011; Cosbey et al., 2013; Elliott and analogue, structurally and pharmacological related to 3,4- Evans, 2014; Loi et al., 2015; Corkery et al., 2017; Papaseit et al., methylenedioxymethamphetamine (MDMA) which it was 2017). The latest results for acute drug toxicity presentations become its legal alternative (Green et al., 2014; Liechti, 2015). In from the European Drug Emergencies Network (Euro-DEN) vitro pharmacological assays have characterized mephedrone as a indicate mephedrone as the most common NPS involved (n = non-selective releaser and inhibitor of their uptake at the 88). The main acute effects include agitation, anxiety, monoamine transporter (Baumann et al., 2012; Simmler et al., palpitations and chest pain. Recently, in Poland an increased 2013; Baumann et al., 2013; Rickli et al., 2015; Saha et al., 2015). number of acute intoxications with mephedrone (binge episodes) Recently, initial data regarding the human pharmacology of in combination primarily with alcohol and also other substances mephedrone have confirmed its psychostimulant-like effects have been detected (Ordak et al., 2018). which were first reported by recreational users in forums, surveys, Despite the potentially added risks of mephedrone combined and naturalistic and observational studies (Dargan et al., 2010; with alcohol, experimental data in humans about interactions Winstock et al., 2011; Freeman et al., 2012; Papaseit et al., 2016; between mephedrone and alcohol are very limited. Experimental Homman et al., 2018). Following controlled oral administration, studies in animal models concerning the effects induced after mephedrone induces cardiovascular-stimulant and euphoric effects single and repeated mephedrone and alcohol administration, with a high abuse liability characterized by earlier onset and shorter demonstrated that alcohol increases stimulant and rewarding duration in comparison to MDMA and other amphetamine effects of mephedrone (Ciudad-Roberts et al., 2015), and can also derivatives (d-amphetamine, methamphetamine and potentiate the neurotoxic properties of mephedrone in methylphenidate) (Mayo and de Wit, 2015; Papaseit et al., 2016; adolescent mice (Ciudad-Roberts et al., 2016). A recent study Dolder et al., 2017; Dolder et al., 2018). has concluded that mephedrone in combination with alcohol In spite of its illicit status, recreational use of mephedrone enhances the psychostimulant effect of mephedrone measured as continues to be present on the drug scene (Winstock et al., 2011; locomotor activity. Given that both serotonin (5-HT) and Deluca et al., 2012; González et al., 2013; Kelly et al., 2013; dopamine are also related with reward and impulsivity, the Mixmag’s Drug Survey: The Results, 2014; CSEW, 2018). In observed effects point to an increased risk of abuse liability 2015, data from the European Drug Report estimated a previous when combining mephedrone with alcohol compared with the year prevalence of 3% among club-goers (EMCDDA, 2015). Based sole administration of these drugs (López-Arnau et al., 2018). A on results from the Crime Survey for England and Wales, previous part of the present study, including the neurocognitive year use of mephedrone among 16- to 34-year-olds was estimated performance effects of mephedrone and alcohol, have been at 0.2%; down from 0.5% in 2015/2016, and 1.1% in 2014/15 formerly published (de Sousa Fernandes Perna et al., 2016). (CSEW, 2018; EMCCDA, 2018). In the United Kingdom, there The results showed that whilst alcohol intoxication generally have also been reports of “slamming”—the intravenous injection impaired performance, mephedrone improved psychomotor of mephedrone and other drugs, such as methamphetamine and performance, impaired spatial memory but it did not affect gamma hydroxybutyrate, immediately before/during sex in groups divided attention performance. Nevertheless, the stimulatory of men who have sex with men at “chemsex” parties (Melendez- effects of mephedrone were not enough to compensate for the Torres et al., 2018). Mephedrone injecting has been reported as impairing effects of alcohol on most performance parameters. occurring mainly among individuals who have previously injected The present study was designed to assess the subjective, other drugs (e.g. heroin users), those who have switched from cardiovascular and hormone effects and pharmacokinetics snorting mephedrone, and among younger users (UNODC, 2017). following the interaction between mephedrone and alcohol Frontiers in Pharmacology | www.frontiersin.org 2 January 2020 | Volume 10 | Article 1588 Papaseit et al. Mephedrone and Alcohol under controlled co-administration in humans. The findings Drugs presented in this paper are part of the previously mentioned Mephedrone was supplied by the Spanish Ministry of Justice and study focusing on the physiological and subjective effects and Ministry of Health. Mephedrone and placebo capsules were pharmacokinetics of both substances. prepared as opaque, white, soft gelatin capsules under the supervision of the Pharmacy Unit of the Hospital del Mar. Acute alcohol intoxication was induced by the ingestion of MATERIALS AND METHODS a beverage containing vodka (Absolut®, Ahus, Sweden) diluted in lemon-flavored water (Fontvella®) to mask the placebo Subjects drink. The alcohol-placebo drink was lemon-flavored Twelve healthy male subjects were recruited by word of mouth. water (Fontvella®). Eligibility criteria required the recreational use of amphetamines, ecstasy, mephedrone, or cathinones with a lifetime minimum of Study Design six times, and on at least two occasions during the year prior to The study was double-blind, double-dummy, randomized, cross- participation, without any serious adverse reaction; recreational over, and placebo-controlled. The four drug conditions consisted use of alcohol (less than four units of alcohol per day) and of a single oral dose: 200 mg of mephedrone plus 0.8 g/kg of previous experience of acute alcohol intoxication; and no history alcohol (combination condition); 200 mg of mephedrone plus of abuse or drug dependence according to the Diagnosis and placebo alcohol (mephedrone condition); placebo mephedrone Statistical Criteria for Mental Disorders IV-R for any other plus 0.8 g/kg of alcohol (alcohol condition); and placebo substances except nicotine (in smokers). mephedrone plus placebo alcohol (placebo condition). The All participants completed the study except for one who dose of mephedrone was selected based on previous results of dropped out due to personal circumstances after participating the first study evaluating pharmacological effects of mephedrone in three experimental sessions (final n = 11). The participants in humans under controlled and experimental administration. had a mean age of 28 years (range 22–39 years), mean weight This dose was selected after a series of pilot studies that included 71.8 kg (range 56.8–83.3 kg), mean height 173.5 cm (range single oral doses of 50, 100, 150, and 200 mg of mephedrone, and 164.0–180.0 cm) and body mass index 23.8 kg/m2 (range 19.0– 100 mg MDMA, being the dose of 200 mg well tolerated and 26.8 kg/m2). They had experience with mephedrone (36.4%), produced similar effects to MDMA (Papaseit et al., 2016). The MDMA (90.9%), and cocaine (100%). The participants drank an dose of alcohol and the combination were selected based in average of 2.4 units of alcohol per day (range 1.0–4.0). All but five previous psychostimulant drug and alcohol interaction studies were smokers (mean five cigarettes/day, range 2–15). (Farré et al., 1993; Hernández-López et al., 2002). Prior to their inclusion the participants underwent a general medical examination, including blood laboratory tests, urinalysis, 12-lead electrocardiogram (ECG), and a Psychiatric Experimental Sessions Research Interview for Substance and Mental Disorders. Subjects were admitted to the Clinical Research Unit facilities at Participants completed a training session to familiarize 07:45 a.m. after an overnight fast. Upon arrival, they were themselves with testing procedures and questionnaires. In questioned about any drug consumption or event that could addition, in order to reduce variability in pharmacokinetics of affect their participation. They had been requested to refrain mephedrone and to avoid the possibility that subject carriers of from using any psychoactive drug for a minimum of seven days allelic variants leading to the PM phenotype for CYP2D6 might prior to the study and throughout it, and from consuming be at increased risk of acute toxicity, only subjects who were caffeinated products for 24 h and alcohol for 48 h. A urine phenotypically CYP2D6 extensive metabolizers were included sample was collected for drug testing (Instant-View®, Multipanel (de la Torre et al., 2005). The protocol was approved by the local 10 Test Drug Screen, Alfa Scientific Designs Inc., Poway, CA- Research Ethics Committee (CEIC-Parc de Salut Mar, Barcelona, USA). They remained in a calm and comfortable laboratory Spain). The study was conducted in accordance with the environment during the entire session. Tobacco smoking was not Declaration of Helsinki and Spanish laws concerning clinical permitted in the sessions. Last cigarette was allowed 2 h trials and registered in ClinicalTrials.gov (number before admission. NCT02294266). The volunteers were financially compensated. At the beginning of each experimental session baseline Safeguard measures taken to ensure participant welfare while measures were performed. Mephedrone or matched placebo they were participating in the study included health controls (one capsule) was administered at 8:30 a.m. in a fasting state before each session (a phone call one day before each session to with 100 milliliters (ml) of bottled water (Fontvella®). Alcohol or ensure health status), during sessions (medical examinations at matched placebo was administered at 9:00 a.m., 30 min after baseline, clinical monitoring with continuous ECG during 10 h mephedrone or matched placebo administration. The total and a psychiatric evaluation) and after sessions (medical volume of the beverage (350 ml) was consumed in 15 min examination 24 h after each session), a final control including (one-third volume every 5 min). medical examination and blood and urine chemistry were done Four, 6, and 10 h after administration a light breakfast, a meal, 3–7 days after the last session, and a final phone call after 3– and a snack were provided to the participants/subjects, 4 weeks. respectively. A psychiatric evaluation was performed 8 h after Frontiers in Pharmacology | www.frontiersin.org 3 January 2020 | Volume 10 | Article 1588 Papaseit et al. Mephedrone and Alcohol administration and adverse effects were assessed during each includes six subscales: sedation (S), psychosomatic anxiety experimental session and the following day. (ANX), changes in perception (CP), pleasure and sociability (SOC), activity and energy (ACT), and psychotic symptoms Physiological Measures (PS) (Poudevida et al., 2003; Papaseit et al., 2016). Non-invasive systolic blood pressure (SBP), diastolic blood The pharmacological class identification questionnaire asks pressure (DBP), heart rate (HR), and oral temperature about the class of drugs the participants believed they had been (T) were repeatedly recorded at: -45 min, 0 h (baseline, given at each administration (Rush et al., 1995). The options immediately prior to capsule administration), 0.25, 0.5 included placebo, benzodiazepine (e.g., valium, diazepam, (immediately prior to beverage administration), 0.75, 1, 1.5, 2, tranxilium, rophipnol), alcohol, stimulant (amphetamine), 3, 4, 6, 8, 10, and 24 h following initial drug administration. All designer drugs (ecstasy), cocaine, hallucinogen (e.g., LSD, assessments were carried out with a DinamapTM 8100-T vital mescaline), cannabinoids (e.g., marijuana, hashish), ketamine signs monitor (Critikon, Tampa, Fla., US). Pupil diameter (PD) (special K), and gamma hydroxybutyrate (liquid ecstasy). and the Maddox-wing device were recorded at: 0 h (baseline, The VAS were administered at: -45 min (baseline), 0.25, 0.5 immediately prior to capsule administration), 0.25, 0.5 (immediately prior to beverage administration), 0.75, 1, 1.5, 2, 3, (immediately prior to beverage administration), 0.75, 1, 1.5, 2, 4, 6, 8, 10, and 24 h following initial drug administration. ARCI 3, 4, 6, 8, 10, and 24 h following initial drug administration. and VESSPA-SEE were administered at: -45 min, 0.25, 0.5 1.5, 2, Pupillary diameter was recorded with a Haab pupil gauge and 3, 4, 6, 8, and 10 h following initial drug administration. The Maddox wing measures were expressed in diopters along the pharmacological class identification questionnaire was given at 8 device’s horizontal scale. The Maddox-wing device measures the h following initial drug administration. balance of extraocular muscles and quantifies exophoria as an indicator of extraocular muscle relaxation, and esophoria as an indicator of extraocular muscle tension. For safety reasons ECG Pharmacokinetics was continuously monitored along 10 h using a DinamapTM Blood samples for the determination of mephedrone were Plus vital signs monitor (Critikon, Tampa, Fla., US). collected during each experimental session at -5 min (0 h, baseline), 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, and 24 h Subjective Effects following drug administration. Urine was collected until 24 h Subjective effects were measured using a set of 23 visual analogue (data not shown). Mephedrone plasma concentrations were scales (VAS), the 49-item Addiction Research Center Inventory quantified by gas chromatography-mass spectrometry (GC- short form (ARCI), the Evaluation of Subjective Effects of MS). A liquid–liquid extraction was performed with tert-butyl Substances with Abuse Potential questionnaire (VESSPA-SEE), methyl etherm and the silylation reagent [N-methyl-N- and a pharmacological class identification questionnaire. (trimethylsilyl) trifluoroacetamide] was used for the VAS (100 mm) were labeled at opposite ends with different derivatization of mephedrone (Papaseit et al., 2016; Olesti adjectives ranging from “not at all” to “extremely” (Farré et al., et al., 2017). 2004; Peiró et al., 2013; Farré et al., 2015; Papaseit et al., 2016). Blood samples for the determination of alcohol were collected Subjects were asked to rate effects from among “high,” during each experimental session at -5 min (0 h, baseline), 0.75, “drunkenness,” “stimulated,” “any effect,” “good effects,” “bad 1, 1.5, 2, 3, 4, 6, 8, and 10 h after drug administration. Urine was effects,” “liking,” “content,” “drowsiness,” “dizziness,” collected until 24 h (data not shown). Alcohol plasma “confusion,” “fear,” “depression or sadness,” “changes in concentrations were determined with an enzymatic test (DRI distances,” “changes in colors,” “changes in shapes,” “changes Ethyl Alcohol Assay, Thermo Scientific) in an autoanalyzer in lights,” “hallucinations-seeing of lights or spots,” “changes in (Indiko Plus, Thermo Fisher Scientific). hearing,” “hallucinations-hearing sounds or voices,” Blood samples for the determination of cortisol were collected “hallucinations-seeing animals, things, insects or people,” during each experimental session at -5 min (0 h, baseline), 1, 2, 4, “different or changed unreal body feeling,” and “different or 6, and 8 h following drug administration. They were centrifuged unreal surroundings”. at 3,000 rpm for 10 min at 4°C, plasma and serum were removed The Spanish validated version of the short-form ARCI and frozen at -220°C until analysis. Cortisol plasma (Lamas et al., 1994), which consists of a true/false 49-item concentrations were determined by fluorescence polarization questionnaire, it is a validated instrument for determining immunoassay (Abbott Laboratories, Chicago, IL) according to subjective drug effects including five subscales: PCAG the manufacturer’s instructions (Kobayashi et al., 1979). (pentobarbital-chlorpromazine-alcohol group, a measure of sedation); MBG (morphine-benzedrine group, a measure of Statistical Analysis euphoria); LSD (lysergic acid diethylamide group, a measure Statistical analysis was performed for eleven participants. of dysphoria and somatic symptoms); BG (benzedrine group, a Values from physiological and subjective effects were stimulant subscale relating to intellectual efficiency and energy); transformed to differences from baseline. The peak effects in and A (amphetamine, a measure of d-amphetamine effects) the first 6 h after first drug administration (maximum absolute (Farré et al., 2015). change from baseline values, Emax), and the 6-h area under the The VESSPA-SEE is a questionnaire that measures changes in curve (AUC) of effects versus time were calculated by the subjective effects caused by different drugs including MDMA. It trapezoidal rule for each variable. Frontiers in Pharmacology | www.frontiersin.org 4 January 2020 | Volume 10 | Article 1588 Papaseit et al. Mephedrone and Alcohol Peak concentration (Cmax) and time to reach peak No serious adverse events were observed. No hallucinations, concentrations (Tmax) from mephedrone, alcohol and cortisol psychotic episodes, or any other psychiatric symptoms were plasma concentrations were determined using PKSolver, a freely experienced during the sessions. None of the participants available add-in program for Microsoft Excel (Joel Usansky, Atul required specific therapy or special care during the study. All Desai, and Diane Tang-Liu, Department of Pharmacokinetics 11 subjects completed the study. and Drug Metabolism, Allergan, Irvine, CA, USA). Area under the concentration-time curve from mephedrone, alcohol and Physiological Effects cortisol (AUC0-6), mephedrone (AUC0-24), and alcohol Regarding physiological effects, the two conditions including (AUC0-10) were calculated by the linear trapezoidal rule. mephedrone (combination condition and mephedrone Firstly, these transformations were analyzed using a two-way condition) produced an increase in SBP, DBP, HR, and PD as analysis of variance (ANOVA) test to study the influence of some compared with the placebo condition (when considering both participant factors as age, body mass index, weight, smoking and peak effects and AUC). The more relevant differences for HR alcohol use in the different parameters calculated. Because the increase appeared between the combination and mephedrone results showed only marginal statistically significant results for conditions. The former produced an increase of 40 ± 17 beats per factors/interactions, the analysis was rejected (11 variables showed min (bpm) at 1 h following the first drug administration whilst significant results for a total number of 200 comparison, for the latter the peak effects was 32 ± 20 bpm at 0.75 h. In percentage 5.5%). Subsequently, the statistical analysis presented statistical terms, only significant differences were detected for was performed without considering those factors. AUC0-6 (142.33 mm × h for the combination condition, and 70.91 Then, these transformations were analyzed by means of one- mm × h for the mephedrone one) and T-C points (1.5–6 h) for way repeated-measures analysis of variance (ANOVA) with drug both. For the remaining physiological variables (SBP, DBP, T, condition as factor. In case of significant differences among and PD), only significant differences in several T-C points were treatment conditions in ANOVA, post-hoc multiple comparisons observed between the combination and mephedrone conditions were performed using the Tukey test. Time course (T-C) of effects (no AUC or peak effects). was analyzed employing two-way repeated-measures ANOVA The alcohol condition produced very slight effects on SBP, with Treatment condition and Time (0–6 h) as factors. When DBP, HR, and T and none on DP compared with the placebo Treatment condition, or the Treatment condition × Time condition (non-statistically significant). Comparing the interaction, were statistically significant, multiple Tukey post-hoc combination and alcohol condition, the mephedrone-alcohol comparisons were performed at each time point. The difference in effects on SBP, DBP, HR and DP were significantly higher than time to reach peak effects (Tmax) values among conditions was those produced by alcohol alone (when considering both peak assessed with the nonparametric Friedman test. When significant effects and AUC) with significant differences at several results among conditions were detected post-hoc multiple T-C points. comparison was performed applying the Wilcoxon signed rank In the Maddox-wing device, the combination and test adjusting the p value to six comparisons (p < 0.008). mephedrone conditions led to an increase in the degree of All statistical tests were performed at each time point using esophoria compared with placebo (when considering peak the PASW Statistics 18.0 (SPSS Inc., Chicago, IL, USA). A value effects and/or AUC). Esophoria induced by the combination of p < 0.05 was considered statistically significant. condition was approximately two to three-fold lower (-1.41 ± 2.91 diopters) in comparison with the mephedrone one (-3.45 ± 2.73 diopters) with only significant differences in several T-C points (1–1.5 h). Alcohol slightly increased exophoria (+1.41 ± RESULTS 1.30 diopters), relaxation of ocular musculature, but only Global Results significant differences in several T-C points were obtained Table 1 shows the summary of physiological and subjective when compared with the placebo. The combination condition effects where at least one statistical difference (peak, AUC) was scored approximately midway between the mephedrone and found in the ANOVA and multiple comparison post-hoc test alcohol conditions. analyses. Furthermore, it includes T-C points that presented significant differences in ANOVA and the multiple-comparison Subjective Effects post-hoc tests. Table 2 shows the Peak effect values of variables The two conditions containing mephedrone caused an increase where at least one statistical difference was found in the ANOVA. in subjective effects (VAS, ARCI, and VESSPA-SEE) compared The T-C (differences from baseline) for the most relevant with placebo (Tables 1 and 2). physiological, psychomotor, and subjective effects are shown in The combination and mephedrone conditions led to significant Figures 1 and 2, respectively. Concentrations over time and changes in the ratings of “high,” “stimulated,” “any effect,” “good pharmacokinetic parameters of mephedrone and alcohol in effects,” “liking,” “content” and also in “change in distances and plasma are presented in Figure 3 and Table 3, respectively. different, changed or unreal body feeling” in comparison with The effects of all drug/experimental conditions on plasma placebo. In general terms, the combination condition produced cortisol levels are depicted in Tables 1 and 2. higher and more prolonged scores with statistically significant Frontiers in Pharmacology | www.frontiersin.org 5 January 2020 | Volume 10 | Article 1588 Frontiers in Pharmacology | www.frontiersin.org Papaseit et al. TABLE 1 | Summary of statistically significant results of the physiological parameters and subjective effects (n = 11) observed after administration of mephedrone plus alcohol (mephedrone–alcohol), mephedrone plus placebo alcohol (mephedrone), placebo mephedrone plus alcohol (alcohol), and placebo mephedrone plus placebo alcohol (placebo condition). Variable Tukey’s Multiple Comparison Test (*p < 0.05; **p < 0.01) ANOVA Placebo Mephedrone Alcohol Parameter F/X2 p Value Alcohol Mephedrone Mephedrone–alcohol Alcohol Mephedrone– Mephedrone–alcohol alcohol Physiological effects SBP AUC (df = 3,30) 22.107 <0.001 NS ** ** ** NS ** Peak (df = 3,30) 38.667 <0.001 NS ** ** ** NS ** Tmax 11.97 0.007 NS NS * NS NS NS T-C (df = 3,270) 13.924 <0.001 0.75*,4* 0.5**,0.75**,1**, 1.5**,2**,3** 0.5**,0.75**,1**, 1.5**,2**,3** 0.5**,0.75**,1**, 2* 0.5**,0.75**,1**,1.5**,2**,3**,4** 1.5**,2**,3**,4** DBP AUC (df = 3,30) 17.087 <0.001 NS ** ** ** NS ** Peak (df = 3,30) 20.138 <0.001 NS ** ** ** NS ** Tmax 9.54 0.023 NS NS NS NS NS NS T-C (df = 3,270) 5.526 <0.001 6* 0.5*,0.75**,1**,1.5**,2** 0.5**,0.75**,1**, 1.5**,2** 0.5**,0.75**,1**, 0.5**,0.75**,1**,1.5**, 2**,3*,4** 1.5**,2**,3*,4**,6** HR AUC (df = 3,30) 25.960 <0.001 NS ** ** NS ** ** Peak (df = 3,30) 23.570 <0.001 NS ** ** ** NS ** Tmax 9.38 0.025 NS * NS NS NS NS T-C (df = 3,270) 10.750 <0.001 0.75**,1**,3*, 4**,6* 0.5**,0.75**,1**, 0.5**,0.75**,1**, 0.5**,0.75**,1**, 1.5**,2**,3** 1.5**,2**,3**,4**,6** 0.5*,0.75**,1**, 1.5**, 1.5**,2**,3**,4**, 6* 1.5**,2**,3**,4**,6** 2**,3**,4**, 6** T AUC (df = 3,30) 6.475 0.002 NS ** NS * NS NS Peak (df = 3,30) 1.420 0.252 Tmax 2.93 0.402 6 T-C (df = 3,270) 4.598 <0.001 0.75** 1.5**,2**,3**,4**, 6** 0.75**,1**,2**,4**,6** 0.75**,1.5**,2**,3**,4**,6** 0.75**,1**,1.5** 0.75**,1**,1.5*,2*,3**,4** PD AUC (df = 3,30) 20.973 <0.001 NS ** ** ** NS ** Peak (df = 3,30) 25.333 <0.001 NS ** ** ** NS ** Tmax 14.24 0.003 NS * NS NS NS NS T-C (df = 3,270) 8.972 <0.001 0.5*,0.75**,1**, 1.5**,2* 0.5**,0.75**,1**,1.5**, 2**, 3** 0.5*,0.75**,1**,1.5**,2* 2*,3** 0.5**,0.75**,1**,1.5**,2**,3** Maddox-wing AUC (df = 3,30) 7.674 0.001 NS NS NS ** NS * Peak (df = 3,30) 13.1320 <0.001 NS ** NS ** NS ** Tmax 6.21 0.102 T-C (df = 3,270) 4.585 <0.001 1**,1.5*,4* 0.75**,1**,1.5**,2* 0.75**,1* 0.5**,0.75**,1**,1.5**,2**,3**,4** 1**, 1.5* 0.75**,1**,1.5**,2**,4** Visual Analogue Scales High AUC (df = 3,30) 14.998 <0.001 NS NS ** NS ** ** Peak (df = 3,30) 27.105 <0.001 NS ** ** ** NS ** Tmax 28.39 <0.001 NS * * NS * * T-C (df = 3,270) 10.564 <0.001 0.5**,0.75**, 1**,1.5**,2* 0.75**, 1**, 1.5**, 2**,3**, 4** 0.5**, 0.75**, 1**, 1.5**, 2* 0.5*, 0.75**, 0.75**, 1**,1.5**,2**,3**,4** 1.5**,2**,3**,4* January 2020 | Volume 10 | Article 1588 Drunkenness AUC (df = 3,30) 20.076 <0.001 ** NS ** ** ** NS Peak (df = 3,30) 30.600 <0.001 ** NS ** ** ** NS Tmax 30.12 <0.001 * NS * * * NS T-C (df = 3,270) 8.780 <0.001 0.75**,1**,1.5**,2**,3** 0.75**,1**,1.5**,2**,3* 0.75**,1**,1.5**, 2**,3** 0.75**,1**,1.5**,2**,3* 0.75**,1**,1.5** Stimulated AUC (df = 3,30) 9.434 <0.001 NS NS ** NS * * Peak (df = 3,30) 17.026 <0.001 * ** ** NS NS ** Mephedrone and Alcohol Tmax 23.69 <0.001 * * * NS NS NS T-C (df = 3,270) 5.568 <0.001 0.75**, 1**,1.5** 0.5**,0.75**,1**,1.5** 0.5**,0.75**,1**,1.5**,2**,3** 0.5**,0.75** 1.5**,2**,3** 0.5**,0.75**,1**,1.5**, 2** Any effect AUC (df = 3,30) 13.702 <0.001 ** NS ** NS ** NS Peak (df = 3,30) 21.813 <0.001 ** ** ** NS NS NS Tmax 24.38 <0.001 * * * NS NS NS T-C (df = 3,270) 6.411 <0.001 0.75**,1**,1.5**,2** 0.5**,0.75**,1**,1.5** 0.5**,0.75**,1**, 1.5**,2**,3** 0.5** 1*,1.5**,2**,3** 0.5**,1.5**,2* (Continued) Frontiers in Pharmacology | www.frontiersin.org Papaseit et al. TABLE 1 | Continued Variable Tukey’s Multiple Comparison Test (*p < 0.05; **p < 0.01) ANOVA Placebo Mephedrone Alcohol 2 Parameter F/X p Value Alcohol Mephedrone Mephedrone–alcohol Alcohol Mephedrone– Mephedrone–alcohol alcohol Good effects AUC (df = 3,30) 14.115 <0.001 NS NS ** NS ** ** Peak (df = 3,30) 22.754 <0.001 NS ** ** * NS ** Tmax 19.21 <0.001 * * * NS NS NS T-C (df = 3,270) 6.173 <0.001 1**.1.5* 0.5**,0.75**,1**, 1.5**,2** 0.25**,0.5**,0.75**, 0.75**,1*,1.5* 0.25**,0.5**,3** 0.25**,0.5**,0.75**, 1**,1.5**,2**,3** 1**,1.5**,2*,3** Bad effects AUC (df = 3,30) 3.426 0.030 * Peak (df = 3,30) 2.384 0.089 Tmax 8.29 0.040 NS NS NS NS NS NS T-C (df = 3,270) 1.164 0.268 Liking AUC (df = 3,30) 12.629 <0.001 NS NS ** NS * ** Peak (df = 3,30) 15.248 <0.001 NS ** ** * NS ** Tmax 17.67 0.001 NS * * NS NS NS T-C (df = 3,270) 5.756 <0.001 0.5*,0.75**,1**, 1.5** 0.5**,0.75**,1**, 1.5**,2**,3** 0.5*,0.75**,1** 1.5**,2**,3** 0.5**,0.75**,1**, 1.5**,2**,3** Content AUC (df = 3,30) 10.094 <0.001 NS NS ** NS ** ** Peak (df = 3,30) 16.287 <0.001 ** ** ** NS NS * Tmax 22.4 <0.001 * * * NS NS NS T-C (df = 3,270) 4.726 <0.001 1**,1.5**,2* 0.75**,1**,1.5**, 2* 0.5**,0.75**,1**, 1.5**,2**,3** 0.75** 1.5**,2**,3** 0.5**,0.75**,1**,1.5**, 2**,3** Drowsiness AUC (df = 3,30) 6.629 0.001 ** NS NS ** NS * Peak (df = 3,30) 5.383 0.004 ** NS NS * NS NS Tmax 14.73 0.002 NS NS NS NS * NS T-C (df = 3,270) 2.117 <0.001 2*,3**,4** 6* 2**,4** 6* 2**,3**,4* 7 Dizziness AUC (df = 3,30) 3.375 0.031 * NS NS NS NS NS Peak (df = 3,30) 3.055 0.043 * NS NS NS NS NS Tmax 13.13 0.004 NS NS NS NS NS NS T-C (df = 3,270) 1.521 0.052 Confusion AUC (df = 3,30) 2.347 0.093 Peak (df = 3,30) 2.395 0.088 Tmax 8.10 0.044 NS NS NS NS NS NS T-C (df = 3,270) 1.679 0.022 0.5*,0.75*,2** 0.5**,0.75**,1**, 1.5**,2* 2** 1.5* Changes in AUC (df = 3,30) 2.071 0.125 Distances Peak (df = 3,30) 1.530 0.226 Tmax 9.87 0.020 NS NS NS NS NS NS T-C (df = 3,270) 1.513 0.054 Changes in Colors AUC (df = 3,30) 3.184 0.038 NS NS * NS NS NS Peak (df = 3,30) 2.310 0.096 Tmax 6.38 0.095 January 2020 | Volume 10 | Article 1588 T-C (df = 3,270) 1.352 0.120 Changes in lights AUC (df = 3,30) 2.880 0.052 Peak (df = 3,30) 2.652 0.067 Tmax 7.03 0.071 T-C (df = 3,270) 1.661 0.024 1* 0.5**,0.75**,1**, 1.5**,2** 0.5**,0.75**,1.5* 0.5**,0.75**,2* Changes in hearing AUC (df = 3,30) 1.102 0.364 Mephedrone and Alcohol Peak (df = 3,30) 1.574 0.216 Tmax 7.32 0.062 T-C (df = 3,270) 1.587 0.037 0.5**,0.75**,1* 0.5**,0.75**,1* 0.5* Different body AUC (df = 3,30) 4.905 0.007 NS NS ** NS NS NS feeling Peak (df = 3,30) 7.766 0.001 NS ** ** NS NS NS Tmax 14.43 0.002 NS NS * NS NS NS T-C (df = 3,270) 3.535 <0.001 0.5**,0.75**,1** 0.5**,0.75**,1**, 1.5**,2**,3** 0.5**,0.75**,1** 1.5*,3** 0.5**,0.75**,1**,1.5**,3* (Continued) Frontiers in Pharmacology | www.frontiersin.org Papaseit et al. TABLE 1 | Continued Variable Tukey’s Multiple Comparison Test (*p < 0.05; **p < 0.01) ANOVA Placebo Mephedrone Alcohol 2 Parameter F/X p Value Alcohol Mephedrone Mephedrone–alcohol Alcohol Mephedrone– Mephedrone–alcohol alcohol Different AUC (df = 3,30) 1.426 0.255 surroundings Peak (df = 3,30) 2.147 0.115 Tmax 9.04 0.029 NS NS NS NS NS NS T-C (df = 3,270) 1.243 0.195 ARCI questionnaire subscales ARCI-PCAG AUC(df = 3,30) 12.704 <0.001 ** NS NS ** NS ** Peak (df = 3,30) 6.992 0.001 ** NS NS * NS ** Tmax 11.64 0.009 * NS NS NS NS NS T-C (df = 3,150) 6.992 <0.001 1.5*,2**,3**,4**,6** 1.5**,2*,6** 1.5**,2**,3**,4**,6** 2**,3**,6** 1.5**,2**,3**,4** ARCI-MBG AUC (df = 3,30) 20.441 <0.001 NS * ** NS ** ** Peak (df = 3,30) 23.217 <0.001 NS ** ** * * ** Tmax 18.88 <0.001 * * * NS NS NS T-C (df = 3,150) 11.162 <0.001 1.5**,2** 1.5**,2**,3**,4* 1.5**,2** 1.5**,2**,3** 1.5**,2**,3** ARCI-LSD AUC (df = 3,30) 3.787 0.020 NS NS * NS NS NS Peak (df = 3,30) 3.290 0.034 NS NS NS NS NS NS Tmax 15.43 0.001 * NS * NS NS NS T-C (df = 3,150) 2.357 0.005/ 1.5**,2**,3** 1.5** 1.5**,2**,3*,4** 3* 4** 0.025 ARCI-BG AUC (df = 3,30) 13.389 <0.001 NS NS * ** NS ** Peak (df = 3,30) 12.759 <0.001 NS NS ** ** NS ** Tmax 14.90 0.001 * NS * NS NS NS 8 T-C (df = 3,150) 5.124 <0.001 3** 1.5** 1.5**,2**,3** 1.5**,2**,3**,4* 2*,3** 1.5**,2**,3**,4** ARCI-A AUC (df = 3,30) 17.750 <0.001 NS ** ** * NS ** Peak (df = 3,30) 24.674 <0.001 NS ** ** * NS ** Tmax 17.60 0.001 NS * * NS NS NS T-C (df = 3,150) 9.197 <0.001 1.5* 1.5**,2**,3* 1.5**,2**,3**,4** 1.5**,2** 2**,3** 1.5**,2**,3**,4** VESSPA questionnaire subscales VESSPA-S AUC (df = 3,30) 7.671 0.001 ** NS NS ** NS NS Peak (df = 3,30) 9.517 <0.001 ** NS * * NS NS Tmax 21.03 <0.001 * * * NS NS NS T-C (df = 3,150) 2.989 <0.001 1.5**, 2**,3**,4**,6** 6** 1.5**, 2**,3**,4*,6** 6** 1.5**,2**,3** VESSPA-ANX AUC (df = 3,30) 28.198 <0.001 NS ** ** * ** ** Peak (df = 3,30) 31.959 <0.001 NS ** ** ** NS ** Tmax 21.51 <0.001 * * * NS NS NS Time Course (df = 20.031 <0.001 1.5**,2**,3** 1.5**,2**,3**,4**,6** 1.5**,2**,3* 2**,3**,4**,6** 1.5**,2**,3**,4**,6** 3,150) January 2020 | Volume 10 | Article 1588 VESSPA-CP AUC (df = 3,30) 3.445 0.029 NS NS NS NS NS NS Peak (df = 3,30) 3.604 0.025 NS NS NS NS NS NS Tmax 8.88 0.031 NS NS NS NS NS NS T-C (df = 3,150) 2.159 0.010 1.5**,2**,3* 1.5**,2**,3** 1.5**,2*,3* 1.5**,2**,3* VESSPA-SOC AUC (df = 3,30) 11.817 <0.001 NS ** ** NS NS ** Peak (df = 3,30) 13.779 <0.001 NS ** ** NS NS ** Mephedrone and Alcohol Tmax 16.53 0.001 NS NS NS NS NS NS T-C (df = 3,150) 8.619 <0.001 1.5**,2* 1.5**,2** 1.5**,2**,3**,4* 1.5** 1.5**,2**,3** 1.5**,2**,3** VESSPA-ACT AUC (df = 3,30) 15.151 <0.001 NS NS ** NS ** ** Peak (df = 3,30) 20.330 <0.001 NS ** ** NS NS ** Tmax 15.03 0.002 * NS NS NS NS NS T-C (df = 3,150) 10.297 <0.001 1.5** 1.5**,2** 1.5**,2**,3**,4* 1.5** 1.5**,2**,3** 1.5**,2**,3** (Continued) Papaseit et al. Mephedrone and Alcohol °C [temperature (T)], mm [pupil diameter (PD); mm visual analog scale (VAS0)], and score [Addiction Research Center Inventory (ARCI), score Evaluation of Subjective Effects of Substances with Abuse Potential questionnaire (VESSPA-SEE)] differences in AUC and several T-C points compared with Mephedrone–alcohol mephedrone alone. In addition, for “stimulated,” “any effect,” “good effects,” “liking,” and “content” scores these differences Alcohol 1.5**,2** 1**,2** were statistically significant for peak effects and, in the case of NS NS NS ** ** * “high,” also for Tmax. As expected, statistically significant differences were detected in drunkenness rating scores between conditions (AUC, peak, Tmax, and T-C, Tables 1 and 2). The alcohol condition produced an increase in the ratings of “drunkenness,” “content,” “stimulated,” “any effect,” “good Mephedrone– effects,” and “liking” compared with placebo (Table 2). alcohol 2**,4* Regarding “drunkenness,” the most characteristic effect for NS NS NS NS NS NS NS alcohol, a no significant lower score was observed for the combination condition in comparison to the alcohol condition Mephedrone (peak effect 34.18 ± 21.53 vs 49.36 ± 25.70 mm, respectively; Tukey’s Multiple Comparison Test (*p < 0.05; **p < 0.01) Table 2), but significant differences, however, were only found at For Peak, AUC and T-C an ANOVA and multiple Tukey post hoc comparisons were used. Statistical differences among conditions are presented as **p < 0.01 and *p < 0.05. several T-C points (0.75–1.5 h). In contrast, significant Alcohol differences were detected for “content” effects in AUC, peak 1**,2** NS NS NS NS ** ** effects, and T-C points (0.5–3 h) with higher scores in the combination condition compared with the alcohol one (Tables 1 and 2). For Tmax a Friedman test and multiple Wilcoxon post-hoc comparisons were used. Differences among conditions are presented as *p < 0.008; **p < 0.002. In the ARCI questionnaire, both conditions including mephedrone produced an increase in the scores of MBG Mephedrone–alcohol Peak = peak effects from 0 to 6 hours measured by mmHg (systolic blood pressure [SBP]; diastolic blood pressure [DBP]), bpm (heart rate [HR]), (euphoria), BG (intellectual efficiency and energy) (Tables 1 1.5**,2**,3*,4* and 2), A (amphetamine-like effects), and LSD (dysphoria) 1**,2** NS NS subscales in comparison to placebo (when considering AUC, ** ** ** * peak effects, and both). The MBG, BG, and A peak scores were 10.36 ± 3.91, 3.82 ± 3.52, and 6.55 ± 2.73 points for the combination condition and 6.45 ± 5.80, 2.64 ± 2.94, and 4.82 ± 2.79 points for the mephedrone condition, respectively. For MBG, significant differences were detected in AUC, peak Placebo Mephedrone effects, and several T-C points (1.5–3h) between conditions. 1**,2** For the other subscales only significant differences were 1.5** NS NS NS NS ** ** detected at several T-C points. Conversely, for the LSD subscale no significant differences were detected between AUC = area under the curve from 0 to 6 h measured by units x hours and expressed as mean. both conditions. Alcohol produced a statistically significant increase in the PCAG-sedation subscale in comparison to placebo. Compared Tmax = time to reach peak effects measured by hours and expressed as median. Alcohol with the alcohol condition, the combination with mephedrone NS NS NS NS NS NS NS reduced the sedation induced by alcohol (peak effect 6.18 ± 3.60 vs 5.26 ± 1.58) with statistical differences in peak effects and AUC that remained statistically significant during 2.5 h in the T-C analysis (1.5–4 h). In contrast, MBG peak difference scores were p Value <0.001 <0.001 <0.001 <0.001 0.028 0.004 0.004 0.001 lower for alcohol compared to the combination condition (2.27 ± 1.62 vs 10.36 ± 3.91, respectively) with statistical differences in peak effects, AUC, and T-C analysis (1.5–3 h) (Tables 1 and 2). 19.266 17.240 17.340 3.463 5.497 13.53 4.025 8.404 2 F/X Regarding the VESSPA-SEE questionnaire, the combination ANOVA and mephedrone conditions increased all the subscales compared with placebo. The combination condition, in T-C (df = 3,150) T-C (df = 3,120) Peak (df = 3,30) Peak (df = 3,30) AUC (df = 3,30) AUC (df = 3,30) Parameter comparison to the mephedrone one, presented statistical Tmax Tmax differences in peak effects for ACT (activity and energy) and ANX (psychosomatic anxiety) subscales and in several T-C and expressed as mean. TABLE 1 | Continued points for all the subscales (Tables 1 and 2). With respect to df = degree of freedom. the alcohol condition, statistical differences in peak effects, AUC, and T-C points for ANX, SOC, (pleasure and sociability) and VESSPA-PS ACT subscales and only in peak effects for CP subscale were Variable Cortisol detected compared to the combination one. Frontiers in Pharmacology | www.frontiersin.org 9 January 2020 | Volume 10 | Article 1588 Papaseit et al. Mephedrone and Alcohol TABLE 2 | Summary of Peak effects (maximal effect, mean value and standard error) for physiological, subjective and cortisol concentrations (Cmax) (n = 11). Placebo Alcohol Mephedrone Mephedrone + alcohol Physiological effects SBP -5.82 ± 2.96 -5.00 ± 4.52 35.45 ± 4.28 40.36 ± 3.57 DBP 0.27 ± 3.10 -1.73 ± 3.86 19.64 ± 2.08 22.45 ± 1.83 HR -7.18 ± 2.28 9.91 ± 4.35 32.27 ± 6.03 40.09 ± 5.25 T -0.94 ± 0.12 -0.47 ± 0.14 -0.56 ± 0.21 -0.56 ± 0.16 PD 0.00 ± 0.00 0.00 ± 0.00 1.64 ± 0.35 1.91 ± 0.33 Maddox-wing 0.00 ± 0.27 1.41 ± 0.39 -3.45 ± 0.82 -1.41 ± 0.88 Visual Analogue Scales High 0.00 ± 0.00 1.55 ± 1.55 47.36 ± 9.31 55.09 ± 7.90 Drunkenness 0.00 ± 0.00 49.36 ± 7.75 0.00 ± 0.00 34.18 ± 6.49 Stimulated 0.00 ± 0.00 23.00 ± 5.77 42.73 ± 8.56 53.18 ± 8.17 Any effect 0.00 ± 0.00 49.91 ± 7.40 51.18 ± 9.27 56.09 ± 7.68 Good effects 0.00 ± 0.00 23.18 ± 5.20 51.09 ± 8.59 63.36 ± 8.49 Bad effects 0.00 ± 0.00 14.18 ± 7.26 3.45 ± 2.06 4.55 ± 2.05 Liking 0.00 ± 0.00 17.18 ± 7.19 44.09 ± 8.59 50.27 ± 8.31 Content 0.00 ± 0.00 30.91 ± 6.73 43.64 ± 8.24 56.09 ± 8.16 Drowsiness 8.45 ± 2.62 29.09 ± 8.14 12.36 ± 4.81 17.00 ± 3.95 Dizziness 0.00 ± 0.00 20.27 ± 8.07 6.18 ± 5.70 11.36 ± 8.00 Confusion 0.00 ± 0.00 20.64 ± 7.67 11.00 ± 5.85 15.73 ± 9.29 Changes in Distances 0.00 ± 0.00 11.55 ± 6.14 11.64 ± 6.53 13.09 ± 6.78 Changes in Colors 0.00 ± 0.00 4.73 ± 2.46 6.27 ± 3.45 8.55 ± 3.84 Changes in lights 0.00 ± 0.00 6.36 ± 2.76 6.64 ± 4.11 11.64 ± 5.73 Changes in hearing 0.00 ± 0.00 3.18 ± 1.58 14.18 ± 9.29 6.27 ± 4.13 Different body feeling 0.00 ± 0.00 13.27 ± 5.21 30.91 ± 9.01 33.18 ± 9.67 Different surroundings 0.00 ± 0.00 9.91 ± 5.39 10.73 ± 3.61 15.36 ± 7.80 ARCI questionnaire subscales ARCI-PCAG 1.00 ± 0.65 6.18 ± 1.09 2.82 ± 1.19 1.73 ± 1.58 ARCI-MBG 0.09 ± 0.09 2.27 ± 0.49 6.45 ± 1.75 10.36 ± 1.18 ARCI-LSD -0.45 ± 0.25 1.55 ± 0.85 0.73 ± 0.74 1.55 ± 0.96 ARCI-BG 0.00 ± 0.23 -2.27 ± 0.54 2.64 ± 0.89 3.82 ± 1.06 ARCI-A 0.27 ± 0.14 2.00 ± 0.47 4.82 ± 0.84 6.55 ± 0.82 VESSPA questionnaire subscales VESSPA-S 1.09 ± 1.09 9.36 ± 2.49 4.27 ± 1.47 6.09 ± 1.40 VESSPA-ANX 0.00 ± 0.00 1.55 ± 0.41 9.00 ± 1.72 12.36 ± 1.46 VESSPA-CP 0.00 ± 0.00 1.64 ± 0.65 0.45 ± 0.25 1.91 ± 0.91 VESSPA-SOC 0.09 ± 0.09 4.09 ± 1.46 7.36 ± 2.22 13.00 ± 1.73 VESSPA-ACT 0.27 ± 0.19 4.36 ± 0.96 8.18 ± 2.14 12.55 ± 1.52 VESSPA-PS 0.00 ± 0.00 0.64 ± 0.39 1.27 ± 0.45 2.45 ± 0.87 Cortisol 14.93 ± 4.50 16.20 ± 4.88 22.23 ± 6.70 23.85 ± 7.19 SBP: systolic blood pressure, mm; DBP: diastolic blood pressure, mmHg; HR: heart rate, beats per minute; T: temperature, °C; PD: pupil diameter, mm; visual analogue scale, mm, ARCI: Addiction Research Center Inventory [ARCI], score; VESSPA: Evaluation of Subjective Effects of Substances with Abuse Potential questionnaire, score. Only statistically significant results are showed (see Table 1 for comparisons). Conditions are mephedrone plus alcohol (mephedrone–alcohol), mephedrone plus placebo alcohol (mephedrone), placebo mephedrone plus alcohol (alcohol), and placebo mephedrone plus placebo alcohol (placebo). In the pharmacologic drug class identification questionnaire, Pharmacokinetic Measures the combination condition (mephedrone plus alcohol) was Mephedrone and Alcohol Concentrations correctly identified by all subjects (100% designer drug and The pharmacokinetic parameters for mephedrone and alcohol 100% alcohol). For the mephedrone condition (mephedrone are summarized in Table 3 and Figure 3. plus placebo), mephedrone was identified by eight subjects as a When the two conditions containing mephedrone were designer drug (72.7%), as a stimulant (18.2%) by two, and as compared, no significant differences were found in the placebo (9.1%) by one, whilst placebo was identified correctly by pharmacokinetic parameters (Table 3). In the combination all the subjects (100%). With respect to the alcohol/placebo condition, mephedrone concentrations peaked at 1.5 h. In the alcohol conditions, the placebo was identified as such by nine mephedrone condition, mephedrone concentrations peaked at subjects (81.8%), and as a designer drug (9.1%) and cannabinoids 1.5 h. In both conditions, at 10 h following first drug (9.1%) by one subject each, whilst alcohol was identified correctly administration, mephedrone concentrations declined to mean by all subjects (100%). values of 15.73 and 12.76 ng/ml until undetectable levels at 24 h, All the active conditions (mephedrone, alcohol, and the respectively (Figure 3). combination) were well tolerated and no mania, hallucinations Regarding alcohol, significant differences in pharmacokinetics or psychotic reactions were observed or reported during were detected. Alcohol concentrations peaked at 2 h (in the the study. combination condition and at 1.5 h in the alcohol one. Frontiers in Pharmacology | www.frontiersin.org 10 January 2020 | Volume 10 | Article 1588 Papaseit et al. Mephedrone and Alcohol FIGURE 1 | Time course of drug effects (n = 11, mean, standard error) on physiological and psychomotor performance (differences from baseline). □ mephedrone + alcohol; ○ mephedrone; ◊ alcohol; D placebo; Significant differences between mephedrone vs mephedrone + alcohol (a: p < 0.05/A: p < 0.01); alcohol vs mephedrone + alcohol (b: p < 0.05/B: p < 0.01). Significant differences in AUC were detected between alcohol and between alcohol and the combination conditions in combination conditions. comparison to mephedrone. Cortisol Concentrations Kinetic parameters for cortisol are summarized in Tables 1 and DISCUSSION 2. Plasma cortisol concentrations were significantly higher (peak and AUC) after the administration of the combination and To the best of our knowledge, this study provides the first mephedrone conditions as compared with placebo. data in humans about the pharmacodynamics and Cortisol concentrations peaked at 2 h with a mean peak of pharmacokinetics of mephedrone and alcohol interactions and 23.85 ± 1.31 µg/dl after the combination condition and 22.23 ± completes previous results on neurocognitive performance 1.23 µg/dl after mephedrone administration. The combination effects (de Sousa Fernandes Perna et al., 2016). Our findings condition also showed significant differences in comparison demonstrate the increased pharmacological effects of the co- to alcohol, cortisol concentrations peaked at 2 h with a administration of mephedrone and alcohol compared to single mean peak of 16.20 ± 0.82 µg/dl. AUC for combination, drug administration. mephedrone and alcohol conditions were 96.05, 87.29 and The 200 mg oral administration of mephedrone reproduced 62.31 ng/ml·h1, respectively. pharmacological effects which concurred with the sole Significant differences in peak, AUC and several T-C points (1 experimental study performed to date in humans (Papaseit and 2 h) were observed between the combination and et al, 2016). Our results demonstrate that mephedrone mephedrone conditions in comparison to placebo, and also produced a significant increase in BP, HR, and PD. It also Frontiers in Pharmacology | www.frontiersin.org 11 January 2020 | Volume 10 | Article 1588 Papaseit et al. Mephedrone and Alcohol FIGURE 2 | Time course of drug effects (n = 11, mean, standard error) on subjective effects (differences from baseline). □ mephedrone + alcohol; ○ mephedrone; ◊ alcohol; D placebo; Significant differences between mephedrone vs mephedrone + alcohol (a: p < 0.05/A: p < 0.01); alcohol vs mephedrone + alcohol (b: p < 0.05/B: p < 0.01). Frontiers in Pharmacology | www.frontiersin.org 12 January 2020 | Volume 10 | Article 1588 Papaseit et al. Mephedrone and Alcohol FIGURE 3 | Plasma concentration over time curves of mephedrone (left) and ethanol (right) (n = 11, mean, standard error). ○ mephedrone; ◊ alcohol; □ mephedrone + alcohol. TABLE 3 | Pharmacokinetics parameters of mephedrone and alcohol in plasma (n = 11). Pharmacokinetic parameters Cmax (ng/ml) AUC0-6 (ng/ml h-1) AUC0-24 (ng/ml h-1) Tmax (h) Ke (h-1) t1/2 (h) Mephedrone Mephedrone and ethanol 175.7 ± 71.1 516.8 ± 264.6 709.8 ± 477.1 1.5 (0.75–2) 0.35 ± 0.14 2.32 ± 1.01 Mephedrone 172.6 ± 82.9 549.0 ± 315.0 778.4 ± 512.9 1.5 (0.5–2) 0.29 ± 0.09 2.68 ± 0.92 p-value NS NS NS NS NS NS Cmax (ng/ml) AUC0-6 (ng/ml h-1) AUC0-10 (ng/ml h-1) Tmax (h) Ethanol Mephedrone and ethanol 103.8 ± 14.1 389.8 ± 73.3 447.0 ± 96.8 2.0 (1.5–2) Ethanol 121.1 ± 14.9 438.4 ± 36.8 512.9 ± 45.2 1.5 (1–2) p-value 0.030 0.048 0.020 0.014 AUC: area under the curve. SD: standard deviation. Tmax is shown as median (range) values. NS: not statistically significant. A value of p < 0.05 was considered statistically significant. induced stimulant-like effects (euphoria, well-being, feelings of Mendelson et al., 1995; Hernández-López et al., 2002; Dumont pleasure) and mild changes in perceptions. All these et al., 2010). physiological and subjective effects were of rapid onset and In addition, the combination of mephedrone and alcohol short duration. The increase in VAS scores (stimulated, high, produced mydriasis and esophoria, an indicator of extraocular good effects, liking) and ARCI (subscales MBG, BG, and A) muscle tension, two specific acute psychostimulant-like effects, caused by mephedrone were within the range of previous studies although slighter in comparison to mephedrone alone. These with psychostimulant drugs with a well-known abuse potential results are in line with those observed after other psycho- (Farré et al., 1993; Hernández-López et al., 2002). Moreover, its stimulant drugs and alcohol co-administration (Mas et al., faster and shorter duration confirmed results obtained in the 1999; de la Torre et al., 2000; Farré et al., 2015). Alcohol, as previous human investigation (Papaseit et al., 2016). The expected from the extrapolation of results obtained from other administration of an oral dose of 0.8 g/kg of alcohol replicated psychostimulant-alcohol interaction studies, attenuated the rise the typical effects of acute alcohol intoxication characterized by in PD and extraocular musculature contraction induced by an increase in the ratings of drunkenness, sedation, and mild mephedrone. Furthermore, the addition of alcohol to effects on physiological responses compared with placebo. mephedrone increased the maximal psychostimulant effects, The co-administration of mephedrone and alcohol amplified maintaining higher measures for euphoria and well-being for a cardiovascular effects, producing a more marked increase in HR in longer period of time in comparison to mephedrone alone, which comparison with mephedrone alone. In turn, these results are maximal effects are faster (Papaseit et al., 2016). Among consistent with a previous description of cardiovascular toxicity conditions, the most remarkable difference was in the associated with mephedrone and alcohol co-ingestion (McGaw and mephedrone-alcohol combination. It produced during 4 h Kankam, 2010). Similar findings have also been observed after the relevant increases in subjective scores which were intense concomitant administration of other psychostimulants drugs during the three first hours compared with 1–2 h following (MDMA, methamphetamine, cocaine) in combination with mephedrone alone. alcohol in healthy volunteers (Pérez-Reyes and Jeffcoat, 1992; Alcohol administration, equivalent four to six alcoholic Farré et al., 1993; Higgins et al., 1993; McCance-Katz et al., 1993; beverages, in combination with mephedrone resulted in Frontiers in Pharmacology | www.frontiersin.org 13 January 2020 | Volume 10 | Article 1588 Papaseit et al. Mephedrone and Alcohol decreased drunkenness and reduced sedative effects producing of this effect is poorly understood. Although the role of cortisol in mixed scores (ARCI-PCAG and VAS drowsiness) between the the acute effects of mephedrone has as yet to be described, it could mephedrone and alcohol alone conditions. Mephedrone be extrapolated to MDMA and related-amphetamines (Mas et al., moderated the effects induced by alcohol in a similar manner 1999). The serotonergic effects of mephedrone might stimulate the to that observed after MDMA experimental administration hypothalamo-pituitary-adrenal axis, leading to an increase in (Hernández-López et al., 2002). cortisol plasma concentrations as previously described by other Overall, the combination of mephedrone and alcohol slightly psychostimulants as MDMA (Seibert et al., 2014; Papaseit et al., delayed peak effects and increased maximal effects which 2016; Strajhar et al., 2019). remained high with no changes in their total duration. Integrating the results of the present study and the previous Cardiovascular and subjective effects after mephedrone-alcohol one on psychomotor performance (de Sousa Fernandes Perna co-administration started at 0.25–5 h, peaked at approximately et al., 2016), it seems that mephedrone reduced some of the 0.75–1.5 h after administration, and returned to pre-dose values subjective feelings of sedation induced by alcohol, but its at 4–8 h after administration with approximately 3–4 h of stimulatory effects were not enough to compensate for the marked effects. In general terms, mephedrone induced lower impairing effects of alcohol on most performance parameters. effects than the combination and almost overlapped with our This dissociation between subjective and objective sedation own observations in a previous study in which the same dose of measures is of interest. Subjects may feel less sedated by alcohol mephedrone was administered (Papaseit et al., 2016). and psychomotor abilities remain impaired or unchanged. The Mephedrone effects were observed between 0.5-1 h and most potential impact of this dissociation in terms of driving safety is returned to baseline 2–3 h after drug administration, but some unknown, but it may be plausible that subjects would consider last more than 3 h (HR, temperature, stimulated or any effect). they are driving better when actual performance continues to be With respect to the pharmacokinetics of the mephedrone- impaired by the effect of alcohol. Similar dissociation has been alcohol combination, the most relevant finding was that alcohol reported when other psychostimulants (e.g. cocaine, MDMA) did not modify the plasma levels of mephedrone. The maximal have been administered simultaneously with alcohol (Farré et al., concentrations of mephedrone after the combination 1993; Farré et al., 1997; Hernández-López et al., 2002). administration were within the range of those obtained after The present study has several limitations which are mainly mephedrone alone and concurred with previously published data associated with its experimental design. Firstly, the moderate (Papaseit et al., 2016). This finding suggests that the sample size. Secondly, a non-representative sample that not pharmacokinetics of mephedrone is not altered when alcohol is allows to generalize the results across gender (absence of concurrently administered. women due to unknown and potential seriously fetal risk). With reference to alcohol pharmacokinetics, statistically Thirdly, the evaluation of only one dose level of mephedrone significant differences were detected in Cmax, AUC, and Tmax. and alcohol (users usually are subjected to repeated drug In this respect, other psychostimulant-alcohol studies have also consumption in a single session). Fourthly, the alcohol blind reported slightly lower alcohol Cmax in combination conditions was potentially insufficient. Nonetheless, we conducted a placebo related to changes in alcohol metabolism and absorption rate controlled study, randomized, within-subjects, various control (Farré et al., 1993; McCance-Katz et al., 1993). In the case of conditions (combination condition, mephedrone condition, mephedrone, initial results suggest a similar kinetic scenario. alcohol condition, and placebo condition). The results provide An adequate pharmacological effect in relation to novel data on pharmacodynamics and pharmacokinetics of pharmacokinetics was thus observed despite the biological mephedrone-alcohol combination. variability among subjects. Our results suggest that the higher In summary, the concomitant administration of mephedrone abuse liability exhibited for mephedrone when concomitantly and alcohol produced a significant increase in cardiovascular consumed with alcohol can be attributed to the early onset and effects and induced more intense and prolonged feelings of maintenance of the subjective/pleasant effects in comparison to euphoria and well-being, in comparison to mephedrone alone. mephedrone alone. It should be emphasized that the short Mephedrone reduced the drunkenness and sedation produced by mephedrone half-life and Tmax following its co-administration alcohol. These effects could encourage the consumption of larger with alcohol could partially explain the mephedrone-alcohol amounts of mephedrone and alcohol, placing the recreational binge pattern among regular users. user at a heightened risk for potentially toxic effects. The results Higher increases in cortisol plasma concentrations were found presented are like those obtained with the combination of after the mephedrone-alcohol co-administration and mephedrone alcohol and other psychostimulants, suggesting that the abuse alone in comparison to the rest of conditions. This pattern of liability of the simultaneous consumption is greater than that response has already been observed after cocaine and alcohol induced by mephedrone alone. (Farré et al., 1997) and MDMA and alcohol administration (Hernández-López et al., 2002). Interestingly, both combinations with alcohol, and also MDMA and cocaine alone, produced DATA AVAILABILITY STATEMENT significant cortisol increases (Farré et al., 1997; Harris et al., 2002; Hernández-López et al., 2002; Kuypers et al., 2013; The datasets generated for this study are available on request to Kuypers et al., 2015). As far as we know, the precise mechanism the senior author (Magi Farré, magi.farre@uab.cat). Frontiers in Pharmacology | www.frontiersin.org 14 January 2020 | Volume 10 | Article 1588 Papaseit et al. Mephedrone and Alcohol ETHICS STATEMENT MF, CP-M, MT, FF, RT, ES, KK, ET, and JR wrote and reviewed the manuscript. The protocol was approved by the local Research Ethics Committee (CEIC-Parc de Salut Mar, Barcelona, Spain). The study was conducted in accordance with the Declaration of FUNDING Helsinki and Spanish laws concerning clinical trials and registered in ClinicalTrials.gov (number NCT02294266). This work was supported in part by grants from Instituto de Salud Carlos III (ISCIII, FIS-FEDER, FIS PI11/01961, FISPI17/ 01962), ISCIII-Red de Trastornos Adictivos (RTA RD16/0017/ 0003 and RD16/0017/0010) and The European Commission AUTHOR CONTRIBUTIONS [HOME/2014/JDRU/AG/DRUG/7082, Predicting Risk of Emerging drugs with In silico and Clinical Toxicology MF, EP, CP-M, ES, RT, KK, ET, and JR conceptualized the (PREDICT)]. EP has a Juan Rodes fellowship (ISC-III, JR16/ study design. EP, CP-M, JM, ES, FF, MT, and MF collected 00020). We are grateful to Esther Menoyo (RN), Marta Pérez the data. RT and EO analyzed mephedrone and alcohol (RN), Soraya Martín (RN), Clara Gibert (RN), and Joan Mestres concentrations. EP, CP-M, EO, and MF analyzed the data. EP, (PsyD) for their valuable assistance throughout the clinical study. REFERENCES de la Torre, R., Farré, M., Roset, P. N., López, C. H., Mas, M., Ortuño, J., et al. (2000). Pharmacology of MDMA in humans. Ann. N. Y. Acad. 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Acute effects of 3,4-methylenedioxymethamphetamine and methylphenidate on circulating steroid levels in healthy subjects. Copyright © 2020 Papaseit, Pérez-Mañá, de Sousa Fernandes Perna, Olesti, Mateus, Neuroendocrinology 100, 17–25. doi: 10.1159/000364879 Kuypers, Theunissen, Fonseca, Torrens, Ramaekers, de la Torre and Farré. This is an Simmler, L. D., Buser, T. A., Donzelli, M., Schramm, Y., Dieu, L. H., Huwyler, J., open-access article distributed under the terms of the Creative Commons Attribution et al. (2013). Pharmacological characterization of designer cathinones in vitro. License (CC BY). The use, distribution or reproduction in other forums is permitted, Br. J. Pharmacol. 168, 458–470. doi: 10.1111/j.1476-5381.2012.02145.x provided the original author(s) and the copyright owner(s) are credited and that the Strajhar, P., Vizeli, P., Patt, M., Dolder, P. C., Kratschmar, D. V., Liechti, M. E., original publication in this journal is cited, in accordance with accepted academic et al. (2019). Effects of lisdexamfetamine on plasma steroid concentrations practice. No use, distribution or reproduction is permitted which does not comply with compared with d-amphetamine in healthy subjects: A randomized, double-blind, these terms. Frontiers in Pharmacology | www.frontiersin.org 17 January 2020 | Volume 10 | Article 1588