pharmaceuticals Article Acute Pharmacological Effects of Oral and Intranasal Mephedrone: An Observational Study in Humans Esther Papaseit 1,2,† , Eulalia Olesti 3,† , Clara Pérez-Mañá 1,2, *,† , Marta Torrens 4 , Francina Fonseca 4 , Marc Grifell 4 , Mireia Ventura 5 , Rafael de la Torre 3 and Magí Farré 1,2 1 Clinical Pharmacology Unit, Hospital Universitari Germans Trias i Pujol (HUGTP-IGTP), 08916 Badalona, Spain; epapaseit.germanstrias@gencat.cat (E.P.); mfarre.germanstrias@gencat.cat (M.F.) 2 Department of Pharmacology, Therapeutics and Toxicology, Universitat Autònoma de Barcelona (UAB), 08193 Cerdanyola del Vallés, Spain 3 Integrative Pharmacology and Systems Neuroscience Research Group, Neurosciences Research Program, Hospital del Mar Medical Research Institute (IMIM) and Universitat Pompeu Fabra (CEXS-UPF), 08003 Barcelona, Spain; eulaliaom@gmail.com (E.O.); rtorre@imim.es (R.d.l.T.) 4 Department of Psychiatry and Forensic Medicine, Institut de Neuropsiquiatria i Adiccions (INAD), Universitat Autònoma de Barcelona (UAB), 08003 Barcelona, Spain; mtorrens@parcdesalutmar.cat (M.T.); mffonseca@parcdesalutmar.cat (F.F.); marcgrifellguardia@gmail.com (M.G.) 5 Energy Control, Associació Benestar i Desenvolupament, 08041 Barcelona, Spain; mireia@energycontrol.org * Correspondence: cperezm.mn.ics@gencat.cat; Tel.: +34-934-978-865 † These authors contributed equally to this work. Abstract: Mephedrone (4-methylmethcathinone) is a synthetic cathinone with psychostimulant  properties which remains one of the most popular new psychoactive substances (NPS). It is fre-  quently used orally and/or intranasally. To date, no studies have evaluated the acute effects and Citation: Papaseit, E.; Olesti, E.; pharmacokinetics after self-administration of mephedrone orally (ingestion) and intranasally (in- Pérez-Mañá, C.; Torrens, M.; Fonseca, sufflation) in naturalistic conditions. An observational study was conducted to assess and compare F.; Grifell, M.; Ventura, M.; de la Torre, the acute pharmacological effects, as well as the oral fluid (saliva) concentrations of mephedrone R.; Farré, M. Acute Pharmacological self-administered orally and intranasally. Ten healthy experienced drug users (4 females and 6 males) Effects of Oral and Intranasal Mephedrone: An Observational self-administered a single dose of mephedrone, orally (n = 5, 100–200 mg; mean 150 mg) or in- Study in Humans. Pharmaceuticals tranasally (n = 5, 50–100 mg, mean 70 mg). Vital signs (blood pressure, heart rate, and cutaneous 2021, 14, 100. https://doi.org/ temperature) were measured at baseline (0), 1, 2, and 4 h after self-administration. Each participant 10.3390/ph14020100 completed subjective effects questionnaires: A set of Visual Analogue Scales (VAS), the 49-item Addiction Research Centre Inventory (ARCI), and Evaluation of the Subjective Effects of Substances Academic Editors: Simona Bertoni with Abuse Potential (VESSPA-SSE) at baseline, 1, 2, and 4 h after self-administration. Oral fluid and and Anna Stary-Weinzinger urine were collected during 4 h. Both routes of mephedrone self-administration enhanced ratings Received: 21 December 2020 of euphoria and well-being effects and increased cardiovascular effects in humans. Although it Accepted: 26 January 2021 was at times assessed that the oral route produced greater and larger effects than the intranasal Published: 28 January 2021 one, concentrations of mephedrone in oral fluid and also the total amount of mephedrone and metabolites in urine showed that concentrations of mephedrone are considerably higher when self- Publisher’s Note: MDPI stays neutral administered intranasally in comparison to orally. Controlled clinical trials are needed to confirm our with regard to jurisdictional claims in observational results. published maps and institutional affil- iations. Keywords: mephedrone (4-methylmethcathinone); novel psychoactive substances (NPS); psychos- timulants; cathinones bath salts; oral administration; intranasal administration Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and Mephedrone (4-methylmethcathinone) is considered to be the most popular syn- conditions of the Creative Commons thetic cathinone drug, resembling the designer drug 3,4-methylenedioxymethamphetamine Attribution (CC BY) license (https:// (MDMA, ecstasy) [1]. It is an amphetamine with an additional beta-ketone group [2,3]. creativecommons.org/licenses/by/ Mephedrone acts as a releaser of monoamines similar to MDMA, but with greater relative 4.0/). potency to release dopamine versus serotonin compared with MDMA [4,5], indicating Pharmaceuticals 2021, 14, 100. https://doi.org/10.3390/ph14020100 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, 100 2 of 13 more stimulant-like properties. After emerging at the new psychoactive substances (NPS) drug market, mephedrone has remained present among certain recreational drug/NPS users and particularly among chemsex participants [6,7]. Mephedrone is most commonly available in powder form, but it is also available as tablets and capsules. Similarly to other psychostimulant drugs, mephedrone can be consumed via different routes. The predominant patterns of use are oral ingestion and nasal insufflation (snorting), although there are also reports of use by rectal insertion and intravenous/intramuscular injection. Because of the common desire to recapture the pleasurable initial high, the use of different routes and re-doses are frequent [8,9]. Users sometimes reported mixing oral and nasal routes, and re-dosing during single-use sessions in which the total doses per session typically reached 0.5–2 g, usually taken in every one or two hours [10]. In this respect, in regular mephedrone recreational users, mephedrone induces some undesirable sub-acute effects such as negative mood, fatigue, and physical symptoms [11]. Additionally, numerous fatal cases and non-fatal mephedrone intoxica- tion cases attributable to high-dose use of mephedrone and to poly-drug use have been documented and attributed to potential interindividual differences in pharmacokinetics– pharmacodynamics [12–16]. Mephedrone and mephedrone metabolites have been detected in human plasma, urine, hair, and nails [17–21]. Until now, pre-clinical self-administration models using mephedrone intravenously and orally have evidenced that mephedrone pro- duces psychomotor speed improvement and abuse liability, both typical psychostimulant properties [22–24]. Different metabolic disposition studies including human specimens suggest that mephedrone is metabolized in part by the CYP2D6 isoenzyme [25–28]. Despite the non-depreciable recreational use of mephedrone over the last years, there is limited scientific knowledge about its acute pharmacological effects and pharmacokinet- ics in humans [26–34] and anecdotal data related to the route of administration. Although as mentioned, mephedrone is frequently used via oral and/or intranasal routes and/or mixing them, no studies have evaluated the acute pharmacological effects of mephedrone in humans comparing both routes of administration. To date, the only three experimental studies conducted with humans have focused primarily on the physiological and subjective effects produced after oral mephedrone, a route of administration least often associated with abuse presumably due to its slow onset of effects [29–31]. After controlled administra- tion, the onset of peak effects (Emax ) produced by oral mephedrone occur about 0.5–0.75 h after [29]. In comparison, recreational users reported that the maximum effects produced by intranasal mephedrone occur within 5 min [34], similarly to other drugs also used in- tranasally [35–37]. Recently, an experimental study in humans was performed after con- trolled intranasal administration of mephedrone (100 mg nasally insufflated) in healthy volunteers describing the profile of pharmacokinetics of mephedrone and its enantiomers, but no data about its acute effects were included in the results published [32,33]. To date, there have been no comparisons of mephedrone using different common routes of administration despite the recreational use of mephedrone. The main objective of the present study was to compare the acute effects after self-administration of oral (inges- tion) and intranasal (insufflation) mephedrone in observational naturalistic conditions. 2. Results Table 1 presents a summary of the physiological and subjective effects where at least one statistical difference in peak effect (Emax ) and/or AUC0–4 h were found and includes time-course (T-C) points that showed significant differences. Pharmaceuticals 2021, 14, 100 3 of 13 Table 1. Summary of statistically significant results on the physiological and subjective effects observed after self- administration of oral (n = 5) and intranasal (n = 5) mephedrone. Mean ± SD T-Student ANOVA Effects Parameter T-Cpoints Oral Intranasal t p-Value F p-Value Emax 0.4 ± 0.6 −0.2 ± 0.2 2.477 0.038 Temperature AUC0–4 0.8 ± 1.1 −0.5 ± 0.4 2.271 0.071 T-C 3.356 0.036 Emax 48 ± 13 25 ± 17 2.376 0.045 Intensity AUC0–4 114 ± 57 37 ± 30 2.700 0.027 T-C 3.940 0.020 b Emax 56 ± 17 22 ± 19 2.976 0.018 Stimulated AUC0–4 141 ± 56 32 ± 32 3.775 0.005 T-C 6.828 0.002 a, b Emax 65 ± 15 25 ± 17 3.952 0.004 High AUC0–4 156 ± 60 33 ± 24 4.238 0.003 T-C 8.645 <0.001 a, b Emax 79 ± 24 26 ± 14 4.168 0.003 Good effects AUC0–4 217 ± 101 32 ± 22 3.954 0.004 T-C 7.120 0.001 a, b Emax 83 ± 21 35 ± 15 4.110 0.003 Liking AUC0–4 246 ± 94 62 ± 34 4.114 0.003 T-C 7.330 0.001 a, b, c Emax 79 ± 25 28 ± 16 3.737 0.006 Content AUC0–4 238 ± 108 45 ± 37 3.766 0.005 T-C 8.210 0.001 a, b, c Emax 12 ± 1.7 6.4 ± 2.4 4.575 0.002 ARCI-MBG AUC0–4 333 ± 14 12 ± 6.7 3.048 0.016 T-C 1.448 0.254 Emax 17 ± 6.0 6.6 ± 4.4 2.999 0.017 VESSPA-SOC AUC0–4 46 ± 24 8.7 ± 8.7 3.237 0.012 T-C 8.901 <0.001 b Emax 13 ± 4.0 5.6 ± 1.5 3.679 0.006 VESSPA-ACT AUC0–4 35 ± 16 7.7 ± 4.9 3.589 0.007 T-C 8.266 0.027 a, b, c Emax 1.8 ± 1.1 0.4 ± 0.9 2.214 0.058 VESSPA-PS AUC0–4 2.6 ± 1.7 0.4 ± 0.9 2.549 0.034 T-C 1.567 0.223 Emax = peak effects 0–4 h (differences from baseline); AUC0–4 = Area under the curve 0–4 h; T-C = temporal course 0–4 h. Emax measured by ◦ C (T (temperature)) mm (visual analog scale (VAS)), and score (Addiction Research Center Inventory (ARCI), Evaluation of Subjective Effects of Substances with Abuse Potential questionnaire (VESSPA-SEE)), and expressed as mean and standard deviation. For Emax and AUC0–4 , a Student’s t-Test for independent sample was used (see Statistical Analysis). A p-value < 0.05 was considered statistically significant. For T-C, a one-way analysis of variance (ANOVA) was used (see Statistical analysis). Statistical differences between oral and intranasal are presented as “a” p < 0.05, “a” p < 0.01 (time 1 h), “b” p < 0.05, “b” p < 0.01 (time 2 h), “c” p < 0.05, “c” p < 0.01 (time 4 h). Background color displays empty cells. Pharmaceuticals 2021, 14, 100 4 of 13 Supplementary Figure S1 presented individual data of systolic blood pressure (SBP) Pharmaceuticals 2021, 14, x FOR PEER REVIEW 4 of 14 in order to show the elevated variability of the acute effects. Supplementary Table S1 shows significant T-C statistical differences of each route of administration in comparison to placebo. All subjects tolerated study procedures well. There Therewere wereneither neithersignificant adverse significant adverse effects including effects hallucinations, including psychotic hallucinations, ep- psychotic isodes, nor any other psychiatric symptoms for oral or intranasal mephedrone self-admin- episodes, nor any other psychiatric symptoms for oral or intranasal mephedrone self- istration during during administration the experimental session.session. the experimental 2.1.Physiological 2.1. PhysiologicalEffects Effects Regarding physiological Regarding physiologicaleffects, effects,both oraloral both andand intranasal mephedrone intranasal self-administration mephedrone self-admin- istration produced an increase in SBP, DBP, HR, and T (see Table 1, SupplementaryS1 produced an increase in SBP, DBP, HR, and T (see Table 1, Supplementary Table and Table Supplementary Figure S1). Comparisons of the two routes of S1 and Supplementary Figure S1). Comparisons of the two routes of administration re-administration revealed no significant vealed differences no significant for Emax differences , AUC for Emax,0–4 AUCh , and 0−4 h, T-C andin vital T-C signssigns in vital except for cutaneous except for cuta- T (Emax neous T).(ET-C max).comparison T-C comparisonto baseline revealed to baseline significant revealed differences significant at 1 and differences at 1 2and h after 2h oral self-administration for SBP, DBP, and HR, whilst after intranasal after oral self-administration for SBP, DBP, and HR, whilst after intranasal self-admin- self-administration differences istration were found differences wereonly foundat 1only h in at comparison to baseline 1 h in comparison for DBP,for to baseline HR, andHR, DBP, T. and T. 2.2. Subjective Effects 2.2. Subjective Effects Both oral and intranasal mephedrone increased subjective drug effects (VAS, ARCI, Both oral and intranasal mephedrone increased subjective drug effects (VAS, ARCI, and VESSPA-SEE) (see Table 1, Supplementary Table S1 and Figure 1). The comparison of and VESSPA-SEE) (see Table 1, Supplementary Table S1 and Figure 1). The comparison the two routes of administration showed significant differences for stimulant-like and plea- of the two routes of administration showed significant differences for stimulant-like and surable effects for both Emax and AUC0–4 h . T-C comparison between oral and intranasal pleasurable effects for both Emax and AUC0−4 h. T-C comparison between oral and intranasal mephedrone showed significant statistical differences for stimulated, high, good effects, mephedrone showed significant statistical differences for stimulated, high, good effects, liking, and content feelings at 1, 2, and/or 4 h after administration. liking, and content feelings at 1, 2, and/or 4 h after administration. 100 100 80 VAS stimulated (score) 80 b VAS high (score) b 60 a 60 a 40 40 20 20 0 0 0 1 2 3 4 0 1 2 3 4 Time (h) Time (h) . 100 100 b b a a VAS good effects (score) 80 80 c VAS liking (score) 60 60 40 40 20 20 0 0 0 1 2 3 4 0 1 2 3 4 Time (h) Time (h) Figure 1. Cont. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 5 of 14 Pharmaceuticals 2021, 14, 100 5 of 13 100 b a 80 VAS content (score) c 60 40 20 0 0 1 2 3 4 Time (h) 20 b 20 VESSPA SOC (score) 15 15 a b VESSPA AE (score) 10 10 c 5 5 0 0 0 1 2 3 4 0 1 2 3 4 Time (h) Time (h) Figure 1. Summary of the course of physiological and subjective effects of mephedrone after oral and intranasal self- Figure 1. Summary of the course of physiological and subjective effects of mephedrone after oral and intranasal self- administration (# oral mephedrone (n = 5);  intranasal mephedrone (n = 5)). Statistical differences between oral and administration (○ oral mephedrone (n = 5); □ intranasal mephedrone (n = 5)). Statistical differences between oral and in- intranasal tranasal areare presented “a”p p< <0.05, presentedasas“a” “a”p p<<0.01 0.05,“a” 0.01(time (time11h), “b”pp<<0.05, h),“b” “b”pp<<0.01 0.05,“b” 0.01(time (time22h), “c” pp << 0.05, h), “c” “c” pp << 0.01 0.05, “c” 0.01 (time 4 h). See text for abbreviations. (time 4 h). See text for abbreviations. After oral After oral mephedrone, mephedrone,T-C T-Ccomparison comparisontotobaseline baselineshowed showed significant significant differences differences for intensity, stimulates, high and good effects at 1 and 2 h, whilst for liking and content, differ- for intensity, stimulates, high and good effects at 1 and 2 h, whilst for liking and content, ences were detected in all times evaluated. In contrast, after intranasal self-administration differences were detected in all times evaluated. In contrast, after intranasal self-admin- T-C comparison to baseline only showed significant differences for intensity, stimulates, istration T-C comparison to baseline only showed significant differences for intensity, high, good effects, and content at 1 h and for liking at 1 and 2 h, respectively. stimulates, high, good effects, and content at 1 h and for liking at 1 and 2 h, respectively. Both oral and intranasal mephedrone produced mild changes in perceptions, but not Both oral and intranasal mephedrone produced mild changes in perceptions, but not hallucinations, although no statistically significant differences were detected among routes hallucinations, although no statistically significant differences were detected among of administration except for different body feeling (AUC h ). routes of administration except for different body feeling0–4 (AUC0−4 h). With respect to the ARCI questionnaire, mephedrone self-administered orally and With respect to the ARCI questionnaire, mephedrone self-administered orally and intranasally produced an increase in all the subscales evaluated. The most marked increases intranasally produced an increase in all the subscales evaluated. The most marked in- were observed in scores for the MBG (euphoria), BG (intellectual efficiency and energy), creases were observed in scores for the MBG (euphoria), BG (intellectual efficiency and and A (amphetamine) subscales. When comparing both routes of administration, statistical energy), differencesandwere A (amphetamine) detected only subscales. for the MBG When comparing subscale in Emaxboth androutes AUC0–4 of administration, h. statistical differencestowere In comparison detected baseline, only fordifferences statistical the MBG subscale were shownin Emax in and AUC several 0−4 h. T-C points In comparison to baseline, statistical differences were shown in after oral self-administration at 1 h and 2 h for MBG and BG subscales, and at 1, 2, and several T-C points 4h after for Aoral self-administration subscales, and also after atintranasal 1 h and 2 hself-administration for MBG and BG subscales, at 1 and 2and h foratPCAG, 1, 2, andBG, 4 hand forAAsubscales, subscales,and andatalso after intranasal 1h for the MBG subscale. self-administration at 1 and 2 h for PCAG, BG, and ARegarding subscales,theandVESSPA-SEE at 1h for the questionnaire, MBG subscale.mephedrone increased all the subscales Regarding regardless of thethe VESSPA-SEE route of administrationquestionnaire, except formephedrone the CP (changesincreased all the subscales in perception) subscale regardless of the route of administration except for the CP (changes for intranasal mephedrone. Comparing both routes, statistical differences were observed in perception) sub- scale for intranasal mephedrone. Comparing both routes, statistical in Emax and AUC0–4 h for the SOC (pleasure and sociability) and ACT (activity and energy)differences were ob- served in E and AUC 0 − for the SOC (pleasure and sociability) subscales and only in AUC0–4 h for PS (psychotic symptoms) subscale. Whilst for T-C, max 4 h and ACT (activity and energy) subscales statistical andwere differences onlyfound in AUC only for 0−4 hin ACTPS (psychotic scores in allsymptoms) points. subscale. Whilst for T-C, statistical differences were found only in ACT scores in all points. Pharmaceuticals Pharmaceuticals2021, 2021,14, 14,x100 FOR PEER REVIEW 6 6ofof1413 T-C comparison at baseline revealed significant differences at 1 h for the PS subscale, T-C comparison at baseline revealed significant differences at 1 h for the PS subscale, at 1 and 2 h for ANX and SOC subscales, and at 1, 2, and 4 h for ATC subscales after oral at 1 and 2 h for ANX and SOC subscales, and at 1, 2, and 4 h for ATC subscales after self-administration, and at 1h for SOC and ACT subscales, and at 1 and 2 h for ANX sub- oral self-administration, and at 1h for SOC and ACT subscales, and at 1 and 2 h for ANX scales after intranasal self-administration. subscales after intranasal self-administration. 2.3. 2.3.Oral Oralfluid concentrations Fluid of mephedrone Concentrations of Mephedrone The The oral fluid (saliva) T-C concentrations oral fluid (saliva) T-C concentrations curve curve for mephedrone is for mephedrone is shown shown in inFigure Figure2 2and andSupplementary SupplementaryFigureFigureS2S2(individual (individualdata). data). 8000 6000 4000 2000 0 0 1 2 4 Time (h) Time-course Figure2.2.Time-course Figure of mephedrone of mephedrone oral oral fluid fluid concentrations concentrations afterand after oral oral and intranasal intranasal self-ad- self- ministration (○ oral administration mephedrone (# oral (n = (n mephedrone 5);=□5); intranasal mephedrone  intranasal (n = 5)). mephedrone (n = 5)). Afterself-administration After self-administration of oforal oralmephedrone, mephedrone, concentrations concentrations of mephedrone of mephedrone in oral fluid in oral increased fluid rapidly, increased reaching rapidly, a peaka2 peak reaching h after 2 ingestion, and decreased h after ingestion, at 4 h. Mean and decreased at 4maximum h. Mean concentration maximum (Cmax ) values concentration (Cmaxof)1571 values± 1367 ng/mL of 1571 (range ± 1367 ng/mL 18–2999 (rangeng/mL) 18–2999 were obtained ng/mL) wereat a T obtained of 2 h following drug administration. max at a Tmax of 2 h following drug administration. The AUC was 3686 ± 3443 ng ·h/mL 0–4 h The AUC0-4h was 3686 ± 3443 (range ng·h/mL ng·h/mL). 61–7593(range At 4ng·h/mL). 61–7593 h, all subjects At 4 presented mephedrone h, all subjects presentedconcentrations except for mephedrone concentra- one subject tions thatone except for hadsubject no detectable that hadconcentrations. no detectable concentrations. Afterself-administration After self-administrationofofintranasal intranasalmephedrone, mephedrone,oral oralfluid fluid concentrations concentrationsofof mephedrone increased rapidly, reaching a peak 1 h after mephedrone increased rapidly, reaching a peak 1 h after ingestion, and theningestion, and thenrapidly rapidlyde- de- creased at 4 h. C values of 4950 ± 5545 ng/mL (range 1091–14,525 creased at 4 h. Cmax values of 4950 ± 5545 ng/mL (range 1091–14,525 ng/mL) were obtained max ng/mL) were atobtained a Tmax ofat1 ha following Tmax of 1 drugh following drug administration. administration. At 4 h, mephedrone At 4 concentration h, mephedrone was con- 9 centration was 9 times lower (4–41 times) in comparison times lower (4–41 times) in comparison to Cmax. The AUC0–4 h was to C max . The AUC 7917 ± 7717 ng·h/mL 0–4 h was 7917 ±1633–20,918 (range 7717 ng·h/mL (rangeOral ng·h/mL). 1633–20,918 ng·h/mL).concentrations fluid mephedrone Oral fluid mephedrone concentra- varied considerably tions varied among oral and considerably among intranasal doses andoral and intranasal subjects. dosesdifferences No significant and subjects. No significant between oral and differences between oral and intranasal mephedrone were intranasal mephedrone were found for Cmax, AUC0–4 h, and Tmax (Table 2). found for Cmax , AUC0–4 h , and Tmax (Table 2). Table 2. Oral fluid pharmacokinetics parameters of oral (n = 5) and intranasal (n = 5) mephedrone. Table 2. Oral fluid pharmacokinetics parameters of oral (n = 5) and intranasal (n = 5) mephedrone. Pharmacokinetic Pharmacokinetic Cmax (ng/mL) AUC0–4 (ng/mL h−1(ng/mL ) h−1 ) Tmax (h) Parameters Parameters Cmax (ng/mL) AUC0–4 Tmax (h) Oral Oral 1571 ± 13671571 ± 1367 3684 ± 3443 3684 ± 3443 2 (1–2)2 (1–2) Intranasal Intranasal 4950 ± 55454950 ± 5545 7917 ± 7717 7917 ± 7717 1 (1–1)1 (1–1) p-value 0.296 0.373 0.083 p-value 0.296 0.373 0.083 Abbreviations: AUC: Area under the curve. SD: Standard deviation. Tmax is shown as median Abbreviations: AUC: Area under the curve. SD: Standard deviation. Tmax is shown as median (range) values. (range) values. 2.4.Urinary 2.4. UrinaryConcentrations ConcentrationsofofMephedrone Mephedroneand andMetabolites Metabolites Recovery of Recovery ofmephedrone mephedrone and and its metabolites nor-mephedrone, its metabolites dihydro-mephedrone, nor-mephedrone, dihydro- mephedrone, 4-carboxy-mephedrone, and succinyl-nor-mephedronethe 4-carboxy-mephedrone, and succinyl-nor-mephedrone in urine in 0–4 hin in urine period post the 0–4 h self-administration is shown in Figure 3. The profile of metabolites recovered in urine period post self-administration is shown in Figure 3. The profile of metabolites recovered was similar for all doses tested, and for the oral doses it was congruent with previous data published [15]. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 7 of 14 Pharmaceuticals 2021, 14, 100 7 of 13 in urine was similar for all doses tested, and for the oral doses it was congruent with pre- vious data published [15]. Figure 3. Urinary Figure concentrations 3. Urinary concentrations ofofmephedrone mephedroneafterafteroral oraland and intranasal intranasal self-administration (un- self-administration (unfilled filled bar:bar: OralOral mephedrone mephedrone (n =(n5);= filled 5); filled bar:bar: Intranasal Intranasal mephedrone mephedrone (n =(n5)). = 5)). 3. 3. Discussion Discussion TheThe overall overall purpose purpose ofof thisstudy this studywas wastotodescribe describethe theacute acuteeffects effectsofoforal oral and and intrana- intranasal mephedrone in naturalistic conditions and to compare the two most sal mephedrone in naturalistic conditions and to compare the two most important routes important routes of its administration. of its administration. The present findings show that mephedrone self-administered in observational natu- The present findings show that mephedrone self-administered in observational nat- ralistic conditions induced acute effects that are similar to those produced under exper- uralistic conditions induced acute effects that are similar to those produced under exper- imental conditions [29,30]. Consistent with these results, mephedrone produced similar imental conditions [29,30]. Consistent with these results, mephedrone produced similar effects on the majority of physiological and subjective measures. Furthermore, both routes effects on the majority of physiological and subjective measures. Furthermore, both routes of mephedrone administration (oral and intranasal self-administration) enhanced ratings of mephedrone administration (oral and intranasal self-administration) enhanced ratings of euphoria and increased cardiovascular effects. of euphoria and increased cardiovascular effects. With respect to the pharmacological effects after oral self-administration of mephedrone, With respect to the pharmacological effects after oral self-administration of the magnitude and maximum intensity of the pharmacological effects are in accordance mephedrone, the magnitude and maximum intensity of the pharmacological effects are in with those observed under controlled conditions. Overall, peak effects were observed accordance with those observed under controlled conditions. Overall, peak effects were between 1–2 h and returned to baseline 3–4 h after drug administration [29,30]. In relation observed between 1–2 h and returned to baseline 3–4 h after drug administration [29,30]. to intranasal mephedrone, as mentioned initially, there is no previously published phar- In macodynamic relation to intranasal data to mephedrone, compare with.asInmentioned initially, general terms, there is noself-administration the intranasal previously pub- lished pharmacodynamic of mephedrone producesdataacuteto pharmacological compare with. Ineffectsgeneral terms,tothe similar intranasal those produced self-ad- by oral ministration of mephedrone produces acute pharmacological effects mephedrone. The most remarkable result of this study showed that, at times assessed similar to those pro- duced by oral mephedrone. The most remarkable result of this study (1 and/or 2 h), mephedrone oral self-administration in comparison to intranasal self- showed that, at times assessed (1 and/orproduced administration 2 h), mephedrone greater andoral larger self-administration effects on some in comparison to intranasal subjective measures (e.g., self-administration produced greater and larger effects on some ratings of VAS and several subscales of ARCI and VESSPA). Nonetheless, as would subjective measures (e.g.,be ratings of VAS expected, and severalsimilarly mephedrone, subscalestoofother ARCI and VESSPA). Nonetheless, psychostimulant drugs that are as also would be usually expected, used by mephedrone, the intranasal similarly to other psychostimulant route (insufflation), drugs that dilated the vascular-rich areofalso areas usually the intranasal used by the cavity andintranasal pulmonary route (insufflation), network, dilated the thus increasing the vascular-rich absorptive surface areas of theand area intranasal allowing cavity and pulmonary network, thus increasing the absorptive surface for more rapid entry of the drug into the bloodstream, producing fast and reinforcing area and allowing foreffects more [37]. rapidThis entry of the drug well-known intocould factor the bloodstream, justify the fact producing fast and reinforcing that the punctuation of subjects effects [37]. This well-known who self-administered factormephedrone intranasal could justifywasthelower fact that thanthe punctuation those of subjects who self-administered who self-administered orally, because the first intranasal evaluation mephedrone (at 1 h) waswas lower than conducted oncethose who self-adminis- the maximum subjective tered orally, effects werebecause induced the first evaluation (several minutes after(at 1self-administration, h) was conducted once which thewas maximum sub- not assessed). jective effects Accordingweretoinduced previous(several published minutes after results self-administration, of mephedrone which wasinnot pharmacokinetics as- plasma sessed). after controlled intranasal administration, mephedrone showed rapid absorption with a mean Tmax of 0.88 ± 0.35 h [32]. Besides, this Tmax in plasma was slightly shorter in comparison to the plasma Tmax of 1.2 h after controlled oral administration of 200 mg of mephedrone by our research group [29]. Again, these data point to faster acute pharmaco- logical effects of intranasal mephedrone compared to oral mephedrone. Pharmaceuticals 2021, 14, 100 8 of 13 Additionally, both results obtained from concentrations of mephedrone in oral fluid and from the total amount of mephedrone and metabolites in urine confirm that con- centrations of mephedrone are considerably higher after intranasal self-administration in comparison to oral self-administration. As expected, mean oral fluid concentrations of mephedrone at 1 h post administration was 4.6 times higher after intranasal than oral administration (4950 ng/mL versus 1070 ng/mL, respectively), achieving by both routes similar concentrations at 2 h. In urine, again mephedrone concentrations were higher after intranasal than oral administration of mephedrone. In relation to mephedrone metabolites after oral self- administration, all metabolites were detected with a similar profile of recovery in compari- son with a previous study [17], whilst there are no previous data for intranasal mephedrone. The relevance of our results for intoxication cases is limited because usually con- centrations in different biological samples have documented great variability. To date, urine concentrations have been analyzed in several intoxications and fatality cases of mephedrone, whilst there is no data about oral fluid ones. Urinary concentrations reported in clinical trials or mild intoxications are in the range of concentrations measured in our study [11,17,38]. Finally, there were a number of limitations presented by this study design. The main limitations associated with the study are the naturalistic-observational design, that doses varied across subjects and were different in subjects using intranasal vs. oral route and the number of time-point measures and their time interval. This last limitation is particularly important for the evaluation of fast acute effects. It did not permit us to accurately know the real maximal or peak effect/concentration times particularly for intranasal mephedrone, which will need more frequent and earlier evaluations. Other limitations to consider are the non-placebo-controlled design, because participants selected the dose and the route of administration according to their preferences and previous experiences (expectancy bias), a limited number of subjects (lack of statistical power in some measures). Furthermore, the effects reported by participants could have influenced the recreational setting. Finally, we did not collect data on genetic polymorphism of CYP2D6 that can influence the pharmacokinetics and effects of the substance. However, the strengths should be remarked on: The participation of female subjects, the dose selection by the participants according to their preferences, the inclusion of two different routes of administration, effects previously experienced with the same or similar psychoactive substances, the recreational scenario, and the use of validated methodology using in controlled studies (rating scales, questionnaires) and analytic techniques. Preliminary data from this observational study have pointed out for the first time that mephedrone profiles in real conditions may vary considerably depending on the route of administration due to the dose administered and the interindividual differences in pharmacodynamic-pharmacokinetics. Thus, it is not possible to make valid conclusions and comparisons, and controlled clinical trials are needed to confirm our observational results. 4. Materials and Methods 4.1. Participants Ten healthy subjects were selected (4 females and 6 males). Participants were recreative drug users who had experience with amphetamines, ecstasy, mephedrone, and/or cathi- nones at least once in their lifetimes without experiencing previous serious adverse reactions. Exclusion criteria included a history of any serious medical or psychopathological disorder including substance use disorder (except for nicotine), a previous serious adverse reaction with users of amphetamines, ecstasy (MDMA), mephedrone, and cathinones, and use of chronic medication. Participants were recruited by word-of-mouth and snowball sampling through the harm reduction, non-governmental organization Energy Control (ABD). The protocol was approved by the Clinical Research Ethics Committee. The study was conducted in accordance with the Declaration of Helsinki recommendations. All the participants were fully informed, both orally and in writing, about the study characteristics. Pharmaceuticals 2021, 14, 100 9 of 13 All of them indicated their agreement to participate and signed an informed consent prior inclusion. Subjects were financially compensated for their participation. 4.2. Design and Treatments The study was conducted according to a non-controlled prospective observational study with minimal intervention in subjects who self-administered mephedrone orally or intranasally. Similarly to previous naturalistic observational studies evaluating acute effects of other NPS, the methodology including evaluations and procedures were similar [39,40]. Each subject participated in one session. Treatment consisted of oral or intranasal self- administration of mephedrone that they brought to the testing site themselves, which they had obtained from an unknown source. Although no information was available about the synthesis of the drug, similar capsules tested by Energy Control, a harm reduction organization that provides a Drug Checking Service for users, showed that the substance contained mephedrone at 95% purity with no toxic adulterants. A gas chromatography associated with mass spectrometry (GC/MS) was previously used by the mephedrone anal- ysis. The method used permits to check for most common drugs of abuse including most of the NPSs and to know the exact purity of mephedrone in the powder to prepare dosing by a precision scale [29]. The dose of oral and intranasal mephedrone self-administered was selected by the participants based presumably on their previous experience. Five subjects self-administered one dose of mephedrone orally, the mean mephedrone dose was 150 mg (100–200 mg) (1 female ingested 100 mg, 2 females and 1 male ingested 150 mg, 1 male ingested 200 mg), and five subjects self-administered one dose of mephedrone intranasally, the mean mephedrone dose was 70 mg (50–100 mg) (3 males insufflated 50 mg and 2 males 100 mg). All the selected doses were well tolerated, and no serious adverse events were observed. No local tissue damage to the nostrils or any other potential acute medical complication after snorting was reported. 4.3. Procedures Prior to the study session, the participants underwent a general medical examination and a psychiatric interview. They received training with respect to questionnaires used in the study. Upon arrival, they were questioned about any event that could affect their participation and any drug use 2 days prior to the session. Participants were not allowed to consume alcohol or beverages containing caffeine the previous 24 h. The session took place on the same day at a private club with ambient music and participants could talk, read, or play table games during the session and interact in exception to the evaluation times. Moreover, they were instructed not to talk about the effects of the substance during the session. Assessments were performed at baseline (pre-dose) and 1, 2, and 4 h after oral or intranasal self-administration of mephedrone. The experiment was conducted from 15:00 to 21:00 h. Earlier assessment (<1 h) could not be carried out due to the setting of consumption. Urine spot samples were collected at baseline (pre-dose) to exclude prior substance drug use (benzodiazepines, barbiturates, morphine, cocaine, amphetamines, methamphetamine, MDMA, marijuana, phencyclidine) with Instant-View, Multipanel 10 Test Drug Screen (Alfa Scientific Designs Inc., Poway, CA, USA). Self-administration of mephedrone took place around 16.00 h. At each time point of the session, the sequence of procedures was: Physiological measures, oral fluid collection, subjective effects questionnaires, and urine recollection. During entire study session, a psychiatrist was present and adverse effects were assessed. 4.4. Physiological Effects Physiological effects including non-invasive systolic blood pressure (SPB), diastolic blood pressure (DBP), and heart rate (HR) were determined with an Omron monitor at baseline and 1, 2, and 4 h after administration. Cutaneous temperature was measured simultaneously. Pharmaceuticals 2021, 14, 100 10 of 13 4.5. Subjective Effects Subjective effects of mephedrone were measured at baseline and at 1, 2, and 4 h after self-administration using different scales and questionnaires. A set of Visual Analog Scales (VAS) (100 mm, from “not at all” to “extremely”) were used to measure rate intensity; stimulated; high; good effects; liking; content; changes in colors; changes in shapes; changes in lights; hallucinations—seeing of lights or spots; hallucinations—seeing animals, things, insects, or people; changes in hearing; hallucinations—hearing sounds or voices; different body feeling; unreal body feeling; changes in distances; different surroundings; unreal surroundings; confusion; fear; depression or sadness; drowsiness; dizziness; bad effects; headache; nausea; vertigo; breathing difficulty; and face flushing [29,39–42]. The 49-item Addiction Research Centre Inventory (ARCI) short form, a validated instrument that includes subscales related to drug sedation (pentobarbital chlorpromazine-alcohol group, PCAG), euphoria (morphine-benzedrine group, MBG), dysphoria and somatic symptoms (lysergic acid diethylamide group, LSD), intellectual efficiency and energy (benzedrine group, BG), and d-amphetamine like effects (A) that evaluate subjective effects produced by psychoactive drugs [38,39]. The Evaluation of the Subjective Effects of Substances with Abuse Potential (VESSPA-SE) questionnaire that includes subscales related to sedation (S), psychosomatic anxiety (ANX), changes in perception (CP), pleasure and sociability (SOC), activity and energy (ACT), and psychotic symptoms (PS) that measures changes in subjective effects caused by different drugs including stimulants and psychedelics [29,43]. 4.6. Urinary Concentrations of Mephedrone and Metabolites Urine samples were collected at baseline (0 h) and during the entire session (0–4 h). Urine was stored at −20 ◦ C until analysis. Urinary samples were analyzed following a previously reported validated method based on liquid chromatography tandem–mass spec- trometry (LC-MS/MS). Mephedrone and its main metabolites, nor-mephedrone, dihydro- mephedrone, nor-succinyl-mephedrone and carboxy-mephedrone were quantified [44,45]. 4.7. Oral Fluid Concentrations of Mephedrone Oral fluid samples were collected with Salivette® tubes at 0 h (baseline), 2, and 4 h after mephedrone self-administration. After collection, samples were centrifuged and frozen at −20 ◦ C until analysis. Mephedrone concentrations were analyzed by a validated LC-MS/MS [39,40]. A Mephedrone chromatogram (one participant that self-administrated orally 150 mg of mephedrone and one participant that self-administrated 50 mg intranasal) and chromatograms of the internal standard used for the previous samples (Mephedrone- d3) were available in Supplementary Figure S2 and linearity parameters of the oral fluid methodology in Supplementary Figure S3. The oral fluid (saliva) T-C concentrations curve for mephedrone is shown in Figure 2 and Supplementary Figure S4 (individual data). 4.8. Statistical Analysis Differences with respect to baseline were calculated for physiological (SBP, DBP, HR, and T) and subjective effects (VAS, ARCI, and VESSPA). Maximum effects (Emax ) were determined and the area under the curve of the effects (AUC0–4 h ) was calculated using the trapezoidal rule by the Pharmacokinetic Functions for Microsoft Excel (Joel Usansky, Atul Desai, and Diane Tang-Liu, Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, CA, USA). To study possible differences between doses, a one-way analysis of variance (ANOVA) test including all doses for each route of administration as a factor was used for Emax and AUC0–4 h .The results showed <15% of statistically significant differences among doses for each route of administration. Therefore, it was decided to consider all doses for each route of administration globally, and Student’s t-Test for paired sample was conducted for Emax and AUC0–4 h. Pharmaceuticals 2021, 14, 100 11 of 13 To compare the time course (T-C) of effects of mephedrone between the two routes of administration, a one-factor repeated measures ANOVA (baseline, 1, 2, and 4 h) was performed. Additionally, to evaluate the mephedrone effects along time of each route of administration a Dunnett multiple comparison post hoc test was conducted to compare the different time points with baseline (times 0–1 h, 0–2 h and 0–4 h) for each route of administration. Differences in time to reach peak effects (Tmax ) values were assessed using a Non- Parametric Test (Wilcoxon test). Statistically analyses were performed using PAWS Statistics version 18 (SPSS Inc., Chicago, IL, USA). Statistically significance was defined as p < 0.05. For mephedrone oral fluid concentrations, the maximum concentration (Cmax ), the time needed to reach the maximum concentration (Tmax ) and the AUC0–4 h was calculated using the Pharmacokinetic Functions for Microsoft Excel (Joel Usansky, Atul Desai, and Diane Tang-Liu, Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, CA, USA). For mephedrone and metabolites urine concentrations, the amount of drug recovered in urine was calculated. 5. Conclusions The study examined for the first time the acute effects of oral and intranasal mephedrone in observational naturalistic conditions. Preliminary data demonstrate that the route of administration of mephedrone could yield some appreciable differences in the acute effects attributed to mephedrone in a sample of young-adult recreational drug users. It is impor- tant to remark that each of the routes of administration carries unique and acute medical associated risks, and clinicians should be prepared to educate patients about the acute risks associated not only with mephedrone use, but also with its route of administration. In conclusion, these results confirm that oral and intranasal mephedrone produced in natural conditions reinforcing and well-being effects in humans. Supplementary Materials: The following are available online at https://www.mdpi.com/1424 -8247/14/2/100/s1, Figure S1: (a). Individual data of systolic blood pressure (SBP) after oral self-administration (n = 5); (b) Individual data of systolic blood pressure (SBP) after intranasal self-administration (n = 5), Figure S2: (a) Mephedrone chromatogram of a participant that self- administrated orally 150 mg of mephedrone (right) and one subject that self-administrated 50 mg intranasal (left) from and oral fluid sample (b) Chromatograms of the internal standard used for the previous samples (Mephedrone-d3), Figure S3: Linearity parameters of the oral fluid methodology, Figure S4: (a) Individual data of time-course of mephedrone oral fluid concentrations after oral self-administration of mephedrone (n = 5); (b) Individual data of time-course of mephedrone oral fluid concentrations after intranasal self-administration of mephedrone (n = 5), Table S1: Summary of time course result on the physiological and subjective effects observed after self-administration of oral (n = 5) and intranasal (n = 5) mephedrone. Author Contributions: Conceptualization, E.O., M.T., M.V., R.d.l.T., and M.F.; formal analysis, E.P., E.O., C.P.-M., M.V., R.d.l.T., and M.F.; investigation, E.P., E.O., M.G., M.V., and M.F.; writing—original draft, E.P., C.P.-M., and M.F.; writing—review & editing, E.P., E.O., C.P.-M., M.T., F.F., M.G., M.V., R.d.l.T., and M.F. All authors have read and agreed to the published version of the manuscript. Funding: The investigation was partially funded by grants from the Instituto de Salud Carlos III (ISCIII, Fondo de Investigación en Salud (FIS)-Fondo Europeo de Desarrollo Regional (FEDER), Grant Numbers: FIS PI14/00715 and FIS PI17/01962, ISCIII-Red de Trastornos Adictivos RTA Grant Number: RD16/0017/0003 and RD16/0017/0010, AGAUR Gencat Suport Grups de Recerca, Grant Number: 2017 SGR 316 and 2017 SGR 530; Ministerio de Sanidad, Política Social e Igualdad (Plan Nacional sobre Drogas-PNSD, Grant Number 2015I054) and The European Commission (Pre- dicting Risk of Emerging Drugs with In silico and Clinical Toxicology [PREDICT, Grant number: HOME/2014/JDRU/AG/DRUG/7082]). Pharmaceuticals 2021, 14, 100 12 of 13 Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of PARC DE SALUT MAR (protocol code 2016/6700 and date of approval on 10 May 2016). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: We are grateful to Pol Quintana (MD) for his valuable assistance throughout the clinical study. Conflicts of Interest: The authors declare no conflict of interest. References 1. Winstock, A.R.; Mitcheson, L.R.; Deluca, P.; Davey, Z.; Corazza, O.; Schifano, F. Mephedrone, new kid for the chop? Addiction 2011, 106, 154–161. [CrossRef] [PubMed] 2. Mephedrone. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/mephedrone (accessed on 20 November 2020). 3. Papaseit, E.; Moltó, J.; Muga, R.; Torrens, M.; de la Torre, R.; Farré, M. Clinical Pharmacology of the Synthetic Cathinone Mephedrone. Curr. Top Behav. Neurosci. 2017, 32, 313–331. [PubMed] 4. Kehr, J.; Ichinose, F.; Yoshitake, S.; Goiny, M.; Sievertsson, T.; Nyberg, F.; Yoshitake, T. 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