All animals were male Wistar rats (Hannover breed, obtained from Konárovice, Czech Republic) weighing 200–250 g and aged 8 weeks at the start of testing. Rats were housed two per cage under controlled temperature (22 ± 2°C) and humidity (30–70%) with food pellets and water freely available. Lights were on from 6:00 to 18:00 h and all experiments were carried out between 7:00 and 13:00 h, except the temperature study where the test lasted until 17:00 h. Animals were allowed 7–10 days to habituate to laboratory conditions before being used in experiments, during which they were weighed twice and handled four times. All behavioral experiments were conducted in the same standard conditions (temperature and humidity) as in the animal housing facility. Each experimental group for behavioral and temperature studies included 10 animals and each animal was tested only once with the exception that (to reduce animal use) rats from behavioral experiments were used for subsequent pharmacokinetic sampling [for pharmacokinetic studies, n = 8 (for methylone) and n = 5 (for nor-methylone) per experimental group]. All procedures were conducted in accordance with the principles of laboratory animal care of the National Committee for the Care and Use of Laboratory Animals (Czech Republic), and according to Guidelines of the European Union (86/609/EU). The protocol was approved by the National Committee for the Care and Use of Laboratory Animals (Czech Republic) under the number: MEYSCR-27527/2012-31.

3,4-Methylenedioxy-N-methylcathinone (methylone) was purchased via the internet and subsequently purified and converted to a hydrochloride (HCl) by Alfarma s.r.o. (Czech Republic). The resulting methylone was certified to be of 99.18% purity (analyzed by infrared spectroscopy) and also served as a reference standard for pharmacokinetic analyses using liquid chromatography. Nor-methylone was synthesized in the Forensic Laboratory of Biologically Active Substances (University of Chemistry and Technology Prague, Czech Republic) in a purity of 99.18%. Internal standards for quantitative liquid chromatography/mass spectrometry (LC/MS) assays were deuterated MDA-D2. HCl with the purity 99.7% (Lipomed, Inc., Switzerland). Reference standards for confirmation of metabolites by LC/HRMS (high-resolution mass spectrometry) and gas chromatography/mass spectrometry were synthesized with purity within 97.5–89.3% (Institute of Chemical Technology, Department of Organic Chemistry, Prague). β-Glucuronidase type HP-2 from Helix Pomatia, EC 3.2.1.31 (184,973 U/ml) was purchased from Sigma-Aldrich, Prague. Extraction columns Bond Elut Certify 50 mg/3 ml were supplied by Labio s.r.o., Olomouc. Other chemicals used for laboratory purposes were of analytical grade purity. Methylone was stored in dry and dark place and dissolved in physiological saline (0.9% NaCl) immediately before experiments.

The methylone doses used in the present study were estimated according to the reported usage by humans and according to our previous studies with entactogens MDMA, para-methoxymethamphetamine (PMMA), and 2C-B (4-bromo-2,5-dimethoxyphenylethylamine). Doses were selected to range from those that: (1) at the lower end are close to those used by humans, to (2) higher doses that might produce significant acute non-lethal toxicity, and (3) with respect to our previous analogous experiments with MDMA, PMMA, and 2C-B (21, 29–31). The treatment range for methylone was set to be 5, 10, 20, and 40 mg/kg, nor-methylone at 10 mg/kg for behavioral experiments. In behavioral experiments (open field, PPI ASR) nor-methylone was tested only in 15 min testing onset. Doses for pharmacokinetic [10 mg/kg subcutaneously (s.c.)] and temperature experiments (10 and 20 mg/kg s.c.) were selected according to the inherent sensitivity of the analytical LC/HRMS procedure utilized and according to the effectiveness in behavioral tasks (effects body temperature). For the pharmacokinetic study, a single bolus of methylone 10 mg/kg s.c. was administered, subsequently animals were decapitated after 30, 60, 120, 240, or 480 min. Additional pharmacokinetic data with the same design were also obtained for nor-methylone 10 mg/kg s.c. Separated sera and whole brains were kept at −20°C until the toxicological analyses. Both drugs were dissolved in a volume of 2 ml/kg and administered s.c. (for comparability with previous studies) in all cases.

All statistical analyses were conducted using IBM SPSS version 22. For the open field, PPI, and temperature analyses, factorial designs for later analysis with analysis of variance (ANOVA) were used.

Significant main effects and interactions ANOVAs were followed with pairwise comparisons using independent t-tests. For repeated measures ANOVAs, where Mauchly’s test of sphericity was significant (and Mauchley’s W < 0.75), Greenhouse–Geisser corrected statistics are reported. For independent t-tests, where Levene’s test for equality of variances was significant, statistics corrected for unequal variances are given p < 0.05 (two tailed) was considered the minimal criterion for statistical significance. For multiple comparisons, t-tests were used with Bonferroni correction. Nor-methylone was not included in ANOVA analyses (only one time of administration was tested, 15 min) and data were analyzed using additional independent t-test.

For methylone, maximum brain and serum concentration were attained within 30 min after the drug administration. The influx into the brain was not delayed and the concentration of methylone in brain was approximately five times higher than serum levels throughout the experiment (serum:brain ratio during the peak was 1:4.54; Figure 1A). Serum levels of nor-methylone were also quantified after methylone administration; they peaked 30 min later than methylone (1 h after administration) and reached about 20% of methylone levels (350 ng/ml). The second most abundant metabolite identified in the serum was 4-OH-MeO-MC (4-hydroxy-3-methoxymethcathinone) (Figures 1C,D), quantification was not possible because of lack of reference standard at the time of analysis. 4-OH-MeO-MC peaked quickly at 30 min after administration of methylone, the peak had bigger area under the curve and then quickly disappeared and nor-methylone became the most abundant at later time points evaluated. After nor-methylone administration, the maximum serum concentrations were reached between 30 min and 1 h, the brain peak appeared at 30 min; the maximum levels were approximately one half when compared with methylone and serum:brain ratio during the peak was 1:7.97 (Figure 1B).

Rats, treated with methylone 40 mg/kg, were tested only in 15 min testing onset in PPI because after 2 h after administration seven rats no longer produced much behavior only lying on the floor. After 5 h rats began moving around the home cage again, however, mortality occurred within 24 h after injection in six rats. In open field testing, only one rat died within 24 h after administration of methylone (40 mg/kg). Behavioral testing at 60 min after administration was not performed since 40 mg/kg was lethal to some animals.

Trajectory length was evaluated using 4 × 2 × 6 mixed factorial ANOVAs with drug treatment (methylone at 5, 10, or 20 mg/kg versus vehicle) and time of administration (15 and 60 min) as independent factor, and blocks (5 min interval) as a repeated measures factor. Mauchly’s test of sphericity was significant and Greenhause–Geisser correction are presented for repeated measures, Mauchly’s W(14) = 0.21, p < 0.001. Degrees of freedom were rounded to whole number for presentational purposes.

Analyses produced significant main effects of drug treatment [F_{(3, 71)} = 22.43, p < 0.001], time of administration [F_{(1, 71)} = 50.68, p < 0.001], and blocks [F_{(3, 211)} = 188.43, p < 0.001]. In addition, there was a significant time of administration × drug treatment interaction [F_{(3, 71)} = 8.37, p < 0.001] and a significant time of administration × blocks interaction [F_{(3, 211)} = 6.81, p < 0.001] no other interactions were observed (Figure 2).

Since no interaction between drug treatment × blocks was observed further pairwise comparisons using independent t-tests were used to explore the significant on total trajectory length; these revealed that compared with vehicle all three doses of methylone significantly increased locomotion at 15 min [minimum t(13) = 5.17, p < 0.001; Figure 3A] as well as at 60 min [minimum t(12) = 2.99, p < 0.05; Figure 3B]. The increase at 60 min was much less pronounced.

Nor-methylone 10 mg/kg compared with vehicle significantly increased total locomotion at 15 min after administration [t(18) = 4.57, p < 0.01], by contrast, compared nor-methylone to methylone 10 mg/kg there was no significant difference (Figure 3A).

Thigmotaxis and T_center of arena were each analyzed with 4 × 2 ANOVAs with drug treatment and time of administration as independent factors.

Habituation, ASR, and PPI data were each analyzed with 4 × 2 independent ANOVAs with drug treatment (methylone at 5, 10, and 20 mg/kg versus vehicle) and time of administration (15 and 60 min) as independent factors.

Habituation data showed a significant main effect of time of administration on habituation [F_{(1, 72)} = 8.17, p < 0.01] but no interaction was observed. Independent t-tests revealed that methylone compared with vehicle did not affect habituation at any of the doses tested (Table 2).

Acoustic startle response data showed a significant main effect of drug treatment [F_{(3, 72)} = 2.83, p < 0.05] and again no interaction was detected. Independent t-tests revealed that methylone compared with vehicle did not affect ASR at any of the doses tested (Table 2).

Independent ANOVA showed a significant main effect of drug treatment on PPI [F_{(3, 72)} = 2.88, p < 0.05], no other interactions were observed. Subsequent independent t-test revealed a trend to decrease for 20 mg/kg at 15 min, compared with control, t(18) = 1.91, p = 0.1 (one-tailed). Nor-methylone did not differ from vehicle (Figure 4).

The effect of methylone on body temperature was analyzed using 3 × 2 × 13 mixed factorial ANOVAs with drug treatment (methylone at 10 and 20 mg/kg versus vehicle) and home-cage conditions (individually and group-housed rats) as independent factors, and time as a repeated measures factor. Mauchly’s test of sphericity was significant and Greenhouse–Geisser correction are presented for repeated measures, Mauchly’s W(44) = 0.05, p < 0.001. Although temperature data before drug administration were significantly different from vehicle, these data were averaged for individual treatment and subtracted from temperature data after drug administration.

Temperature data showed a significant main effect of drug treatment [F_{(2, 54)} = 5.29, p < 0.05], home-cage conditions [F_{(1, 54)} = 4.41, p < 0.05], and time [F_{(5, 289)} = 161.58, p < 0.001]. The interaction of drug treatment × time [F_{(11, 289)} = 6.87, p < 0.001] and the three-way interaction of drug treatment × time × home-cage conditions [F_{(11, 289)} = 4.3, p < 0.001] were significant.

Independent t-tests revealed that under individually conditions, methylone significantly increased body temperature half an hour (9.30 h) after administration for both doses (10 and 20 mg/kg), an effect that was maintained until 13.00 for 10 mg/kg groups, minimum t(18) = 2.15, p < 0.05, and to 14.00 in the case of 20 mg/kg, minimum t(18) = 2.07, p = 0.05, Figure 5A.

In rats housed under group-housed condition, the temperature started to increase at 30 min after methylone administration after each of the doses. Methylone 10 mg/kg significantly increased body temperature from 9.30 to 10.30 h, minimum t(18) = 2.6, p < 0.05. At 20 mg/kg dose, temperature maintained elevated until 11.00, minimum t(18) = 2.46, p < 0.05, Figure 5B.

Compared with our data, Elmore et al. (32) found the peak serum of methylone levels even earlier at 15 min using the same route of administration. Interestingly, Lopez-Arnau et al. (33) found maximum plasma levels at 30 min after oral administration of methylone in rats which is indicative of a very fast gastrointestinal absorption. Additionally, only our experiments indicate a very fast and effective crossing of blood–brain barrier as methylone levels in the brain were more than five times those in serum. The incorporation of methylone into the brain may be associated with high lipophilicity, as we have already suggested for other compounds, e.g., PMMA or MDAI (5,6-methylenedioxy-2-aminoindane) (21, 27). Similarly, as methylone, its metabolite nor-methylone showed similar serum:brain ratio. The other important and major metabolites 4-OH-MeO-MC (4-hydroxy-3-methoxymethcathinone) (1, 34) were also detected in serum and brain. The rapid decrease of its dominance in the analytical spectrum at 60 min may be related to its fast conjugation, which would explain its lower plasmatic concentrations compared with nor-methylone. Even though we did not perform enzymatic hydrolysis, we might assume that the rapid decrease of its levels might be related to its fast conjugation with glucuronic and/or sulfuric acid that is typical for fenolic metabolites (35). Compared with MDMA where peak MDMA concentrations are achieved within 1 h after subcutaneous or oral administration both methylone and its metabolite nor-methylone showed a more rapid kinetic profile (36, 37) which is in line with the reported shorter duration of effects in humans (and might lead to more frequent re-dosing by users).

According to our knowledge, there is no evidence about determination of lethal methylone dose in animals. In our study, we obtained unexpected findings on the lethal effects of the highest dose of methylone (40 mg/kg) in the rats. The symptoms observed in this case (i.e., hyperventilation, seizures) were similar to symptoms detected in MDAI (27) and may be associated with serotonin syndrome, mainly hyperthermia which is one of the core symptom caused by 5-HT release (38).

In accordance with its kinetic profile, the overall locomotor stimulatory activity was more pronounced 15 min after administration and was also comparable with other studies in rats (16, 19) and mice (17, 18). In mice, after methylone 30 mg/kg, locomotor activity was lower compared with 10 mg/kg (18) indicative of an inverted U shaped curve of locomotor effects. This inverted U shaped locomotor curve is also typical for most of the stimulants and characteristically linked to an increase in stereotyped behavior (e.g., circling) (21, 39). It is well established that the stimulatory versus hallucinogenic potency of cathinones and other related compounds is related to their DAT:SERT inhibition ratio. As stated above, methylone has been reported to have similar DAT:SERT inhibition ratio to cocaine (3, 12), and in contrast to other related cathinones, e.g., mephedrone, naphyrone, and methylenedioxypyrovalerone (MDPV) methylone has lower selectivity over DAT making it less stimulatory (3, 40). As reported in comparable behavioral studies of our currently submitted manuscripts, its stimulatory potency is slightly lower compared with mephedrone and much less potent compared with MDPV (unpublished observations Horsley et al.and Sichova et al.).

According to the temporal and spatial patterns in locomotor activity, methylone disrupted habituation, increased exploration, and stimulated activity at lower doses, however, high doses induced stereotyped behavior. In this respect, methylone behaves in a very similar manner to other stimulants and entactogens tested in identical (or near-identical) paradigms in our laboratory (21, 31).

Methylone has a relatively weak potency to disturb sensorimotor processing. In line with this, our recent experiments with mephedrone, nor-mephedrone, and MDPV showed comparable weak or negative effects on PPI in rats (unpublished observations Horsley et al. and Sichova et al.). Interestingly amphetamine, which is approximately 10 times more potent in disrupting PPI in rodents, in humans also failed to have disruptive effect on PPI (41). However, this might be related to the fact that it was used in much lower dose (0.45 mg/kg) in humans compared with rodents (typically 1–4 mg/kg). On the contrary drugs affecting mainly SERT, e.g., MDMA, PMMA, or MDAI seem to have much stronger ability to disrupt sensorimotor gating in animals as well as in humans (26, 31, 42). Since PPI is typically used as a model of psychotomimetic potential in animals with translational validity, we may conclude that methylone has only mild psychotomimetic effects. Apart from PPI, and similarly like with the open field, the habituation to startle was attenuated during the peak of methylone effect (i.e., in 15 min time of administration). This can be theoretically also related to the overall stimulatory effect or to anxiety, since with the highest dose also the decreased time spent in the center was present.

As expected and in accordance with previous studies with methylone (22, 43, 44), MDMA (45, 46) as well as our comparable studies with phenethylamine PMMA, and aminoindane MDAI (21, 27) the hyperthermic reaction was more pronounced in group-housed condition where it increased up to 1.5°C. The temperature increase was rapid and was not associated with visible perspiration as has been described with PMMA and MDAI (21, 27). In animals housed separately, the increase in temperature lasted for a 1 h longer compared with animals housed in groups. This is surprising since the opposite would be expected. This might be explained by accelerated metabolism due to higher increase in body temperature (cca 1°C) in animals housed in groups. Serotonergic drugs have more pronounced hyperthermic effects compared with drugs with dopaminergic actions. Since serotonin is a critical neuromodulator involved in the thermoregulation, with 5-HT2A receptors being a key mechanism responsible for hyperthermia (47). It is therefore very probable that this is also the case for methylone where the 5-HT2A receptor is stimulated via indirect mechanisms related to the increased serotonergic tone (44). On the other hand, in study of Javadi-Paydar et al. (19) was shown that mean body temperature did not vary more than 0.5°C from baseline temperature after methylone application. These findings of different (negative) results could be caused by methodological differences, where they measured temperature using radiotelemetry with lower doses of methylone than us.

Also in some of the cathinones, e.g., mephedrone have been also reported to induce hypothermia in rats but not mice (43, 48, 49). This effect is typically stimulated by activity at 5-HT1A receptors, and sometimes drugs that induce serotonin release might have biphasic effects on temperature or bidirectional depending on pharmacodynamics and the stimulation of these receptors (50).

Since here rats in group-housed conditions exhibited greater elevations in temperature (than under individually housed rats), this provides more support for the idea that environmental conditions that are crowded and/or hot (e.g., people dancing in a crowded clubs) increase the risk of hyperthermia and acute toxicity associated with methylone (51).