The designer methcathinone analogs, mephedrone and methylone, are substrates for monoamine transporters in brain tissue

Abstract

The nonmedical use of 'designer' cathinone analogs, such as 4-methylmethcathinone (mephedrone) and 3,4-methylenedioxymethcathinone (methylone), is increasing worldwide, yet little information is available regarding the mechanism of action for these drugs. Here, we employed in vitro and in vivo methods to compare neurobiological effects of mephedrone and methylone with those produced by the structurally related compounds, 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine. In vitro release assays using rat brain synaptosomes revealed that mephedrone and methylone are nonselective substrates for plasma membrane monoamine transporters, similar to MDMA in potency and selectivity. In vivo microdialysis in rat nucleus accumbens showed that i.v. administration of 0.3 and 1.0 mg/kg of mephedrone or methylone produces dose-related increases in extracellular dopamine and serotonin (5-HT), with the magnitude of effect on 5-HT being greater. Both methcathinone analogs were weak motor stimulants when compared with methamphetamine. Repeated administrations of mephedrone or methylone (3.0 and 10.0 mg/kg, s.c., 3 doses) caused hyperthermia but no long-term change in cortical or striatal amines, whereas similar treatment with MDMA (2.5 and 7.5 mg/kg, s.c., 3 doses) evoked robust hyperthermia and persistent depletion of cortical and striatal 5-HT. Our data demonstrate that designer methcathinone analogs are substrates for monoamine transporters, with a profile of transmitter-releasing activity comparable to MDMA. Dopaminergic effects of mephedrone and methylone may contribute to their addictive potential, but this hypothesis awaits confirmation. Given the widespread use of mephedrone and methylone, determining the consequences of repeated drug exposure warrants further study.

Figure 1

Chemical structures of designer methcathinone analogs and related compounds.

Figure 2

Dose-response effects of test drugs on the release of [³H]MPP⁺ and [³H]5-HT from rat brain synaptosomes in vitro, under conditions optimized for NET (a), DAT (b), and SERT (c). Dose-response curves were constructed by incubating various concentrations of each test drug with synaptosomes that had been preloaded with tritiated substrate, either [³H]MPP⁺ or [³H]5-HT. Test drugs were mephedrone (4MMC), methylone (MDMC), 3,4-methylenedioxymethamphetamine (MDMA), and methamphetamine (MA). Data are mean±SD for N=6 (for NET assays) or N=3 (DAT and SERT assays) separate experiments.

Figure 3

Dose-response effects of mephedrone (4MMC) on neurochemistry and locomotor behavior in male rats undergoing in vivo microdialysis in nucleus accumbens. 4MMC-treated rats received i.v. injections of 0.3 mg/kg at time zero, followed by 1.0 mg/kg 60 min later. Control rats received i.v. saline injections (1 ml/kg) on the same schedule. Data are mean±SEM for N=7 rats per group, expressed as a percentage of preinjection baseline values (% basal) for dopamine (DA, panel a), serotonin (5-HT, panel b), horizontal locomotor activity (HLA, panel c), and stereotypic movements (Stereo, panel d). Basal concentrations of dialysate DA and 5-HT were 2.39±0.51 and 0.25±0.03 pg/5 μl, respectively. Basal levels of HLA and Stereo were 170±15 cm/20 min and 131±15 moves/20 min, respectively. ^**p<0.01 or ^*p<0.05 vs saline control at the corresponding time point (Bonferroni post hoc test).

Figure 4

Dose-response effects of methylone (MDMC) on neurochemistry and locomotor behavior in male rats undergoing in vivo microdialysis in nucleus accumbens. MDMC-treated rats received i.v. injections of 0.3 mg/kg at time zero, followed by 1.0 mg/kg 60 min later. Control rats received i.v. saline injections (1 ml/kg) on the same schedule. Data are mean±SEM for N=7 rats per group, expressed as a percentage of preinjection baseline values (% basal) for dopamine (DA, panel a), serotonin (5-HT, panel b), horizontal locomotor activity (HLA, panel c), and stereotypic movements (Stereo, panel d). Basal concentrations of dialysate DA and 5-HT were 2.63±0.49 and 0.36±0.09 pg/5 μl, respectively. Basal levels of HLA and Stereo were 181±18 cm/20 min and 132±17 moves/20 min, respectively. ^**p<0.01 vs saline control at the corresponding time point (Bonferroni post hoc test).

Figure 5

Dose-response effects of 3,4-methylenedioxymethamphetamine (MDMA) on neurochemistry and locomotor behavior in male rats undergoing in vivo microdialysis in nucleus accumbens. MDMA-treated rats received i.v. injections of 0.3 mg/kg at time zero, followed by 1.0 mg/kg 60 min later. Control rats received i.v. saline injections (1 ml/kg) on the same schedule. Data are mean±SEM for N=7 rats per group, expressed as a percentage of preinjection baseline values (% basal) for dopamine (DA, panel a), serotonin (5-HT, panel b), horizontal locomotor activity (HLA, panel c), and stereotypic movements (Stereo, panel d). Basal concentrations of dialysate DA and 5-HT were 2.00±0.52 pg/5 μl and 0.44±0.09 pg/5 μl, respectively. Basal levels of HLA and Stereo were 134 ± 22 cm/20 min and 109±10 moves/20 min, respectively. ^**p<0.01 or ^*p<0.05 vs saline control at the corresponding time point (Bonferroni post hoc test).

Figure 6

Dose-response effects of methamphetamine (MA) on neurochemistry and locomotor behavior in male rats undergoing in vivo microdialysis in nucleus accumbens. MA-treated rats received i.v. injections of 0.3 mg/kg at time zero, followed by 1.0 mg/kg 60 min later. Control rats received i.v. saline injections (1 ml/kg) on the same schedule. Data are mean±SEM for N=7 rats per group, expressed as a percentage of preinjection baseline values (% basal) for dopamine (DA, panel a), serotonin (5-HT, panel b), horizontal locomotor activity (HLA, panel c), and stereotypic movements (Stereo, panel d). Basal concentrations of dialysate DA and 5-HT were 2.01±0.54 pg/5 μl and 0.31±0.05 pg/5 μl, respectively. Basal levels of HLA and Stereo were 151±29 cm/20 min and 129±29 moves/20 min, respectively. ^**p<0.01 or ^*p<0.05 vs saline control at the corresponding time point (Bonferroni post hoc test).

Figure 7

Acute and long-term effects of repeated administrations of mephedrone (4MMC) in single-housed male rats. 4MMC-treated rats received s.c. injections of 3.0 or 10.0 mg/kg at 0, 2, and 4 h (denoted by arrows in panel a). Control rats received s.c. saline injections (1 ml/kg) on the same schedule. Core temperature and motor behaviors were assessed every hour beginning at time zero, whereas post-mortem brain tissue amines were assessed 2 weeks after binge dosing. Data are mean±SEM for N=6–7 rats per group, expressed as core temperature in °C (panel a) and summed behavioral scores for ambulation (Amb), rearing (Rear), and forepaw treading (Tread) (panel b). Tissue amine concentrations in cortex (panel c) and striatum (panel d) are expressed as percentage of values for saline-treated rats (% control). Control amine levels in cortex were 241±15, 25±4, and 212±24 pg/mg for norepinephrine (NE), dopamine (DA), and serotonin (5-HT), respectively. Control amine levels in striatum were 840±68, 12691±363, and 254±48 pg/mg for NE, DA, and 5-HT, respectively. In panel (a), filled symbols indicate significant differences with respect to saline-treated controls at a given time point, p<0.05. In panels (b–d), ^*p<0.05 compared with saline control group (Newman–Keul's post hoc test).

Figure 8

Acute and long-term effects of repeated administrations of methylone (MDMC) in single-housed male rats. MDMC-treated rats received s.c. injections of 3.0 or 10.0 mg/kg at 0, 2, and 4 h (denoted by arrows in panel a). Control rats received s.c. saline injections (1 mL/kg) on the same schedule. Core temperature and motor behavior were assessed every hour beginning at time zero, whereas post-mortem brain tissue amines were assessed 2 weeks after binge dosing. Data are mean±SEM for N=6–7 rats per group, expressed as core temperature in °C (panel a) and summed behavioral scores for ambulation (Amb), rearing (Rear), and forepaw treading (Tread) (panel b). Tissue amine concentrations in cortex (panel c) and striatum (panel d) are expressed as percentage of values for saline-treated rats (% control). Control amine levels in cortex were 273±12, 29±3, and 284±23 pg/mg for norepinephrine (NE), dopamine (DA), and serotonin (5-HT), respectively. Control amine levels in striatum were 605±45, 11511±708, and 206±18 pg/mg for NE, DA and 5-HT, respectively. In panel (a), filled symbols indicate significant differences with respect to saline-treated control at a given time point, p<0.05. In panels (b–d), ^*p<0.05 compared with saline control group (Newman–Keul's post hoc test).

Figure 9

Acute and long-term effects of repeated administrations of 3,4-methylenedioxymethamphetamine (MDMA) in single-housed male rats. MDMA-treated rats received s.c. injections of 2.5 or 7.5 mg/kg at 0, 2, and 4 h (denoted by arrows in panel a). Control rats received s.c. saline injections (1 ml/kg) on the same schedule. Core temperature and motor behavior were assessed every hour beginning at time zero, whereas post-mortem brain tissue amines were assessed 2 weeks after binge dosing. Data are mean±SEM for N=5–6 rats per group, expressed as core temperature in °C (panel a) and summed behavioral scores for ambulation (Amb), rearing (Rear), and forepaw treading (Tread) (panel b). Tissue amine concentrations in cortex (panel c) and striatum (panel d) are expressed as percentage of values for saline-treated rats (% control). Control amine levels in cortex were 275±7, 31±3, and 308±33 pg/mg for norepinephrine (NE), dopamine (DA), and serotonin (5-HT), respectively. Control amine levels in striatum were 753±75, 10 701±1335, and 313±20 pg/mg for NE, DA and 5-HT, respectively. In panel (a), filled symbols indicate significant differences with respect to saline-treated control at a given time point, p<0.05. In panels (b–d), ^*p<0.05 compared with saline control group (Newman–Keul's post hoc test).