Pharmacokinetic Profiles and Pharmacodynamic Effects for Methylone and Its Metabolites in Rats

Abstract

3,4-Methylenedioxy-N-methylcathinone (methylone) is a new psychoactive substance and the β-keto analog of 3,4-methylenedioxy-N-methylamphetamine (MDMA). It is well established that MDMA metabolism produces bioactive metabolites. Here we tested the hypothesis that methylone metabolism in rats can form bioactive metabolites. First, we examined the pharmacokinetics (PKs) of methylone and its metabolites after subcutaneous (sc) methylone administration (3, 6, 12 mg/kg) to male rats fitted with intravenous (iv) catheters for repeated blood sampling. Plasma specimens were assayed by liquid chromatography tandem mass spectrometry to quantify methylone and its phase I metabolites: 3,4-methylenedioxycathinone (MDC), 3,4-dihydroxy-N-methylcathinone (HHMC), and 4-hydroxy-3-methoxy-N-methylcathinone (HMMC). The biological activity of methylone and its metabolites was then compared using in vitro transporter assays and in vivo microdialysis in rat nucleus accumbens. For the PK study, we found that methylone and MDC peaked early (T_max=15-45 min) and were short lived (t_1/2=60-90 min), while HHMC and HMMC peaked later (T_max=60-120 min) and persisted (t_1/2=120-180 min). Area-under-the-curve values for methylone and MDC were greater than dose-proportional, suggesting non-linear accumulation. Methylone produced significant locomotor activation, which was correlated with plasma methylone, MDC, and HHMC concentrations. Methylone, MDC, and HHMC were substrate-type releasers at monoamine transporters as determined in vitro, but only methylone and MDC (1, 3 mg/kg, iv) produced significant elevations in brain extracellular dopamine and 5-HT in vivo. Our findings demonstrate that methylone is extensively metabolized in rats, but MDC is the only centrally active metabolite that could contribute to overall effects of the drug in vivo.

Figure 1

Pathways of methylone metabolism. Methylone, 3,4-methylenedioxy-N-methylcathinone; MDC, 3,4-methylenedioxycathinone; HHMC, 3,4-dihydroxy-N-methylcathinone; HMMC, 4-hydroxy-3-methoxy-N-methylcathinone.

Figure 2

Concentration–time profiles for methylone, MDC, HHMC, and HMMC after sc administration of methylone. Data are mean±SEM for N=6/7 rats per group. Rats received a sc dose of 0 (saline), 3, 6, or 12 mg/kg methylone, and blood was collected at 15, 30, 60, 120, 240, and 480 min postinjection. Plasma was separated and analyzed by LC–MS/MS.

Figure 3

Comparison of expected vs observed area-under-the-curve values for methylone, MDC, HHMC, and HMMC. Expected AUCs for each analyte at 6 and 12 mg/kg methylone doses were determined by multiplying the values observed at 3 mg/kg by a factor of 2 and 4, respectively. Data are mean±SEM for N=6/7 rats per group. *p<0.05 vs expected value at the corresponding dose (Bonferroni's post hoc test).

Figure 4

Pharmacodynamic data for rats receiving sc injection of 0 (saline), 3, 6, and 12 mg/kg methylone. Behavioral score and core temperature were determined at 15, 30, 60, 120, 240, and 480 min postinjection as described in Materials and Methods section. Data are mean±SEM for N=6/7 rats per group. For temperature data, the filled symbols represent significant effects when compared with saline-injected control rats at corresponding time points (p<0.05, Bonferroni's post hoc test).

Figure 5

Dose–response effects of methylone, MDC, HHMC, and HMMC on extracellular dopamine (DA) in male rats undergoing in vivo microdialysis in nucleus accumbens. Drug-treated rats received iv injections of 1 mg/kg at time 0, followed by 3 mg/kg 60 min later. Control rats received iv saline injections (1 ml/kg) on the same schedule. Data are mean±SEM for N=6/7 rats per group, expressed as a percentage of preinjection baseline values (% basal). Mean basal dialysate DA concentration for all the treatment groups was 1.55±0.35 pg/5 μl (N=34 rats). *p<0.05 vs saline control at the corresponding time point (Bonferroni's post hoc test).

Figure 6

Dose–response effects of methylone, MDC, HHMC, and HMMC on extracellular serotonin (5-HT) in male rats undergoing in vivo microdialysis in nucleus accumbens. Drug-treated rats received iv injections of 1 mg/kg at time 0, followed by 3 mg/kg 60 min later. Control rats received iv saline injections (1 ml/kg) on the same schedule. Data are mean±SEM for N=6/7 rats per group, expressed as a percentage of preinjection baseline values (% basal). Mean basal dialysate 5-HT concentration for all the groups was 0.42±0.11 pg/5 μl (N=34 rats). *p<0.05 vs saline control at the corresponding time point (Bonferroni's post hoc test).

Figure 7

Dose–response effects of methylone, MDC, HHMC, and HMMC on forward locomotion (activity) in male rats undergoing in vivo microdialysis in nucleus accumbens. Drug-treated rats received iv injections of 1 mg/kg at time 0, followed by 3 mg/kg 60 min later. Control rats received iv saline injections (1 ml/kg) on the same schedule. Data are mean±SEM for N=6/7 rats per group, expressed as a percentage of preinjection baseline values (% basal). Mean basal activity was 86±22 cm/20 min bin (N=34 rats). *p<0.05 vs saline control at the corresponding time point (Bonferroni's post hoc test).