Methylone, a rapid acting entactogen with robust anxiolytic and antidepressant-like activity

Major depressive disorder (MDD) and anxiety are highly prevalent and frequently co-morbid conditions for which selective serotonin reuptake inhibitors (SSRIs) are first-line pharmacological treatments. SSRIs and other traditional slow-acting antidepressants (SAADs) have a delayed onset of efficacy (commonly 4–8 weeks), produce side effects that can limit adherence, and leave a substantial proportion of patients (up to about 40%) without an adequate response. Rapid-acting antidepressants (RAADs) such as esketamine, psilocybin and MDMA are of growing interest because they may produce faster, durable clinical effects and permit shorter or less frequent dosing regimens. The researchers focus on methylone (3,4-methylenedioxy-N-methylcathinone; MDMC, βk-MDMA, M1), a beta‑ketone analogue of MDMA that has shown preliminary tolerability in Phase I studies and signal of benefit in retrospective clinical case series of PTSD and MDD. Preclinical data on methylone are limited and often reflect binge-dosing paradigms. The present study therefore tested the hypothesis that methylone produces rapid-onset antidepressant- and anxiolytic-like effects in rats, comparing methylone with the SSRI fluoxetine in the forced swim test (FST) and open field test (OFT), probing dose–response, durability, interactions with SSRI co-treatment, and pharmacokinetic exposure in plasma and brain.

Overall design and animals: The experiments used male Sprague Dawley rats (6–8 weeks old). Distinct cohorts were used for individual experiments. Behavioural testing was conducted at Melior Discovery and pharmacokinetic (PK) work at WuXi Apptec; all procedures were compliant with institutional animal care protocols. Sample sizes varied across experiments (the extraction reports group Ns of 6–16 for behavioural tests); PK terminal samples used N = 3 rats per time point per dose. Drug treatments: Methylone HCl (0.5–30 mg/kg, intraperitoneal, IP) and fluoxetine HCl (10 mg/kg, IP) were prepared in 0.9% saline. Methylone or saline was given 30 min before FST or OFT sessions. Fluoxetine or saline were administered at 23.5, 5 and 1 h before testing (three doses) to match prior FST protocols. Separate vehicle control groups were used for methylone (single injection) and fluoxetine (three injections) to account for injection stress effects. Pharmacokinetics: For PK, terminal blood and brain samples were collected at 0.25, 0.5, 1, 2, 4 and 8 h after single IP doses of methylone at 5, 10 or 15 mg/kg (three rats per time point). Plasma was processed and analysed by LC‑MS/MS with an assay LLOQ of 1 ng/mL and ULOQ of 3000 ng/mL. Non-compartmental analysis using linear/log trapezoidal methods in Phoenix WinNonlin was used to calculate PK parameters (Cmax, AUC, Tmax, half-life, MRT). Behavioural assays: The Forced Swim Test (FST) followed a standard two-day, modified protocol: a 15‑min training swim on Day 1 and a 5‑min test 24 h later. Rats were scored every 5 s for immobility, swimming and climbing; results are reported as percent time in each behaviour over the 5‑min session. The Open Field Test (OFT) assessed locomotion and anxiety-like behaviour in a 30‑min session, with ambulatory distance recorded in 5‑min bins and time spent in the predefined central area recorded as an anxiolytic measure. Experimenters were blind to treatment. Statistics: Data are presented as mean ± SEM. Two‑group comparisons used unpaired t-tests; comparisons among more than two groups used one‑way ANOVA with post hoc tests specified in figure legends. For drug × time designs, two‑way ANOVA with Bonferroni post hoc tests was used. A significance threshold of p ≤ 0.05 was applied and analyses were performed in GraphPad Prism v9.3.1.

Antidepressant-like effects in the FST: Single IP doses of methylone ranging from 0.5 to 30 mg/kg were tested 30 min before the FST. Vehicle controls showed 63.9 ± 2.7% time immobile. A low effective dose (5 mg/kg) reduced immobility to 31.9 ± 4.7%. Moderate doses (10–15 mg/kg) produced a robust reduction in immobility to 4.2 ± 1.3% compared with vehicle (63.9 ± 2.7%), with the overall dose–response yielding a highly significant effect [ANOVA reported: F (7,67) = 23.32, p < 0.0001]. At the highest doses (20–30 mg/kg) there was a non‑significant trend toward less reduction in immobility, suggesting a possible U‑shaped dose–response. Fluoxetine, given as three doses (10 mg/kg each) prior to testing, produced an approximately 50% reduction in immobility relative to its own vehicle control (different control group due to repeated injections). The magnitude of effect from three fluoxetine doses was comparable to the single low methylone dose (5 mg/kg) but markedly smaller than the maximal methylone effect at 10–15 mg/kg. Durability: Pharmacokinetic and repeated testing experiments showed a durable antidepressant-like effect after a single methylone dose. Following a single 15 mg/kg dose administered 30 min before the initial FST, methylone reduced immobility significantly for at least 72 h compared with vehicle [two‑way ANOVA: Drug: F (1,36) = 173.9, p < 0.0001; Time: F (2,36) = 33.82, p < 0.0001; Drug × Time: F (2,36) = 8.548, p < 0.001]. In contrast, fluoxetine showed a significant effect on immobility only at 1 h post‑dose and not at later time points. Pharmacokinetics: After single IP administration at 5, 10 and 15 mg/kg, plasma Cmax values were reported as 1983, 4507 and 8470 ng/mL, respectively, with Tmax at 15 min post‑dose. Reported AUC0‑last values were 908, 3242 and 7320 (units in the extracted text are given as h/mL), the terminal half‑life (T1/2) was 0.6–0.8 h, and MRT0‑last was 0.7–1.13 h. The brain-to-plasma AUC ratio was approximately 1.8, indicating brain penetration. (If precise PK units or full tabulated parameters are required, the extracted text does not unambiguously report all units.) Locomotion and anxiety measures (OFT): Locomotor activity during the first 5 min of the OFT (the interval corresponding to the FST test duration) showed that fluoxetine reduced distance traveled 1 h post‑dose [t (10) = 2.882, p < 0.05]. Methylone increased ambulatory distance at higher doses (15–30 mg/kg) but lower doses (0.5–10 mg/kg), which include the effective FST doses, did not affect locomotor activity [reported ANOVA: F (7,72) = 5.816, p < 0.0001]. When retested 24 h later (a timepoint at which FST antidepressant effects persisted), locomotor effects had resolved while antidepressant-like effects remained, supporting a dissociation between locomotion and FST immobility. Anxiolytic-like effects: In the OFT, methylone (5–30 mg/kg) significantly increased time spent in the centre of the arena 30 min after dosing compared with controls [ANOVA: F (7,71) = 4.184, p < 0.001], consistent with an anxiolytic-like effect. By contrast, acute fluoxetine significantly reduced time in centre [t (10) = 3.587, p < 0.01], consistent with an anxiogenic-like effect of acute SSRI dosing reported elsewhere. Combined treatment with fluoxetine: To test for interaction between SSRIs and methylone (relevant because SSRIs can blunt MDMA effects), rats received fluoxetine (three doses) plus either a low effective methylone dose (5 mg/kg) or a maximal methylone dose (15 mg/kg). Fluoxetine pre‑treatment did not alter methylone's reduction of immobility in the FST. For example, methylone (15 mg/kg) + fluoxetine produced 3.3 ± 2.4% time immobile versus vehicle 61.6 ± 5.2% (p < 0.0001), similar to methylone alone (4.2 ± 1.8% vs vehicle 63.9 ± 2.7%).

The authors interpret their data as the first preclinical demonstration that methylone produces rapid, robust and durable antidepressant‑ and anxiolytic‑like effects in rats. They note that a single methylone dose produced large reductions in FST immobility (reported reductions of 78–98% relative to vehicle), greater than many antidepressants reported in the FST literature and larger than the acute effects observed with fluoxetine in their protocol. The antidepressant-like effect manifested rapidly (within 30 min) and persisted up to 72 h after one dose, a profile the authors liken to other RAADs such as ketamine, MDMA and psilocybin. Pharmacologically, the authors emphasise that methylone is a monoamine uptake inhibitor and releaser at DAT, NET and SERT but is reported to be 3–4× less potent at inhibiting serotonin uptake than MDMA and to have much lower potency at VMAT2. Behavioural indicators in the FST (increases in climbing at lower doses and increased swimming only at higher doses) are consistent with a relatively greater noradrenergic versus serotonergic contribution compared with MDMA. The PK data show rapid plasma and brain exposure (Cmax at ~15 min) and a brain:plasma AUC ratio of about 1.8, supporting central nervous system availability. The authors discuss prior conflicting preclinical reports that used binge‑dosing paradigms and different dose ranges/timings; they attribute discrepancies to those methodological differences, noting that binge regimens produced depression- or anxiety-like outcomes in other studies, while their single lower‑dose, therapeutically oriented regimen produced antidepressant and anxiolytic effects. They also raise the potentially practical advantage that methylone's efficacy in the FST was not reduced by concurrent fluoxetine, contrasting with reports that SSRIs can blunt MDMA's effects — a feature that, if replicated in humans, could avoid the need for prolonged SSRI washout before entactogen‑assisted therapy. Limitations acknowledged by the authors include the limitations of the FST as a screening tool and the potential confound of locomotor stimulation; they argue against a locomotor artefact because effective FST doses did not change locomotion, and antidepressant-like effects persisted after locomotor effects resolved. They note that experiments were performed in naïve animals and that future work should test chronically stressed models to better model clinical depression. Mechanistic explanations (for example, a role for SERT inhibition given an in vitro SERT IC50 of 5.75 µM) are presented as speculative and in need of further study. Finally, the authors use an allometric scaling estimate to suggest rat effective doses (10–15 mg/kg) roughly correspond to 100–150 mg in humans, doses that have been evaluated in a Phase I study and reported as tolerable, but they emphasise the need for additional rodent and human research to define clinical potential and safety.