Serotonin-releasing agents with reduced off-target effects

Abstract

Increasing extracellular levels of serotonin (5-HT) in the brain ameliorates symptoms of depression and anxiety-related disorders, e.g., social phobias and post-traumatic stress disorder. Recent evidence from preclinical and clinical studies established the therapeutic potential of drugs inducing the release of 5-HT via the 5-HT-transporter. Nevertheless, current 5-HT releasing compounds under clinical investigation carry the risk for abuse and deleterious side effects. Here, we demonstrate that S-enantiomers of certain ring-substituted cathinones show preference for the release of 5-HT ex vivo and in vivo, and exert 5-HT-associated effects in preclinical behavioral models. Importantly, the lead cathinone compounds (1) do not induce substantial dopamine release and (2) display reduced off-target activity at vesicular monoamine transporters and 5-HT_2B-receptors, indicative of low abuse-liability and low potential for adverse events. Taken together, our findings identify these agents as lead compounds that may prove useful for the treatment of disorders where elevation of 5-HT has proven beneficial.

Fig. 1. Chemical structures and inhibition of transporter-mediated uptake by cathinones.

a–e display the synthesis and the chemical structures of the stereoisomers of MC, 4-MMC, 4-MC and 4-TFMMC, respectively. f–i display inhibition of human DAT-mediated uptake of [³H]MPP⁺ and j–m depict inhibition of [³H]5-HT transport via human SERT in HEK293 cells. Data are shown as mean and standard deviation. N > 3 independent experiments. Detailed sample sizes are given in the Supplementary Information.

Fig. 2. Cathinone-induced 5-HT efflux and electrophysiology.

a Schematic representation of substrate-type releaser induced efflux of preloaded [³H]5-HT from HEK293 cells stably expressing human SERT. Representative traces showing the effect of the two stereoisomers of b MC, c 4-MMC, d 4-MC and e 4-TFMMC on SERT-mediated efflux at 10 µM. f-i Cathinone-induced efflux at t = 8–14 min was normalized to basal efflux at t = 0–4 min and plotted against the applied concentrations of the stereoisomers of f MC, g 4-MMC, h 4-MC, i 4-TFMMC. j Schematics of whole-cell patch clamp experiments used to identify cathinone-induced inwardly directed currents in SERT-expressing HEK293 cells. Representative single-cell traces showing currents elicited by 10 µM of k S- and R-MC, l S- and R-4-MMC, m S- and R-4-MC, n S- and R-4-TFMMC. o–r Cathinone-induced currents were normalized to the current elicited by bath-application of 10 µM 5-HT and plotted against the applied concentrations. Data in f–i and o–r are shown as mean and standard deviation. Data shown in d and h are replotted from 10.1016/j.neuropharm.2018.12.032. N > 3 independent observations per compound and concentration. Detailed sample sizes are given in the Supplementary Information.

Fig. 3. Stereospecific effect of cathinones on behavior.

Total time spent immobile in the forced-swim test following systemic (intraperitoneal) administration of vehicle or a S-4-MMC, S-4-MC and S-4-TFMMC or b R-4-MMC, R-4-MC and R-4-TFMMC at the indicated doses (1, 5 and 10 mg kg⁻¹). c Total distance traveled, d vertical counts and e distance traveled in the center in the open field test following intraperitoneal injections of S-4-MMC, S-4-MC, S-4-TFMMC at the indicated doses (1, 5 and 10 mg kg⁻¹) and R-4-MMC (10 mg kg⁻¹). f Total time spent immobile in the forced-swim test in male and female mice after systemic administration (intraperitoneal) of S-4-MMC (5 mg kg⁻¹), S-4-MC (5 mg kg⁻¹) and S-4-TFMMC (10 mg kg⁻¹). Data are shown as the mean and standard deviation (bars) with the individual mice being represented by gray symbols. a, c–e were analyzed using Brown–Forsythe and Welch ANOVA, followed by Dunnet’s T3 multiple comparison test. b, f were analyzed with ordinary one-way ANOVA and Dunnett’s multiple comparison test to identify differences versus vehicle. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. N = at least 6 animals per compound and dose. Detailed sample sizes are given in the Supplementary Information.

Fig. 4. Stereospecific effect of cathinones on SERT-dependent 5-HT release in vivo.

a Schematics of optic probe placement in the NAc of freely moving mice to assess extracellular 5-HT using a genetically encoded sensor. CPu = caudate putamen. b Representative single traces displaying the effect of saline (SAL) or D-FEN (3 or 10 mg kg⁻¹, intraperitoneal injections at t = 120 s) on the relative fluorescence emitted by the 5-HT sensor. Data are shown as mean and standard deviation (N = 4 per condition). c Comparison of the effect of 3 and 10 mg kg⁻¹ D-FEN on the sensor-emitted fluorescence versus saline control. Representative single traces showing changes in sensor-derived fluorescence following intraperitoneal injections (at t = 600 s) of d SAL, fluoxetine (FLX, 10 mg kg⁻¹), D-FEN (10 mg kg⁻¹), e S-4-MC (5 mg kg⁻¹) plus vehicle (=S-4-MC, dark green) or S-4-MC (5 mg kg⁻¹) plus FLX (10 mg kg⁻¹) (=S-4-MC + FLX, light green), f S-4-TFMMC (10 mg kg⁻¹) plus vehicle (=S-4-TFMMC, dark blue) or S-4-TFMMC (10 mg kg⁻¹) plus FLX (10 mg kg⁻¹) (=S-4-TFMMC + FLX, light blue) and g R-4-MC (5 mg kg⁻¹) and R-4-TFMMC (10 mg kg⁻¹). h Comparison of drug induced changes in fluorescence (N = 5 per condition). i Cartoon depicting guide cannula (blue column) and active microdialysis membrane (red tip, active length of 1 mm) placement in the NAc. j, k Effect of intraperitoneal S-4-MC (5 mg kg⁻¹) on extracellular 5-HT and dopamine (DA) in presence or absence of co-administered fluoxetine (FLX, 10 mg kg⁻¹) or vehicle. * denotes p ≤ 0.05 (Bonferroni’s) at the corresponding timepoint l fold increase in extracellular 5-HT and DA at t = 160 min relative to baseline (t = 0–80 min) as shown in j and k. One-sample t-test versus the hypothetical mean of 1 (*p ≤ 0.05). Data are shown as mean and standard deviation. Data in b, c, h, j, k and l are given as mean and standard deviation. Data in c and h were analyzed using Kruskal–Wallis, followed by Dunn’s multiple comparison test. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 versus SAL. Detailed sample sizes are given in the Supplementary Information.