Fluorinated phenmetrazine "legal highs" act as substrates for high-affinity monoamine transporters of the SLC6 family

Abstract

A variety of new psychoactive substances (NPS) are appearing in recreational drug markets worldwide. NPS are compounds that target various receptors and transporters in the central nervous system to achieve their psychoactive effects. Chemical modifications of existing drugs can generate NPS that are not controlled by current legislation, thereby providing legal alternatives to controlled substances such as cocaine or amphetamine. Recently, 3-fluorophenmetrazine (3-FPM), a derivative of the anorectic compound phenmetrazine, appeared on the recreational drug market and adverse clinical effects have been reported. Phenmetrazine is known to elevate extracellular monoamine concentrations by an amphetamine-like mechanism. Here we tested 3-FPM and its positional isomers, 2-FPM and 4-FPM, for their abilities to interact with plasma membrane monoamine transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). We found that 2-, 3- and 4-FPM inhibit uptake mediated by DAT and NET in HEK293 cells with potencies comparable to cocaine (IC₅₀ values < 2.5 μM), but display less potent effects at SERT (IC₅₀ values >80 μM). Experiments directed at identifying transporter-mediated reverse transport revealed that FPM isomers induce efflux via DAT, NET and SERT in HEK293 cells, and this effect is augmented by the Na⁺/H⁺ ionophore monensin. Each FPM evoked concentration-dependent release of monoamines from rat brain synaptosomes. Hence, this study reports for the first time the mode of action for 2-, 3- and 4-FPM and identifies these NPS as monoamine releasers with marked potency at catecholamine transporters implicated in abuse and addiction. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.'

Keywords: Amphetamine; Legal high; Monoamine transporter; New psychoactive substances; Phenmetrazine.

Figure 1:

Chemical structures of fluorophenmetrazine isomers, 2-, 3- and 4-FPM

Figure 2:

Effects of fluorinated phenmetrazines on transporter-mediated uptake in HEK293 cells expressing human MATs. Cells were incubated with 0.02 µM [³H]MPP⁺ for hDAT and hNET assays or 0.1 µM [³H]5HT for hSERT assays, along with various concentrations of fluorinated phenmetrazines. Non-specific uptake was determined in the presence of 10 µM mazindol for DAT and NET or 10 µM paroxetine for SERT. All data are presented as mean ± SEM from 3 experiments performed in triplicate.

Figure 3:

Effects of fluorinated phenmetrazines on transporter-mediated efflux of tritiated substrates in HEK293 cells expressing human MATs. Cells were preloaded with [³H]MPP⁺ for hDAT and hNET assays, or [³H]5HT for hSERT assays, and subsequently perfused. After three basal fractions monensin or control buffer was added at t = −8 min (MON, 10 µM, indicated by black bar). Subsequently, the cells were exposed to FPMs at t = 0 min (5 µM, addition at t = 0 min indicated by arrow). All data are represented as mean ± SEM. Data were analyzed by two-way ANOVA followed by Bonferroni’s test. * denotes p <0.05 when compared to the corresponding control buffer condition.

Figure 4:

Representative traces of ionic currents mediated via hSERT, recorded from the same cell (3 different batches of cells were recorded). The three upper panels show currents induced by 30 µM 2-FPM (−4 pA; n = 7), 10 µM 3-FPM (−3.8 pA; n = 6) and 10 µM 4-FPM (−2.5 pA; n = 7), respectively. These currents were inwardly directed and resembled the current induced by 30 µM 5-HT (−7 pA; n = 3) (left lower panel). Application of cocaine (10 µM; n = 4) gave rise to an outwardly directed current, which presumably reflected blockage of a substrate independent leak conductance through hSERT.

Figure 5:

Effects of fluorinated phenmetrazines on transporter-mediated release of tritiated substrates from rat brain synaptosomes. Rat brain synaptosomes were preloaded with [³H]MPP⁺ for DAT and NET assays, or [³H]5HT for SERT assays, and subsequently exposed to various concentrations of 2-, 3- or 4-FPM. Release assays were optimized for DAT, NET or SERT using unlabeled inhibitors as outlined in the materials and methods section. Data are mean ± SEM obtained from 3 experiments performed in triplicate.