The use of new psychoactive substances (NPS) is steadily increasing. One commonly used NPS is methoxetamine (MXE), a ketamine analogue. Several adverse effects have been reported following MXE exposure, while only limited data are available on its neuropharmacological modes of action.

We investigated the effects of MXE and ketamine on several endpoints using multiple in vitro models. These included rat primary cortical cells, human SH-SY5Y cells, human induced pluripotent stem cell (hiPSC)-derived iCell® Neurons, DopaNeurons and astrocyte co-cultures, and human embryonic kidney (HEK293) cells. We investigated effects on several neurotransmitter receptors using single cell intracellular calcium [Ca2+] i imaging, effects on neuronal activity using micro-electrode array (MEA) recordings and effects on human monoamine transporters using a fluorescence-based plate reader assay.

In rat primary cortical cells, 10 μM MXE increased the glutamate-evoked increase in [Ca2+] i , whereas 10 μM ketamine was without effect. MXE and ketamine did not affect voltage-gated calcium channels (VGCCs), but inhibited spontaneous neuronal activity (IC 50 0.5 μM and 1.2 μM respectively). In human SH-SY5Y cells, 10 μM MXE slightly inhibited the K+- and acetylcholine-evoked increase in [Ca2+] i . In hiPSC-derived iCell®(Dopa)Neurons, only the ATP-evoked increase in [Ca2+] i was slightly reduced. Additionally, MXE inhibited spontaneous neuronal activity (IC 50 between 10 and 100 μM). Finally, MXE potently inhibits uptake via monoamine transporters (DAT, NET and SERT), with IC 50 values in the low micromolar range (33, 20, 2 μM respectively).

Our combined in vitro data provide an urgently needed first insight into the multiple modes of action of MXE. The use of different models and different (neuronal) endpoints can be complementary in pharmacological profiling. Rapid in vitro screening methods as those presented here, could be of utmost importance for gaining a first mechanistic insight to aid the risk assessment of emerging NPS.