In vitro inhibition of monoamine transport by amphetamine-like pre-workout supplement ingredients

Phenethylamine (PEA) and alkylamine (AA) analogues are a prominent group of pre-workout food supplement ingredients. They are structurally related to the stimulant amphetamine and to the endogenous catecholamines noradrenaline and dopamine, implying potential cardiovascular and psychological effects. This study systematically investigated the inhibitory potential of 12 PEAs and 4 AAs identified in pre-workout supplements on the human dopamine transporter (hDAT), human noradrenaline transporter (hNET) and human serotonin transporter (hSERT) that are stably overexpressed in HEK 293 cells. All PEAs and AAs tested, except DMAE, inhibited substrate uptake by one or more monoamine transporters. Overall, the substances displayed the highest potency and efficacy at hNET, followed by hDAT and with considerably weaker effects on hSERT. At hNET, potency values (IC₅₀) ranged from 0.5 µM to 123 µM, with maximal inhibition (E_max) ranging from -59.2% to -120%. Inhibition of substrate uptake by hDAT occurred with IC₅₀ values between 4.0 and 95.8 µM and E_max values between -66.8% and -135%. For hSERT 50% inhibition was observed at concentrations ranging from 2.6 µM to 131 µM, with maximal effect between 85.3% and -64.8%. These findings indicate a potential for sympathetic activation and behavioral rewarding and reinforcing effects. Notably, the in vitro potency and efficacy of several PEAs and AAs were comparable to those of the well-known illicit stimulants amphetamine and cocaine. Together, these findings highlight the urgent need to further characterize pharmacokinetic and pharmacodynamic properties of these pre-workout supplement ingredients to support robust risk assessment and informed regulatory decision-making regarding the safety of pre-workout supplement ingredients.