Characterisation of the monoamine transport inhibition of 28 new psychoactive stimulants

Abstract

The objective of this study was to characterize new psychoactive stimulants’ effects on the inhibition of the monoamine transporters for dopamine (DAT), serotonine (SER) and norepinephrine (NET). Still, new psychoactive substances (NPS) are continuously emerging in Europe and globally. A large group of NPS is stimulants, drugs including cathinones, phenylethylamines, MDMA-analogues and derivatives of cocaine. Many stimulants affect the reuptake and storage of neurotransmitters in neurons. A major site of stimulant action is inhibition of the reuptake transporters DAT, SERT and NET. These proteins transport monoamine neurotransmittors across the nerve synaptic membrane, thereby ceasing the synaptic activity. Inhibition of the reuptake of the neurotransmitter will prolong the synaptic activity. It has been shown that the relative potency in inhibiting the DAT, SERT and NET transporters correlate with the observed drug effects and also their abuse potential. Especially the DAT/SERT inhibition ratio gives an indication of the abuse potential. The DAT/NET/SERT inhibition potency for a set of NPS stimulant drugs were determined by a rapid and automated method to measure inhibition of neurotransmitter transport, using a fluorescent dye mix. Transporter inhibition studies were conducted using cell lines expressing either the human DAT (in CHO-K1 cells), SERT (in HEK293 cells) or NET (in MDCK cells) transporters. The signal from the dye transported into the cells was measured using a TECAN Spark plate reader. Inhibition was measured at 15 concentrations in triplicate and repeated in three independent experiments. In total eighteen cathinones, six phenylethylamines and four arylcyclohexylamines were characterized. DAT, NET and SERT transport inhibiting curves were generated and IC50 values were determined for all substances. For DAT the potency varied between an IC50 of 1.03 nM for the cathinone MDPiHP to > 50,000 nM for the phenytlethylamine TMA-6. For SERT inhibition the potency was in general lower. The phenylethylamines were the extremes with a range from 79.6 nM for methamnetamine to > 36,000 nM for TMA-6. The DAT/SERT inhibition ratios for the tested substances varied from 0.4 to 3080. Four substances exhibited an MDMA-like ratio below 1. Two substances had an inhibition ratio varying between > 1 and < 10, close to cocaine at 3.37. Nineteen of the tested substances had a ratio between > 10 and 330. The highest inhibition ratios were identified for MDPiHP (705) and 3F-α-PiHP (3080). For the cathinone N-butyl Hexedrone, it was not possible to determine the ratio. With the exception of TH-PVP all other cathinones had a higher DAT/SERT inhibition ratio than cocaine and ten even more than amphetamine, indicating a high abuse potential. The substance with the highest potency for NET transport inhibition was MDPiHP at IC50 27 nM, which was the only one with higher potency than amphetamine. Three cathinones, MDPT (tBuoNE), N-butyl Hexedrone and TH-PVP had IC50 value for NET > 11,000 nM. For most of the characterized substances, the potency preference was DAT > NET > SERT, but there are exceptions [deschloroketamine, 3F-PCP, TMA-6, methamnetamine, 5-MMPA, TH-PVP, eutylone, N-ethyl heptylone, MDPT (tBuONE) and 3,4-methylenedioxy PV8]. With the large variation among the stimulants in potency, it is therefore, essential to characterize DAT, SERT and NET inhibition. These results show that many of the emerging NPS stimulants are potent inhibitors of the monoamine transporters, although some lack activity or have a partial inhibitory effect.