In vitro characterization of new psychoactive substances at the μ-opioid, CB1, 5HT1A, and 5-HT2A receptors—On-target receptor potency and efficacy, and off-target effects

Sixty new psychoactive substances (NPS) and their metabolites, including opioids, cannabinoids, and serotonergic hallucinogens, were screened for their ability to activate μ-opioid, CB1, 5-HT1A, and 5-HT2A receptors. Most substances activated their intended target receptor. Among μ-opioid agonists, 2-fluorofentanyl (EC50 = 1.0 nM), carfentanil (EC50 = 2.7 nM), and acrylfentanyl (EC50 = 2.8 nM) were the most potent, with a >1500-fold potency range across compounds. Furanylfentanyl, 4-methoxybutyrylfentanyl, and valerylfentanyl acted as partial agonists. On the 5-HT2A receptor, bromo-dragonfly was the most potent (EC50 = 0.05 nM, 400 times more potent than LSD), followed by NBOMe compounds (EC50 0.11–1.3 nM). Off-target μ-opioid activation occurred for piperazines, phenethylamines, and tryptamines. The synthetic cannabinoid metabolite 3-carboxy indole PB-22 activated 5-HT2A. Bromo-dragonfly activated all four receptors. These findings highlight complex, often overlapping targets among NPS.

Study at a glance

Abstract

New psychoactive substances (NPS) appear on the recreational market on a monthly basis, with unclear toxicology, resulting in an increasing number of fatalities. Identification of drug targets and potencies is crucial for understanding and treating intoxications and for scheduling processes. In this study 60 NPS and metabolites belonging to opioids, cannabinoids and serotonergic hallucinogens classes were screened for in vitro activation of the μ-opioid, CB1, 5-HT1A and 5-HT2A receptors using the AequoZen cell system. Fentanyl and NBOMe analogues were chosen for full dose-response characterization of the μ-opioid and 5-HT2A receptors, respectively. Most substances activated their corresponding target receptor. The most potent μ-opioid receptor agonists were 2-fluorofentanyl (EC50 = 1.0 nM), carfentanil (EC50 = 2.7 nM) and acrylfentanyl (EC50 = 2.8 nM) and in total a >1500-fold difference was seen among the tested compounds. Moreover, furanylfentanyl, 4-methoxybutyrylfentanyl and valerylfentanyl acted as partial agonists of the μ-receptor. On the 5-HT2A receptor, bromo-dragonfly showed the highest potency (EC50 = 0.05 nM, 400 times more potent than LSD), followed by most NBOMe compounds with EC50 values ranging from 0.11 nM (for 25N-NBOMe) to 1.3 nM (for 25T4-NBOMe)). Off-target activation of the μ-opioid receptor was identified for piperazines, phenethylamines (in particular NBOMe and 2C compounds) and tryptamines. Moreover, the synthetic cannabinoid metabolite 3-carboxy indole PB-22 activated the 5-HT2A receptor. Bromo-dragonfly was the only compound that activated all four receptors. These results highlight the possible interplay of known and unknown NPS targets and unveil its complexity. Moreover, the detailed, quantitative information presented facilitates our further understanding of NPS toxicology.