In vitro pharmacological characterization of recent non-fentanyl synthetic opioids including 2-benzylbenzimidazole ‘nitazene’ opioids

New synthetic opioids (NSOs) constitute one of the fastest growing groups of new psychoactive substances, and structurally diverse non-fentanyl opioids are increasingly emerging. Amid this highly dynamic landscape, insight into the pharmacology of NSOs has become an important tool to estimate their harm potential. In this work, we determined the MOR activation potential of a selection of non-fentanyl NSOs monitored in Europe between 2018-2021. The in vitro MOR activation potential of tianeptine, piperidylthiambutene, cinnamylpiperazines (2-methyl-AP-237/AP-237/AP-238/para-methyl-AP-237), 2F-viminol, brorphine, and all previously reported 2-benzylbenzimidazoles/’nitazenes’ was studied by means of a cell-based β-arrestin2 (βarr2) recruitment assay. In short, activation of human MOR, fused to one subunit of a nanoluciferase enzyme, leads to recruitment of βarr2, fused to the complementing subunit. This results in functional complementation of the enzyme, restoring its luciferase activity. Upon addition of a substrate, a bioluminescent signal is generated (NanoBiT®). From this, potency and efficacy (% versus hydromorphone) values were calculated. Fentanyl was included as comparator. Concentration-response curves were generated for all tested NSOs. Compared to fentanyl (EC50=14.3 nM/Emax=163%), tianeptine (EC50=3262 nM/Emax=166%), piperidylthiambutene (EC50=180 nM/Emax=130%) and 2F-viminol (EC50=2151 nM/Emax=21.2%) were considerably less potent, whereas brorphine (EC50=31.1 nM/Emax=226%) showed a more comparable potency and higher efficacy. AP-238 was the most potent (EC50=248 nM/Emax=91.3%) cinnamylpiperazine tested, whereas 2-methyl-AP-237 was the most efficacious (EC50=749 nM/Emax=125%). However, all cinnamylpiperazines were less active than fentanyl. In contrast, nitazenes were generally highly active, with potencies and efficacies of several analogues – e.g., isotonitazene (EC50=1.63 nM/Emax=179%), protonitazene (EC50=3.95 nM/Emax=167%), N-piperidinyl etonitazene (EC50=2.49 nM/Emax=183%) – exceeding those of fentanyl. N-pyrrolidino etonitazene (EC50=0.348/Emax=298%) is among the most potent NSOs described to date. Non-fentanyl NSOs are increasingly emerging on the recreational drug market, each new member carrying a substantial risk of toxicity. In vitro research on MOR activation offers a first estimation of the potential danger these compounds may evoke in vivo.