Pharmacological evaluation and forensic case series of N -pyrrolidino etonitazene (etonitazepyne), a newly emerging 2-benzylbenzimidazole ‘nitazene’ synthetic opioid

Novel synthetic opioids continue to emerge on recreational drug markets worldwide. In response to legislative bans on fentanyl analogues, non-fentanyl structural templates, such as 2-benzylbenzimidazoles (‘nitazenes’), are being exploited to create new μ-opioid receptor (MOR) agonists. Here, we pharmacologically characterize an emerging cyclic analogue of etonitazene, called N- pyrrolidino etonitazene (etonitazepyne), using in vitro and in vivo methods. A series of analytically confirmed fatalities is described to complement preclinical findings. Radioligand binding assays in rat brain tissue revealed that N- pyrrolidino etonitazene has high affinity for MOR (Ki = 4.09 nM) over δ-opioid (Ki = 959 nM) and κ-opioid (Ki = 980 nM) receptors. In a MOR-β-arrestin2 activation assay, N- pyrrolidino etonitazene displayed high potency (EC 50 = 0.348 nM), similar to etonitazene (EC 50 = 0.360 nM), and largely exceeding the potencies of fentanyl (EC 50 = 14.9 nM) and morphine (EC 50 = 290 nM). When administered s.c. to male Sprague Dawley rats, N- pyrrolidino etonitazene induced opioid-like antinociceptive, cataleptic, and thermic effects. Its potency in the hot plate test (ED 50 = 0.0017 mg/kg) was tenfold and 2,000-fold greater than fentanyl (ED 50 = 0.0209 mg/kg) and morphine (ED 50 = 3.940 mg/kg), respectively. Twenty-one overdose fatalities associated with N- pyrrolidino etonitazene were found to contain low blood concentrations of the drug (median = 2.2 ng/mL), commonly in the context of polysubstance use. N- Pyrrolidino etonitazene was reported as a cause of death in at least two cases, demonstrating toxicity in humans. We demonstrate that N- pyrrolidino etonitazene is an extremely potent MOR agonist that is likely to present high risk to users. Continued vigilance is required to identify and characterize emergent 2-benzylbenzimidazoles, and other non-fentanyl opioids, as they appear in the marketplace.