Human metabolism and pharmacological profiling of protonitazepyne and metonitazepyne, two highly potent nitazenes: prediction of main metabolite activity based on µ-opioid receptor docking simulations

: Nitazenes have recently surfaced the illicit opioid market, causing numerous intoxications and fatalities. N-Pyrrolidino derivatives, protonitazepyne and metonitazepyne, have circulated since 2023 and have been involved in overdose intoxications. Their pharmacological properties remain largely unknown. However, pharmacokinetic/dynamic data are crucial for clinicians and toxicologists to manage intoxications and interpret legal cases. Protonitazepyne and metonitazepyne metabolism was assessed using human hepatocyte incubations and blood/urine from an intoxication case; samples were analyzed with liquid chromatography-high-resolution mass spectrometry and software-aided data mining. µ-(MOR), κ-(KOR), and δ-(DOR) opioid receptor activation was assessed using a GTP Gi binding assay. MOR docking was simulated with UCSF Chimera and AutoDockSuite. Pharmacological relevance of major metabolites was predicted through in silico MOR docking. Major metabolites were produced through nitroreduction, pyrrolidine N-dealkylation, and oxidation to N-butanoic acid and O-dealkylation. Protonitazepyne and metonitazepyne potencies at MOR were 3.7 and 11.5 nmol L-1, respectively; efficacies were 154 and 101%. Partial agonism and low potency were observed at KOR/DOR. In silico inhibition constants at MOR for protonitazepyne, 5-amino-protonitazepyne, metonitazepyne, and 5-amino-metonitazepyne were 0.68, 11.45, 1.98, and 2,050 nmol L-1, respectively. Protonitazepyne and metonitazepyne are MOR-selective full agonists, with potencies about seven and two times higher than fentanyl. These nitazenes present significant health risks through central nervous system/respiratory depression. Their primary metabolites showed lower/marginal in silico MOR affinity, suggesting they might be pharmacologically active, albeit to a much lesser extent than the parent compounds. We propose 5-amino derivatives (blood) and N-butanoic acid derivatives (urine) as biomarkers for detecting consumption.