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

Abstract 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 G i 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.