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Rationale Novel synthetic opioids (NSOs) are emerging in recreational drug markets worldwide. In particular, 2-benzylbenzimidazole ‘nitazene’ compounds are problematic NSOs associated with serious clinical consequences, including fatal respiratory depression. Evidence from in vitro studies shows that alkoxy chain length can influence the potency of nitazenes at the mu-opioid receptor (MOR). However, structure–activity relationships (SARs) of nitazenes for inducing opioid-like effects in animal models are not well understood compared to relevant opioids contributing to the ongoing opioid crisis (e.g., fentanyl). Objectives Here, we examined the in vitro and in vivo effects of nitazene analogues with varying alkoxy chain lengths (i.e., metonitazene, etonitazene, isotonitazene, protonitazene, and butonitazene) as compared to reference opioids (i.e., morphine and fentanyl). Methods and Results Nitazene analogues displayed nanomolar affinities for MOR in rat brain membranes and picomolar potencies to activate MOR in transfected cells. All compounds induced opioid-like effects on locomotor activity, hot plate latency, and body temperature in male mice, and alkoxy chain length markedly influenced potency. Etonitazene, with an ethoxy chain, was the most potent analogue in MOR functional assays (EC 50  = 30 pM, E max  = 103%) and across all in vivo endpoints (ED 50  = 3–12 μg/kg). In vivo SARs revealed that ethoxy, isopropoxy, and propoxy chains engendered higher potencies than fentanyl, whereas methoxy and butoxy analogues were less potent. MOR functional potencies, but not MOR affinities, were positively correlated with in vivo potencies to induce opioid effects. Conclusions Overall, our data show that certain nitazene NSOs are more potent than fentanyl as MOR agonists in mice, highlighting concerns regarding the high potential for overdose in humans who are exposed to these compounds.

Rationale Isotonitazene is an illicit synthetic opioid associated with many intoxications and fatalities. Recent studies show that isotonitazene is a potent µ-opioid receptor (MOR) agonist in vitro, but little information is available about its in vivo effects. Objectives The aims of the present study were to investigate the pharmacokinetics of isotonitazene in rats, and relate pharmacokinetic parameters to pharmacodynamic effects. Methods Isotonitazene and its metabolites were identified and quantified by liquid chromatography tandem quadrupole mass spectrometry (LC-QQQ-MS). Male Sprague–Dawley rats with jugular catheters and subcutaneous (s.c.) temperature transponders received isotonitazene (3, 10, 30 µg/kg, s.c.) or its vehicle. Blood samples were drawn at 15, 30, 60, 120, and 240 min post-injection, and plasma was assayed using LC-QQQ-MS. At each blood draw, body temperature, catalepsy scores, and hot plate latencies were recorded. Results Maximum plasma concentrations of isotonitazene rose in parallel with increasing dose (range 0.2–9.8 ng/mL) and half-life ranged from 23.4 to 63.3 min. The metabolites 4′-hydroxy nitazene and N -desethyl isotonitazene were detected, and plasma concentrations were below the limit of quantitation (0.5 ng/mL) but above the limit of detection (0.1 ng/mL). Isotonitazene produced antinociception (ED 50  = 4.22 µg/kg), catalepsy-like symptoms (ED 50  = 8.68 µg/kg), and hypothermia (only at 30 µg/kg) that were significantly correlated with concentrations of isotonitazene. Radioligand binding in rat brain tissue revealed that isotonitazene displays nM affinity for MOR (Ki = 15.8 nM), while the N -desethyl metabolite shows even greater affinity (Ki = 2.2 nM). Conclusions In summary, isotonitazene is a potent MOR agonist whose pharmacodynamic effects are related to circulating concentrations of the parent drug. The high potency of isotonitazene portends substantial risk to users who are exposed to the drug.

•Isotonitazene quantification in human post-mortem tissues, blood and hair.•Isotonitazene accumulation in brain and hearth and very low concentration in liver.•Isotonitazene powder determination using GC-MS, NMR.•Very low isotonitazene concentration in blood can be fatal. The paper describes the first three deaths reported in Europe involved in isotonitazene consumption, a potent benzimidazole derivate opioid consumed in the recreational drug scene. Isotonitazene powder and purity determination was performed on the sample collected in the first death scene by NMR, HRMS, GC-FTIR, ATR-FTIR and GC–MS. Isotonitazene purity was determined by GC–MS analysis and proton NMR, and was defined to be above 95 % and 98 %, respectively. Quantification of isotonitazene in biological samples was performed using a targeted analysis based on SPE extraction and ultra-high performance liquid chromatography tandem mass spectrometry. The isotonitazene median concentration in femoral whole blood was 1.20ng/mL. Isotonitazene concentration in hair was similar or even lower compared to that seen in fentanyl abusers. Isotonitazene distribution in tissues converges in the brain, lungs and heart, respectively. Surprisingly, isotonitazene concentration in liver is the lowest measured for all tissues and fluids analyzed. Based on circumstantial evidence, autopsy findings and the results of the toxicological analysis, the medical examiner concluded that the cause of all three deaths was an acute intoxication with isotonitazene. Since isotonitazene toxic concentration levels are very low, the consumption of this new psychoactive drug is a real hazard for human health.

The recent re-emergence and the increasing popularity of nitazenes, a group of new synthetic opioids (NSO) that belong to the benzimidazole chemical class, has raised public health concerns. As a class of potential opioid analgesic agents whose development was discontinued in the 1960s due to their high potential for abuse, very little is known about their metabolism and physiologic disposition. In the current study, three nitazenes–butonitazene, isotonitazene and protonitaze were incubated in human liver microsomes (HLM), human S9 (HS9) fractions and recombinant cytochrome P450 enzymes. All three nitazenes were rapidly metabolized in both HLM and HS9 with over 95% depletion within 60 min. In HLM, butonitazene, isotonitazene and protonitazene had in vitro intrinsic clearance (CLint) (µL/min/mg protein) values of 309, 221 and 216 respectively compared to 150 of verapamil, the positive control. In HS9, CLint values were 217, 139, and 150 for butonitazene, isotonitazene and protonitazene respectively compared to only 35 for testosterone, the control probe substrate. Putative metabolite identified from this study include products of hydroxylation, desethylation, dealkylation, desethylation followed by dealkylation, and desethylation followed by hydroxylation. The metabolic phenotyping showed CYP2D6, CYP2B6 and CYP2C8 and the major hepatic enzymes responsible for the metabolism of nitazenes. Within 30 min of incubation, CYP2D6 depleted butonitazene (99%), isotonitazene (72%) and butonitazene (100%) significantly. The rapid metabolism of nitazenes may be an important factor in accurate and timely detections and quantitation of the unchanged drugs in human matrices following intoxication or in forensic analysis. The involvement of multiple polymorphic CYPs in their metabolism may play important roles in the susceptibility to intoxication and/or addiction, depending on the activity of the metabolites.

Nitazenes are potent synthetic opioids that were never marketed as analgesics, due to the high overdose risk with rapid respiratory arrest and death. This study aims to present the nitazene concentrations and toxicological findings in all nitazene-related deaths registered in Norway during 2021–2024. De-identified data from the LIMS database (Oslo University Hospital) and the Beaker database (St. Olavs Hospital) were extracted for all forensic autopsy cases in Norway with nitazenes detected in blood from January 2021 to October 2024, including nitazene and drug concentrations in peripheral blood and urine, age, gender, location of autopsy, date of death, and sampling date. Analysis of nitazenes was performed using liquid chromatography-mass spectrometry methods. Altogether, 36 nitazene-related deaths were registered in Norway; one death in 2021 (etonitazepyne (N-pyrrolidino etonitazene)), one death in 2022 (protonitazene), 13 deaths in 2023 (metonitazene n = 11, metonitazepyne (N-pyrrolidino metonitazene) n = 1, N-desethyl isotonitazene n = 1), and 21 deaths in 2024 (metonitazene n = 18, protonitazepyne (N-pyrrolidino protonitazene) n = 3). The nitazene-related deaths occurred in all regions of Norway, the majority were males (94.4 %), and median age was 29 years (range 19–56 years). Over 90 % of the cases also had one or more of other psychoactive drugs detected in blood, and 50 % had one or more designer benzodiazepines detected. The median (range) concentrations in post-mortem peripheral blood were 7.3 ng/ml (0.8–114.7 ng/ml) for metonitazene (n = 28) and 3.4 ng/ml (2.0–3.7 ng/ml) for protonitazepyne (n = 3). In summary, since June 2023 the prevalence of nitazene-related deaths escalated in Norway, and the majority were males in their twenties or younger. Our findings indicate that inexperienced drug users need better information about the extreme risk of accidental overdose when using such potent opioids. •36 nitazene-related deaths in Norway 2021–2024.•Most were men under 30 years.•Over 90 % polydrug use.•50 % had designer benzodiazepine(s) detected in blood.•Inexperienced users need information about overdose risk when using potent opioids.

Isotonitazene (IZN) is a potent synthetic opioid associated with a growing number of fatal intoxications worldwide. Despite its increasing presence in forensic cases, postmortem data regarding the distribution of IZN and its metabolites in human tissues remain limited. We report the first documented case of fatal IZN intoxication in France, involving a 39-year-old man with a history of heroin use. Comprehensive postmortem toxicological analysis was conducted using a LC-MS/MS quantification method. Quantification of IZN and its three active metabolites: N-desethyl-isotonitazene, 4’hydroxy-nitazene, and 5-amino-isotonitazene was performed in multiple matrices, including blood, urine, bile, and solid organs. IZN was detected in femoral and cardiac blood, with concentrations of 1.20 ng/mL and 1.74 ng/mL, respectively. High concentrations were observed in the heart (20 ng/g), lungs (32.6 ng/g), and brain (7.9 ng/g), consistent with marked postmortem redistribution. Active metabolites showed variable distribution: N-desethyl isotonitazene was detected in lung tissue and brain, 5-amino isotonitazene in both brain and lungs, while 4’-hydroxy-nitazene appeared to be predominantly eliminated via the biliary route. A high concentration of IZN at the injection site (343.2 ng/mL) indicated intravenous administration. Ethanol and cetirizine were also present at non-lethal concentrations. To the best of our knowledge, this is the first reported fatal IZN intoxication with comprehensive postmortem analysis, including quantification of active metabolites in solid organs. The case is marked by low peripheral blood levels, extensive redistribution, and selective tissue accumulation. Active metabolites: N-desethyl IZN, 4′-hydroxy-nitazene, and 5-amino IZN showed distinct distribution and elimination profiles. These findings highlight the high potency, rapid metabolism, and complex toxicokinetic of IZN. [Display omitted] •First toxicokinetic investigation of isotonitazene and its metabolites in post-mortem samples.•Very low blood concentration of isotonitazene can be lethal.•Isotonitazene undergoes rapidly metabolism to pharmacologically active metabolites.•Accumulation of isotonitazene and its active metabolites occurs in vital organs such as the brain, heart, and lungs.

Objectives: This study aimed to develop and validate a sensitive and reliable liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the quantification of flualprazolam and isotonitazene in serum samples to address critical gaps in forensic and clinical toxicology. Materials and Methods: A single-center validation study was conducted using serum samples spiked with analyte standards. Analytical parameters, including linearity, precision, recovery, the limit of detection (LOD), and the limit of quantification (LOQ), were evaluated in accordance with international guidelines. The LC-MS/MS method employed a Shimadzu Triple Quadrupole™ MS 8045 system with solid-phase extraction (SPE) for sample preparation. Results: The method exhibited high linearity for flualprazolam (r2 = 0.997) and isotonitazene (r2 = 0.999) over a concentration range of 1–100 ng/mL. The LODs were determined as 0.608 ng/mL and 0.192 ng/mL, and the LOQs were 1.842 ng/mL and 0.584 ng/mL for flualprazolam and isotonitazene, respectively. Recovery tests yielded results within the acceptable range of 70–120%. Flualprazolam demonstrated recovery rates of 98.0% and 97.0% at theoretical concentrations of 10 ng/mL and 50 ng/mL, respectively. In contrast, the isotonitazene recovery rates were slightly lower, measuring 75.5% at 10 ng/mL and 71.9% at 50 ng/mL, suggesting minor matrix effects that could influence its quantification. Precision analysis, including both repeatability and reproducibility, highlighted the reliability of the method. The %RSD values for flualprazolam were consistently below 7.07%, with mean concentrations closely aligning with theoretical values across fortification levels. For isotonitazene, the %RSD values remained below 6.24%, although recoveries at higher concentrations indicated potential challenges in matrix interaction. Conclusions: This validated LC-MS/MS method offers high sensitivity, precision, and recovery for detecting flualprazolam and isotonitazene in serum, filling a critical need in toxicological investigations. Further validation in other biological matrices is recommended to broaden its applicability.