Buprenorphine has been successfully used for decades in the treatment of opioid use disorder, yet there are complexities to its use that warrant attention to maximize its utility. While the package insert of the combination product buprenorphine\\naloxone continues to recommend a maximum dose of 16 mg daily for maintenance, the emergence of fentanyl and synthetic analogs in the current drug supply may be limiting the effectiveness of this standard dose. Many practitioners have embraced and appropriately implemented novel practices to mitigate the sequelae of our current crisis. It has become common clinical practice to stabilize patients with 24 - 32 mg of buprenorphine daily at treatment initiation. Many of these patients, however, are maintained on these high doses (>16 mg/d) indefinitely, even after prolonged stability. Although this may be a necessary strategy in the short term, there is little evidence to support its safety and efficacy, and these high doses may be exposing patients to more complications and side effects than standard doses. Commonly known side effects of buprenorphine that are likely dose-related include hyperhidrosis, sedation, decreased libido, constipation, and hypogonadism. There are also complications related to the active metabolite of buprenorphine (norbuprenorphine) which is a full agonist at the mu opioid receptor and does not have a ceiling on respiratory suppression. Such side effects can lead to medical morbidity as well as decreased medication adherence, and we, therefore, recommend that after a period of stabilization, practitioners consider a trial of decreasing the dose of buprenorphine toward standard dose recommendations. Some patients\' path of recovery may never reach this stabilization phase (i.e., several months of adherence to medications, opioid abstinence, and other clinical indicators of stability). Side effects of buprenorphine may not have much salience when patients are struggling for survival and safety, but for those who are fortunate enough to advance in their recovery, the side effects become more problematic and can limit quality of life and adherence.

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.

From 2019, in the United States and Europe, the synthetic opioid market has diversified with the appearance of the 2-benzylbenzimidazole family, commonly named \"nitazenes\". In vitro studies show that these synthetic opioids have much higher affinities on μ-opioid receptors: 100 times more than morphine, and slightly higher than fentanyl for isotonitazene, increasing the risk of overdose. In south of France, isotonitazene (IZN) was identified for the first time in March 2023. In this context, there were 9 reports concerning the use of IZN in the south of France over a short period (March-April 2023), with identification of IZN in 4 cases and suspicion in others. They concerned 6 men and 3 women, with a mean age of 44.9±2 years. When available (2 cases), the product had been purchased from a dealer. IZN was identified on sample in 2 cases of overdose. Isotonitazene was also identified in biological samples in 2 cases: 1 case of overdose and coma requiring hospitalization with a favorable outcome (urinary analysis), and a death with post-mortem identification. This was the first identification of this product in France. The immediate broadcast of the alert limited the risks for users and made it possible to quickly inform regional and national health authorities. IZN is under intensive surveillance by the EMCDDA and classified as a narcotic in France since 2021. The analysis of the literature made it possible to identify cases of overdoses requiring very high doses of naloxone and deaths. The emergence of these synthetic opioids constitutes an important signal, due to their superior effects to heroin, their incomplete response to naloxone and the current difficulty in identifying them (devices for analyzing products in the reduction of risks, toxicology laboratories).

Protonitazene is a synthetic benzoimidazole opioid of the nitazenes class, developed in the 1950s as an effective analgesic, but never released on the market due to severe side effects and possible dependence. Despite its increasing use as a new psychoactive substance starting in 2019, its detection in human hair of intoxicated and deceased consumers has never been reported. We present the development and validation of a specific procedure to identify protonitazene in hair by LC-MS-MS. Drugs were incubated overnight at 40°C in 1 mL borate buffer, pH 9.5 with 20 mg pulverized hair and 1 ng/mg fentanyl-d5 used as internal standard. Drugs were then extracted with a mixture of organic solvents. The chromatographic separation was performed using a HSS C18 column with a 15 min gradient elution. Linearity was verified from 1 to 100 pg/mg. The limit of detection was estimated at 0.1 pg/mg. No interference was noted from a large panel of natural and synthetic opioids, fentanyl derivatives or other new synthetic opioids. Protonitazene was identified at 70 and at > 7600 pg/mg in the whole head hair specimens of two male subjects deceased from acute drug overdose in jail. Protonitazene was also identified at 14 and 54 pg/mg in two living co-prisoners. As nitazenes represent a growing threat to public health in various parts of the world, this method was developed in response to the challenges posed by the identification of this class of substances.

暂无摘要。

Following isotonitazene scheduling in 2019, the availability of alternative 2-benzylbenzimidazole opioids (nitazenes) on the global drug market increased, resulting in many fatalities worldwide. Nitazenes are potent µ-opioid receptor agonists with strong narcotic/analgesic effects, and their concentrations in biological matrices are low, making the detection of metabolite biomarkers of consumption crucial to document use in clinical and forensic settings. However, there is little to no data on the metabolism of the most recently available nitazenes, especially desnitro-analogues. The aim of the research was to assess isotonitazene, metonitazene, etodesnitazene, and metodesnitazene human metabolism and identify specific metabolite biomarkers of consumption. The four analogues were incubated with 10-donor-pooled human hepatocytes, and the incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry and data mining with Compound Discoverer (Thermo Scientific); the analysis was supported by in silico metabolite predictions with GLORYx open-access software. Metabolites were identified in postmortem blood and/or urine samples from two metonitazene-positive and three etodesnitazene-positive cases following the same workflow, with and without glucuronide hydrolysis in urine, to confirm in vitro results. Twelve, nine, twenty-two, and ten metabolites were identified for isotonitazene, metonitazene, etodesnitazene, and metodesnitazene, respectively. The main transformations were N-deethylation at the N,N-diethylethanamine side chain, O-dealkylation, and further O-glucuronidation. In vitro and autopsy results were consistent, demonstrating the efficacy of the 10-donor-pooled human hepatocyte model to predict human metabolism. We suggest the parent and the corresponding O-dealkyl- and N-deethyl-O-dealkyl metabolites as biomarkers of exposure in urine after glucuronide hydrolysis, and the corresponding N-deethyl metabolite as additional biomarker in blood.

The detection of nitazenes in biological fluids is increasingly needed as they are repeatedly reported in intoxication and overdose cases. A simple method for the quantification of low levels of nine nitazene analogs and brorphine in Dried Blood Spots (DBS) was developed and validated. 10 μL of spiked whole blood is deposited on a Capitainer®B card and allowed to dry. The spot is punched out, and extracted with 500 μL methanol:acetonitrile (3:1 v/v) added with 1.5 μL of fentanyl-D5 as the internal standard. After stirring, sonication, and centrifugation of the vial, the solvent is dried under nitrogen, the extract is reconstituted in 30 μL methanol, and 1 μL is injected into a UHPLC-MS/MS instrument. The method validation showed linear calibration in the 1-50 ng/mL range, LOD values ranging between 0.3 ng/mL (isotonitazene) and 0.5 ng/mL (brorphine), average CV% and bias% within 15 % and 10 % for all compounds, respectively. The matrix effect due to blood and filter paper components was within 85-115 % while recovery was between 15-20 %. Stability tests against time and temperature showed no significant variations for storage periods up to 28 days. Room temperature proved to represent the best samples storage conditions. UHPLC-MS/MS proved capable to reliably identify all target analytes at low concentration even in small specimen volumes, as those obtained from DBS cards, which in turn confirmed to be effective and sustainable micro-sampling devices. This procedure improves the efficiency of toxicological testing and provides an innovative approach for the identification of the nitazene class of illicit compounds.

Benzimidazole opioids, often referred to as nitazenes, represent a subgroup of new psychoactive substances with a recent increase in fatal overdoses in the USA and Europe. With a variety of analogs emerging on the illicit drug market, forensic laboratories are challenged to identify these potent drugs. We here present a simple quantitative approach for the determination of nine nitazene analogs, namely, clonitazene, etodesnitazene, etonitazene, etonitazepyne, flunitazene, isotonitazene, metodesnitazene, metonitazene and protonitazene in whole blood using liquid-phase microextraction and electromembrane extraction in a 96-well format and liquid chromatography-tandem mass spectrometry. Green and efficient sample preparation was accomplished by liquid-phase microextraction in a 96-well format and resulted in high extraction yields for all analytes (>81%). Here, blood diluted with buffer (1:1, %v) was extracted from a donor compartment across a thin organic liquid membrane and into an aqueous acceptor solution. The acceptor solution was collected and directly injected into the analysis platform. Chromatographic separation was accomplished with a biphenyl column, allowing for a baseline separation of the structural isomers isotonitazene and protonitazene before detection by multiple reaction monitoring. Validation was performed according to Scientific Working Group of Forensic Toxicology guidelines. The calibration range was from 0.5 to 50 nM (except for protonitazene and clonitazene from 0.1 nM) with good linearity and limits of detection down to 0.01 nM. An AGREEprep assessment was performed to evaluate sample preparation greenness, with a final score of 0.71. Nitazenes represent a current threat to public health, and analytical methods that cover a wide range of these analogs are limited. Here, the described method may assist in the detection of nitazenes in whole blood and prevent these substances from being missed in postmortem investigations.

BACKGROUND: 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).
OBJECTIVE: 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).
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 (EC50 = 30 pM, Emax = 103%) and across all in vivo endpoints (ED50 = 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.

Nitazenes are a group of compounds developed in the 1950s as opioid analgesics, but they were never approved to market. As such, they are not well known outside of academic research laboratories. A characteristic of nitazenes is their high potency (e.g., hundreds to thousands fold more potent than morphine and other opioids and tenfold more potent than fentanyl). In the past few years, several nitazenes, including \"designer analogs,\" have been detected in the illicit drug supply and have been implicated in overdose mortality, primarily due to their exceptionally high potency. In the street drug supply, nitazenes are often found mixed with fentanyl or other agents but their presence is not always disclosed to drug buyers, who may not even be familiar with nitazenes. These drugs pose a particular challenge since there is little experience in how to reverse a nitazene overdose or potential drug-drug or drug-alcohol interactions. Public health efforts are needed to better inform street drug consumers, first responders, healthcare professionals, and the general public about these \"new old drugs\" that are infiltrating the recreational drug supply.