Identification and biophysical characterization of a novel domain-swapped camelid antibody specific for fentanyl.

Opioid use disorders (OUD) and overdoses are ever-evolving public health threats that continue to grow in incidence and prevalence in the United States and abroad. Current treatments consist of opioid receptor agonists and antagonists, which are safe and effective but still suffer from some limitations. Murine and humanized monoclonal antibodies (mAb) have emerged as an alternative and complementary strategy to reverse and prevent opioid-induced respiratory depression. To explore antibody applications beyond traditional heavy-light chain mAbs, we identified and biophysically characterized a novel single-domain antibody specific for fentanyl from a camelid variable-heavy-heavy (VHH) domain phage display library. Structural data suggested that VHH binding to fentanyl was facilitated by a unique domain-swapped dimerization mechanism, which accompanied a rearrangement of complementarity-determining region loops leading to the formation of a fentanyl-binding pocket. Structure-guided mutagenesis further identified an amino acid substitution that improved the affinity and relaxed the requirement for dimerization of the VHH in fentanyl binding. Our studies demonstrate VHH engagement of an opioid and inform on how to further engineer a VHH for enhanced stability and efficacy, laying the groundwork for exploring the in vivo applications of VHH-based biologics against OUD and overdose.

Intoxications involving methoxyacetylfentanyl and U-47700: a study of 3 polydrug fatalities.

Novel synthetic opioids (NSOs) represent an emerging group of novel psychoactive substances, acting as agonists at the opioid receptors. NSOs include fentanyl-related compounds, e.g. methoxyacetylfentanyl (MeACF), and non-fentanyl analogs, e.g. "U compounds" including U-47700. Here we present three cases of death involving MeACF and U-47700, with particular reference to preliminary data on pharmacokinetics and tissue distribution.After a complete post-mortem examination, general unknown screenings and analysis of drugs of abuse were performed on postmortem samples by immunoassays, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. To quantify the analytes of interest in post-mortem blood and tissues, the standard addition method was used. A toxicological significance score (TSS), weighing the role of the NSO in each death case, was assigned.Case 1 died at the hospital after consumption of U-47700, methadone (serum levels: 2,600 ng/ml and 37 ng/ml), tilidine and benzodiazepines. In case 2, U-47700 (204 ng/ml) together with methadone (290 ng/ml), flubromazepam (480 ng/ml) and diazepam (300 ng/ml) were detected in peripheral blood. In case 3, methoxyacetylfentanyl (266 ng/ml), furanylfentanyl (4.3 ng/ml) 4-ANPP (15 ng/ml) and alprazolam (69 ng/ml) were quantified in femoral blood. In all cases, the NSO likely contributed to the death (TSS = 3).NSOs appear to be often consumed in the setting of polydrug intoxications, especially in combination with other opioids and benzodiazepines, which often exert synergistic effects. The standard addition method remains the most reliable in post-mortem analysis and toxicological results should always be evaluated together with circumstantial and autopsy data.

Human metabolism of four synthetic benzimidazole opioids: isotonitazene, metonitazene, etodesnitazene, and metodesnitazene.

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.

In vitro structure-activity relationships and forensic case series of emerging 2-benzylbenzimidazole 'nitazene' opioids.

2-Benzylbenzimidazole 'nitazene' opioids are presenting a growing threat to public health. Although various nitazenes were previously studied, systematic comparisons of the effects of different structural modifications to the 2-benzylbenzimidazole core structure on µ-opioid receptor (MOR) activity are limited. Here, we assessed in vitro structure-activity relationships of 9 previously uncharacterized nitazenes alongside known structural analogues. Specifically, we focused on MOR activation by 'ring' substituted analogues (i.e., N-pyrrolidino and N-piperidinyl modifications), 'desnitazene' analogues (lacking the 5-nitro group), and N-desethyl analogues. The results from two in vitro MOR activation assays (ß-arrestin 2 recruitment and inhibition of cAMP accumulation) showed that 'ring' modifications overall yield highly active drugs. With the exception of 4'-OH analogues (which are metabolites), N-pyrrolidino substitutions were generally more favorable for MOR activation than N-piperidine substitutions. Furthermore, removal of the 5-nitro group on the benzimidazole ring consistently caused a pronounced decrease in potency. The N-desethyl modifications showed important MOR activity, and generally resulted in a slightly lowered potency than comparator nitazenes. Intriguingly, N-desethyl isotonitazene was the exception and was consistently more potent than isotonitazene. Complementing the in vitro findings and demonstrating the high harm potential associated with many of these compounds, we describe 85 forensic cases from North America and the United Kingdom involving etodesnitazene, N-desethyl etonitazene, N-desethyl isotonitazene, N-pyrrolidino metonitazene, and N-pyrrolidino protonitazene. The low-to-sub ng/mL blood concentrations observed in most cases underscore the drugs' high potencies. Taken together, by bridging pharmacology and case data, this study may aid to increase awareness and guide legislative and public health efforts.

Nitazene test strips: a laboratory evaluation.

BACKGROUND: 2-Benzylbenzimidazole 'nitazene' opioids pose a growing threat to public health. Nitazene analogues are increasingly found mixed with or (mis)sold as heroin and in falsified (non-)opioid medications, posing a great risk of intoxication in users (un)knowingly exposed to these potent opioids. Lateral flow immunoassay nitazene test strips (NTS; BTNX Rapid Response™) became commercially available in Q1 2024, with the aim to enable rapid detection of nitazene analogues in drug samples. As only limited independent data is available on the performance of these strips, this lab-based study aimed at evaluating their potential for drug checking applications. METHODS: Following dilution of drug standards in water, the NTS readouts were analyzed independently by two individuals and by ImageJ. The limit of detection for isotonitazene was determined using two manufacturing lots of NTS. Cross-reactivity with 32 other nitazene analogues was evaluated. Six sourced drug samples were tested to explore the ability of NTS to detect the presence of a nitazene analogue in authentic samples. RESULTS: The limits of detection for isotonitazene were 2000 or 3000 ng/mL, depending on the lot. Twenty-four of the 33 tested nitazene analogues cross-reacted with the NTS at concentrations ≤ 9000 ng/mL. Structural analysis indicated that either substitution or removal of the 5-nitro group, or lengthening the linker between the two aromatic rings, generally hampered detection. All six authentic drug samples consistently tested positive, with no observed false negatives. CONCLUSIONS: This study provides a better understanding of the potential of NTS for drug checking purposes. Our findings indicate that NTS can theoretically alert to the presence of most nitazene analogues that have emerged on recreational drug markets. However, 'desnitazenes' (lacking the 5-nitro group) may yield false negative results due to low cross-reactivity. Although factors like specificity, lot-to-lot variability, nitazene analogue content in drug samples, solubility, and different testing conditions should be considered, our study results indicate that, at least under the conditions evaluated here (using reference standards and sourced powders), NTS are capable of detecting the presence of a wide range of nitazene analogues. Hence, NTS may alert users of the presence of nitazene analogues in drug samples.

[The challenges for psychiatric care posed by synthetic drugs]. / Die Herausforderungen für die psychiatrische Versorgung durch synthetische Drogen.

BACKGROUND: In addition to the drugs that have been known for decades, several hundred mainly synthetic substances have been identified as drugs for the first time in the last 20 years. AIM OF THE WORK: Presentation of the various groups of substances and their psychotropic effects, the epidemiology of their use and the legal and social background of this development. MATERIAL: Narrative literature review. RESULTS: The most important new psychoactive substances (NPS) are synthetic cannabinoids, synthetic stimulants (cathinones), halluginogens and new synthetic opioids (NSO), in particular fentanyl and related substances. The new substances do not have any qualitatively new psychotropic effects. They were brought onto the market in particular as substitutes for substances subject to the Narcotics Act but are often associated with dangerous side effects and even mortality. The increasing availability of these substances has gone hand in hand with the establishment of the Internet as a source of knowledge (e.g. for synthesis routes) and as a marketplace. Substance group-related regulations have also been established in Germany (New Psychoactive Substances Act). In Germany the prevalence of NPS use is significantly lower than that of cannabis; however, there are indications that the production and distribution of synthetic drugs is more profitable for drug dealers than with conventional plant-based drugs, such as heroin. In the USA, for example, NSOs are the primarily drugs used for opioid addiction. DISCUSSION: It remains to be seen whether NPS and NSOs will replace conventional drugs. The availability of synthetic drugs is more difficult to reduce than that of plant-based drugs. Harm reduction measures should be expanded, e.g., early warning systems for new drugs, drug checking and naloxone programs.

Detection, chemical analysis, and pharmacological characterization of dipyanone and other new synthetic opioids related to prescription drugs.

The emergence of structurally diverse new synthetic opioids (NSOs) has caused the opioid crisis to spiral to new depths. Little information is available about the pharmacology of most novel opioids when they first emerge. Here, using a ß-arrestin 2 recruitment assay, we investigated the in vitro µ-opioid receptor (MOR) activation potential of dipyanone, desmethylmoramide, and acetoxymethylketobemidone (O-AMKD) - recent NSOs that are structurally related to the prescription opioids methadone and ketobemidone. Our findings indicate that dipyanone (EC50=39.9 nM; Emax=155% vs. hydromorphone) is about equally active as methadone (EC50=50.3 nM; Emax=152%), whereas desmethylmoramide (EC50=1335 nM; Emax=126%) is considerably less active. A close structural analogue of ketobemidone (EC50=134 nM; Emax=156%) and methylketobemidone (EC50=335 nM; Emax=117%), O-AMKD showed a lower potency (EC50=1262 nM) and efficacy (Emax=109%). Evaluation of the opioid substitution product buprenorphine and its metabolite norbuprenorphine confirmed the increased in vitro efficacy of the latter. In addition to in vitro characterization, this report details the first identification and full chemical analysis of dipyanone in a seized powder, as well as a postmortem toxicology case from the USA involving the drug. Dipyanone was quantified in blood (370 ng/mL), in which it was detected alongside other NSOs (e.g., 2-methyl AP-237) and novel benzodiazepines (e.g., flualprazolam). While dipyanone is currently not commonly encountered in forensic samples worldwide, its emergence is worrisome and representative of the dynamic NSO market. Graphical Abstract.

URINE TOXICOLOGY PROFILES OF EMERGENCY DEPARTMENT PATIENTS WITH UNTREATED OPIOID USE DISORDER: A MULTI-SITE VIEW.

BACKGROUND: Opioid overdose deaths in 2021 were the highest ever, driven by fentanyl and polysubstance use. OBJECTIVE: The aim of the study was to characterize drug use, assessed by urine drug screens (UDSs), in patients with untreated opioid use disorder (OUD) presenting to 28 emergency departments (EDs) nationally and by region. METHODS: We analyzed UDSs from patients enrolled in the CTN-0099 ED-INNOVATION (Emergency Department-Initiated Buprenorphine Validation) trial between July 12, 2020 and March 9, 2022. Participants were adult ED patients with OUD not engaged in addiction treatment with a UDS positive for an opioid, but negative for methadone. Sites were divided into "East" and "West" regions. RESULTS: A UDS was available for all 925 enrolled participants, 543 from East and 382 from West. Fentanyl was in 702 specimens (76%) (n = 485 [89%] East vs. n = 217 [57%] West; p < 0.01) and was the only opioid in 269 (29%). After fentanyl, the most common opioids were morphine (presumably heroin; n = 411 [44%]; n = 192 [35%] East vs. n = 219 [57%] West; p < 0.01) and buprenorphine (n = 329 [36%]; n = 186 [35%] East vs. n = 143 [37%] West; p = 0.32). The most common drugs found with opioids were stimulants (n = 545 [59%]), tetrahydrocannabinol (n = 417 [45%]), and benzodiazepines (n = 151 [16%]). Amphetamine-type stimulants were more common in West (n = 209 [55%] vs. East (n = 125 [23%]). Cocaine was more common in East (n = 223 [41%]) vs. West (n = 82 [21%]). The presence of multiple drugs was common (n = 759 [82%]). CONCLUSIONS: Most participants had UDS specimens containing multiple substances; a high proportion had fentanyl, stimulants, and buprenorphine. Regional differences were noted. Given the increased risk of death with fentanyl and polysubstance use, ED providers should be providing risk reduction counseling, treatment, and referral.

Identification of the Putative Binding Site of a Benzimidazole Opioid (Etazene) and Its Metabolites at µ-Opioid Receptor: A Human Liver Microsomal Assay and Systematic Computational Study.

The synthetic benzimidazole opioid etazene (which has a 70-times higher analgesic activity than morphine), a recreational drug, has gained popularity as a novel psychoactive substance (NPS) on the illegal/darknet market; however, no experimental information is available at the molecular level on the binding mechanism and putative binding site of etazene and its metabolites at the µ-opioid receptor (MOR). In the present study, we investigated the metabolism of etazene in human liver microsomes using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). We also explored the possibilities of MOR activation by etazene and its metabolites by studying their binding mechanisms and interaction profiles at an active-state MOR model via molecular docking, binding free energy calculations, and all-atom molecular dynamics (MD) simulations. The putative metabolites of etazene were also predicted using the ADMET Predictor 10.1. The molecular docking studies and free energy calculations showed that etazene and its metabolites (M1, M2, and M5-M7) exhibited strong predicted binding affinity at MOR and showed overlapped binding orientation with MOR-bound agonist BU72, which was co-crystallized in the MOR X-ray crystal structure (PDB ID: 5C1M). MD also confirmed the stability of the MOR-etazene and MOR-M6 complexes. These results suggest that etazene and its metabolites may act as strong MOR agonists, highlighting the necessity of experimental validation. The insights from this study, such as key interactions between etazene and its metabolites and the MOR, will allow authorities to predict potential analogs and clarify the target-protein interactions associated with this illicit substance, granting advanced or rapid reactions to confiscating or banning potential emerging drugs.

Fentanyl and other New Psychoactive Synthetic Opioids. Challenges to Prevention and Treatment.

Synthetic opioids have played a significant role in the current opioid crisis in the United States (U.S.) and Canada and are a matter of concern worldwide. New psychoactive opioids (NPOs) are classified in the internationally recognized new psychoactive substances (NPSs) category. This group comprises compounds that may have been synthesized decades ago but appeared only recently in the illicit drug market. Such is the case of fentanyl, fentanyl analogs, and non-fentanyl opioids. Most NPOs have effects similar to morphine, including euphoria and analgesia, and can produce fatal respiratory depression. Here, we present an overview of the systemic and molecular effects of main NPOs, their classification, and their pharmacological properties. We first review the fentanyl group of NPOs, including the four compounds of clinical use (fentanyl, alfentanil, sufentanil, and remifentanil) and the veterinary drug carfentanil. We also provide essential information on non-medical fentanyl analogs and other synthetic opioids such as brorphine, etonitazene, and MT-45, used as adulterants in commonly misused drugs. This paper also summarizes the scarce literature on the use of NPOs in Mexico. It concludes with a brief review of the challenges to prevention and treatment posed by NPOs and some recommendations to face them.

Blood concentrations of new synthetic opioids.

PURPOSE: Over the last decade, there has been a significant growth in the market and number of new psychoactive substances (NPS). One of the NPS groups that has grown rapidly in recent years, bringing a new set of problems, consists of new synthetic opioids. The extreme potency of these compounds poses a high risk of acute poisoning, as an overdose can cause respiratory depression. Most of the information regarding human pharmacokinetics of new opioids is based on toxicological case reports and the data on concentrations of new opioids in human blood are scarce. The interpretation of results usually requires a comparison to previously published cases; therefore, a referenced compilation of previously published concentration data would be useful. METHODS: The data were collected by searching the PubMed and Scopus databases and by using the Google search engine. All the available data from articles and reports that measured new opioid concentrations in plasma, serum, or whole blood were included in the data analysis. RESULTS: The presented tables list the observed concentrations in fatal and nonfatal cases involving 37 novel synthetic opioids. CONCLUSIONS: Blood levels of new opioids are extremely difficult to interpret. Low blood concentrations of these substances do not rule out acute poisoning as their high potency creates a risk of respiratory depression even at low doses. Opioid tolerance, frequent presence of other drugs, and additional diseases make it impossible to define concentration ranges, especially the minimum fatal concentrations. This report provides quick access to the source articles quantifying novel synthetic opioids.

Toxicological and pharmacological characterization of novel cinnamylpiperazine synthetic opioids in humans and in vitro including 2-methyl AP-237 and AP-238.

The recent scheduling actions of fentanyl-related substances in both the United States and China have sparked the emergence and proliferation of other generations of "legal" opioids that are structurally distinct from fentanyl, including the recently emerged class of cinnamylpiperazines. In contrast to fentanyl, which contains a piperidine core and a phenethyl moiety, the primary structural components of cinnamylpiperazines are the piperazine core and a cinnamyl moiety. This manuscript reports on the toxicological profile for antemortem and postmortem cases where a cinnamylpiperazine was detected. Samples were quantitatively confirmed using liquid chromatography tandem mass spectrometry. The cases were received between February 2020 and April 2021. Concentrations of 2-methyl AP-237 from four postmortem cases ranged from 820 to 5800 ng/mL, and concentrations of AP-238 from two postmortem cases were 87 and 120 ng/mL. µ-Opioid receptor (MOR) activation potential for 2-methyl AP-237, AP-237, para-methyl AP-237, and AP-238 were studied using a ßarr2 recruitment assay. Efficacies (Emax, relative to hydromorphone) and potencies (EC50) were derived and of the compounds tested AP-238 was the most potent compound in the panel with an EC50 of 248 nM. 2-Methyl AP-237 was found to be the most efficacious drug (Emax = 125%) of the tested cinnamylpiperazines; however, it had substantially less efficacy than fentanyl. The in vitro MOR activation potential of the studied cinnamylpiperazines was lower than that of fentanyl and other novel synthetic opioids (NSOs), in line with the relatively higher concentrations observed in postmortem toxicology samples-an important observational link between in vitro pharmacology and in vivo toxicology.

Characterization of recent non-fentanyl synthetic opioids via three different in vitro µ-opioid receptor activation assays.

New synthetic opioids (NSOs) are one of the fastest growing groups of new psychoactive substances. Amid this dynamic landscape, insight into the pharmacology of NSOs is important to estimate the harm potential of newly emerging drugs. In this work, we determined the µ-opioid receptor (MOR) affinity and activation potential of seven poorly characterized non-fentanyl NSOs (N-ethyl-U-47700, 3,4-difluoro-U-47700, U-47931E/bromadoline, 2,4-difluoro-U-48800, U-62066/spiradoline, 2F-viminol, ketobemidone) and a panel of nine reference opioids. MOR affinity was determined via [3H]-DAMGO binding in rat brain tissue homogenates, and was found to correlate well with different functional parameters. MOR activation potential was studied at different levels of receptor signaling using three distinct assays (NanoBiT® MOR-ß-arrestin2/mini-Gαi and AequoScreen®). The most active compounds were ketobemidone (EC50 32.8-528 nM; Emax 105-271%, relative to hydromorphone) and N-ethyl-U-47700 (EC50 241-767 nM; Emax 139-247%). The same opioids showed the strongest MOR affinity. As most of the other NSOs only weakly activated MOR in the three assays (EC50 values in the high nM-µM range), they likely do not pose a high overdose risk. 2F-viminol (EC50 2.2-4.5 µM; Emax 21.2-61.5%) and U-47931E/bromadoline (EC50 0.55-2.9 µM; Emax 52.8-85.9%) were partial agonists compared to hydromorphone, and maximum receptor activation was not reached for 2,4-difluoro-U-48800 (EC50 > 22 µM). We further highlight the importance of considering specific assay characteristics upon interpretation of potencies, efficacies and biased agonism. As absolute values may greatly differ between assays with varying experimental set-ups, a comparison of functional parameters to those of well-characterized reference agonists is considered the most informative.

First identification, chemical analysis and pharmacological characterization of N-piperidinyl etonitazene (etonitazepipne), a recent addition to the 2-benzylbenzimidazole opioid subclass.

N-Piperidinyl etonitazene ('etonitazepipne') represents a recent addition to the rapidly expanding class of 2-benzylbenzimidazole 'nitazene' opioids. Following its first identification in an online-sourced powder and in biological samples from a patient seeking help for detoxification, this report details its in-depth chemical analysis and pharmacological characterization. Analysis of the powder via different techniques (LC-HRMS, GC-MS, UHPLC-DAD, FT-IR) led to the unequivocal identification of N-piperidinyl etonitazene. Furthermore, we report the first activity-based detection and analytical identification of N-piperidinyl etonitazene in authentic samples. LC-HRMS analysis revealed concentrations of 1.21 ng/mL in serum and 0.51 ng/mL in urine, whereas molecular networking enabled the tentative identification of various (potentially active) urinary metabolites. In addition, we determined that the extent of opioid activity present in the patient's serum was equivalent to the in vitro opioid activity exerted by 2.5-10 ng/mL fentanyl or 10-25 ng/mL hydromorphone in serum. Radioligand binding assays in rat brain tissue revealed that the drug binds with high affinity (Ki = 14.3 nM) to the µ-opioid receptor (MOR). Using a MOR-ß-arrestin2 activation assay, we found that N-piperidinyl etonitazene is highly potent (EC50 = 2.49 nM) and efficacious (Emax = 183% versus hydromorphone) in vitro. Pharmacodynamic evaluation in male Sprague Dawley rats showed that N-piperidinyl etonitazene induces opioid-like antinociceptive, cataleptic, and thermic effects, its potency in the hot plate assay (ED50 = 0.0205 mg/kg) being comparable to that of fentanyl (ED50 = 0.0209 mg/kg), and > 190 times higher than that of morphine (ED50 = 3.940 mg/kg). Taken together, our findings indicate that N-piperidinyl etonitazene is a potent opioid with the potential to cause harm in users.

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-ß-arrestin2 activation assay, N-pyrrolidino etonitazene displayed high potency (EC50 = 0.348 nM), similar to etonitazene (EC50 = 0.360 nM), and largely exceeding the potencies of fentanyl (EC50 = 14.9 nM) and morphine (EC50 = 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 (ED50 = 0.0017 mg/kg) was tenfold and 2,000-fold greater than fentanyl (ED50 = 0.0209 mg/kg) and morphine (ED50 = 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.

New Synthetic Opioids Use among Patients in Treatment for an Opioid Use Disorder in Barcelona.

INTRODUCTION: New synthetic opioids (NSO), a class of new psychoactive substances (NPS), have recently emerged and pose an upcoming global public health challenge. The effects produced by NSO are similar to those from morphine, but they present greater pharmacological potency and abuse potential. Due to the increasing number of fatal overdoses and seizures in which NSO have been detected as heroin substitutes or adulterants, individuals with Opioid Use Disorder (OUD) represent a vulnerable population. The aim of our study was to describe and characterize from a gender perspective a Spanish cohort of potential conscious or unconscious NSO users. METHODS: A cross-sectional study was conducted in a cohort of OUD participants under treatment in addiction care services in Barcelona and Badalona, Spain. Clinical evaluation was performed through an ad hoc survey, a scale to evaluate reasons to use an opioid without prescription (range 0-4) and the Wellbeing Index (WHO-5) (range 0-100). Objective consumption of NSO was assessed by urinalysis carried out by two validated methods: high-sensitivity gas chromatography-mass spectrometry (MS) and ultra-high-performance liquid chromatography-high-resolution MS. RESULTS: A total of 154 participants with OUD were enrolled. They were mainly men (72.7%), mean age 47.8 years. Methadone was the predominant medication for opioid agonist treatment (mean dose 61.25 mg/day). A total of 32 (20.8%) participants reported having consumed some opioid to become "high" in the previous 3 months. The principal reasons for consuming illicit opioids were Replacing other drugs (mean 2.03) and Availability (mean 1.62), although Low price, was more highly valued by men (p = 0.045) and Shorter effect duration, most highly rated by women (p = <0.001). In the WHO-5, the mean score was 55 (SD = 30.1) without differences by gender. Fentanyl and derivatives or/and metabolites were detected in 7 (6.1%) participants, but illicit/non-prescribed NSOs were found in 5 out of 114 patients (4.4%), and other non-fentanyl opioids in 36 participants (26 men and 10 women). CONCLUSION: A non-negligible consumption of NSO-fentanyl's (positive detection in 6.1% of biological samples) was detected. The reasons for using these substances and also the well-being differed between the genders. There is therefore both voluntary and involuntary NSO consumption in our country which highlights the importance of approaching this potential public health problem.

How much illegally manufactured fentanyl could the U.S. be consuming?

The spread of illegally manufactured opioids, including fentanyl, has brought unprecedented levels of drug overdose deaths in North America. In some markets, illegally manufactured fentanyl (IMF) is essentially displacing heroin, not just being used to adulterate it. It is not possible at this time to provide an accurate point estimate of the amount of IMF consumed in the United States. Yet for various purposes (e.g. assessing changes in production levels and the appropriate role for various supply reduction efforts), it is important to have a sense of scale. This article provides guidance through two thought experiments that provide a hypothetical upper bound on U.S. consumption. The first considers a scenario in which IMF replaces heroin in all illegal opioid markets. The second starts with the number of individuals with an opioid use disorder and considers what total consumption would be if IMF was the only opioid they consumed. Both calculations suggest it is unlikely that the annual consumption of IMF in 2021 could have been more than single digit pure metric tons. For comparison, the most recent best estimates of the amount of cocaine and heroin consumed in the U.S. are 145 and 47 pure metric tons, respectively. The article also raises questions about the limitations of using traditional equianalgesic morphine equivalent dose conversions to estimate the total market consumption of IMF.

The Genotoxicity of Acrylfentanyl, Ocfentanyl and Furanylfentanyl Raises the Concern of Long-Term Consequences.

Three fentanyl analogues Acrylfentanyl, Ocfentanyl and Furanylfentanyl are potent, rapid-acting synthetic analgesics that recently appeared on the illicit market of new psychoactive substances (NPS) under the class of new synthetic opioids (NSO). Pharmacotoxicological data on these three non-pharmaceutical fentanyl analogues are limited and studies on their genotoxicity are not yet available. Therefore, the aim of the present study was to investigate this property. The ability to induce structural and numerical chromosomal aberrations in human lymphoblastoid TK6 cells was evaluated by employing the flow cytometric protocol of the in vitro mammalian cell micronucleus test. Our study demonstrated the non-genotoxicity of Fentanyl, i.e., the pharmaceutical progenitor of the class, while its illicit non-pharmaceutical analogues were found to be genotoxic. In particular, Acrylfentanyl led to a statistically significant increase in the MNi frequency at the highest concentration tested (75 µM), while Ocfentanyl and Furanylfentnyl each did so at both concentrations tested (150, 200 µM and 25, 50 µM, respectively). The study ended by investigating reactive oxygen species (ROS) induction as a possible mechanism linked to the proved genotoxic effect. The results showed a non-statistically significant increase in ROS levels in the cultures treated with all molecules under study. Overall, the proved genotoxicity raises concern about the possibility of serious long-term consequences.

Toxicokinetics of U-47700, tramadol, and their main metabolites in pigs following intravenous administration: is a multiple species allometric scaling approach useful for the extrapolation of toxicokinetic parameters to humans?

New synthetic opioids (NSOs) pose a public health concern since their emergence on the illicit drug market and are gaining increasing importance in forensic toxicology. Like many other new psychoactive substances, NSOs are consumed without any preclinical safety data or any knowledge on toxicokinetic (TK) data. Due to ethical reasons, controlled human TK studies cannot be performed for the assessment of these relevant data. As an alternative animal experimental approach, six pigs per drug received a single intravenous dose of 100 µg/kg body weight (BW) of U-47700 or 1000 µg/kg BW of tramadol to evaluate whether this species is suitable to assess the TK of NSOs. The drugs were determined in serum and whole blood using a fully validated method based on solid-phase extraction and LC-MS/MS. The concentration-time profiles and a population (pop) TK analysis revealed that a three-compartment model best described the TK data of both opioids. Central volumes of distribution were 0.94 L/kg for U-47700 and 1.25 L/kg for tramadol and central (metabolic) clearances were estimated at 1.57 L/h/kg and 1.85 L/h/kg for U-47700 and tramadol, respectively. The final popTK model parameters for pigs were upscaled via allometric scaling techniques. In comparison to published human data, concentration-time profiles for tramadol could successfully be predicted with single species allometric scaling. Furthermore, possible profiles for U-47700 in humans were simulated. The findings of this study indicate that unlike a multiple species scaling approach, pigs in conjunction with TK modeling are a suitable tool for the assessment of TK data of NSOs and the prediction of human TK data.

Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry and High-Sensitivity Gas Chromatography-Mass Spectrometry Screening of Classic Drugs and New Psychoactive Substances and Metabolites in Urine of Consumers.

The use of the new psychoactive substances is continuously growing and the implementation of accurate and sensible analysis in biological matrices of users is relevant and fundamental for clinical and forensic purposes. Two different analytical technologies, high-sensitivity gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) were used for a screening analysis of classic drugs and new psychoactive substances and their metabolites in urine of formed heroin addicts under methadone maintenance therapy. Sample preparation involved a liquid-liquid extraction. The UHPLC-HRMS method included Accucore™ phenyl Hexyl (100 × 2.1 mm, 2.6 µm, Thermo, USA) column with a gradient mobile phase consisting of mobile phase A (ammonium formate 2 mM in water, 0.1% formic acid) and mobile phase B (ammonium formate 2 mM in methanol/acetonitrile 50:50 (v/v), 0.1% formic acid) and a full-scan data-dependent MS2 (ddMS2) mode for substances identification (mass range 100-1000 m/z). The GC-MS method employed an ultra-Inert Intuvo GC column (HP-5MS UI, 30 m, 250 µm i.d, film thickness 0.25 µm; Agilent Technologies, Santa Clara, CA, USA) and electron-impact (EI) mass spectra were recorded in total ion monitoring mode (scan range 40-550 m/z). Urine samples from 296 patients with a history of opioid use disorder were examined. Around 80 different psychoactive substances and/or metabolites were identified, being methadone and metabolites the most prevalent ones. The possibility to screen for a huge number of psychotropic substances can be useful in suspected drug related fatalities or acute intoxication/exposure occurring in emergency departments and drug addiction services.