Search

Hypoxia-inducible factors (HIF) are interesting targets for multiple therapeutic indications. While HIF activation is desired for the treatment of anemia-related and ischemic diseases, HIF inhibition is of tremendous interest to anti-cancer drug development. Different signaling events within the HIF pathway are being targeted by drug discovery programs, with a special interest in HIF-selective (possibly also HIF1/2 isoform-selective) compounds. In this study, we applied recently developed cell-based split-nanoluciferase HIF heterodimerization assays to study the effects of compounds, targeting HIF activity by various mechanisms of action. This study shows that the application of similar or diverse assay protocols allows to detect various influences on HIF heterodimerization as a key signaling event in the oxygen sensing pathway: increased HIF heterodimerization (roxadustat, MG-132), decreased HIF heterodimerization (PX-478, ibuprofen) and direct (HIF isoform-selective) heterodimerization inhibiting effects (PT-2385). Changes in treatment time and in the assay protocol allowed to assess direct and indirect effects on HIFα-HIFβ heterodimerization. In addition to the evaluation of applications of these new bioassays regarding pharmacological characterizations, benefits and considerations are discussed related to the use of cellular, luminescent-based bioassays. Briefly, benefits include the bidirectional nature of the biological readout, the upstream mechanism of detection, the differentiation between HIF1 and HIF2 effects and the simulation of various conditions. Specific and general considerations include cell-based, technical and disease/drug-related aspects (e.g., non-specific effects, color interference). In summary, the versatility of these bioassays offers benefits in widespread applications regarding drug discovery and pharmacological characterization of various therapeutics, applying either the same or optimized experimental protocols., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Opioid use disorder - particularly involving fentanyl - has precipitated a public health crisis characterized by a significant increase in addiction and overdose-related deaths. Fentanyl-specific immunotherapy, which aims at inducing fentanyl-specific antibodies capable of binding fentanyl molecules in the bloodstream, preventing their entry in the central nervous system, is therefore gaining momentum. Conventional opioid designs rely on the covalent conjugation of fentanyl analogues to immunogenic carrier proteins that hold the inherent capacity of mounting immunodominant responses. Here, we present an alternative fentanyl vaccine design that utilizes a non-covalent assembly of lipid nanoparticles (LNPs) to deliver fentanyl haptens in conjunction with a CD4+ T-helper peptide epitope and an imidazoquinoline TLR7/8 agonist. Our results demonstrate that a single intramuscular administration of the LNP-based nanovaccine elicits fentanyl-specific antibodies, significantly mitigating the effects of opioid overdose in preclinical mouse models. Furthermore, we analyzed the immunobiological behavior of the vaccine in vivo in mouse models, providing evidence that covalent attachment of a fentanyl hapten to a carrier proteins or peptide epitope is not necessary for inducing an effective immune response. However, co-delivery - specifically, the physical assembly of all immune cues into an LNP - remains essential for inducing hapten-specific immunity., (© 2024 Wiley‐VCH GmbH.)

After the Scottish Prison Service (SPS) introduced mail photocopying procedures in December 2021, a shift in smuggling methods was observed for synthetic cannabinoid receptor agonists (SCRAs) and other new psychoactive substances (NPS) from drug-infused papers back to traditional sample matrices (e.g., tablets and powders), although new matrices also emerged. This study reports on waxy- or putty-like materials as a novel drug preparation for SCRAs and other drugs seized from UK prisons. In 2023, 22 of these new preparations were seized from Scottish prisons, with eight found in sealed vape pods. The materials were positive for SCRAs, phytocannabinoids, novel benzodiazepines, and/or gabapentinoids. Additionally, 11 preparations were seized from an English prison, all containing the SCRAs MDMB-4en-PINACA and MDMB-INACA. MDMB-INACA was pharmacologically characterized using in vitro CB 1 and CB 2 bioassays, revealing moderate efficacy but low potency at CB 1 . Furthermore, the in vitro CB 1 bioassay was also used to evaluate the CB 1 activating potential of extracts from eight seized samples. Six of these showed high CB 1 activity, whereas the samples lacking SCRAs or containing only MDMB-INACA showed no or only weak CB 1 activity, respectively. Lastly, applying the bioassay as an activity-based "untargeted" screening method effectively identified the presence of SCRAs in one waxy preparation, which was initially not detected by gas chromatography-mass spectrometry (GC-MS). This underscores the effectiveness of the bioassay for evaluating these new waxy- or putty-like materials for the presence of SCRAs., (© 2024 The Author(s). Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Synthetic cannabinoid receptor agonists (SCRAs) are a class of synthetic drugs that mimic and greatly surpass the effect of recreational cannabis. Acute SCRA intoxications are in general difficult to assess due to the large number of compounds involved, differing widely in both chemical structure and pharmacological properties. The rapid pace of emergence of unknown SCRAs hampers on one hand the timely availability of methods for identification and quantification to confirm and estimate the extent of the SCRA intoxication. On the other hand, lack of knowledge about the harm potential of emerging SCRAs hampers adequate interpretation of serum concentrations in intoxication cases. In the present study, a novel comparative measure for SCRA intoxications was evaluated, focusing on the cannabinoid activity (versus serum concentrations), which can be measured in serum extracts with an untargeted bioassay assessing ex vivo CB 1 activity. Application of this principle to a series of SCRA intoxication cases (n = 48) allowed for the determination of activity equivalents, practically entailing a conversion from different SCRA serum concentrations to a JWH-018 equivalent. This allowed for the interpretation of both mono- (n = 34) and poly-SCRA (n = 14) intoxications, based on the intrinsic potential of the present serum levels to exert cannabinoid activity (cf. pharmacological/toxicological properties). A non-distinctive toxidrome was confirmed, showing no relation to CB 1 activity. The JWH-018 equivalent was partly related to the poison severity score (PSS) and causality of the clinical intoxication elicited by the SCRA. Altogether, this equivalent concept allows to comparatively and timely interpret (poly-)SCRA intoxications based on CB 1 activity., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 (β-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., (© 2024. The Author(s).)

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS) and new structural scaffolds have emerged on the recreational drug market since the enactment of Chinese SCRA analog controls in 2021. This study reports the first SCRAs to be detected with a bromide at the 5 position (5'Br) on the phenyl ring of the indazole core and without a tail moiety. ADB-5'Br-INACA (ADMB-5'Br-INACA) and MDMB-5'Br-INACA were detected in seized samples from Scottish prisons, Belgian customs, and US forensic casework. The brominated analog with a tail moiety, ADB-5'Br-BUTINACA (ADMB-5'Br-BUTINACA), was also detected in Scottish prisons and US forensic casework. The metabolites of these compounds and the predicted compound MDMB-5'Br-BUTINACA were identified through incubation with primary human hepatocytes to aid in their toxicological identification. The bromide on the indazole remains intact on metabolites, allowing these compounds to be easily distinguished in toxicological samples from their non-brominated analogs. Glucuronidation was more common for tail-less analogs than their butyl tail-containing counterparts. Forensic toxicologists are advised to update their analytical methods with the characteristic ions for these compounds, as well as their anticipated urinary markers: amide hydrolysis and monoOH at tert-butyl metabolites (after β-glucuronidase treatment) for ADB-5'Br-INACA; monoOH at tert-butyl and amide hydrolysis metabolites for ADB-5'Br-BUTINACA; and ester hydrolysis metabolites with additional metabolites for MDMB-5'Br-INACA and MDMB-5'Br-BUTINACA. Toxicologists should remain vigilant to the emergence of new SCRAs with halogenation of the indazole core and tail-less analogs, which have already started to emerge., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

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., (© 2024. The Author(s).)

Cultivation of industrial low-Δ 9 -tetrahydrocannabinol (Δ 9 -THC) hemp has created an oversupply of cannabidiol (CBD)-rich products. The fact that phytocannabinoids, including CBD, can be used as precursors to synthetically produce a range of THC variants-potentially located in a legal loophole-has led to a diversification of cannabis recreational drug markets. 'Hemp-compliant', 'hemp-derived' and 'semisynthetic' cannabinoid products are emerging and being advertised as (legal) alternatives for Δ 9 -THC. This study included a large panel (n = 30) of THC isomers, homologs, and analogs that might be derived via semisynthetic procedures. As a proxy for the abuse potential of these compounds, we assessed their potential to activate the CB 1 cannabinoid receptor with a β-arrestin2 recruitment bioassay (picomolar-micromolar concentrations). Multiple THC homologs (tetrahydrocannabihexol, THCH; tetrahydrocannabiphorol, THCP; tetrahydrocannabinol-C8, THC-C8) and THC analogs (hexahydrocannabinol, HHC; hexahydrocannabiphorol, HHCP) were identified that showed higher potential for CB 1 activation than Δ 9 -THC, based on either higher efficacy (E max ) or higher potency (EC 50 ). Structure-activity relationships were assessed for Δ 9 -THC and Δ 8 -THC homologs encompassing elongated alkyl chains. Additionally, stereoisomer-specific differences in CB 1 activity were established for various THC isomers (Δ 7 -THC, Δ 10 -THC) and analogs (HHC, HHCP). Evaluation of the relative abundance of 9(S)-HHC and 9(R)-HHC epimers in seized drug material revealed varying epimeric compositions between batches. Increased abundance of the less active 9(S)-HHC epimer empirically resulted in decreased potency, but sustained efficacy for the resulting diastereomeric mixture. In conclusion, monitoring of semisynthetic cannabinoids is encouraged as the dosing and the relative composition of stereoisomers can impact the harm potential of these drugs, relative to Δ 9 -THC products., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Blood microsampling has increasingly attracted interest in the past decades as a more patient-centric sampling approach, offering the possibility to collect a minimal volume of blood following a finger or arm prick at home. In addition to conventional dried blood spots (DBS), many different devices allowing self-sampling of blood have become available. Obviously, the success of home-sampling can only be assured when (inexperienced) users collect samples of good quality. Therefore, the feasibility of six different microsampling devices to collect capillary blood by inexperienced adolescents at home was evaluated. Participants (n = 95) were randomly assigned to collect blood (dried or liquid) at different time points using four of six different self-sampling devices (i.e., DBS, Mitra volumetric absorptive microsampling (VAMS), Capitainer B, Tasso M20, Minicollect tube and Tasso + serum separator tube (SST)). The quality of the samples was visually inspected and analytically determined. Moreover, the participants' satisfaction was assessed via questionnaires. Although a majority succeeded based on the visual inspection, the success rate differed largely between the different devices. In general, the lowest success rate was obtained for the Minicollect tubes, although there is an opportunity and need for improvement for the other self-sampling devices as well. Hence, this also emphasizes the importance to assess the quality of samples collected by the target population prior to study initiation. In addition, visual classification by a trained individual was confirmed based on assessment of the analytical variability between replicates. Finally, self-sampling at home was overall (very) positively received by the participants., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Background: Since late 2019, fortification of 'regular' cannabis plant material with synthetic cannabinoid receptor agonists (SCRAs) has become a notable phenomenon on the drug market. As many SCRAs pose a higher health risk than genuine cannabis, recognizing SCRA-adulterated cannabis is important from a harm reduction perspective. However, this is not always an easy task as adulterated cannabis may only be distinguished from genuine cannabis by dedicated, often expensive and time-consuming analytical techniques. In addition, the dynamic nature of the SCRA market renders identification of fortified samples a challenging task. Therefore, we established and applied an in vitro cannabinoid receptor 1 (CB 1 ) activity-based procedure to screen plant material for the presence of SCRAs., Methods: The assay principle relies on the functional complementation of a split-nanoluciferase following recruitment of β-arrestin 2 to activated CB 1 . A straightforward sample preparation, encompassing methanolic extraction and dilution, was optimized for plant matrices, including cannabis, spiked with 5 µg/mg of the SCRA CP55,940., Results: The bioassay successfully detected all samples of a set (n = 24) of analytically confirmed authentic Spice products, additionally providing relevant information on the 'strength' of a preparation and whether different samples may have originated from separate batches or possibly the same production batch. Finally, the methodology was applied to assess the occurrence of SCRA adulteration in a large set (n = 252) of herbal materials collected at an international dance festival. This did not reveal any positives, i.e. there were no samples that yielded a relevant CB 1 activation., Conclusion: In summary, we established SCRA screening of herbal materials as a new application for the activity-based CB 1 bioassay. The simplicity of the sample preparation, the rapid results and the universal character of the bioassay render it an effective and future-proof tool for evaluating herbal materials for the presence of SCRAs, which is relevant in the context of harm reduction., (© 2024. The Author(s).)

2-Arylethynyl (N)-methanocarba adenosine 5'-methylamides are selective A 3 adenosine receptor (AR) agonists containing a preestablished receptor-preferred pseudoribose conformation. Here, we compare analogues having bulky 2-substitution, either containing or lacking an ethynyl spacer between adenine and a cyclic group. 2-Aryl compounds 9 - 11 , 13 , 14 , 19 , 22 , 23 , 27 , 29 , 31 , and 34 , lacking a spacer, had human (h) A 3 AR K i values of 2-30 nM, and others displayed lower affinity. Mouse (m) A 3 AR affinity varied, with 2-arylethynyl having a higher affinity than 2-aryl analogues ( 7 , 8 > 3c , 3d > 3b ). However, 2-aryl-4'-truncated derivatives had greatly reduced hA 3 AR affinity, even containing affinity-enhancing N 6 -dopamine-derived substituents. Molecular modeling, including molecular dynamics simulation, predicted stable poses in the canonical A 3 AR agonist binding site, but 2-aryl (ECL2 interactions) and 2-arylethynyl (TM2 interactions) substituents have different conformations and environments. In a hA 3 AR miniGα i recruitment assay, 31 (MRS8062) was (slightly) more potent compared to a β-arrestin2 recruitment assay, both in engineered HEK293T cells, and its maximal efficacy ( E max ) was much higher (165%) than reference agonist NECA's. Thus, in the 2-aryl series, A 3 AR affinity and selectivity were variable and generally reduced compared to the 2-arylethynyl series, with a greater dependence on the specific aryl group present. Selected compounds were studied in vivo in an ischemic model of peripheral artery disease (PAD). Rigidified 2-arylethynyl analogues 3a - 3c were protective in this model of skeletal muscle ischemia-reperfusion injury/claudication, as previously shown only for moderately A 3 AR-selective ribosides or (N)-methanocarba derivatives. Thus, we have expanded the A 3 AR agonist SAR for (N)-methanocarba adenosines., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

Following the enactment of a generic ban in China in 2021, the synthetic cannabinoid market has been evolving, now encompassing even wider structural diversity. Compounds carrying a brominated core such as ADB-5'Br-BUTINACA (ADMB-B-5Br-INACA) and tail-less analogs, such as ADB-5'Br-INACA (ADMB-5Br-INACA), MDMB-5'Br-INACA, and ADB-INACA (ADMB-INACA), have been detected since late 2021. This study investigated the cannabinoid receptor (CB) activation potential of synthesized (S)-enantiomers of these substances, as well as of two predicted analogs MDMB-5'Br-BUTINACA (MDMB-B-5Br-INACA) and ADB-5'F-BUTINACA (ADMB-B-5F-INACA), using CB 1 and CB 2 β-arrestin 2 recruitment assays and a CB 1 intracellular calcium release assay. Surprisingly, the tail-less (S)-ADB-5'Br-INACA and (S)-MDMB-5'Br-INACA retained CB activity, albeit with a decreased potency compared to their tailed counterparts (S)-ADB-5'Br-BUTINACA and (S)-MDMB-5'Br-BUTINACA, respectively, which were potent and efficacious CB 1 agonists. Also, at CB 2 , tail-less analogs showed a lower potency but increased efficacy. Removing the bromine substitution ((S)-ADB-INACA) resulted in a reduced activity at CB 1 ; however, this effect was less prominent at CB 2 . Looking at tailed analogs, replacing the bromine with a fluorine substitution ((S)-ADB-5'F-BUTINACA) resulted in an increased potency and efficacy at both receptors. Furthermore, as ADB-5'Br-INACA and MDMB-5'Br-INACA have been frequently detected together in Scottish prisons, this study also evaluated the CB 1 receptor activation potential of different mixtures of their respective reference standards, showing no unexpected cannabimimetic effect of combining both substances. Lastly, two powders seized by Belgian Customs and confirmed to contain ADB-5'Br-INACA and MDMB-5'Br-INACA, respectively, were assessed for CB activity. Based on the comparison with their reference standards, varying degrees of purity were suspected., (© 2023 John Wiley & Sons Ltd.)

The rapidly evolving synthetic cannabinoid receptor agonist (SCRA) market poses significant challenges for forensic scientists. Since the enactment of a generic ban in China, a variety of new compounds have emerged capable of evading the legislation by carrying new structural features. One recent example of a SCRA with new linker and head moieties is CH-PIATA (CH-PIACA, CHX-PIATA, CHX-PIACA). CH-PIATA bears an additional methylene spacer in the linker moiety between the indole core and the traditional carbonyl component of the linker. This study describes detections in 2022 of this new SCRA in the United States, Belgium, and Scottish prisons. CH-PIATA was detected once in a seized powder by Belgian customs and 12 times in Scottish prisons in infused papers or resin. The metabolites of CH-PIATA were investigated via in vitro human liver microsome (HLM) incubations and eight metabolites were identified, dominated by oxidative biotransformations. A blood sample from the United States was confirmed to contain a mixture of SCRAs including CH-PIATA via presence of the parent and at least five of the metabolites identified from HLM incubations. Furthermore, this paper evaluates the intrinsic in vitro cannabinoid 1 and 2 (CB 1 and CB 2 ) receptor activation potential of CH-PIATA reference material and the powder seized by Belgian customs by means of β-arrestin 2 recruitment assays. Both the reference and the seized powder showed a weak activity at both CB receptors with signs of antagonism found. Based on these results, the expected harm potential of this newly emerging substance remains limited., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

New synthetic opioids (NSOs) with diverse chemical structures continue to appear on recreational drug markets worldwide. U-type opioids have become one of the largest groups of non-fentanyl-related NSOs. Starting in 2020, a previously unreported U-compound coined "β-U10" (2-naphthyl U-47700; N-[2-(dimethylamino)cyclohexyl]-N-methylnaphthalene-2-carboxamide) was identified in Australia and the United States. β-U10 is a positional isomer of α-U10 (1-naphthyl U-47700), more commonly known as "U10." Here, the first comparative in vitro pharmacological characterization of naphthyl U-47700 (U10 and β-U10), together with the structural analogue U-47700 and fentanyl, is reported. Application of a cell-based μ-opioid receptor (MOR) activation (β-arrestin 2 recruitment) assay demonstrated β-U10 (EC 50  = 348 nM; E max  = 150% vs. hydromorphone) to be less potent than U-47700 (EC 50  = 116 nM; E max  = 154%) and fentanyl (EC 50  = 9.35 nM; E max  = 146%) but considerably more active than the α-isomer (EC 50 value in the μM range). For the latter, maximum receptor activation could not be reached at 100 μM. The difference in MOR activation potential for U10 and β-U10 stresses the importance of (analytical) differentiation between closely related analytes. The emergence of β-U10 on the recreational drug market is an example of the continuing emergence of non-fentanyl-related NSOs and further emphasizes the need to closely monitor fluctuations in the drug supply., (© 2023 John Wiley & Sons, Ltd.)

Lisuride is a non-psychedelic serotonin (5-HT) 2A receptor (5-HT 2A ) agonist and analogue of the psychedelic lysergic acid diethylamide (LSD). Lisuride also acts as an agonist at the serotonin 1A receptor (5-HT 1A ), a property known to counter psychedelic effects. Here, we tested whether lisuride lacks psychedelic activity due to a dual mechanism: (1) partial agonism at 5-HT 2A and (2) potent agonism at 5-HT 1A . The in vitro effects of lisuride, LSD, and related analogues on 5-HT 2A signaling were characterized by using miniGα q and β-arrestin 2 recruitment assays. The 5-HT 1A - and 5-HT 2A -mediated effects of lisuride and LSD were also compared in male C57BL/6J mice. The in vitro results confirmed that LSD is an agonist at 5-HT 2A , with high efficacy and potency for recruiting miniGα q and β-arrestin 2. By contrast, lisuride displayed partial efficacy for both functional end points (6-52% of 5-HT or LSD E max ) and antagonized the effects of LSD. The mouse experiments demonstrated that LSD induces head twitch responses (HTRs)(ED 50 = 0.039 mg/kg), while lisuride suppresses HTRs (ED 50 = 0.006 mg/kg). Lisuride also produced potent hypothermia and hypolocomotion (ED 50 = 0.008-0.023 mg/kg) that was blocked by the 5-HT 1A antagonist WAY100635 (3 mg/kg). Blockade of 5-HT 1A prior to lisuride restored basal HTRs, but it failed to increase HTRs above baseline levels. HTRs induced by LSD were blocked by lisuride (0.03 mg/kg) or the 5-HT 1A agonist 8-OH-DPAT (1 mg/kg). Overall, our findings show that lisuride is an ultrapotent 5-HT 1A agonist in C57BL/6J mice, limiting its use as a 5-HT 2A ligand in mouse studies examining acute drug effects. Results also indicate that the 5-HT 2A partial agonist-antagonist activity of lisuride explains its lack of psychedelic effects., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

Dried blood microsampling is increasingly used for home-sampling and epidemiological studies because of its multiple advantages, including an often greatly improved analyte stability. However, a critical assessment of the stability under realistic conditions should always be performed as part of the validation, especially for unstable molecules like folates (vitamin B9). Here, the objective was to determine whether folate stability in dried blood microsamples is sufficient to allow the set-up of home-sampling studies for the monitoring of folate status in e.g. , women of reproductive age. An extensive set of stability experiments was performed to evaluate the stability of the main folate vitamer 5-methyltetrahydrofolate (5MTHF), its oxidation product MeFOX and the minor non-methyl folate vitamers 10-formylfolic acid (10FoFA), 5,10-methenyltetrahydrofolate (5,10CH+THF) and tetrahydrofolate (THF) in dried blood microsamples using volumetric absorptive microsampling (VAMS) or regular dried blood spots (DBS). The evaluations included (EDTA-anticoagulated blood was collected from a single donor measured in four replicates per condition and time point): (i) the effect of temperature (-20 °C, 4 °C, ambient temperature and 37 °C), (ii) the effect of light (during drying and storage) and humidity, and (iii) the effect of storage under vacuum and pretreatment of the microsamples with stabilizing agents on folate stability. At -20 °C and 4 °C, all folate levels were within 85 to 115% of the baseline value up till two weeks of storage in both VAMS samples and DBS. However, at room temperature the stability of the analyzed folates was only consistently observed up till three days in VAMS samples, and for none of the folates at 37 °C. Humidity had a major impact on 5,10CH+THF stability, but this could be easily improved by using desiccant. Both vacuum treatment and pretreatment of microsamples with 0.1% DL-dithiothreitol and 5% butylated hydroxytoluene improved the stability at room temperature in VAMS samples, but these effects were limited at 37 °C and in DBS. Overall, the stability of the individual folate vitamers proved to be challenging and strongly temperature- and time-dependent. Nonetheless, if controlled transport (temperature and duration) can be assured, the set-up of home-sampling studies to evaluate the folate status using dried blood microsamples can still be beneficial.

Hypoxia-inducible factor (HIF) stabilizers are listed in the World Anti-Doping Agency's prohibited list as they can increase aerobic exercise capacity. The rapid pace of emergence of highly structurally diverse HIF stabilizers could pose a risk to conventional structure-based methods in doping control to detect new investigational drugs. Therefore, we developed a strategy that is capable of detecting the presence of any HIF stabilizer, irrespective of its structure, by detecting biological activity. Previously developed cell-based HIF1/2 assays were optimized to a stable format and evaluated for their screening potential toward HIF stabilizers. Improved pharmacological characterization was established by the stable cell-based formats, and broad specificity was demonstrated by pharmacologically characterizing a diverse set of HIF stabilizers (including enarodustat, IOX2, IOX4, MK-8617, JNJ-42041935). The methodological (in solvent) limit of detection of the optimal HIF1 stable bioassay toward detecting the reference compound roxadustat was 100 nM, increasing to 50-100 ng/mL (corresponding to 617-1233 nM in-well) in matching urine samples, owing to strong matrix effects. In a practical context, a urinary limit of detection of 1.15 μg/mL (95% detection rate) was determined, confirming the matrix-dependent detectability of roxadustat in urine. Pending optimization of a universal sample preparation strategy and/or a methodology to correct for the matrix effects, this untargeted approach may serve as a complementing method in antidoping control, as theoretically, it would be capable of detecting any unknown substance with HIF stabilizing activity.

The cannabinoid 2 receptor (CB 2 R) has high therapeutic potential for multiple pathogenic processes, such as neuroinflammation. Pathway-selective ligands are needed to overcome the lack of clinical success and to elucidate correlations between pathways and their respective therapeutic effects. Herein, we report the design and synthesis of a photoswitchable scaffold based on the privileged structure of benzimidazole and its application as a functionally selective CB 2 R "efficacy-switch". Benzimidazole azo-arenes offer huge potential for the broad extension of photopharmacology to a wide range of optically addressable biological targets. We used this scaffold to develop compound 10 d, a "trans-on" agonist, which serves as a molecular probe to study the β-arrestin2 (βarr2) pathway at CB 2 R. βΑrr2 bias was observed in CB 2 R internalization and βarr2 recruitment, while no activation occurred when looking at Gα 16 or mini-Gα i . Overall, compound 10 d is the first light-dependent functionally selective agonist to investigate the complex mechanisms of CB 2 R-βarr2 dependent endocytosis., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Quality of life is often reduced in patients with sleep-wake disorders. Insomnia is commonly treated with benzodiazepines, despite their well-known side effects. Pellotine ( 1 ), a Lophophora alkaloid, has been reported to have short-acting sleep-inducing properties in humans. In this study, we set out to evaluate various in vitro and in vivo properties of 1 . We demonstrate that 1 undergoes slow metabolism; e.g. in mouse liver microsomes 65% remained, and in human liver microsomes virtually no metabolism was observed after 4 h. In mouse liver microsomes, two phase I metabolites were identified: 7-desmethylpellotine and pellotine- N -oxide. In mice, the two diastereomers of pellotine -O -glucuronide were additionally identified as phase II metabolites. Furthermore, we demonstrated by DESI-MSI that 1 readily enters the central nervous system of rodents. Furthermore, radioligand-displacement assays showed that 1 is selective for the serotonergic system and in particular the serotonin (5-HT) 1D , 5-HT 6 , and 5-HT 7 receptors, where it binds with affinities in the nanomolar range (117, 170, and 394 nM, respectively). Additionally, 1 was functionally characterized at 5-HT 6 and 5-HT 7 , where it was found to be an agonist at the former (EC 50 = 94 nM, E max = 32%) and an inverse agonist at the latter (EC 50 = 291 nM, E max = -98.6). Finally, we demonstrated that 1 dose-dependently decreases locomotion in mice, inhibits REM sleep, and promotes sleep fragmentation. Thus, we suggest that pellotine itself, and not an active metabolite, is responsible for the hypnotic effects and that these effects are possibly mediated through modulation of serotonergic receptors., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

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 β-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 (EC 50 =39.9 nM; E max =155% vs. hydromorphone) is about equally active as methadone (EC 50 =50.3 nM; E max =152%), whereas desmethylmoramide (EC 50 =1335 nM; E max =126%) is considerably less active. A close structural analogue of ketobemidone (EC 50 =134 nM; E max =156%) and methylketobemidone (EC 50 =335 nM; E max =117%), O-AMKD showed a lower potency (EC 50 =1262 nM) and efficacy (E max =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., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

The A 3 adenosine receptor (A 3 AR) is implicated in a variety of (patho)physiological conditions. While most research has focused on agonists and antagonists, inverse agonism at A 3 AR has been scarcely studied. Therefore, this study aimed at exploring inverse agonism, using two previously engineered cell lines (hA 3 ARLgBiT-SmBiTβarr2 and hA 3 ARLgBiT-SmBiTminiGα i ), both employing the NanoBiT technology. The previously established inverse agonist PSB-10 showed a decrease in basal signal in the β-arrestin 2 (βarr2) but not the miniGα i recruitment assay, indicative of inverse agonism in the former assay. Control experiments confirmed the specificity and reversibility of this observation. Evaluation of a set of presumed neutral antagonists (MRS7907, MRS7799, XAC, and MRS1220) revealed that all displayed concentration-dependent signal decreases when tested in the A 3 AR-βarr2 recruitment assay, yielding EC 50 and E max values for inverse agonism. Conversely, in the miniGα i recruitment assay, no signal decreases were observed. To assess whether this observation was caused by the inability of the ligands to induce inverse agonism in the G protein pathway, or rather by a limitation inherent to the employed A 3 AR-miniGα i recruitment assay, a GloSensor cAMP assay was performed. The outcome of the latter also suggests inverse agonism by the presumed neutral antagonists in this latter assay. These findings emphasize the importance of prior characterization of ligands in the relevant test system. Moreover, it showed the suitability of the NanoBiT βarr2 recruitment and the GloSensor cAMP assays to capture inverse agonism at the A 3 AR, as opposed to the NanoBiT miniGα i recruitment assay., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Serotonergic psychedelics are described to have activation of the serotonin 2A receptor (5-HT 2A ) as their main pharmacological action. Despite their relevance, the molecular mechanisms underlying the psychedelic effects induced by certain 5-HT 2A agonists remain elusive. One of the proposed hypotheses is the occurrence of biased agonism, defined as the preferential activation of certain signaling pathways over others. This study comparatively monitored the efficiency of a diverse panel of 4-position-substituted (and N -benzyl-derived) phenylalkylamines to induce recruitment of β-arrestin2 (βarr2) or miniGα q to the 5-HT 2A , allowing us to assess structure-activity relationships and biased agonism. All test compounds exhibited agonist properties with a relatively large range of both EC 50 and E max values. Interestingly, the lipophilicity of the 2C-X phenethylamines was correlated with their efficacy in both assays but yielded a stronger correlation in the miniGα q - than in the βarr2-assay. Molecular docking suggested that accommodation of the 4-substituent of the 2C-X analogues in a hydrophobic pocket between transmembrane helices 4 and 5 of 5-HT 2A may contribute to this differential effect. Aside from previously used standard conditions (lysergic acid diethylamide (LSD) as a reference agonist and a 2 h activation profile to assess a compound's activity), serotonin was included as a second reference agonist, and the compounds' activities were also assessed using the first 30 min of the activation profile. Under all assessed circumstances, the qualitative structure-activity relationships remained unchanged. Furthermore, the use of two reference agonists allowed for the estimation of both "benchmark bias" (relative to LSD) and "physiology bias" (relative to serotonin).

Background: The emergence of novel synthetic opioids (NSOs) is contributing to the opioid overdose crisis. While fentanyl analogs have historically dominated the NSO market, a shift towards non-fentanyl compounds is now occurring., Methods: Here, we examined the neuropharmacology of structurally distinct non-fentanyl NSOs, including U-47700, isotonitazene, brorphine, and N-desethyl isotonitazene, as compared to morphine and fentanyl. Compounds were tested in vitro using opioid receptor binding assays in rat brain tissue and by monitoring forskolin-stimulated cAMP accumulation in cells expressing the human mu-opioid receptor (MOR). Compounds were administered subcutaneously to male Sprague-Dawley rats, and hot plate antinociception, catalepsy score, and body temperature changes were measured., Results: Receptor binding results revealed high MOR selectivity for all compounds, with MOR affinities comparable to those of morphine and fentanyl (i.e., nM). All drugs acted as full-efficacy MOR agonists in the cyclic AMP assay, but nitazene analogs had greater functional potencies (i.e., pM) compared to the other drugs (i.e., nM). When administered to rats, all compounds induced opioid-like antinociception, catalepsy, and body temperature changes, but nitazenes were the most potent. Similar to fentanyl, the nitazenes had faster onset and decline of in vivo effects when compared to morphine. In vivo potencies to induce antinociception and catalepsy (i.e., ED 50 s) correlated with in vitro functional potencies (i.e., EC 50 s) but not binding affinities (i.e., K i s) at MOR., Conclusions: Collectively, our findings indicate that non-fentanyl NSOs pose grave danger to those individuals who use opioids. Continued vigilance is needed to identify and characterize synthetic opioids as they emerge in clandestine drug markets., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest., (Published by Elsevier B.V.)

The collection of capillary blood microsamples via finger-prick has several advantages over traditional blood collection. It is considered convenient and more patient-centric, enabling collection of the sample by the patient at her/his home with subsequent analysis in the lab following postal shipment. Determination of the diabetes biomarker HbA 1c in self-collected microsamples to remotely monitor diabetes patients seems to be a very promising option which could eventually lead to better treatment adaptations and disease control. This is especially convenient/relevant for patients living in areas where venipuncture is impractical, or to support virtual consultations using telemedicine. Over the years, a substantial numbers of reports on HbA 1c and microsampling have been published. However, the heterogeneity of the applied study designs and data evaluation is remarkable. This review provides a general and critical overview of these papers, along with specific points of attention that should be dealt with when aiming at implementing microsampling for reliable HbA 1c determination. We focus on the used (dried) blood microsampling techniques, collection conditions, stability of the microsamples, sample extraction, analytical methods, method validation, correlation studies with conventional venous blood samples and patient satisfaction. Lastly, the possibility of using liquid instead of dried blood microsamples is discussed. Liquid blood microsampling is expected to have similar advantages as dried blood microsampling and several studies suggest it to be a suitable approach to collect samples remotely for subsequent HbA 1c analysis in the lab., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

We present the synthesis and characterization of merged human butyrylcholinesterase ( h BChE) inhibitor/cannabinoid receptor 2 ( h CB 2 R) ligands for the treatment of neurodegeneration. In total, 15 benzimidazole carbamates were synthesized and tested for their inhibition of human cholinesterases, also with regard to their pseudoirreversible binding mode and affinity toward both cannabinoid receptors in radioligand binding studies. After evaluation in a calcium mobilization assay as well as a β-arrestin 2 (βarr2) recruitment assay, two compounds with balanced activities on both targets were tested for their immunomodulatory effect on microglia activation and regarding their pharmacokinetic properties and blood-brain barrier penetration. Compound 15d , containing a dimethyl carbamate motif, was further evaluated in vivo, showing prevention of Aβ 25-35 -induced learning impairments in a pharmacological mouse model of Alzheimer's disease for both short- and long-term memory responses. Additional combination studies proved a synergic effect of BChE inhibition and CB 2 R activation in vivo.

New psychoactive substances (NPS) are introduced on the illicit drug market at a rapid pace. Their molecular targets are often inadequately elucidated, which contributes to the delayed characterization of their pharmacological effects. Inspired by earlier findings, this study set out to investigate the µ opioid receptor (MOR) activation potential of a large set of psychedelics, substances which typically activate the serotonin (5-HT 2A ) receptor as their target receptor. We observed that some substances carrying the N-benzyl phenethylamine (NBOMe) structure activated MOR, as confirmed by both the NanoBiT® βarr2 recruitment assay and the G protein-based AequoScreen® Ca 2+ release assay. The use of two orthogonal systems proved beneficial as some aspecific, receptor independent effects were found for various analogs when using the Ca 2+ release assay. The specific 'off-target' effects at MOR could be blocked by the opioid antagonist naloxone, suggesting that these NBOMes occupy the same common opioid binding pocket as conventional opioids. This was corroborated by molecular docking, which revealed the plausibility of multiple interactions of 25I-NBOMe with MOR, similar to those observed for opioids. Additionally, structure-activity relationship findings seen in vitro were rationalized in silico for two 25I-NBOMe isomers. Overall, as MOR activity of these psychedelics was only noticed at high concentrations, we consider it unlikely that for the tested compounds there will be a relevant opioid toxicity in vivo at physiologically relevant concentrations. However, small modifications to the original NBOMe structure may result in a panel of more efficacious and potent MOR agonists, potentially exhibiting a dual MOR/5-HT 2A activation potential., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Fully automated dried blood spot (DBS) analysis for therapeutic drug monitoring (TDM) of the immunosuppressants tacrolimus, sirolimus, everolimus and cyclosporin A suffers from a so-called hematocrit (hct) effect. This effect is related to the analysis of a partial DBS punch and extractability differences imposed by blood with different hcts. As this is intrinsic to automated DBS analysis, this poses a serious drawback for accurate immunosuppressant quantification. Knowledge of a sample's hct allows to correct the derived immunosuppressant concentrations for this effect. Unfortunately, when using the DBS approach for sampling at patients' homes, this hct will typically not be available. The aim of this study was to investigate the validity of a correction algorithm during fully automated DBS analysis of immunosuppressants, based on knowledge of the DBS' hct, obtained via two distinct non-contact hematocrit prediction strategies, using either near-infrared (NIR) or ultra-violet/visible (UV/VIS) spectroscopy. For tacrolimus, sirolimus, everolimus, and cyclosporin A, 48, 47, 58 and 48 paired venous whole blood and venous DBS patient samples were collected, respectively, and analyzed using an automated DBS-MS 500 HCT extraction unit coupled to a liquid chromatography tandem mass spectrometry system. Additionally, for all 201 samples the hct of the DBS was predicted based on NIR and UV/VIS spectroscopy. For tacrolimus and cyclosporin A, both hct prediction strategies allowed for adequate correction of the hct effect. Also for sirolimus and everolimus the results greatly improved after hct correction, although a hct bias remained for sirolimus and for everolimus a slightly significant hct effect was observed after NIR- and UV/VIS-based correction. Application of both hct prediction strategies ensured that clinical acceptance limits (i.e. ≥ 80% of the samples within 20% difference compared to whole blood) were met for all analytes. In conclusion, we demonstrated that non-contact hct prediction strategies, applied in tandem with fully automated DBS analysis, can be used to adequately correct immunosuppressant concentrations, yielding a good agreement with whole blood., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In recent years, a lot of attention has been given to a more patient-centric therapeutic drug monitoring (TDM) of immunosuppressant drugs (tacrolimus, sirolimus, everolimus and cyclosporin A) by the use of microsampling techniques. By adopting Dried Blood Spots (DBS) after a finger prick, instead of conventional venous blood draws, follow-up can (partially) be established from patients' homes. Despite the many advantages of DBS, one of the major disadvantages associated with this technique is the well described hematocrit (hct) effect. In order to overcome the hct area bias, different strategies have been proposed, amongst which the use of dried blood sampling techniques based on the volumetric collection of blood. The aim of this study was to evaluate the use of the Capitainer® qDBS (quantitative Dried Blood Spot) device for the combined TDM of four immunosuppressants and creatinine from a single qDBS. The set-up of an adequate sample preparation allowing both immunosuppressants and creatinine quantification was one of the key challenges in the method development due to device-specific interferences. Liquid chromatography tandem-mass spectrometry methods for the quantification of tacrolimus, sirolimus, everolimus, cyclosporin A and creatinine from qDBS (10 μL) were developed and validated based on international guidelines, also taking into account DBS-specific parameters. The methods proved to be accurate and reproducible, with absolute biases below 10% and within-run CVs (%) below 8% over a calibration range from 1 to 50 ng/mL for tacrolimus, sirolimus and everolimus, 20-1500 ng/mL for cyclosporin A, and 15-700 μmol/L for creatinine. Reproducible (CV < 15%) IS-compensated relative recovery values were obtained, showing no hematocrit-dependence (compared to a hct of 0.37), except for cyclosporin A at higher hct values. Application on venous blood left-over patient samples showed good agreement between the results of Capitainer® qDBS and whole blood with 98% (47/48), 93% (41/44), 89% (41/46), 88% (38/43) and 89% (116/131) of the samples lying within 20% of the whole blood result for tacrolimus, sirolimus, everolimus, cyclosporin A and plasma/serum for creatinine, respectively. For creatinine a blood/plasma ratio of 0.85 was found and used to convert qDBS results to plasma/serum results. As a next step, capillary finger prick samples will need to demonstrate the clinical applicability of the method in a real life setting., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The synthetic cannabinoid receptor agonist (SCRA) market is undergoing important changes since the enactment of the 2021 class-wide generic SCRA ban in China, one of the most important source countries for new psychoactive substances (NPS). Recently, various compounds with new structural features, synthesized to bypass this legislation, have entered the recreational drug market. Certain monocyclic pyrazole-carrying "FUPPYCA" SCRAs have been sporadically detected since 2015 without gaining further popularity. However, as evidenced by their recent detection in Scottish prisons, 5F-3,5-AB-PFUPPYCA and 3,5-ADB-4en-PFUPPYCA have re-emerged, potentially triggered by the new legislative ban. The aim of this study was to characterize the in vitro intrinsic CB 1 and CB 2 receptor activation potential of 5F-3,5-AB-PFUPPYCA and 3,5-ADB-4en-PFUPPYCA, as well as 4 analogs (5F-3,5-ADB-PFUPPYCA, 3,5-AB-CHMFUPPYCA, 5,3-AB-CHMFUPPYCA and 5,3-ADB-4en-PFUPPYCA) using live cell β-arrestin 2 recruitment assays. Most analogs were essentially inactive at either CB 1 or CB 2 , with only 3,5-AB-CHMFUPPYCA, 5,3-AB-CHMFUPPYCA and 5,3-ADB-4en-PFUPPYCA showing a limited activation potential at CB 1 . Furthermore, the importance of the position of the tail structure was demonstrated, with 5,3 regioisomers being more active than their 3,5 analogs. Moreover, all compounds exhibited antagonistic behavior at both receptors, which may be associated with their structural resemblance to cannabinoid antagonists and inverse agonists. Although the 3,5 regioisomers of these "FUPPYCA" SCRAs circumvent the Chinese ban, it is unlikely that these SCRAs will pose a major threat to public health, given the lack of pronounced CB receptor activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In the field of solid organ transplantation, chemotherapy and autoimmune disorders, treatment with immunosuppressant drugs requires intensive follow-up of the blood concentrations via therapeutic drug monitoring (TDM) because of their narrow therapeutic window and high intra- and inter-subject variability. This requires frequent hospital visits and venepunctures to allow the determination of these analytes, putting a high burden on the patients. In the context of patient-centric thinking, it is becoming increasingly established that at least part of these conventional blood draws could be replaced by microsampling, allowing home-sampling and increasing the quality of life for these patients. In this review we discuss the published methods - mostly using liquid chromatography coupled to tandem mass spectrometry - that have utilized (volumetric) dried blood samples as an alternative for conventional liquid whole blood for the TDM of immunosuppressant drugs. Furthermore, some pre-analytical considerations using DBS or volumetric alternatives are considered, as well as the applicability on clinical samples. The implementation status in clinical practice is also discussed, including (1) the cost-effectiveness of this approach compared to venepuncture, (2) the availability of multiplexed methods, (3) the status of harmonization and (4) patient perception. A brief perspective on potential future developments for the dried blood-based TDM of immunosuppressant drugs is provided, by considering how obstacles for the implementation of these strategies into clinical practice might be overcome., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Over 200 synthetic cannabinoid receptor agonists (SCRAs) have been identified as new psychoactive substances. Effective monitoring and characterization of SCRAs are hindered by the rapid pace of structural evolution. Ahead of possible appearance on the illicit drug market, new SCRAs were synthesized to complete a systematic library of cumyl-indole- ( e.g. , CUMYL-CPrMICA, CUMYL-CPMICA) and cumyl-indazole-carboxamides ( e.g. , CUMYL-CPrMINACA, CUMYL-CPMINACA), encompassing butyl, pentyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl tails. Comprehensive pharmacological characterization was performed with three assay formats, monitoring the recruitment of either wild-type or C-terminally truncated (βarr2d366) β-arrestin2 to the activated cannabinoid 1 receptor (CB 1 ) or monitoring G βγ -mediated membrane hyperpolarization. Altered compound characterization was observed when comparing derived potency (EC 50 ) and efficacy ( E max ) values from both assays monitoring the same or a different signaling event, whereas ranges and ranking orders were similar. Structure-activity relationships (SAR) were assessed in threefold, resulting in the identification of the pendant tail as a critical pharmacophore, with the optimal chain length for CB 1 activation approximating an n- pentyl ( e.g. , cyclopentylmethyl or cyclohexylmethyl tail). The activity of the SCRAs encompassing cyclic tails decreased with decreasing number of carbons forming the cyclic moiety, with CUMYL-CPrMICA showing the least CB 1 activity in all assay formats. The SARs were rationalized via molecular docking, demonstrating the importance of the optimal steric contribution of the hydrophobic tail. While SAR conclusions remained largely unchanged, the differential compound characterization by both similar and different assay designs emphasizes the importance of detailing specific assay characteristics to allow adequate interpretation of potencies and efficacies.

The pandemic readiness toolbox needs to be extended, targeting different biomolecules, using orthogonal experimental set-ups. Here, we build on our Cov-MS effort using LC-MS, adding SISCAPA technology to enrich proteotypic peptides of the SARS-CoV-2 nucleocapsid (N) protein from trypsin-digested patient samples. The Cov 2 MS assay is compatible with most matrices including nasopharyngeal swabs, saliva, and plasma and has increased sensitivity into the attomole range, a 1000-fold improvement compared to direct detection in a matrix. A strong positive correlation was observed with qPCR detection beyond a quantification cycle of 30-31, the level where no live virus can be cultured. The automatable sample preparation and reduced LC dependency allow analysis of up to 500 samples per day per instrument. Importantly, peptide enrichment allows detection of the N protein in pooled samples without sensitivity loss. Easily multiplexed, we detect variants and propose targets for Influenza A and B detection. Thus, the Cov 2 MS assay can be adapted to test for many different pathogens in pooled samples, providing longitudinal epidemiological monitoring of large numbers of pathogens within a population as an early warning system.

Background: The pharmacokinetic (PK) profile of a drug is influenced by several factors, which can lead to a suboptimal dosing regimen in specific patient populations. As obesity becomes increasingly prevalent, it is important that optimized dosing schemes are available for these patients. To set up such dosing schemes, PK studies should be performed in this population. Regarding paracetamol (acetaminophen [APAP]), obese patients would benefit from a tailored dosing scheme, as both the volume of distribution and metabolism are increased compared with non-obese patients. This includes metabolism by cytochrome P450 2E1, which is involved in APAP-associated hepatotoxicity. To decrease the burden for patients in these PK studies, finger-prick sampling could be used., Objective: The aim of this study was to compare the quantitative determination of APAP and four metabolites in different blood-based matrices and to determine if capillary dried blood samples, collected directly following finger-prick, could serve as a tool to investigate APAP PK in obese and non-obese patients., Methods: In this study, we performed a clinical validation of methods for the determination of APAP and four of its metabolites (APAP-glucuronide, APAP-sulfate, APAP-mercapturate, and APAP-cysteine) in blood, plasma, and dried blood. The latter was obtained by volumetric absorptive microsampling (VAMS), either starting from the venous blood or collected directly following a finger-prick. Results were compared between the different matrices and, in addition, blood:plasma (B:P) ratios were determined for the different analytes., Results: Liquid and dried venous blood results were in good agreement. Furthermore, differences between capillary (finger-prick) and venous VAMS blood samples remained limited for most analytes. However, for APAP-cysteine, caution should be paid to the interpretation of concentrations in (dried) blood. With the exception of APAP, concentrations were higher in plasma compared with blood, with B:P ratios ranging between 0.52 and 0.65. A time-dependent change in median B:P ratio was observed for APAP and APAP-cysteine. Additionally, a time-dependent trend was seen for APAP, as well as for APAP-glucuronide and APAP-mercapturate, for the distribution between capillary and venous blood., Conclusions: We demonstrated that finger-prick sampling is a viable alternative to conventional venous blood sampling to investigate the PK of APAP and its metabolites in obese and non-obese patients., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Dried blood spot(s) (DBS) microsampling has increasingly attracted interest as a patient-centric alternative to conventional blood withdrawal. Despite the many advantages associated with DBS sampling, its widespread use in clinical practice is still hampered, which is mainly caused by the hematocrit (Hct) effect. One approach to cope with this issue is the Hct prediction of DBS using ultraviolet-visible (UV-Vis) spectroscopy. Recently, a UV-Vis-based Hct prediction module has been incorporated into the automated CAMAG® DBS-MS 500 HCT system. However, although a proof-of-principle yielded promising results, there is no formal in-depth evaluation of the performance of this module. Hence, it remained to be established to what extent automated Hct prediction of DBS via this module can universally be applied and generates acceptable results. Using authentic patient samples, we set up and validated a calibration model and evaluated whether this could serve as a 'generic' calibration model for different, independent Hct prediction modules. A quadratic calibration curve with 1/ x 2 weighting was established. The bias, intra-day and total precision were below 0.025 L L -1 , 2.2% and 2.7%, respectively. Additionally, the influence of storage and the robustness of the method was evaluated. Moreover, a lab-lab comparison of the performance of the Hct module of two independently operated instruments demonstrated that the validated model can be used as a generic calibration model. Finally, application of the method to venous DBS ( n = 48) prepared from patient samples in the context of therapeutic drug monitoring of tacrolimus revealed a good concordance between the actual ( i.e. Sysmex-based) and UV-Vis-based predicted Hct.

4-Phosphoryloxy- N , N -dimethyltryptamine (psilocybin) is a naturally occurring tertiary amine found in many mushroom species. Psilocybin is a prodrug for 4-hydroxy- N , N -dimethyltryptamine (psilocin), which induces psychedelic effects via agonist activity at the serotonin (5-HT) 2A receptor (5-HT 2A ). Several other 4-position ring-substituted tryptamines are present in psilocybin-containing mushrooms, including the secondary amine 4-phosphoryloxy- N -methyltryptamine (baeocystin) and the quaternary ammonium 4-phosphoryloxy- N , N , N -trimethyltryptamine (aeruginascin), but these compounds are not well studied. Here, we investigated the structure-activity relationships for psilocybin, baeocystin, and aeruginascin, as compared to their 4-acetoxy and 4-hydroxy analogues, using in vitro and in vivo methods. Broad receptor screening using radioligand binding assays in transfected cells revealed that secondary and tertiary tryptamines with either 4-acetoxy or 4-hydroxy substitutions display nanomolar affinity for most human 5-HT receptor subtypes tested, including the 5-HT 2A and the serotonin 1A receptor (5-HT 1A ). The same compounds displayed affinity for 5-HT 2A and 5-HT 1A in mouse brain tissue in vitro and exhibited agonist efficacy in assays examining 5-HT 2A -mediated calcium mobilization and β-arrestin 2 recruitment. In mouse experiments, only the tertiary amines psilocin, psilocybin, and 4-acetoxy- N , N -dimethyltryptamine (psilacetin) induced head twitch responses (ED 50 0.11-0.29 mg/kg) indicative of psychedelic-like activity. Head twitches were blocked by 5-HT 2A antagonist pretreatment, supporting 5-HT 2A involvement. Both secondary and tertiary amines decreased body temperature and locomotor activity at higher doses, the effects of which were blocked by 5-HT 1A antagonist pretreatment. Across all assays, the pharmacological effects of 4-acetoxy and 4-hydroxy compounds were similar, and these compounds were more potent than their 4-phosphoryloxy counterparts. Importantly, psilacetin appears to be a prodrug for psilocin that displays substantial serotonin receptor activities of its own., Competing Interests: The authors declare the following competing financial interest(s): A.R.C. has an ownership stake in CaaMTech, Inc., which owns patent applications covering new tryptamine compounds, their compositions, formulations, novel crystalline forms, methods of treatment, and methods for synthesis. No other authors report any competing financial interests related to the present work., (© 2022 American Chemical Society.)

Synthetic cannabinoid receptor agonists (SCRAs) pose a danger to public health. This study focused on individuals experiencing recreational drug toxicity who had used 5F-MDMB-PICA.Patient records were evaluated regarding vital signs, Glasgow Coma Scale (GCS) and clinical features. Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) confirmed and quantified the presence of 5F-MDMB-PICA (and/or metabolites) as the only SCRA present in the serum of 71 patients. Cannabinoid activity was evaluated by a cannabinoid receptor (CB 1 ) bioassay, to assess the relationship between serum concentrations and ex vivo human CB 1 activation potential. Furthermore, a link with the clinical presentation was appraised.5F-MDMB-PICA and five metabolites were pharmacologically profiled in vitro, revealing theoretically possible contributions of two active in vivo metabolites to overall cannabinoid activity. Serum concentrations of 5F-MDMB-PICA were correlated to the ex vivo cannabinoid activity, revealing a sigmoidal relationship. The latter could also be predicted based on pharmacological characterization of 5F-MDMB-PICA and its metabolites and an in-depth investigation of the bioassay outcome. Clinically, the GCS showed a significant trend (decrease) with increasing ex vivo cannabinoid activity.This is the first study to evaluate possible toxic effects of 5F-MDMB-PICA in a unique large patient cohort. It allows a better understanding of 5F-MDMB-PICA and metabolites in humans, suggesting a negligible contribution by 5F-MDMB-PICA metabolites to the overall cannabinoid activity in serum. Additionally, this work shows that in vitro pharmacological characterization allows close prediction of an individual's ex vivo CB 1 activity, the latter showing a relationship with the level of consciousness., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The serotonin 2A receptor (5-HT 2A R) is the mediator of the psychedelic effects of serotonergic psychedelics, which have shown promising results in clinical studies for several neuropsychiatric indications. The 5-HT 2A R is able to signal through the Gα q and β-arrestin effector proteins, but it is currently not known how the different signaling pathways contribute to the therapeutic effects mediated by serotonergic psychedelics. In the present work, we have evaluated the subtype-selective 5-HT 2A R agonist 25CN-NBOH and a series of close analogues for biased signaling at this receptor. These ligands were designed to evaluate the role of interactions with Ser159 3×36 . The lack of interaction between this hydroxyl moiety and Ser159 3×36 resulted in detrimental effects on potency and efficacy in both βarr2 and miniGα q recruitment assays. Remarkably, Gα q -mediated signaling was considerably more affected. This led to the development of the first efficacious βarr2-biased 5-HT 2A R agonists 4a-b and 6e-f , βarr2 preferring, relative to lysergic acid diethylamide (LSD).

In recent years, several nations have implemented various measures to control the surge of new synthetic cannabinoid receptor agonists (SCRAs) entering the recreational drug market. In July 2021, China put into effect a new generic legislation, banning SCRAs containing one of seven general core scaffolds. However, this has driven manufacturers towards the synthesis of SCRAs with alternative core structures, exemplified by the recent emergence of "OXIZID SCRAs." Here, using in vitro β-arrestin2 recruitment assays, we report on the CB 1 and CB 2 potency and efficacy of five members of this new class of SCRAs: BZO-HEXOXIZID, BZO-POXIZID, 5-fluoro BZO-POXIZID, BZO-4en-POXIZID, and BZO-CHMOXIZID. All compounds behaved as full agonists at CB 1 and partial agonists at CB 2 . Potencies ranged from 84.6 to 721 nM at CB 1 and 2.21 to 25.9 nM at CB 2 . Shortening the n-hexyl tail to a pentyl tail enhanced activity at both receptors. Fluorination of this pentyl analog did not yield a higher receptor activation potential, whereas an unsaturated tail resulted in decreased potency and efficacy at CB 1 . The cyclohexyl methyl analog BZO-CHMOXIZID was the most potent compound at both receptors, with EC 50 values of 84.6 and 2.21 nM at CB 1 and CB 2 , respectively. Evaluation of the activity of a seized powder containing BZO-4en-POXIZID suggested a high purity, in line with high-performance liquid chromatography coupled to diode-array detection (HPLC-DAD), gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography coupled to time-of-flight mass spectrometry (LC-QTOF-MS), and Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) analysis. Furthermore, all tested compounds showed a preference for CB 2 , except for BZO-POXIZID. Overall, these findings inform public health officials, law enforcement agencies, and clinicians on these newly emerging SCRAs., (© 2022 John Wiley & Sons Ltd.)

Following the class-wide ban of synthetic cannabinoid receptor agonists (SCRAs) in China, SCRAs carrying new core and linker structures, aimed at circumventing the recent Chinese generic legislation, have appeared on the recreational drug market. A very recent example is (S)-2-(2-(1-(4-fluorobenzyl)-1H-indol-3-yl)acetamido)-3,3-dimethylbutanamide (ADB-FUBIATA), which is structurally closely related to the potent SCRA ADB-FUBICA, but carries an additional methylene in the linker region of the molecule. ADB-FUBIATA has recently been identified in seized materials in China, Russia, the United States, and also Belgium; however, its pharmacological characteristics were unknown. The aim of this study was to evaluate the intrinsic cannabinoid receptor (hCB 1 and hCB 2 ) activation potential of this previously unknown substance via two distinct yet similar in vitro β-arrestin2 recruitment assays, based on the NanoLuc Binary Technology®. At CB 1 , a potency of 635 nM (EC 50 ) was found, with an efficacy (E max ) of 141% relative to the reference compound CP55,940. On the other hand, ADB-FUBIATA had almost no activity at CB 2 , indicative of a clear CB 1 selectivity. Interestingly, this activation pattern differs markedly from that observed for ADB-FUBICA, which was previously found to be potent and efficacious at both cannabinoid receptors. Additionally, the bioassays were applied to a seized powder containing ADB-FUBIATA, as analytically confirmed by high-performance liquid chromatography coupled to diode-array detection (HLPC-DAD), gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography couple to time-of-flight mass spectrometry (LC-QTOF-MS), Fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance (NMR). The EC 50 and E max values obtained for this powder were very similar to those of the ADB-FUBIATA analytical standard, suggesting a high purity of the powder, although analytical techniques did reveal that the sample was not entirely pure., (© 2022 John Wiley & Sons Ltd.)

Activation of the human cannabinoid receptor type 1 ( h CB 1 R) with high spatiotemporal control is useful to study processes involved in different pathologies related to nociception, metabolic alterations, and neurological disorders. To synthesize new agonist ligands for h CB 1 R, we have designed different classes of photoswitchable molecules based on an indole core. The modifications made to the central core have allowed us to understand the molecular characteristics necessary to design an agonist with optimal pharmacological properties. Compound 27a shows high affinity for CB 1 R ( K i ( cis- form) = 0.18 μM), with a marked difference in affinity with respect to its inactive " trans -off" form (CB 1 R K i trans/cis ratio = 5.4). The novel compounds were evaluated by radioligand binding studies, receptor internalization, sensor receptor activation (GRABeCB2.0), Western blots for analysis of ERK1/2 activation, NanoBiT βarr2 recruitment, and calcium mobilization assays, respectively. The data show that the novel agonist 27a is a candidate for studying the optical modulation of cannabinoid receptors (CBRs), serving as a new molecular tool for investigating the involvement of h CB 1 R in disorders associated with the endocannabinoid system.

Background: Synthetic cannabinoid receptor agonists (SCRAs) are amongst the largest groups of new psychoactive substances (NPS). Their often high activity at the CB1 cannabinoid receptor frequently results in intoxication, imposing serious health risks. Hence, continuous monitoring of these compounds is important, but challenged by the rapid emergence of novel analogues that are missed by traditional targeted detection strategies. We addressed this need by performing an activity-based, universal screening on a large set (n = 968) of serum samples from patients presenting to the emergency department with acute recreational drug or NPS toxicity., Methods: We assessed the performance of an activity-based method in detecting newly circulating SCRAs compared with liquid chromatography coupled to high-resolution mass spectrometry. Additionally, we developed and evaluated machine learning models to reduce the screening workload by automating interpretation of the activity-based screening output., Results: Activity-based screening delivered outstanding performance, with a sensitivity of 94.6% and a specificity of 98.5%. Furthermore, the developed machine learning models allowed accurate distinction between positive and negative patient samples in an automatic manner, closely matching the manual scoring of samples. The performance of the model depended on the predefined threshold, e.g., at a threshold of 0.055, sensitivity and specificity were both 94.0%., Conclusion: The activity-based bioassay is an ideal candidate for untargeted screening of novel SCRAs. The combination of this universal screening assay and a machine learning approach for automated sample scoring is a promising complement to conventional analytical methods in clinical practice., (© American Association for Clinical Chemistry 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Serotonergic psychedelics are substances that induce alterations in mood, perception, and thought, and have the activation of serotonin (5-HT) 2A receptors (5-HT 2A Rs) as a main pharmacological mechanism. Besides their appearance on the (illicit) drug market, e.g. as new psychoactive substances, their potential therapeutic application is increasingly explored. This group of substances demonstrates a broad structural variety, leading to insufficiently described structure-activity relationships, hence illustrating the need for better functional characterization. This review therefore elaborates on the in vitro molecular techniques that have been used the most abundantly for the characterization of (psychedelic) 5-HT 2A R agonists. More specifically, this review covers assays to monitor the canonical G protein signaling pathway (e.g. measuring G protein recruitment/activation, inositol phosphate accumulation, or Ca 2+ mobilization), assays to monitor non-canonical G protein signaling (such as arachidonic acid release), assays to monitor β-arrestin recruitment or signaling, and assays to monitor receptor conformational changes. In particular, focus lies on the mechanism behind the techniques, and the specific advantages and challenges that are associated with these. Additionally, several variables are discussed that one should consider when attempting to compare functional outcomes from different studies, both linked to the specific assay mechanism and linked to its specific execution, as these may heavily impact the assay outcome., (© 2022 International Society for Neurochemistry.)

New psychoactive substances (NPS), formerly also referred to as "designer drugs", are often synthetic derivatives of existing psychoactive drugs, their diverse structures aiming at circumventing legislation and detection while their effects mimic those of traditional drugs of abuse. Of these, the group of new synthetic opioids (NSOs) has been one of the fastest growing NPS subclasses in the last couple of years, with over 70 new compounds detected in Europe since 2009. Apart from effects such as euphoria and analgesia, opioid use is associated with severe side effects such as constipation and respiratory depression. The μ-opioid receptor (MOR), a class A G protein-coupled receptor, is responsible for most of the therapeutic and adverse opioid effects. Insight into the pharmacology of opioids can aid the implementation of proactive harm reduction strategies, as well as the development of safer opioid analgesics. This review aims at assembling the available information on in vitro MOR agonism of the emerging class of new synthetic opioids, with a special focus on functional assays monitoring G protein and β-arrestin pathways., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background and Aims: Phosphatidylethanol 16:0/18:1 (PEth) is a biomarker for alcohol intake. It has a half-life of 7.9 days. Chronic alcohol consumption causes high PEth values. It can take weeks before PEth values fall below the decision limit for 'alcohol abstinence'. Our aim was to validate whether alcohol abstinence can be determined based on two consecutive PEth results above the decision limit., Design: Observational study., Setting: Belgium, February 2019. The study was linked to a social initiative in Belgium, 'Tournée Minérale'., Participants: Adults (aged > 18 years, n = 796) with varying drinking habits who self-reportedly refrained from alcohol consumption during the study., Measurements: A validated liquid chromatography-tandem mass spectrometry method was used to quantify PEth in participants' dried blood samples, collected at three time-points via remote fingerprick-based self-sampling., Findings: A population-based algorithm to evaluate abstinence based on 95% prediction limits was developed by fitting a linear mixed-effect model to discern patterns in PEth elimination over time. It took intra- and inter-individual variability into consideration. The algorithm was included in a two-step decision tree, assessing whether (i) PEth values fell within the prediction interval and (ii) the slope between two PEth values was consistent with no alcohol consumption. Data for 74 participants reporting no alcohol intake during the study were used for validation. With a detection limit of 'four units spread over 14 days', the sensitivity and specificity of the decision tree was 89%., Conclusions: Claims of alcohol abstinence can be verified using a two-step decision tree for phosphatidylethanol 16:0/18:1 values, even when those values are above the limit for 'alcohol abstinence'., (© 2022 Society for the Study of Addiction.)

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-β-arrestin2 activation assay, N-pyrrolidino etonitazene displayed high potency (EC 50  = 0.348 nM), similar to etonitazene (EC 50  = 0.360 nM), and largely exceeding the potencies of fentanyl (EC 50  = 14.9 nM) and morphine (EC 50  = 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 (ED 50  = 0.0017 mg/kg) was tenfold and 2,000-fold greater than fentanyl (ED 50  = 0.0209 mg/kg) and morphine (ED 50  = 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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)