Untargeted Detection of HIF Stabilizers in Doping Samples: Activity-Based Screening with a Stable In Vitro Bioassay.

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.

Remote HbA1c testing via microsampling: fit for purpose?

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 HbA1c 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 HbA1c 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 HbA1c 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 HbA1c analysis in the lab.

Interpreting mono- and poly-SCRA intoxications from an activity-based point of view: JWH-018 equivalents in serum as a comparative measure.

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 CB1 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 CB1 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 CB1 activity.

Investigation of the intrinsic cannabinoid activity of hemp-derived and semisynthetic cannabinoids with ß-arrestin2 recruitment assays-and how this matters for the harm potential of seized drugs.

Cultivation of industrial low-Δ9-tetrahydrocannabinol (Δ9-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 Δ9-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 CB1 cannabinoid receptor with a ß-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 CB1 activation than Δ9-THC, based on either higher efficacy (Emax) or higher potency (EC50). Structure-activity relationships were assessed for Δ9-THC and Δ8-THC homologs encompassing elongated alkyl chains. Additionally, stereoisomer-specific differences in CB1 activity were established for various THC isomers (Δ7-THC, Δ10-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 Δ9-THC products.

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.

Activity-based detection of synthetic cannabinoid receptor agonists in plant materials.

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 (CB1) 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 ß-arrestin 2 to activated CB1. 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 CB1 activation. CONCLUSION: In summary, we established SCRA screening of herbal materials as a new application for the activity-based CB1 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.

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.

A Fentanyl Hapten-Displaying Lipid Nanoparticle Vaccine that Non-Covalently Encapsulates a TLR7/8 Agonist and T-helper Epitope Induces Protective Anti-Fentanyl Immunity.

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.

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.

Off-target activity of NBOMes and NBOMe analogs at the µ opioid receptor.

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-HT2A) 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® ßarr2 recruitment assay and the G protein-based AequoScreen® Ca2+ release assay. The use of two orthogonal systems proved beneficial as some aspecific, receptor independent effects were found for various analogs when using the Ca2+ 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-HT2A activation potential.

Visible-Light Photoswitchable Benzimidazole Azo-Arenes as ß-Arrestin2-Biased Selective Cannabinoid 2 Receptor Agonists.

The cannabinoid 2 receptor (CB2 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 CB2 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 ß-arrestin2 (ßarr2) pathway at CB2 R. ßΑrr2 bias was observed in CB2 R internalization and ßarr2 recruitment, while no activation occurred when looking at Gα16 or mini-Gαi . Overall, compound 10 d is the first light-dependent functionally selective agonist to investigate the complex mechanisms of CB2 R-ßarr2 dependent endocytosis.

In vitro assays for the functional characterization of (psychedelic) substances at the serotonin receptor 5-HT2A R.

Serotonergic psychedelics are substances that induce alterations in mood, perception, and thought, and have the activation of serotonin (5-HT) 2A receptors (5-HT2A 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-HT2A 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 Ca2+ mobilization), assays to monitor non-canonical G protein signaling (such as arachidonic acid release), assays to monitor ß-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.

Cov2MS: An Automated and Quantitative Matrix-Independent Assay for Mass Spectrometric Measurement of SARS-CoV-2 Nucleocapsid Protein.

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 Cov2MS 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 Cov2MS 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.

Machine Learning to Assist in Large-Scale, Activity-Based Synthetic Cannabinoid Receptor Agonist Screening of Serum Samples.

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.

In-depth evaluation of automated non-contact reflectance-based hematocrit prediction of dried blood spots.

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/x2 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.

Linking in vitro and ex vivo CB1 activity with serum concentrations and clinical features in 5F-MDMB-PICA users to better understand SCRAs and their metabolites.

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 (CB1) bioassay, to assess the relationship between serum concentrations and ex vivo human CB1 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 CB1 activity, the latter showing a relationship with the level of consciousness.

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.

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.

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.