The 'ABC' of split-nanoluciferase HIF heterodimerization bioassays: Applications, Benefits & Considerations.

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-HIFß 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.

Detection of N-desethyl etonitazene in a drug checking sample: Chemical analysis and pharmacological characterization of a recent member of the 2-benzylbenzimidazole "nitazene" class.

The emergence of 2-benzylbenzimidazole "nitazene" opioids is stirring up the recreational synthetic opioid market. Many nitazene analogues act as potent agonists at the µ­opioid receptor (MOR), as demonstrated in various in vitro and in vivo studies. Severe intoxication and overdose deaths associated with nitazene analogues are increasingly being reported. Nitazene opioids are classified as a public health threat, stressing the need for close monitoring of new developments on the recreational drug market. This study reports on the detection of N-desethyl etonitazene in a sample handed in by a recreational drug user at a Swiss drug checking service in August 2023. The person bought the sample through an internet source where it was stated to contain isotonitazene. Chemical analyses were conducted to characterize the sample, i.e. nuclear magnetic resonance (NMR), capillary electrophoresis (CE), and high-resolution mass spectrometry (HRMS). The sample was additionally investigated using two different in vitro MOR activation assays. NMR and high-performance liquid chromatography (HPLC) coupled to HRMS confirmed the presence of N-desethyl etonitazene at a high purity and in the absence of isotonitazene and etonitazene. N-Desethyl nitazene analogues have been detected before as metabolites of isotonitazene and etonitazene. However, as first seen with N-desethyl isotonitazene, they are now emerging as standalone drugs. The applied bioassays demonstrated increased efficacy and approximately 6-9-fold higher potency of N-desethyl etonitazene at MOR compared to fentanyl. N-Desethyl etonitazene showed EC50 values of 3.35 nM and 0.500 nM in the ß-arrestin 2 recruitment and Aequoscreen® assays, respectively. The opioid activity present in the collected sample was additionally evaluated using the bioassays and showed good overlap with the reference standard, in line with the analytical purity assessment. This demonstrates the potential of these bioassays to provide a rapid opioid activity assessment of authentic samples. The emergence of other N-desethyl nitazene analogues must be considered during forensic and clinical toxicology casework, to avoid misclassification of intake of such analogues as metabolites. Finally, drug checking services enable the close monitoring of market developments and trends and are of great value for early warning and harm reduction purposes.

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.

Elucidating the harm potential of brorphine analogues as new synthetic opioids: Synthesis, in vitro, and in vivo characterization.

The emergence of new synthetic opioids (NSOs) has added complexity to recreational opioid markets worldwide. While NSOs with diverse chemical structures have emerged, brorphine currently remains the only NSO with a piperidine benzimidazolone scaffold. However, the emergence of new generations of NSOs, including brorphine analogues, can be anticipated. This study explored the pharmaco-toxicological, opioid-like effect profile of brorphine alongside its non-brominated analogue (orphine) and three other halogenated analogues (fluorphine, chlorphine, iodorphine). In vitro, radioligand binding assays in rat brain tissue indicated that all analogues bind to the µ-opioid receptor (MOR) with nM affinity. While analogues with smaller-sized substituents showed the highest MOR affinity, further in vitro characterization via two cell-based (HEK 293T) MOR activation (ß-arrestin 2 and mini-Gαi recruitment) assays indicated that chlorphine, brorphine, and iodorphine were generally the most active MOR agonists. None of the compounds showed significant in vitro biased agonism compared to hydromorphone. In vivo, we investigated the effects of intraperitoneal (IP) administration of the benzimidazolones (0.01-15 mg/kg) on mechanical and thermal antinociception in male CD-1 mice. Chlorphine and brorphine overall induced the highest levels of antinociception. Furthermore, the effects on respiratory changes induced by a fixed dose (15 mg/kg IP) of the compounds were investigated using non-invasive plethysmography. Fluorphine-, chlorphine-, and brorphine-induced respiratory depressant effects were the most pronounced. For some compounds, pretreatment with naloxone (6 mg/kg IP) could not reverse respiratory depression. Taken together, brorphine-like piperidine benzimidazolones are opioid agonists that have the potential to cause substantial harm to users should they emerge as NSOs. This article is part of the Special Issue on "Novel Synthetic Opioids (NSOs)".

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.

Self-Sampling by Adolescents at Home: Assessment of the Feasibility to Successfully Collect Blood Microsamples by Inexperienced Individuals.

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.

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.

2-Substituted (N)-Methanocarba A3 Adenosine Receptor Agonists: In Silico, In Vitro, and In Vivo Characterization.

2-Arylethynyl (N)-methanocarba adenosine 5'-methylamides are selective A3 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) A3AR K i values of 2-30 nM, and others displayed lower affinity. Mouse (m) A3AR 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 hA3AR affinity, even containing affinity-enhancing N 6-dopamine-derived substituents. Molecular modeling, including molecular dynamics simulation, predicted stable poses in the canonical A3AR agonist binding site, but 2-aryl (ECL2 interactions) and 2-arylethynyl (TM2 interactions) substituents have different conformations and environments. In a hA3AR miniGαi recruitment assay, 31 (MRS8062) was (slightly) more potent compared to a ß-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, A3AR 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 A3AR-selective ribosides or (N)-methanocarba derivatives. Thus, we have expanded the A3AR agonist SAR for (N)-methanocarba adenosines.

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.

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.

Comparative Pharmacological Effects of Lisuride and Lysergic Acid Diethylamide Revisited.

Lisuride is a non-psychedelic serotonin (5-HT) 2A receptor (5-HT2A) agonist and analogue of the psychedelic lysergic acid diethylamide (LSD). Lisuride also acts as an agonist at the serotonin 1A receptor (5-HT1A), 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-HT2A and (2) potent agonism at 5-HT1A. The in vitro effects of lisuride, LSD, and related analogues on 5-HT2A signaling were characterized by using miniGαq and ß-arrestin 2 recruitment assays. The 5-HT1A- and 5-HT2A-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-HT2A, with high efficacy and potency for recruiting miniGαq and ß-arrestin 2. By contrast, lisuride displayed partial efficacy for both functional end points (6-52% of 5-HT or LSD Emax) and antagonized the effects of LSD. The mouse experiments demonstrated that LSD induces head twitch responses (HTRs)(ED50 = 0.039 mg/kg), while lisuride suppresses HTRs (ED50 = 0.006 mg/kg). Lisuride also produced potent hypothermia and hypolocomotion (ED50 = 0.008-0.023 mg/kg) that was blocked by the 5-HT1A antagonist WAY100635 (3 mg/kg). Blockade of 5-HT1A 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-HT1A agonist 8-OH-DPAT (1 mg/kg). Overall, our findings show that lisuride is an ultrapotent 5-HT1A agonist in C57BL/6J mice, limiting its use as a 5-HT2A ligand in mouse studies examining acute drug effects. Results also indicate that the 5-HT2A partial agonist-antagonist activity of lisuride explains its lack of psychedelic effects.

In vitro cannabinoid receptor activity, metabolism, and detection in seized samples of CH-PIATA, a new indole-3-acetamide synthetic cannabinoid.

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 (CB1 and CB2) receptor activation potential of CH-PIATA reference material and the powder seized by Belgian customs by means of ß-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.

In vitro µ-opioid receptor activation potential of U10 and ß-U10, positional isomers of the synthetic opioid naphthyl U-47700.

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 "ß-U10" (2-naphthyl U-47700; N-[2-(dimethylamino)cyclohexyl]-N-methylnaphthalene-2-carboxamide) was identified in Australia and the United States. ß-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 ß-U10), together with the structural analogue U-47700 and fentanyl, is reported. Application of a cell-based µ-opioid receptor (MOR) activation (ß-arrestin 2 recruitment) assay demonstrated ß-U10 (EC50 = 348 nM; Emax = 150% vs. hydromorphone) to be less potent than U-47700 (EC50 = 116 nM; Emax = 154%) and fentanyl (EC50 = 9.35 nM; Emax = 146%) but considerably more active than the α-isomer (EC50 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 ß-U10 stresses the importance of (analytical) differentiation between closely related analytes. The emergence of ß-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.

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.

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.

Detection in seized samples, analytical characterization, and in vitro metabolism of the newly emerged 5-bromo-indazole-3-carboxamide synthetic cannabinoid receptor agonists.

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 ß-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.

In vitro cannabinoid activity profiling of generic ban-evading brominated synthetic cannabinoid receptor agonists and their analogs.

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 CB1 and CB2 ß-arrestin 2 recruitment assays and a CB1 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 CB1 agonists. Also, at CB2 , tail-less analogs showed a lower potency but increased efficacy. Removing the bromine substitution ((S)-ADB-INACA) resulted in a reduced activity at CB1 ; however, this effect was less prominent at CB2 . 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 CB1 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.

In Vitro and In Vivo Evaluation of Pellotine: A Hypnotic Lophophora Alkaloid.

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-HT6, and 5-HT7 receptors, where it binds with affinities in the nanomolar range (117, 170, and 394 nM, respectively). Additionally, 1 was functionally characterized at 5-HT6 and 5-HT7, where it was found to be an agonist at the former (EC50 = 94 nM, Emax = 32%) and an inverse agonist at the latter (EC50 = 291 nM, Emax = -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.

Alkoxy chain length governs the potency of 2-benzylbenzimidazole 'nitazene' opioids associated with human overdose.

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 (EC50 = 30 pM, Emax = 103%) and across all in vivo endpoints (ED50 = 3-12 µg/kg). In vivo SARs revealed that ethoxy, isopropoxy, and propoxy chains engendered higher potencies than fentanyl, whereas methoxy and butoxy analogues were less potent. MOR functional potencies, but not MOR affinities, were positively correlated with in vivo potencies to induce opioid effects. CONCLUSIONS: Overall, our data show that certain nitazene NSOs are more potent than fentanyl as MOR agonists in mice, highlighting concerns regarding the high potential for overdose in humans who are exposed to these compounds.

Assay-Dependent Inverse Agonism at the A3 Adenosine Receptor: When Neutral Is Not Neutral.

The A3 adenosine receptor (A3AR) is implicated in a variety of (patho)physiological conditions. While most research has focused on agonists and antagonists, inverse agonism at A3AR has been scarcely studied. Therefore, this study aimed at exploring inverse agonism, using two previously engineered cell lines (hA3ARLgBiT-SmBiTßarr2 and hA3ARLgBiT-SmBiTminiGαi), both employing the NanoBiT technology. The previously established inverse agonist PSB-10 showed a decrease in basal signal in the ß-arrestin 2 (ßarr2) but not the miniGαi 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 A3AR-ßarr2 recruitment assay, yielding EC50 and Emax values for inverse agonism. Conversely, in the miniGαi 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 A3AR-miniGαi 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 ßarr2 recruitment and the GloSensor cAMP assays to capture inverse agonism at the A3AR, as opposed to the NanoBiT miniGαi recruitment assay.