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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 [ 3 H]-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-β-arrestin2/mini-G αi and AequoScreen ® ). The most active compounds were ketobemidone (EC 50 32.8–528 nM; E max 105–271%, relative to hydromorphone) and N -ethyl-U-47700 (EC 50 241–767 nM; E max 139–247%). The same opioids showed the strongest MOR affinity. As most of the other NSOs only weakly activated MOR in the three assays (EC 50 values in the high nM–µM range), they likely do not pose a high overdose risk. 2F-viminol (EC 50 2.2–4.5 µM; E max 21.2–61.5%) and U-47931E/bromadoline (EC 50 0.55–2.9 µM; E max 52.8–85.9%) were partial agonists compared to hydromorphone, and maximum receptor activation was not reached for 2,4-difluoro-U-48800 (EC 50  > 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.

Recently, a number of fentanyl analogs have been implicated in overdose deaths in Europe and in the US. So far, little is known of the molecular behavior of the structurally related subgroup; the alicyclic fentanyls. In this study, reference standards of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and 2,2,3,3-tetramethylcyclopropyl fentanyl (TMCPF) at a final concentration of 5 µM were incubated with cryopreserved human hepatocytes (1 × 106 cells/mL) for 0, 1, 3 and 5 h. The metabolites formed were identified by liquid chromatography–quadrupole time-of-flight mass spectrometry analysis. The most abundant biotransformation found was N-dealkylation (formation of normetabolites) and oxidation of the alicyclic rings. As ring size increased, the significance of N-dealkylation decreased in favor of alicyclic ring oxidation. An example of this was cyclopropyl fentanyl, with a three-carbon ring, whose normetabolite covered 82% of the total metabolic peak area and no oxidation of the alicyclic ring was observed. In contrast, TMCPF, with a seven-carbon ring structure, rendered as much as 85% of its metabolites oxidized on the alicyclic ring. Other biotransformations found included oxidation of the piperidine ethyl moiety and/or the phenethyl substructure, glucuronidation as well as amide hydrolysis to form metabolites identical to despropionyl fentanyl. Taken together, this study provides a base for understanding the metabolism of a number of structurally related fentanyl analogs formed upon intake.

Combination treatment with low-dose thiopurine and allopurinol (AP) has successfully been used in patients with inflammatory bowel disease with a so called skewed thiopurine metabolite profile. In red blood cells in vivo, it reduces the concentration of methylated metabolites and increases the concentration of the phosphorylated ones, which is associated with improved therapeutic efficacy. This study aimed to investigate the largely unknown mechanism of AP on thiopurine metabolism in cells with an active thiopurine metabolic pathway using HepG2 and HEK293 cells. Cells were treated with 6-mercaptopurine (6MP) and AP or its metabolite oxypurinol. The expression of genes known to be associated with thiopurine metabolism, and the concentration of thiopurine metabolites were analyzed. Gene expression levels were only affected by AP in the presence of 6MP. The addition of AP to 6MP affected the expression of in total 19 genes in the two cell lines. In both cell lines the expression of the transporter SLC29A2 was reduced by the combined treatment. Six regulated genes in HepG2 cells and 8 regulated genes in HEK293 cells were connected to networks with 18 and 35 genes, respectively, present at known susceptibility loci for inflammatory bowel disease, when analyzed using a protein-protein interaction database. The genes identified as regulated as well as the disease associated interacting genes represent new candidates for further investigation in the context of combination therapy with thiopurines and AP. However, no differences in absolute metabolite concentrations were observed between 6MP+AP or 6MP+oxypurinol vs. 6MP alone in either of the two cell lines. In conclusion; the effect of AP on gene expression levels requires the presence of 6MP, at least in vitro. Previously described AP-effects on metabolite concentrations observed in red blood cells in vivo could not be reproduced in our cell lines in vitro. AP's effects in relation to thiopurine metabolism are complex. The network-identified susceptibility genes represented biological processes mainly associated with purine nucleotide biosynthetic processes, lymphocyte proliferation, NF-KB activation, JAK-STAT signaling, and apoptotic signaling at oxidative stress.

Single nucleotide polymorphisms (SNPs) in the gene for multidrug resistance protein ABCG2, an erlotinib transporter, is a possible contributor to the interindividual variation observed in erlotinib pharmacokinetics and toxicity. Therefore, the aim was to study erlotinib efflux by ABCG2 wild‐type (wt) and ABCG2 polymorphic variants in the K562 cell line. The chronic myeloid leukemia K562 cell line, neither expressing EGFR nor ABCG2, was transduced with vectors containing the ABCG2 wt, the SNPs: 34 G > A and 421 C > A, or with empty vector (K562/ve). ABCG2‐expressing cells were enriched using magnetic sorting and the expression was verified using flow cytometry. Intracellular erlotinib concentrations were analyzed by LC–MS/MS after incubation with 1 µmol/L erlotinib for 60 minutes. All recombinant cell lines were confirmed carriers of the vector and expressed ABCG2. Differences in intracellular erlotinib concentrations were observed between K562/ve and K562 ABCG2 wt and between K562/ve and K562 ABCG2 34G > A (both P ≤ .001, one‐way ANOVA with Tukey HSD post hoc test), indicating that the cell lines carrying ABCG2 wt and ABCG2 34G > A actively transports erlotinib out of the cells. The ABCG2 34G > A cell line had a higher transport capacity compared with ABCG2 wt after adjusting for ABCG2 expression (P = .024, t test). No differences were observed between K562/ve and K562 ABCG2 421 C > A. Genetic polymorphism in the ABCG2 gene has an influence on the transport of erlotinib which can contribute to the observed variation in erlotinib pharmacokinetics and toxicity.

Purpose Norflurazepam, also a metabolite of other prescription benzodiazepines, appeared on the new psychoactive substances (NPS) drug market recently, complicating the interpretation of NPS findings. The aims of the study were to tentatively identify potential metabolites of norflurazepam and structural analogues (flurazepam, fludiazepam and cinolazepam) produced by hepatocytes and in authentic human samples and to discuss the possibility to differentiate drug consumption. Methods Each drug (5 µmol/L) was incubated with pooled human hepatocytes, and metabolites were identified using liquid chromatography–high-resolution mass spectrometry (LC–HRMS). Similarly, urine with/without hydrolysis and blood/serum from three flurazepam and seven presumptive norflurazepam cases were analysed by LC–HRMS. Results No metabolites were detected for norflurazepam in hepatocytes, but six metabolites for flurazepam, two for fludiazepam and three for cinolazepam were found. In human specimens collected after flurazepam ingestion, a total of eight metabolites, in good agreement with hepatocyte metabolites, were detected. In specimens of presumptive norflurazepam intake, norflurazepam and its metabolites (four hydroxy metabolites and one glucuronide of a hydroxy metabolite) were found. Conclusions Based on the results, hydroxy metabolites for norflurazepam, N -(hydroxyethyl), desethyl and didesethyl for flurazepam, hydroxy for fludiazepam and glucuronides and N -(hydroxyethyl) for cinolazepam are recommended for monitoring. While flurazepam, fludiazepam and cinolazepam were metabolised by hepatocytes at side chain, norflurazepam was not, which seems to indicate that hepatocytes have difficulty in modifying the benzene/diazepine rings of some 1,4-benzodiazepines. As for confirming the intake of norflurazepam, the urine ratio of 3-hydroxy-norflurazepam/norflurazepam might be the key; a high ratio might be correlated to norflurazepam intake, thereby enabling the differentiation.

Tobacco use is strongly associated with cardiovascular disease and the only avoidable risk factor associated with development of aortic aneurysm. While smoking is the most common form of tobacco use, snuff and other oral tobacco products are gaining popularity, but research on potentially toxic effects of oral tobacco use has not kept pace with the increase in its use. Here, we demonstrate that cigarette smoke and snuff extracts are highly toxic to developing zebrafish embryos. Exposure to such extracts led to a palette of toxic effects including early embryonic mortality, developmental delay, cerebral hemorrhages, defects in lymphatics development and ventricular function, and aneurysm development. Both cigarette smoke and snuff were more toxic than pure nicotine, indicating that other compounds in these products are also associated with toxicity. While some toxicities were found following exposure to both types of tobacco product, other toxicities, including developmental delay and aneurysm development, were specifically observed in the snuff extract group, whereas cerebral hemorrhages were only found in the group exposed to cigarette smoke extract. These findings deepen our understanding of the pathogenic effects of cigarette smoking and snuff use on the cardiovascular system and illustrate the benefits of using zebrafish to study mechanisms involved in aneurysm development.

•Flualprazolam was frequently detected in fatalities in Sweden and Finland.•Flualprazolam was implicated in the cause of death in 40 % of the studied cases.•Flualprazolam was the only intoxicant found in two (6 %) poisoning cases.•The age in the Finnish cases was significantly lower than in the Swedish cases. Flualprazolam is a novel designer benzodiazepine, structurally related to alprazolam, flubromazolam and triazolam. In the last couple of years, it has been frequently detected in seizures and in forensic cases in Sweden and Finland. However, there is a lack of published blood concentrations for the drug, which presents difficulties when assessing its relevance for the cause of death. A quantitative method for the determination of flualprazolam in post-mortem blood was developed and validated, and subsequently used to analyse samples from 33 deaths previously screened as testing positive for flualprazolam in Sweden and Finland. Most of the cases in the study were accidental deaths (61 %) or suicides (18 %). The median (range) flualprazolam concentration was 18.0 (3.0–68) ng/g. The majority of the deceased were male (82 %) and the median age was 30 years. The median age in the Swedish cases was significantly higher (35 years) than in the Finnish cases (23 years) (p< 0.05). Poly-drug use and particularly the concomitant use of flualprazolam and opioids were very common in the study population. Most of the cases that were positive for flualprazolam were fatal poisonings by a drug (N=23), and in 13 cases, flualprazolam was implicated in the cause of death. Combining the resources of two countries in which all post-mortem toxicology is centralised provided a more comprehensive insight into the toxicology of flualprazolam. Research on novel psychoactive substances, such as flualprazolam, is required in order to be able to provide scientific evidence on the risks of these new substances for drug administration and potential users.

•Activity at μ–opioid, CB1, 5-HT2A & 5-HT1A receptor is shown for 60 NPS.•The results show high variability in potency for fentanyl analogues & hallucinogens.•NPS off-target effects is evident & needs consideration when interpreting toxicity. New psychoactive substances (NPS) appear on the recreational market on a monthly basis, with unclear toxicology, resulting in an increasing number of fatalities. Identification of drug targets and potencies is crucial for understanding and treating intoxications and for scheduling processes. In this study 60 NPS and metabolites belonging to opioids, cannabinoids and serotonergic hallucinogens classes were screened for in vitro activation of the μ-opioid, CB1, 5-HT1A and 5-HT2A receptors using the AequoZen cell system. Fentanyl and NBOMe analogues were chosen for full dose-response characterization of the μ-opioid and 5-HT2A receptors, respectively. Most substances activated their corresponding target receptor. The most potent μ-opioid receptor agonists were 2-fluorofentanyl (EC50 = 1.0 nM), carfentanil (EC50 = 2.7 nM) and acrylfentanyl (EC50 = 2.8 nM) and in total a >1500-fold difference was seen among the tested compounds. Moreover, furanylfentanyl, 4-methoxybutyrylfentanyl and valerylfentanyl acted as partial agonists of the μ-receptor. On the 5-HT2A receptor, bromo-dragonfly showed the highest potency (EC50 = 0.05 nM, 400 times more potent than LSD), followed by most NBOMe compounds with EC50 values ranging from 0.11 nM (for 25N-NBOMe) to 1.3 nM (for 25T4-NBOMe)). Off-target activation of the μ-opioid receptor was identified for piperazines, phenethylamines (in particular NBOMe and 2C compounds) and tryptamines. Moreover, the synthetic cannabinoid metabolite 3-carboxy indole PB-22 activated the 5-HT2A receptor. Bromo-dragonfly was the only compound that activated all four receptors. These results highlight the possible interplay of known and unknown NPS targets and unveil its complexity. Moreover, the detailed, quantitative information presented facilitates our further understanding of NPS toxicology.

New psychoactive substances (NPS) appear on the recreational market on a monthly basis, with unclear toxicology, resulting in an increasing number of fatalities. Identification of drug targets and potencies is crucial for understanding and treating intoxications and for scheduling processes. In this study 60 NPS and metabolites belonging to opioids, cannabinoids and serotonergic hallucinogens classes were screened for in vitro activation of the mu-opioid, CB1, 5-HT1A and 5-HT2A receptors using the AequoZen cell system. Fentanyl and NBOMe analogues were chosen for full dose-response characterization of the mu-opioid and 5-HT2A receptors, respectively. Most substances activated their corresponding target receptor. The most potent mu-opioid receptor agonists were 2-fluorofentanyl (EC50 = 1.0 nM), carfentanil (EC50 = 2.7 nM) and acrylfentanyl (EC50 = 2.8 nM) and in total a &gt;1500-fold difference was seen among the tested compounds. Moreover, furanylfentanyl, 4-methoxybutyrylfentanyl and valerylfentanyl acted as partial agonists of the mu-receptor. On the 5-HT2A receptor, bromo-dragonfly showed the highest potency (EC50 = 0.05 nM, 400 times more potent than LSD), followed by most NBOMe compounds with EC50 values ranging from 0.11 nM (for 25N-NBOMe) to 1.3 nM (for 25T4-NBOMe)). Off-target activation of the mu-opioid receptor was identified for piperazines, phenethylamines (in particular NBOMe and 2C compounds) and tryptamines. Moreover, the synthetic cannabinoid metabolite 3-carboxy indole PB-22 activated the 5-HT2A receptor. Bromo-dragonfly was the only compound that activated all four receptors. These results highlight the possible interplay of known and unknown NPS targets and unveil its complexity. Moreover, the detailed, quantitative information presented facilitates our further understanding of NPS toxicology. (c) 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).