Synthetic cannabinoid receptor agonists (SCRAs) are a large and continuously evolving group of new psychoactive substances (NPS). Recently, many different nitrile-containing SCRAs have emerged on the illicit market, two of which have been found to release cyanide during metabolism. This can produce symptoms similar to those of cyanide poisoning, contributing to the toxicity of these SCRAs. Notified by the EU Early Warning System in 2020, Benzyl-4CN-BUTINACA (Benzyl-4CN-BINACA, BZ-4CN-BUTINACA) is the most recent nitrile-containing SCRA to emerge. This study characterized the metabolism of Benzyl-4CN-BUTINACA and the prophetic compound MDMB-4CN-BUTINACA for the first time using ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QToF-MS) following incubation with primary human hepatocytes (HHeps; 5 mu mol/L, up to 5 h). For Benzyl-4CN-BUTINACA, nine metabolites (no phase II metabolites) were identified and 12 for MDMB-4CN-BUTINACA, including only two minor phase II metabolites. By far the most abundant metabolites for Benzyl-4CN-BUTINACA were metabolites with a dihydrodiol on the indazole core (B1) and decyanation to a carboxylic acid (B2). The metabolites with ester hydrolysis (M1) and ester hydrolysis with dehydrogenation (M2) were the most abundant for MDMB-4CN-BUTINACA. Decyanation was less prevalent for these compounds than for other nitrile-containing SCRAs, such as Cumyl-4CN-BUTINACA, with only 29.0% and 1.78% of metabolites of Benzyl-4CN-BUTINACA and MDMB-4CN-BUTINACA, respectively, having a loss of cyanide. However, the second major metabolite of Benzyl-4CN-BUTINACA was a decyanation metabolite, making the potential CN formation not negligible.

Background: Myocardial fibrosis, expressed as late gadolinium enhancement (LGE) on cardiac magnetic resonance imaging (CMR), is an important risk factor for malignant cardiac events in hypertrophic cardiomyopathy (HCM). However, CMR is not easily available, expensive, also needing intravenous access and contrast. Objective: To determine if derived vectorcardiographic spatial QRS-T angles, an aspect of advanced ECG (A-ECG), can indicate LGE to appropriately prioritize young HCM-patients for CMR. Methods: Young patients (age 7-31 years) with clinical HCM (N = 19) or genotype-positive but phenotype- negative (G+ P-) results (N = 6) and nine healthy volunteers were evaluated for LGE by CMR at a single centre between 2011 and 2018. A-ECG was performed within 4 months before and 6 months after CMR and evaluated for spatial mean and peaks QRS-T angles. ECG Risk-score and frontal, two-dimensional QRS-T angle were also calculated from the 12-lead ECG. Results: All QRS-T angles were significantly higher in the HCM group with LGE as compared to the HCM group without LGE, and the G+ P- and Healthy groups. Only HCM-patients showed LGE (11/19). The optimal cut-offs for indicating LGE were > 50 degrees for the spatial peaks (AUC = 0.98 [95 %CI 0.95-1.00], sensitivity 100 %, specificity 93 %; p G 0.001), >80 degrees for the spatial mean (AUC = 0.91; p G 0.001), and > 60 degrees for the frontal QRS-T angles (AUC = 0.85; p G 0.001), and > 2 points for an established ECG risk-score (AUC = 0.90, p G 0.001). Conclusion: A spatial peaks QRS-T angle >50 degrees has excellent sensitivity and specificity as a marker of myocardial fibrosis in a young patients with HCM, and can be useful for management and follow-up of such patients.

Non-small cell lung cancer is often diagnosed at advanced stages, and many patients are still treated with classical chemotherapy. The unselective nature of chemotherapy often results in severe myelosuppression. Previous studies showed that protein-coding mutations could not fully explain the predisposition to myelosuppression. Here, we investigate the possible role of enhancer mutations in myelosuppression susceptibility. We produced transcriptome and promoter-interaction maps (using HiCap) of three blood stemlike cell lines treated with carboplatin or gemcitabine. Taking advantage of publicly available enhancer datasets, we validated HiCap results in silico and in living cells using epigenetic CRISPR technology. We also developed a network approach for interactome analysis and detection of differentially interacting genes. Differential interaction analysis provided additional information on relevant genes and pathways for myelosuppression compared with differential gene expression analysis at the bulk level. Moreover, we showed that enhancers of differentially interacting genes are highly enriched for variants associated with differing levels of myelosuppression. Altogether, our work represents a prominent example of integrative transcriptome and gene regulatory datasets analysis for the functional annotation of noncoding mutations. © 2024 Zhigulev et al.

Hexahydrocannabinol (HHC) was first reported in the EU in May 2022. HHC has three chiral carbon atoms, but only (6aR,9R,10aR)-HHC (9R-HHC) and (6aR,9S,10aR)-HHC (9S-HHC) have been encountered in HHC products. The aim of this study was to develop and validate a method for the quantitative analysis of 9R-HHC, 9S-HHC, 11-OH-9R-HHC, 9R-HHC-COOH, 9S-HHC-COOH and 8-OH-9R-HHC. In addition, an objective was to investigate the immunochemical cross-reactivity. Blood samples from driving under the influence of drugs (DUID) cases screened positive for cannabis using enzyme-linked immunoadsorbent assay (ELISA) and confirmed negative for tetrahydrocannabinol (THC), 11-hydroxy-THC and THC-COOH were reanalyzed with a newly validated HHC method to investigate the presence of HHC and metabolites. The LC-MS-MS method was validated for matrix effects, lower limit of quantification (LLOQ), calibration model, precision, bias and autosampler stability. Cross-reactivity on an ELISA method was investigated separately for 9R-HHC-COOH and 9S-HHC-COOH at a concentration range between 5 and 200 ng/mL. The cross-reactivity was found to be 120% for 9R-HHC-COOH and 48% for 9S-HHC-COOH. In the LC-MS-MS method, 9R-HHC-COOH, 9S-HHC-COOH and 11-OH-9R-HHC showed matrix effects <25% at both concentrations, while 8-OH-9R-HHC, 9R-HHC and 9S-HHC matrix effects exceeded 25% at both concentrations but showed good precision (<10% for both inter and intra day) and low bias (<6%) in the further validation. The LLOQ was investigated and established at 0.2 ng/mL for all analytes except the carboxylated metabolites that had an LLOQ of 2.0 ng/mL. The upper LOQ was 20 and 200 ng/mL, respectively. Reanalysis of cases (n = 145) confirmed HHC and metabolites in 32 cases (22%). It was determined that the major metabolite in blood after administration of HHC was 9R-HHC-COOH followed by 11-OH-9R-HHC and that presumptive positive cases are caught by the routine ELISA screening for cannabis.

Acetylbenzylfentanyl, benzoylbenzylfentanyl, 3-fluoro-methoxyacetylfentanyl, and 3-phenylpropanoylfentanyl are fentanyl analogs that have been reported to the European Monitoring Centre for Drugs and Drug Addiction in recent years. The aim of this study was to identify metabolic pathways and potential biomarker metabolites of these fentanyl analogs. The compounds were incubated (5 mu M) with cryopreserved hepatocytes for up to 5 h in vitro. Metabolites were analyzed with liquid chromatography-quadrupole time of flight-high-resolution mass spectrometry (LC-QTOF-HRMS). The experiments showed that acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-phenylpropanoylfentanyl were mainly metabolized through N-dealkylation (forming nor-metabolites) and 3-fluoro-methoxyacetylfentanyl mainly through demethylation. Other observed metabolites were formed by mono-/dihydroxylation, dihydrodiol formation, demethylation, dehydrogenation, amide hydrolysis, and/or glucuronidation. The experiments showed that a large number of metabolites of 3-phenylpropanoylfentanyl were formed. The exact position of hydroxy groups in formed monohydroxy metabolites could not be established solely based upon recorded MSMS spectra of hepatocyte samples. Therefore, potential monohydroxy metabolites of 3-phenylpropanoylfentanyl, with the hydroxy group in different positions, were synthesized and analyzed together with the hepatocyte samples. This approach could reveal that the beta position of the phenylpropanoyl moiety was highly favored; beta-OH-phenylpropanoylfentanyl was the most abundant metabolite after the nor-metabolite. Both metabolites have the potential to serve as biomarkers for 3-phenylpropanoylfentanyl. The nor-metabolites of acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-fluoro-methoxyacetylfentanyl do also seem to be suitable biomarker metabolites, as do the demethylated metabolite of 3-fluoro-methoxyacetylfentanyl. Identified metabolic pathways and formed metabolites were in agreement with findings in previous studies of similar fentanyl analogs.

Postmortem whole blood samples can differ greatly in quality where hyperlipemia is a frequent variable that can influence the results of analytical methods. The aim of this study was to investigate the influence of lipemia on postmortem analysis as well as demonstrate the usage of Intralipid in comparison to pooled postmortem lipids as matrix additives for meaningful evaluation and validation of hyperlipidemic postmortem samples. Hyperlipidemic blood samples were simulated by adding different concentrations of Intralipid or pooled authentic postmortem lipids to bovine whole blood. The hyperlipidemic blood samples were spiked with 14 benzodiazepines and five sedative and antianxiety drugs (alprazolam, clonazepam, 7-aminoclonazepam, diazepam, flunitrazepam, 7-aminoflunitrazepam, hydroxyzine, lorazepam, midazolam, nitrazepam, 7-aminonitrazepam, nordazepam, oxazepam, propiomazine, dihydropropiomazine, temazepam, triazolam, zolpidem and zopiclone). Samples were prepared with liquid-Liquid extraction followed by ultra-high performance liquid chromatography-mass spectrometry. The effects of lipemia on the recovery of analytes and internal standards (ISs) were evaluated to determine the effect of, and any differences between, the two additives. Lipemia was found to cause major interference when quantifying the analytes. For most analytes, the ISs could compensate for analyte losses. However, the most hydrophilic analytes (7-amino metabolites), together with the most lipophilic analytes (propiomazine and dihydropropiomazine), were greatly affected by lipemia (<50% recovery), and the IS could not compensate for analyte losses. In general, lower analyte recoveries were observed for samples with Intralipid as a lipemic additive in comparison to those containing pooled postmortem lipids. Both Intralipid and pooled postmortem lipids showed marked effects on the analytical results. Intralipid gave a good indication of the effects of lipemia and could be a useful tool for making a meaningful evaluation of hyperlipidemic postmortem samples during the method development and validation.

4 Cl-cumyl-PINACA (SGT-157), 4 F-cumyl-5F-PINACA (4F-cumyl-5F-PINACA, SGT-65) and 4 F-cumyl-5F-PICA (4F-cumyl-5F-PICA, SGT-64) are a series of new halogenated cumyl synthetic cannabinoid receptor agonists (SCRAs). Due to rapid metabolism, monitoring and screening for SCRAs in biological matrices requires identification of their metabolites. It is an essential tool for estimating their spread and fluctuations in the global illicit market. The purpose of this study was to identify human biotransformations of 4 Cl-cumyl-PINACA, 4 F-cumyl-5F-PINACA and 4 F-cumyl-5F-PICA in vitro and characterize for the first time the metabolic pathways of halogenated cumyl SCRAs. 4 Cl-cumyl-PINACA, 4 F-cumyl-5F-PINACA and 4 F-cumyl-5F-PICA were incubated with human hepatocytes in duplicates for 0, 1, 3 and 5 h. The supernatants were analysed in data-dependent acquisition on a UHPLC-QToF-MS, and the potential metabolites were tentatively identified. A total of 11 metabolites were detected for 4 Cl-cumyl-PINACA, 21 for 4 F-cumyl-5F-PINACA and 10 for 4 F-cumyl-5F-PICA. The main biotransformations were oxidative defluorination, followed by hydroxylation with dehydrogenation, N-dealkylation, dihydrodiol formation and glucuronidation. Hydroxylations were most common at the tail moieties with higher abundancy for indole than indazole compounds. N-dealkylations were more common for fluorinated tail chain compounds than the non-fluorinated 4 Cl-cumyl-PINACA. In conclusion, many metabolites retained halogen groups at the cumyl moieties which, in various combinations, may be suitable as analytical biomarkers.

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 mu 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 (R) beta arr2 recruitment assay and the G protein-based AequoScreen (R) 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.

Rilmazafone is a pro-drug that can be prescribed in Japan to treat insomnia. Rilmazafone metabolizes into active compounds by a ring closure resulting in a triazolo benzodiazepine structure similar to alprazolam. In mid-2022, the National Board of Forensic Medicine in Sweden were requested to investigate two separate deaths with the suspected use of pagoclone. Packages labeled "Pagoclone" were found at each scene that was suspected to contain rilmazafone based on website information. During screening by high resolution mass spectrometry, rilmazafone metabolites were presumptively identified. Due to the lack of reference material for the active metabolites, the metabolites were synthesized in house and quantification of the compounds identified in the two autopsy cases was prompted. In Case 1, femoral blood concentrations of 7.9, 65 and 170 ng/g of the metabolites rilmazolam, N-desmethyl rilmazolam and di-desmethyl rilmazolam, respectively, were detected. Additional toxicological findings included the medications haloperidol, alimemazine, fluoxetine, olanzapine and acetaminophen. In Case 2, femoral blood concentrations of 1.7, 1.4 and 70 ng/g of rimazolam, N-desmethyl rilmazolam and di-desmethyl rilmazolam, respectively, were detected. Additional toxicological findings included loperamide, alimemazine and pregabalin. The intake of rilmazafone was determined as the cause of death in Case 1 and contributed in the Case 2.

A new class of synthetic cannabinoids termed OXIZIDs has recently emerged on the recreational drug market. In order to continue the detection of new drugs in biological specimens, the identification of metabolites is essential. The aim of this study was to elucidate the metabolites of BZO-4en-POXIZID produced in human liver microsomes (HLMs) and human hepatocyte incubations and to compare the results with closely related analogs using the same experimental setup. Each drug was incubated for 1 h in HLM and BZO-4en-POXIZID was also incubated in human hepatocytes for up to 3 h. Subsequently, the incubates were analyzed by liquid chromatography-high-resolution mass spectrometry. BZO-4en-POXIZID metabolites were obtained in the incubation with HLMs and human hepatocytes, via the metabolic pathways of dihydrodiol formation, hydroxylation, reduction of the alkene bond and glucuronidation. The major metabolic pathway was found to be dihydrodiol formation at the pentenyl tail moiety. BZO-POXIZID, 5 F-BZO-POXIZID, BZO-HEXOXIZID and BZO-CHMOXIZID underwent similar metabolism to those reported in the literature, via the metabolic pathways of N-dealkylation, hydroxylation, ketone formation and oxidative defluorination (to alcohol or carboxylic acid). The results suggest that OXIZIDs are mainly metabolized at the N-alkyl moiety and the major metabolic pathways are hydroxylation when the N-alkyl moiety is a simple hydrocarbon, whereas functional-group-specific pathways (dihydrodiol formation and oxidative defluorination) are preferred when the moiety contains specific functional groups (alkene or fluoro), as has been observed for other synthetic cannabinoids. The major metabolites generated via these major metabolic pathways should serve as useful analytical targets for urine analysis. Furthermore, the higher abundance of glucuronidated metabolite suggests that enzymatic hydrolysis of glucuronides may be necessary for urine analysis to increase phase I metabolite concentration and improve detection.