Studies on the in vitro and in vivo metabolism of the synthetic opioids U-51754, U-47931E, and methoxyacetylfentanyl using hyphenated high-resolution mass spectrometry

New Synthetic Opioids (NSOs) are one class of New Psychoactive Substances (NPS) enjoying increasing popularity in Europe. Data on their toxicological or metabolic properties have not yet been published for most of them. In this context, the metabolic fate of three NSOs, namely, trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzenacetamide (U-51754), trans-4-bromo-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzamide (U-47931E), and 2-methoxy-N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl] acetamide (methoxyacetylfentanyl), was elucidated by liquid chromatography high-resolution mass spectrometry after pooled human S9 fraction (phS9) incubations and in rat urine after oral administration. The following major reactions were observed: demethylation of the amine moiety for U-51754 and U-47931E, N-hydroxylation of the hexyl ring, and combinations thereof. N-dealkylation, O-demethylation, and hydroxylation at the alkyl part for methoxyacetylfentanyl. Except for U-47931E, parent compounds could only be found in trace amounts in rat urine. Therefore, urinary markers should preferably be metabolites, namely, the N-demethyl-hydroxy and the hydroxy metabolite for U-51754, the N-demethylated metabolite for U-47931E, and the N-dealkylated metabolite as well as the O-demethylated one for methoxyacetylfentanyl. In general, metabolite formation was comparable in vitro and in vivo, but fewer metabolites, particularly those after multiple reaction steps and phase II conjugates, were found in phS9. These results were consistent with those of comparable compounds obtained from human liver microsomes, human hepatocytes, and/or human case studies

Toxicokinetics and analytical toxicology of the abused opioid U-48800 - in vitro metabolism, metabolic stability, isozyme mapping, and plasma protein binding

Due to the risk of new synthetic opioids (NSOs) for human health, the knowledge of their toxicokinetic characteristics is important for clinical and forensic toxicology. U‐48800 is an NSO structurally non‐related to classical opioids such as morphine or fentanyl and offered for abuse. As toxicokinetic data of U‐48800 is not currently available, the aims of this study were to identify the in vitro metabolites of U‐48800 in pooled human liver S9 fraction (pS9), to map the isozymes involved in the initial metabolic steps, and to determine further toxicokinetic data such as metabolic stability, including the in vitro half‐life (t1/2), and the intrinsic (CLint) and hepatic clearance (CLh). Furthermore, drug detectability studies in rat urine should be done using hyphenated mass spectrometry. In total, 13 phase I metabolites and one phase II metabolite were identified. N‐Dealkylation, hydroxylation, and their combinations were the predominant metabolic reactions. The isozymes CYP2C19 and CYP3A4 were mainly involved in these initial steps. CYP2C19 poor metabolizers may suffer from an increased U‐48800 toxicity. The in vitro t1/2 and CLint could be rated as moderate, compared to structural related compounds. After administration of an assumed consumer dose to rats, the unchanged parent compound was found only in very low abundance but three metabolites were detected additionally. Due to species differences, metabolites found in rats might be different from those in humans. However, phase I metabolites found in rat urine, the parent compound, and additionally the N‐demethyl metabolite should be used as main targets in toxicological urine screening approaches

Phenethylamine-derived new psychoactive substances 2C-E-FLY, 2C-EF-FLY, and 2C-T-7-FLY: Investigations on their metabolic fate including isoenzyme activities and their toxicological detectability in urine screenings

Psychoactive substances of the 2C‐series are phenethylamine‐based designer drugs that can induce psychostimulant and hallucinogenic effects. The so‐called 2C‐FLY series contains rigidified methoxy groups integrated in a 2,3,6,7‐tetrahydrobenzo[1,2‐b:4,5‐b']difuran core. The aim of the presented work was to investigate the in vivo and in vitro metabolic fate including isoenzyme activities and toxicological detectability of the three new psychoactive substances (NPS) 2C‐E‐FLY, 2C‐EF‐FLY, and 2C‐T‐7‐FLY to allow clinical and forensic toxicologists the identification of these novel compounds. Rat urine, after oral administration, and pooled human liver S9 fraction (pS9) incubations were analyzed by liquid chromatography−high‐resolution tandem mass spectrometry (LC−HRMS/MS). By performing activity screenings, the human isoenzymes involved were identified and toxicological detectability in rat urine investigated using standard urine screening approaches (SUSAs) based on gas chromatography (GC)−MS, LC−MSn, and LC−HRMS/MS. In total, 32 metabolites were tentatively identified. Main metabolic steps consisted of hydroxylation and N‐acetylation. Phase I metabolic reactions were catalyzed by CYP2D6, 3A4, and FMO3 and N‐acetylation by NAT1 and NAT2. Methoxyamine was used as a trapping agent for detection of the deaminated metabolite formed by MAO‐A and B. Interindividual differences in the metabolism of the 2C‐FLY drugs could be caused by polymorphisms of enzymes involved or drug–drug interactions. All three SUSAs were shown to be suitable to detect an intake of these NPS but common metabolites of 2C‐E‐FLY and 2C‐EF‐FLY have to be considered during interpretation of analytical findings

Adherence to Antihypertensive Drugs Assessed by Hyphenated High-Resolution Mass Spectrometry Analysis of Oral Fluids

Background It is currently unknown if antihypertensive drugs can be monitored in oral fluid (OF) using liquid chromatography coupled to high-resolution mass spectrometry. Methods and Results We assessed adherence using liquid chromatography coupled to high-resolution mass spectrometry in OF, plasma, and urine of 56 consecutive patients with hypertension referred to a tertiary hypertension unit. Of these patients, 59% were completely adherent (all drugs detectable in urine), whereas 29% and 13% were partially adherent (1 drug not detectable in urine) or nonadherent (>1 drug not detectable in urine), respectively. Adherent patients were on fewer antihypertensive drugs (P=0.001), had fewer daily drug doses (P=0.012), and had lower 24-hour ambulatory systolic (P=0.012) and diastolic (P=0.009) blood pressures than nonadherent or partially adherent patients. Most drugs were detected in urine compared with plasma and OF (181 versus 119 versus 88; P=0.001). Compared with urine and plasma, detection rates of angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and diuretics were lower in OF. There was no difference in the frequency of detecting β blockers (P=1.0) and calcium channel blockers (P=0.063) when comparing OF with urine. There was no difference in the number of calcium channel blockers (P=0.727), β blockers (P=1.000), thiazide diuretics (P=0.125), and α-2 agonists (P=0.125) identified between OF and plasma. Conclusions This study shows the feasibility of drug adherence testing for several antihypertensive drugs, especially those without acidic components, in OF, with a similar recovery compared with plasma. Therefore, drug adherence testing in OF should be further explored as a noninvasive approach, which can easily be performed in an "out-of-office" setting

Calcium Flux in Neutrophils Synchronizes β2 Integrin Adhesive and Signaling Events that Guide Inflammatory Recruitment

Intracellular calcium flux is an early step in the signaling cascade that bridges ligation of selectin and chemokine receptors to activation of adhesive and motile functions during recruitment on inflamed endothelium. Calcium flux was imaged in real time and provided a means of correlating signaling events in neutrophils rolling on E-selectin and stimulated by chemokine in a microfluidic chamber. Integrin dependent neutrophil arrest was triggered by E-selectin tethering and ligation of IL-8 seconds before a rapid rise in intracellular calcium, which was followed by the onset of pseudopod formation. Calcium flux on rolling neutrophils increased in a shear dependent manner, and served to link integrin adhesion and signaling of cytoskeletally driven cell polarization. Abolishing calcium influx through membrane expressed store operated calcium channels inhibited activation of high affinity β2 integrin and subsequent cell arrest. We conclude that calcium influx at the plasma membrane integrates chemotactic and adhesive signals, and functions to synchronize signaling of neutrophil arrest and migration in a shear stress dependent manner

In vitro toxicokinetics and analytical toxicology of three novel NBOMe derivatives - Phase I and II metabolism, plasma protein binding, and detectability in standard urine screening approaches studied by means of hyphenated mass spectrometry

Purpose Toxicokinetic studies are essential in clinical and forensic toxicology to understand drug-drug interactions, influence of individual polymorphisms, and elimination routes, as well as to evaluate targets for toxicological screening procedures. An N-(2-methoxybenzyl)-substituted phenethylamines (NBOMe analogues) intake has been associated with severe adverse reactions including deaths. 1-(1-Benzofuran-5-yl)-N-[(2-methoxyphenyl)methyl]propan-2-amine (5-APB-NBOMe), 2-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b′]difuran-4-yl)-N-[(5-chloro-2-ethoxyphenyl)methyl]ethan-1-amine (2C-B-FLY-NB2EtO5Cl), and 2-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b′]difuran-4-yl)-N-[(2-methoxyphenyl)methyl]ethan-1-amine (2C-BFLY-NBOMe) are three emerging NBOMe analogues, which have encountered on the drugs of abuse market. So far, their toxicokinetic data are completely unexplored. Methods The study included mass spectrometry-based identification of phase I and II metabolites following exposure to the terminally differentiated human hepatocellular carcinoma cells (HepaRG). The determination of enzymes involved in the major phase I/II metabolic steps and determination of plasma protein binding (PPB) was done. Finally, the evaluation of the toxicological detectability by different hyphenated mass spectrometry techniques in standard urine screening approaches (SUSAs) was investigated. Results The compounds were extensively metabolized in HepaRG cells mainly via O-dealkylation, hydroxylation, glucuronidation, and combinations thereof. CYP1A2, 2D6, 2C8, 2C19, and 3A4, were involved in the initial reactions of all investigated compounds. Glucuronidation of the phase I metabolites – when observed - was mainly catalyzed by UGT1A9. The PPB of all compounds was determined to be > 85%. Only the high-resolution mass spectrometry-based SUSA allowed detection of all compounds in rat urine but only via metabolites. Conclusions The toxicokinetic data provided by this study will help forensic and clinical toxicologists to reliably identify these substances in case of abuse and/or intoxication and will allow them a thorough risk assessment

Construction and test of a fine-grained liquid argon preshower prototype

A separate liquid argon preshower detector consisting of two layers featuring a fine granularity of 2.5~10$^{\mathrm{-3}}$ was studied by the RD3 collaboration. A prototype covering approximately 0.8 in pseudo-rapidity and 9 degrees in azimuth was built and tested at CERN in July 94. CMOS and GaAs VLSI preamplifiers were designed and tested for this occasion. The combined response of this detector and an accordion electromagnetic calorimeter prototype to muons, electrons and photons is presented. For minimum ionizing tracks a signal-to-noise ratio of 4.5 per preshower layer was measured. Above 150~GeV the space resolution for electrons is better than 250~$\mu$m in both directions. The precision on the electromagnetic shower direction, determined together with the calorimeter, is better than 4 mrad above 50~GeV. It is concluded that the preshower detector would adequately fulfil its role for future operation at CERN Large Hadron Collider

Tools for studying the metabolism of new psychoactive substances for toxicological screening purposes - A comparative study using pooled human liver S9, HepaRG cells, and zebrafish larvae.

New psychoactive substances (NPS) are an emerging topic amongst abused compounds. New varieties appear constantly on the market, without any knowledge about their toxicodynamic and/or -kinetic properties and knowledge of their metabolism is crucial for the development of analytical methods employed for their detection. Controlled human studies would of course be best suited but due to ethical reasons and lack of preclinical safety data, they are usually not available. Often, in vitro models are used to evaluate similarities to human in vivo hepatic phase I and II metabolism and systems explored include primary human hepatocytes, pooled human S9 fraction, and HepaRG, a human hepatic cell line. All these in vitro models have considerable limitations and drug distribution, reabsorption, enterohepatic circulation, and renal elimination cannot be studied. In the recent years, zebrafish (Danio rerio) larvae (embryos) were discussed as a potential in vivo model to overcome these limitations. To date, no studies demonstrating its suitability for studying NPS metabolism in the context of analytical toxicology are available. The aim of this study was to elucidate whether zebrafish larvae can serve as a surrogate for human hepatic metabolism of NPS to develop toxicological screening procedures. Here, we used methyl 2-(1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)-3,3-dimethylbutanoate (7'N-5F-ADB), a new synthetic cannabinoid, whose human metabolism was recently described in the literature, as a model compound to evaluate zebrafish larvae as a new tool for metabolism studies. Different conditions for zebrafish larvae and HepaRG protocols were tested. As zebrafish larvae and HepaRG cell incubations provided the highest number of metabolites and the most authentic spectrum of human metabolites. The most suitable larvae protocol was the incubation via medium and the analysis of the extracted zebrafish larvae. The zebrafish larvae model might be a promising preclinical surrogate for human hepatic metabolism of NPS

Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches.

Desomorphine is an opioid misused as "crocodile", a cheaper alternative to heroin. It is a crude synthesis product homemade from codeine with toxic byproducts. The aim of the present work was to investigate the metabolic fate of desomorphine in vivo using rat urine and in vitro using pooled human liver microsomes and cytosol as well as human liver cell lines (HepG2 and HepaRG) by Orbitrap-based liquid chromatography-high resolution-tandem mass spectrometry or hydrophilic interaction liquid chromatography. According to the identified metabolites, the following metabolic steps could be proposed: N-demethylation, hydroxylation at various positions, N-oxidation, glucuronidation, and sulfation. The cytochrome P450 (CYP) initial activity screening revealed CYP3A4 to be the only CYP involved in all phase I steps. UDP-glucuronyltransferase (UGT) initial activity screening showed that UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 formed desomorphine glucuronide. Among the tested in vitro models, HepaRG cells were identified to be the most suitable tool for prediction of human hepatic phase I and II metabolism of drugs of abuse. Finally, desomorphine (crocodile) consumption should be detectable by all standard urine screening approaches mainly via the parent compound and/or its glucuronide assuming similar kinetics in rats and humans