scholarly journals Phase I-metabolism studies of the synthetic cannabinoids PX-1 and PX-2 using three different in vitro models

2021 ◽  
Author(s):  
Patrick Dahm ◽  
Andreas Thomas ◽  
Markus A. Rothschild ◽  
Mario Thevis ◽  
Katja Mercer-Chalmers-Bender
Keyword(s):  
Cytochrome P450 ◽  
Metabolic Pathways ◽  
High Resolution Mass Spectrometry ◽  
Synthetic Cannabinoids ◽  
Liver Microsomes ◽  
In Vitro Models ◽  
In Vitro Assays ◽  
Cunninghamella Elegans ◽  
Rat Urine

Abstract Purpose Synthetic cannabinoids (SCs), highly metabolized substances, are rarely found unmodified in urine samples. Urine screening relies on SC metabolite detection, requiring metabolism knowledge. Metabolism data can be acquired via in vitro assays, e.g., human hepatocytes, pooled human liver microsomes (pHLM), cytochrome P450 isoforms and a fungal model; or in vivo by screening, e.g., authentic human samples or rat urine. This work describes the comprehensive study of PX-1 and PX-2 in vitro metabolism using three in vitro models. 5F-APP-PICA (PX-1) and 5F-APP-PINACA (PX-2) were studied as they share structural similarity with AM-2201, THJ-2201 and 5F-AB-PINACA, the metabolism of which was described in the literature. Methods For SC incubation, pHLM, cytochrome P450 isoenzymes and the fungal model Cunninghamella elegans LENDNER (C. elegans) were used. PX-1 and PX-2 in vitro metabolites were revealed comprehensively by liquid chromatography–high-resolution mass spectrometry measurements. Results In total, 30 metabolites for PX 1 and 15 for PX-2 were detected. The main metabolites for PX-1 and PX-2 were the amide hydrolyzed metabolites, along with an indole monohydroxylated (for PX-1) and a defluorinated pentyl-monohydroxylated metabolite (for PX-2). Conclusions CYP isoforms along with fungal incubation results were in good agreement to those obtained with pHLM incubation. CYP2E1 was responsible for many of the metabolic pathways; particularly for PX-1. This study shows that all three in vitro assays are suitable for predicting metabolic pathways of synthetic cannabinoids. To establish completeness of the PX-1 and PX-2 metabolic pathways, it is not only recommended but also necessary to use different assays.

Hydroxylation and dimerization of zearalenone: comparison of chemical, enzymatic and electrochemical oxidation methods

2017 ◽  
Vol 10 (4) ◽  
pp. 297-307 ◽  
Author(s):  
J. Keller ◽  
H. Haase ◽  
M. Koch
Keyword(s):  
Mass Spectrometry ◽  
High Resolution Mass Spectrometry ◽  
Liver Microsomes ◽  
Transformation Products ◽  
In Vitro Assays ◽  
Rat Liver Microsomes ◽  
Boron Doped Diamond ◽  
Natural Processes ◽  
Increasing Demand

Investigations of the metabolic pathway of mycotoxins by microsomal techniques are often laborious, causing an increasing demand for easy and rapid simulation methods. Thus, the non-microsomal oxidation technique of electrochemistry coupled online to mass spectrometry (EC/MS) was applied to simulate phase I biotransformation of the Fusarium mycotoxin zearalenone (ZEA). The obtained transformation products were identified by high resolution mass spectrometry (FT-ICR) and HPLC-MS/MS. Transformation products (TPs) from EC/MS were compared to those of other oxidative methods, such as Fenton-like and Ce(IV) reactions and metabolites derived from in vitro assays (human and rat liver microsomes). Electrochemical oxidisation of ZEA was achieved by applying a potential between 0 and 2,500 mV vs Pd/H2 using a flow-through cell with a boron-doped diamond working electrode. Several mono-hydroxylated TPs were generated by EC/MS and Fenton-like reaction, which could also be found in microsomal in vitro assays. EC and Ce(IV) led to the formation of structurally different ZEA dimers and dimeric quinones probably connected over covalent biaryl C-C and C-O-C bonds. Although the dimerization of phenolic compounds is often observed in natural processes, ZEA dimers have not yet been reported. This is the first report on the formation of stable ZEA dimers and their related quinones. The tested non-microsomal methods, in particular EC/MS, could be useful in order to predict the biotransformation products of mycotoxins, even in cases where one to one simulation is not always feasible.

scholarly journals In Vitro Toxicokinetics and Phase I Biotransformation of the Mycotoxin Penitrem A in Dogs

Toxins ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 293 ◽  
Author(s):  
Silvio Uhlig ◽  
Lada Ivanova ◽  
Pauline Voorspoels ◽  
Christiane Kruse Fæste
Keyword(s):  
Phase I ◽  
High Resolution Mass Spectrometry ◽  
Liver Microsomes ◽  
Absorbed Dose ◽  
Metabolite Profile ◽  
Oxidation Products ◽  
In Vitro Assays ◽  
Penicillium Species

The tremorgenic mycotoxin penitrem A is produced by Penicillium species as a secondary metabolite on moldy food and feed. Dogs are sometimes exposed to penitrem A by consumption of spoiled food waste or fallen fruit. The lipophilic toxin crosses the blood-brain barrier and targets neuroreceptors and neurotransmitter release mechanisms in the central and peripheral nervous systems. Typical symptoms of penitrem A intoxication are periodical or continuous tremors, which can be passing, persistent or lethal, depending on the absorbed dose. There is presently no information on the biotransformation and toxicokinetics of penitrem A in dogs. The aim of the present study was therefore to identify potential metabolites of the toxin by performing in vitro biotransformation assays in dog liver microsomes. Analyses by liquid chromatography coupled to high-resolution mass spectrometry led to the provisional identification of eleven penitrem A phase I metabolites, which were tentatively characterized as various oxidation products. Furthermore, elimination parameters determined in in vitro assays run under linear kinetics were used for in vitro-to-in vivo extrapolation of the toxicokinetic data, predicting a maximal bioavailability of more than 50%. The metabolite profile detected in the in vitro assays was similar to that observed in the plasma of an intoxicated dog, confirming the predictive capability of the in vitro approach.

scholarly journals Cytotoxicity, metabolism, and isozyme mapping of the synthetic cannabinoids JWH-200, A-796260, and 5F-EMB-PINACA studied by means of in vitro systems

2021 ◽  
Author(s):  
Tanja M. Gampfer ◽  
Lea Wagmann ◽  
Anouar Belkacemi ◽  
Veit Flockerzi ◽  
Markus R. Meyer
Keyword(s):  
Phase I ◽  
Synthetic Cannabinoids ◽  
Liver Microsomes ◽  
In Vitro Assays ◽  
New Psychoactive Substances ◽  
Minor Role ◽  
Screening Assay ◽  
Mediated Effects ◽  
A Minor

AbstractIntake of synthetic cannabinoids (SC), one of the largest classes of new psychoactive substances, was reported to be associated with acute liver damage but information about their hepatotoxic potential is limited. The current study aimed to analyze the hepatotoxicity including the metabolism-related impact of JWH-200, A-796260, and 5F-EMB-PINACA in HepG2 cells allowing a tentative assessment of different SC subclasses. A formerly adopted high-content screening assay (HCSA) was optimized using a fully automated epifluorescence microscope. Metabolism-mediated effects in the HCSA were additionally investigated using the broad CYP inhibitor 1-aminobenzotriazole. Furthermore, phase I metabolites and isozymes involved were identified by in vitro assays and liquid chromatography–high-resolution tandem mass spectrometry. A strong cytotoxic potential was observed for the naphthoylindole SC JWH-200 and the tetramethylcyclopropanoylindole compound A-796260, whereas the indazole carboxamide SC 5F-EMB-PINACA showed moderate effects. Numerous metabolites, which can serve as analytical targets in urine screening procedures, were identified in pooled human liver microsomes. Most abundant metabolites of JWH-200 were formed by N-dealkylation, oxidative morpholine cleavage, and oxidative morpholine opening. In case of A-796260, most abundant metabolites included an oxidative morpholine cleavage, oxidative morpholine opening, hydroxylation, and dihydroxylation followed by dehydrogenation. Most abundant 5F-EMB-PINACA metabolites were generated by ester hydrolysis plus additional steps such as oxidative defluorination and hydroxylation. To conclude, the data showed that a hepatotoxicity of the investigated SC cannot be excluded, that metabolism seems to play a minor role in the observed effects, and that the extensive phase I metabolism is mediated by several isozymes making interaction unlikely.

scholarly journals 25CN-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C.elegans—Structure Determination and Synthesis of the Most Abundant Metabolites

Metabolites ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 212
Author(s):  
Anna Šuláková ◽  
Jitka Nykodemová ◽  
Petr Palivec ◽  
Radek Jurok ◽  
Silvie Rimpelová ◽  
...  
Keyword(s):  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Cunninghamella Elegans ◽  
Rat Urine ◽  
C Elegans ◽  
Liquid Chromatography Tandem Mass ◽  
Phase Ii Metabolites

N-Benzylphenethylamines are novel psychedelic substances increasingly used for research, diagnostic, or recreational purposes. To date, only a few metabolism studies have been conducted for N-2-methoxybenzylated compounds (NBOMes). Thus, the available 2,5-dimethoxy-4-(2-((2-methoxybenzyl)amino)ethyl)benzonitrile (25CN-NBOMe) metabolism data are limited. Herein, we investigated the metabolic profile of 25CN-NBOMe in vivo in rats and in vitro in Cunninghamella elegans (C. elegans) mycelium and human liver microsomes. Phase I and phase II metabolites were first detected in an untargeted screening, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of the most abundant metabolites by comparison with in-house synthesized reference materials. The major metabolic pathways described within this study (mono- and bis-O-demethylation, hydroxylation at different positions, and combinations thereof, followed by the glucuronidation, sulfation, and/or N-acetylation of primary metabolites) generally correspond to the results of previously reported metabolism of several other NBOMes. The cyano functional group was either hydrolyzed to the respective amide or carboxylic acid or remained untouched. Differences between species should be taken into account in studies of the metabolism of novel substances.

scholarly journals 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

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Frederike Nordmeier ◽  
Lilian H. J. Richter ◽  
Peter H. Schmidt ◽  
Nadine Schaefer ◽  
Markus R. Meyer
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
High Resolution Mass Spectrometry ◽  
Liver Microsomes ◽  
Rat Urine ◽  
Synthetic Opioids ◽  
High Resolution Mass ◽  
Resolution Mass

Abstract 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.

In Vitro Metabolic Profile Elucidation of Synthetic Cannabinoid APP-CHMINACA (PX-3)

2019 ◽  
Vol 44 (3) ◽  
pp. 226-236 ◽  
Author(s):  
Brandon C Presley ◽  
Barry K Logan ◽  
Susan A Jansen-Varnum
Keyword(s):  
Method Development ◽  
High Resolution Mass Spectrometry ◽  
Synthetic Cannabinoids ◽  
Liver Microsomes ◽  
Synthetic Cannabinoid ◽  
New Psychoactive Substances ◽  
Amide Hydrolysis ◽  
Causative Agents ◽  
Hydroxylated Metabolite

Abstract Indazole carboxamide synthetic cannabinoids remain the most prevalent subclass of new psychoactive substances (NPS) reported internationally. However, the metabolic and pharmacological properties of many of these compounds remain unknown. Elucidating these characteristics allows members of the clinical and forensic communities to identify causative agents in patient samples, as well as render conclusions regarding their toxic effects. This work presents a detailed report on the in vitro phase I metabolism of indazole carboxamide synthetic cannabinoid APP-CHMINACA (PX-3). Incubation of APP-CHMINACA with human liver microsomes, followed by analysis of extracts via high-resolution mass spectrometry, yielded 12 metabolites, encompassing 7 different metabolite classes. Characterization of the metabolites was achieved by evaluating the product ion spectra, accurate mass and chemical formula generated for each metabolite. The predominant biotransformations observed were hydrolysis of the distal amide group and hydroxylation of the cyclohexylmethyl (CHM) substituent. Nine metabolites were amide hydrolysis products, of which five were monohydroxylated, one dihydroxylated and two were ketone products. The metabolites in greatest abundance in the study were products of amide hydrolysis with no further biotransformation (M1), followed by amide hydrolysis with monohydroxylation (M2.1). Three APP-CHMINACA-specific metabolites were generated, all of which were hydroxylated on the CHM group; one mono-, di- and tri-hydroxylated metabolite each was produced, with dihydroxylation (M6) present in the greatest abundance. The authors propose that metabolites M1, M2.1 and M6 are the most appropriate markers to determine consumption of APP-CHMINACA. The methods used in the current study have broad applicability and have been used to determine the in vitro metabolic profiles of multiple synthetic cannabinoids and other classes of NPS. This research can be used to guide analytical scientists in method development, synthesis of reference material, pharmacological testing of proposed metabolites and prediction of metabolic processes of compounds yet to be studied.

The Molecular and Enzyme Kinetic Basis for Altered Activity of Three Cytochrome P450 2C19 Variants Found in the Chinese Population

2020 ◽  
Vol 13 (3) ◽  
pp. 233-244
Author(s):  
Amelia Nathania Dong ◽  
Nafees Ahemad ◽  
Yan Pan ◽  
Uma Devi Palanisamy ◽  
Beow Chin Yiap ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Molecular Docking ◽  
Active Site ◽  
Chinese Population ◽  
Accessible Surface Area ◽  
Solvent Accessible Surface Area ◽  
In Vitro Assays ◽  
Access Channel ◽  
Cytochrome P450 2C19

Background: There is a large inter-individual variation in cytochrome P450 2C19 (CYP2C19) activity. The variability can be caused by the genetic polymorphism of CYP2C19 gene. This study aimed to investigate the molecular and kinetics basis for activity changes in three alleles including CYP2C19*23, CYP2C19*24 and CYP2C19*25found in the Chinese population. Methods: The three variants expressed by bacteria were investigated using substrate (omeprazole and 3- cyano-7-ethoxycoumarin[CEC]) and inhibitor (ketoconazole, fluoxetine, sertraline and loratadine) probes in enzyme assays along with molecular docking. Results: All alleles exhibited very low enzyme activity and affinity towards omeprazole and CEC (6.1% or less in intrinsic clearance). The inhibition studies with the four inhibitors, however, suggested that mutations in different variants have a tendency to cause enhanced binding (reduced IC50 values). The enhanced binding could partially be explained by the lower polar solvent accessible surface area of the inhibitors relative to the substrates. Molecular docking indicated that G91R, R335Q and F448L, the unique mutations in the alleles, have caused slight alteration in the substrate access channel morphology and a more compact active site cavity hence affecting ligand access and binding. It is likely that these structural alterations in CYP2C19 proteins have caused ligand-specific alteration in catalytic and inhibitory specificities as observed in the in vitro assays. Conclusion: This study indicates that CYP2C19 variant selectivity for ligands was not solely governed by mutation-induced modifications in the active site architecture, but the intrinsic properties of the probe compounds also played a vital role.

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