<i>In vitro</i> metabolism of the synthetic cannabinoids PX‐1, PX‐2, and PX‐3 by high‐resolution mass spectrometry and their clearance rates in human liver microsomes

Cooman, Travon; Bell, S. A.

doi:10.1002/rcm.8543

Cited by 12 publications

(18 citation statements)

References 19 publications

(24 reference statements)

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“…A fluorine‐containing metabolite was identified by (Fabregat‐Safont et al (; M3); however, this metabolite is potentially shared with 5F‐MPP‐PICA (Figure ) as outlined below. M1–M4 reported in our report (non‐specific metabolites) were also identified by Cooman and Bell (); these were the only metabolites reported. Fabregat‐Safont et al identified five Phase II metabolites corresponding to the Phase I metabolites identified in their study.…”

Section: Discussionsupporting

confidence: 83%

Phase I metabolism of synthetic cannabinoid receptor agonist PX‐1 (5F‐APP‐PICA) via incubation with human liver microsomes and UHPLC–HRMS

Presley

Logan

Jansen‐Varnum

2020

Biomedical Chromatography

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Studies of the metabolic and pharmacological profiles of indole carboxamide synthetic cannabinoids (a prevalent class of new psychoactive substances) are critical in ensuring that their use can be detected through bioanalytical testing. We have determined the in vitro Phase I metabolism of one such compound, PX‐1 (5F‐APP‐PICA), and appropriate markers to demonstrate human consumption. PX‐1 was incubated with human liver microsomes, followed by analysis of the extracts via high‐resolution mass spectrometry. A total of 10 metabolites were identified, with simultaneous defluorination and monohydroxylation of the pentyl side chain as the primary biotransformation product (M1). Additional metabolites formed were hydroxylation products of the indole and benzyl moieties, distal amide hydrolysis, N‐desfluoropentyl, and carboxypentyl metabolites. Three monohydroxylated metabolites specific to PX‐1 were identified and are reported for the first time in this study. The primary metabolite, M1, was further oxidized to M5, a carboxypentyl metabolite. M8 is PX‐1 specific, possessing an intact fluoropentyl side chain. These three metabolites are the most suitable for implementation into bioanalytical assays for demonstrating PX‐1 consumption. The findings of this study can be used by analytical scientists and medical professionals to determine PX‐1 ingestion and predict the metabolites of synthetic cannabinoids sharing structural elements.

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Section: Discussionsupporting

confidence: 83%

Phase I metabolism of synthetic cannabinoid receptor agonist PX‐1 (5F‐APP‐PICA) via incubation with human liver microsomes and UHPLC–HRMS

Presley

Logan

Jansen‐Varnum

2020

Biomedical Chromatography

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“…Fragment m/z 233.11 is the unchanged fluoropentylindazole acylium ion, and removal of carboxamide group is demonstrated by m/z 352.18. For the observed product ion m/z 251.12, two structures can be considered: (a) a fluoropentylindazole acylium ion with addition of a water molecule as described by Fabregat-Safon et al [13] and (b) a dealkylation of the fluoropentylindazole acylium ion with a shift of the phenyl group to the second indazole nitrogen as described by Cooman et al [11]. Since a shift of the phenyl group to the second indazole nitrogen was considered to be less likely than the addition of water, configuration (a) was concluded for the first structure.…”

Section: Characteristic Fragments Of Px-2mentioning

confidence: 99%

“…The number of phase I metabolites which they found ranged from six to ten. They all found monohydroxylation of PX-1 and PX-2 and also further hydroxylations, such as dihydroxylation, amide hydrolysis, carboxylation and dealkylation [11][12][13]; monohydroxylation, oxidative defluorination, and defluorination with subsequent carboxylation [14][15][16][17][18][19][20][21][22][23][24] were the most dominant alterations. Dihydrodiol metabolites, detected for 5F-AB-PINACA and THJ-2201 were not observed for PX-1 and PX-2 [14,17,18].…”

Section: Introductionmentioning

confidence: 99%

“…In vitro metabolisms of THJ-2201, 5F-AB-PINACA and AM-2201 have been extensively studied. To date, in vitro studies on the metabolism of PX-1, applying human hepatocyte assays and pooled human liver microsomes (pHLM), have been published by Cooman et al [11], Presley et al [12] and Fabregat-Safont et al [13]. Their findings showed an SC's typical metabolism.…”

Section: Introductionmentioning

confidence: 99%

“…Their findings showed an SC's typical metabolism. Only Cooman et al [11] investigated PX-2 while Fabregat-Safont et al [13] studied also the phase II metabolism of PX-1. The number of phase I metabolites which they found ranged from six to ten.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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Detection and characterization of the new synthetic cannabinoid APP‐BINACA in forensic casework

Krotulski

Mohr

Diamond

et al. 2019

Drug Testing and Analysis

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New psychoactive substances (NPS) continue to emerge around the world. APP‐BINACA (or APP‐BUTINACA), a novel synthetic cannabinoid, was first reported in Europe in January 2019 and later in the United States in March 2019. APP‐BINACA was identified in the United States for the first time in blood sample extracts from forensic casework by liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC‐QTOF‐MS). To date, APP‐BINACA has been identified in 11 forensic toxicology cases from five states and in both medicolegal death investigations and drug impaired driving investigations. APP‐BINACA was commonly found in combination with 4F‐MDMB‐BINACA. Subsequent to its discovery in biological samples, APP‐BINACA was detected and characterized in seized drug material by gas chromatography mass spectrometry (GC–MS), LC‐QTOF‐MS, and nuclear magnetic resonance (NMR) spectroscopy. Further analysis of biological specimens resulted in the identification of five metabolites, including 4‐HO‐APP‐BINACA and APP‐BINACA 3‐phenylpropanoic acid. The frequency of APP‐BINACA detection appears to be increasing and this new synthetic cannabinoid has been identified as a possible contributory factor in adverse events, including death. This is the first literature report regarding the characterization of the new synthetic cannabinoid APP‐BINACA in humans. Since it is not widely tested for, it is not yet known the extent to which APP‐BINACA is contributing to morbidity and mortality, but forensic scientists, public health officials, and others should be aware of its possible presence and impact. Laboratories should incorporate APP‐BINACA into testing workflows for detection and confirmation, where possible.

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In vitro metabolism of the synthetic cannabinoids PX‐1, PX‐2, and PX‐3 by high‐resolution mass spectrometry and their clearance rates in human liver microsomes

Cited by 12 publications

References 19 publications

Phase I metabolism of synthetic cannabinoid receptor agonist PX‐1 (5F‐APP‐PICA) via incubation with human liver microsomes and UHPLC–HRMS

Phase I metabolism of synthetic cannabinoid receptor agonist PX‐1 (5F‐APP‐PICA) via incubation with human liver microsomes and UHPLC–HRMS

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

Detection and characterization of the new synthetic cannabinoid APP‐BINACA in forensic casework

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