Hide and Seek: Overcoming the Masking Effect of Opioid Antagonists in Activity-Based Screening Tests

Cannaert, Annelies; Deventer, Marie H; Fogarty, Melissa F; Mohr, Amanda L A; Stove, Christophe

doi:10.1373/clinchem.2019.309443

Cited by 15 publications

(18 citation statements)

References 2 publications

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“…The stability of the cell line (i.e. the expression levels of fusion proteins) was monitored by flow cytometric analysis of co‐expressed markers . The cells were routinely maintained at 37°C, 5% CO 2 , under a humidified atmosphere in DMEM (GlutaMAX™) supplemented with 10% heat‐inactivated FBS, 100 IU/mL penicillin, 100 mg/L streptomycin, and 0.25 mg/L amphotericin B.…”

Section: Methodsmentioning

confidence: 99%

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Report on a novel emerging class of highly potent benzimidazole NPS opioids: Chemical and in vitro functional characterization of isotonitazene

Blanckaert

Cannaert

Uytfanghe

et al. 2020

Drug Testing and Analysis

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This paper reports on the identification and full chemical characterization of isotonitazene (N,N‐diethyl‐2‐[5‐nitro‐2‐({4‐[(propan‐2‐yl)oxy]phenyl}methyl)‐1H‐benzimidazol‐1‐yl]ethan‐1‐amine), a potent NPS opioid and the first member of the benzimidazole class of compounds to be available on online markets. Interestingly, this compound was sold under the name etonitazene, a structural analog. Identification of isotonitazene was performed by gas chromatography mass spectrometry (GC–MS) and liquid chromatography time‐of‐flight mass spectrometry (LC‐QTOF‐MS), the latter identifying an exact‐mass m/z value of 411.2398. All chromatographic data indicated the presence of a single, highly pure compound. Confirmation of the specific benzimidazole regio‐isomer was performed using 1H and 13C NMR spectroscopy, after which the chemical characterization was finalized by recording Fourier‐transform (FT‐IR) spectra. A live cell‐based reporter assay to assess the in vitro biological activity at the μ‐opioid receptor (MOR) revealed that isotonitazene has a high potency (EC50 of 11.1 nM) and efficacy (Emax 180% of that of hydromorphone), thus confirming that this substance is a strong opioid. Isotonitazene has not been previously detected, either in powder form, or in biological fluids. The high potency and efficacy of isotonitazene, combined with the fact that this compound was being sold undiluted, represents an imminent danger to anyone aiming to use this powder.

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Section: Methodsmentioning

confidence: 99%

“…the expression levels of fusion proteins) was monitored by flow cytometric analysis of coexpressed markers. 30 The cells were routinely maintained at 37 C, 5%…”

Section: Determination Of Biological Activity At the μ-Opioid Recepmentioning

confidence: 99%

Report on a novel emerging class of highly potent benzimidazole NPS opioids: Chemical and in vitro functional characterization of isotonitazene

Blanckaert

Cannaert

Uytfanghe

et al. 2020

Drug Testing and Analysis

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“…Both βarr2 and MOR are fused to an inactive part of nanoluciferase. When MOR is activated by a ligand, βarr2 is recruited to the receptor, allowing interaction of the complementary nanoluciferase subunits, yielding a functional enzyme that generates a bioluminescent signal in the presence of the substrate furimazine (Cannaert et al 2019). In vitro MOR activity analysis of 4F-Cy-BAP and Fu-BAP revealed that these compounds were only able to activate MOR to a limited extent.…”

Section: In Vitro Mor Receptor Activitymentioning

confidence: 99%

Toxicokinetics and toxicodynamics of the fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine and furanoyl-1-benzyl-4-anilinopiperidine

et al. 2020

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The two fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine (4F-Cy-BAP) and furanoyl-1-benzyl-4-anilinopiperidine (Fu-BAP) have recently been seized as new psychoactive substances (NPS) on the drugs of abuse market. As their toxicokinetic and toxicodynamic characteristics are completely unknown, this study focused on elucidating their in vitro metabolic stability in pooled human liver S9 fraction (pHLS9), their qualitative in vitro (pHLS9), and in vivo (zebrafish larvae) metabolism, and their in vitro isozyme mapping using recombinant expressed isoenzymes. Their maximum-tolerated concentration (MTC) in zebrafish larvae was studied from 0.01 to 100 µM. Their µ-opioid receptor (MOR) activity was analyzed in engineered human embryonic kidney (HEK) 293 T cells. In total, seven phase I and one phase II metabolites of 4F-Cy-BAP and 15 phase I and four phase II metabolites of Fu-BAP were tentatively identified by means of liquid chromatography high-resolution tandem mass spectrometry, with the majority detected in zebrafish larvae. N-Dealkylation, N-deacylation, hydroxylation, and N-oxidation were the most abundant metabolic reactions and the corresponding metabolites are expected to be promising analytical targets for toxicological analysis. Isozyme mapping revealed the main involvement of CYP3A4 in the phase I metabolism of 4F-Cy-BAP and in terms of Fu-BAP additionally CYP2D6. Therefore, drug-drug interactions by CYP3A4 inhibition may cause elevated drug levels and unwanted adverse effects. MTC experiments revealed malformations and changes in the behavior of larvae after exposure to 100 µM Fu-BAP. Both substances were only able to produce a weak activation of MOR and although toxic effects based on MOR activation seem unlikely, activity at other receptors cannot be excluded. Keywords In vitro and in vivo metabolism • Metabolic stability • LC-HRMS/MS • Zebrafish larvae • In vitro µ-opioid receptor activity Electronic supplementary material The online version of this article (

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“…The in vitro activity of brorphine at MOR was evaluated using a previously reported, live cell-based assay (20,25). The efficacy of brorphine (Emax = 209%) was found to be double that of the reference agonist hydromorphone (Emax = 97.5%), indicating potentially higher levels of opioid effects with brorphine.…”

Section: Discussionmentioning

confidence: 99%

“…The biological activity of brorphine at MOR was evaluated by means of a previously reported, live cell-based MOR reporter assay (20,25). The employed assay monitors MOR activation via its interaction with β-arrestin 2 (βarr2) using the NanoLuc Binary Tech-nology® (Promega).…”

Section: Determination Of In Vitro Biological Activity At Mormentioning

confidence: 99%

First Report on Brorphine: The Next Opioid on the Deadly New Psychoactive Substance Horizon?

Verougstraete

Vandeputte

Lyphout

et al. 2020

Journal of Analytical Toxicology

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New psychoactive substances (NPS) continue to appear on the drug market. Until recently, new synthetic opioids, which are amongst the most dangerous NPS, primarily encompassed analogues of the potent analgesic fentanyl. Lately, also other new synthetic opioids have increasingly started to surface. This is the first report on the identification and full chemical characterization of brorphine, a novel potent synthetic opioid with a piperidine benzimidazolone structure. Brorphine was identified in a powder and in the serum of a patient seeking medical help for detoxification. Liquid chromatography–high resolution mass spectrometry (LC–HRMS) identified an exact mass of m/z 400.1020 and 402.1005 for the compound, corresponding to both bromine isotopes. Further chemical characterization was performed by gas chromatography–mass spectrometry (GC–MS), LC–diode array detection (DAD) and Fourier-transform infrared (FT-IR) spectroscopy analyses. Finally, the structure was confirmed by performing 1H- and 13C-NMR spectroscopy. In vitro biological activity of brorphine was determined by a cell-based µ-opioid receptor (MOR) activation assay, resulting in an EC50 of 30.9 nM (13.5 ng/mL) and an Emax of 209% relative to hydromorphone, confirming the high potency and efficacy of this compound. In a serum sample of the patient, brorphine and a hydroxy-metabolite were found using the LC–HRMS screening method. The presence of opioid activity in the serum was also confirmed via the activity-based opioid screening assay. The occurrence of brorphine is yet another example of how the illicit drug market is continuously evolving in an attempt to escape international legislation. Its high potency poses a serious and imminent health threat for any user.

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Hide and Seek: Overcoming the Masking Effect of Opioid Antagonists in Activity-Based Screening Tests

Cited by 15 publications

References 2 publications

Report on a novel emerging class of highly potent benzimidazole NPS opioids: Chemical and in vitro functional characterization of isotonitazene

Report on a novel emerging class of highly potent benzimidazole NPS opioids: Chemical and in vitro functional characterization of isotonitazene

Toxicokinetics and toxicodynamics of the fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine and furanoyl-1-benzyl-4-anilinopiperidine

First Report on Brorphine: The Next Opioid on the Deadly New Psychoactive Substance Horizon?

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