Brodifacoum does not modulate human cannabinoid receptor-mediated hyperpolarization of AtT20 cells or inhibition of adenylyl cyclase in HEK 293 cells

Abstract

BackgroundSynthetic cannabinoids are a commonly used class of recreational drugs that can have significant adverse effects. There have been sporadic reports of co-consumption of illicit drugs with rodenticides such as warfarin and brodifacoum (BFC) over the past 20 years but recently, hundreds of people have been reported to have been poisoned with a mixture of synthetic cannabinoids and BFC. We have sought to establish whether BFC directly affects cannabinoid receptors, or their activation by the synthetic cannabinoid CP55940 or the phytocannabinoid Δ9-tetrahydrocannabinol (Δ9-THC).MethodsThe effects of BFC on the hyperpolarization of wild type AtT20 cells, or AtT20 cells stably expressing human CB1- or CB2- receptors, were studied using a fluorescent assay of membrane potential. The effect of BFC on CB1- and CB2-mediated inhibition of forskolin-stimulated adenylyl cyclase (AC) activation was measured using a BRET assay of cAMP levels in HEK 293 cells stably expressing human CB1or CB2.ResultsBFC did not activate CB1or CB2receptors, or affect the hyperpolarization of wild type AtT20 cells produced by somatostatin. BFC (1 µM) did not affect the hyperpolarization of AtT20-CB1or AtT20-CB2cells produced by CP55940 or Δ9-THC. BFC (1 µM) did not affect the inhibition of forskolin-stimulated AC activity by CP55940 in HEK 293 cells expressing CB1or CB2. BFC (1 µM) also failed to affect the desensitization of CB1and CB2signaling produced by prolonged (30 min) application of CP55940 or Δ9-THC to AtT20 cells.DiscussionBFC is not a cannabinoid receptor agonist, and appeared not to affect cannabinoid receptor activation. Our data suggests there is no pharmacodynamic rationale for mixing BFC with synthetic cannabinoids; however, it does not speak to whether BFC may affect synthetic cannabinoid metabolism or biodistribution. The reasons underlying the mixing of BFC with synthetic cannabinoids are unknown, and it remains to be established whether the “contamination” was deliberate or accidental. However, the consequences for people who ingested the mixture were often serious, and sometimes fatal, but this seems unlikely to be due to BFC action at cannabinoid receptors.

Do gabapentin or pregabalin directly modulate the µ receptor?

“…Bioluminescence based cAMP assay was performed in HEK-µ cells to investigate the effect of pregabalin or gabapentin on morphine mediated inhibition of cAMP production (Sachdev et al, 2019;Manandhar, Sachdev & Santiago, 2020). The cells for the assay were detached from the flask with trypsin/EDTA and seeded in a 10 cm plate at a density of 6 million cells to reach 60-70% confluency in 24 h. Next day, pcDNA3L-His-CAMYEL plasmid was transiently transfected into the cells using linear polyethyleneimine (PEI) at a ratio of 1:6 (DNA: PEI).…”

Superwarfarin (Long-Acting Anti-coagulant Rodenticides) Poisoning: from Pathophysiology to Laboratory-Guided Clinical Management

“…16 Limited experimental data suggests that there is no pharmacodynamics interaction between brodifacoum and synthetic cannabinoids, though pharmacokinetic interactions have not been ruled out. 72 It remains unclear as to whether the presence of superwarfarins in synthetic cannabinoids represents malicious adulteration or inadvertent contamination.…”

Monitoring Cannabinoid CB2 -Receptor Mediated cAMP Dynamics by FRET-Based Live Cell Imaging

“…Previous studies of CB2-mediated cAMP signaling have exclusively used multicell approaches to determine the inhibition of cAMP production after CB2 activation as either endpoint [ 13 , 21 , 35 ] or as live measures [ 19 , 23 ]. Similar to this study, FSK and, often, also the PDE inhibitor IBMX (3-isobutyl-1-methylxanthine), are added to the stimulation with the receptor ligands to increase basal cAMP levels.…”

“…Over the last few years, advancements in the measurement of intracellular cAMP levels have significantly contributed to the understanding of the spatial and temporal control of cAMP-dependent signaling in cells. Transfectable biosensors that report cellular cAMP concentrations by bioluminescence resonance energy transfer (BRET) has been utilized to elucidate the temporal nature of agonists and allosteric modulators of the cannabinoid receptor CB1 [ 22 ] and CB2 [ 19 , 23 ].…”