Brodifacoum (BFC) is an inhibitor of vitamin K epoxide reductase and active ingredient of rodenticides (King & Tran, 2015). There have been sporadic reports of brodifacoum consumption with drugs such as cocaine and cannabis (La Rosa, Clarke & Lefkowitz, 1997; Waien, Hayes Jr & Leonardo, 2001; Spahr, Maul & Rodgers, 2007), however, a large number of people were recently hospitalized with poisoning by brodifacoum and related compounds following ingestion of what are believed to be synthetic cannabinoid receptor agonists (SCRAs) (Kelkar et al., 2018; Riley et al., 2019; Moritz et al., 2018; Panigrahi, Jones & Rowe, 2018). There is limited evidence to suggest that people have on occasions deliberately combined brodifacoum with cannabis (La Rosa, Clarke & Lefkowitz, 1997; Spahr, Maul & Rodgers, 2007), and the apparent mixing of brodifacoum with a variety of different SCRA could be a deliberate attempt to enhance the effects of the drugs through either a pharmacokinetic or pharmacodynamic mechanism. In this study, we have examined the effects of brodifacoum on the acute signalling of human CB1 and CB2 receptors in AtT20 and HEK 293 cells. In AtT20 cells, activation of heterologously expressed CB1 or CB2 produces a hyperpolarization, mediated by activation of G protein-gated inwardly rectifying K channels (Mackie et al., 1995; Banister et al., 2016). In CB1- or CB2-expressing HEK 293 cells, we measured the real time modulation of forskolin-stimulated cAMP accumulation (Cawston et al., 2013). We found that cannabinoid-induced signaling was not affected by brodifacoum, indicating that combining SCRA with brodifacoum is not likely to enhance user experience through interactions with cannabinoid receptors.
(-) CP 55940 was from Cayman Chemical (#90084; Ann Arbor MI, USA), Δ9-tetrahydrocannabinol (THC) was from THCPharm (Frankfurt, Germany) and was a kind gift from the Lambert Initiative for Cannabis Therapeutics (University of Sydney). Brodifacoum was from Sigma-Aldrich (#46036), and forskolin was from Ascent Scientific Ltd.
Experiments on AtT20FlpIn cells stably transfected with human CB1 (AtT20-CB1) or CB2 (AtT20-CB2) were carried out essentially as described in Banister et al. (2016). The AtT20FlpIn cells were created in our laboratory from wild type AtT20 cells we purchased from the American Type Culture Collection (ATCC CRL-1795). The assay method is based on that outlined in detail in Knapman et al. (2013). Cells were grown in DMEM (#D6429; Sigma-Aldrich, Castle Hill, NSW) supplemented with 10% fetal bovine serum (FBS, #12003C; SAFC Biosciences, Brooklyn, Victoria, Australia), 100 units penicillin/100 µg ml−1 streptomycin (1%, #15140122; Life Technologies, Scoresby, Victoria, Australia), hygromycin gold (80 µg ml−1, #ant-hg; Invivogen, San Diego, CA). Cells were grown in 75 cm2 flasks and passaged when 80–90% confluent. On the evening before experiments, cells were detached using trypsin/EDTA solution (#T3924; Sigma-Aldrich), resuspended in L-15 media (#11415064; Life Technologies) supplemented with 1% FBS, penicillin/streptomycin, and glucose (15 mM, SIGMA #G7021) and plated onto 96 well black walled, clear bottomed, culture plates which had been previously coated with poly-D-lysine (SIGMA #P6407). Cells were incubated overnight at 37 °C in a humidified incubator in room air.
Proprietary FLIPR membrane potential dye (blue, #R8034, Molecular Devices, Sunnyvale CA) was dissolved in Hank’s Balanced Salt Solution (HBSS) of composition (mM) NaCl 145, HEPES 22, Na2HPO4 0.338, NaHCO3 4.17, KH2PO4 0.441, MgSO4 0.407, MgCl2 0.493, CaCl2 1.26, glucose 5.56 (pH 7.4, osmolarity 315 ± 15) and added to the cells an hour before fluorescence reading began. Dye was used at 50% of the manufacturers recommended concentration, and cells were incubated at 37 °C in humidified room air for loading. Plates were read using a Flexstation 3 (Molecular Devices) plate reader at 37 °C. Plates were excited at a wavelength of 530 nm, emission was measured at 565 nm, with cut-off filter at 550 nm. Drugs were added using the pipetting function of the Flexstation in a volume of 20 µl after recording 60–120 s of baseline fluorescence. Readings were made every 2 s. Drug stocks were made up in DMSO (#D8418, Sigma-Aldrich) and diluted on the day of experiment, the final concentration of DMSO in the assay was 0.1%.
Data were expressed as the percentage change in baseline fluorescence produced by drug addition. The change in fluorescence produced by vehicle (0.1% DMSO) addition was subtracted from the traces before this calculation. Data is expressed as the mean ± SEM of at least 5 independent determinations performed in duplicate, unless otherwise noted. Pooled data was fit to a four-parameter logistic equation in Graphpad PRISM 7 (GraphPad Software, San Diego CA, USA).
Assay of cAMP levels
Human embryonic kidney (HEK) 293 FlpIn cells stably transfected with human CB1 or CB2 receptors tagged with three haemagglutinin epitopes at the amino terminus and human G protein gated inwardly rectifying potassium channel 4 (GIRK4) were used (the construction of these cells will be described in another place, and we did not assay CB receptor coupling to GIRK4 in this study). Cells were grown in DMEM containing 10% FBS and 100 units/ml/penicillin, 100 µg/ml streptomycin and were maintained under selection with hygromycin (80 µg ml−1) and G418 (400 µg ml−1). HEK 293 FlpIn cells were originally obtained from Life Technologies (now Thermofisher, #75007).
Cellular cAMP levels were measured using the pcDNA3L-His-CAMYEL plasmid, which encodes the cAMP sensor YFP-Epac-RLuc (CAMYEL), (Cawston et al., 2013). The pcDNA3L-His-CAMYEL was a kind gift from Dr. Angela Finch (The University of New South Wales, NSW, Australia), and originally obtained from American Type Culture Collection (Manassas, VI, USA). Cells were seeded in 10 cm dishes at a density of 6,000,0000 such that they would be 60–70% confluent the next day. The day after seeding, pcDNA3L-His-CAMYEL plasmid was transiently transfected into cells using linear polyethyleneimine (PEI, m.w. 25 kDa) (#23966, Polysciences, Warrington, PA, USA). The DNA-PEI complex mixture was added to the cells at the ratio of 1:6, and incubated for 24 h in 5% CO2 at 37 °C. After the incubation, cells were detached from the dish using trypsin/EDTA and the pellet was resuspended in 10 ml Leibovitz’s L-15, no phenol red (#21083027; Gibco) media supplemented with 1% FBS, 100 units/ml/penicillin, 100 µg/ml streptomycin and 15 mM glucose. The cells were seeded at a density of 100,000 cells per well in poly D-lysine (Sigma-Aldrich) coated, white wall, clear bottom 96 well microplates. Cells were incubated overnight at 37 °C in ambient CO2.
On the following day, drugs were prepared in HBSS containing 0.1 mg ml−1 BSA. For measurement of cAMP inhibition, all the drugs were made in 3 µM of forskolin. Coelenterazine-h substrate (2.5 µM) (#S2011; Promega, Madison, WI, USA) was added to the cells, and incubated for 5 mins prior to the addition of drugs or vehicle. Luminescence was measured using a Flexstation 3 (Molecular Devices) microplate reader at 37 °C at an emission wavelength of 461 nm and 542 nm simultaneously, with an integration time of 1 s. Drugs were added in a volume of 10 µl (10×) to each well to give the desired concentration. The final concentration of DMSO in each well was always 0.1%. Raw data are presented as inverse BRET ratio of emission at 461/542. Background reading (no substrate) was subtracted from raw values before calculating ratios. For convenience, values are expressed such that an increase in ratio correlates with increase in cAMP production. Area under the curve (AUC) analysis was performed in GraphPad prism (Graph Pad Software Inc., San Diego, CA, USA), and data were expressed as percentage of the difference between basal (vehicle, 0%) and forskolin (100%) values over a 5-minute period after forskolin addition.
For experiments examining the potential interaction between brodifacoum and cannabinoids, the cells were pre-treated with 1 µM of brodifacoum (or vehicle) and the response to a subsequent addition of SCRAs was measured. The concentration of DMSO (0.1%) was kept constant for the brodifacoum-treated and control cells. Data was normally distributed (D’Agostino and Pearson normality test, PRISM), differences between groups were tested using unpaired Student’s t-Test (PRISM). Statistical significance was defined as P < 0.05.
Acute application of brodifacoum for 5 min at concentrations up to 30 µM did not significantly affect the fluorescence of AtT20 cells expressing CB1 or CB2 receptors (Fig. 1). Prolonged exposure to brodifacoum at concentrations greater than 10 µM produced decreases in fluorescence in AtT20 cells expressing CB receptors as well as wild type cells, and so for experiments examining the potential interaction between brodifacoum and cannabinoids we used a concentration of 1 µM.
We generated concentration–response curves for the high efficacy cannabinoid agonist CP55940 and the lower efficacy agonist THC after 5 min of exposure to brodifacoum (Fig. 2). In AtT20-CB1 cells, application of CP55940 produced a maximum change in fluorescence of 33 ± 1%, with a pEC50 of 7.7 ± 0.04; with the addition of brodifacoum the maximum change in fluorescence was 33 ± 1%, with a pEC50 of 7.7 ± 0.06 (P = 0.97). In AtT20-CB2 cells, application of CP55940 produced a maximum change in fluorescence of 29 ± 1.1%, with a pEC50 of 7.3 ± 0.1; with the addition of brodifacoum the maximum change in fluorescence was 31 ± 1.2%, with a pEC50 of 7.4 ± 0.1 (Fig. 2, P = 0.85). Brodifacoum failed to affect the hyperpolarization produced by THC in AtT20-CB1 cells (control, pEC50 6.4 ± 0.6, maximum change in fluorescence 18 ± 5%; in brodifacoum, pEC50 6.5 ± 0.5, max 18 ± 5%, P = 0.95). In AtT20-CB2 cells THC only produced a small hyperpolarization, the response to 10 µM THC was unchanged in the presence of brodifacoum (6.4 ± 1.2% in control, 7.4 ± 1.8% in brodifacoum, P = 0.65) (Fig. 2). Application of brodifacoum (10 µM) or CP55940 (1 µM) for 5 min produced very small changes in the fluorescence of wild type AtT20 cells, and neither drug affected the response to subsequently applied somatostatin (100 nM), which activates native SST receptors in AtT20 cells (Günther, Culler & Schulz, 2016) (Fig. S1).
Inhibition of adenylyl cyclase activity is another significant biological effect of cannabinoid receptor activation. Brodifacoum (300 nM–30 µM) co-applied with forskolin (3 µM) for 10 min did not affect increases in cAMP levels in HEK 293 cells expressing CB1 or CB2 (Fig. 3). Brodifacoum (1 µM) incubation for 5 min also failed to affect the CP55940 inhibition of forskolin-stimulated cAMP elevation. In cells expressing CB1, CP55940 inhibited cAMP with a pEC50 of 7.5 ± 0.3, to a minimum of 52 ± 12% of forskolin alone; in the presence of brodifacoum these were pEC50 7.4 ± 0.2 and minimum of 52 ± 7% of the forskolin response. Brodifacoum also did not affect forskolin-stimulated cAMP levels in HEK293 cells expressing CB2 (Fig. 3), or CP55940 inhibition of cAMP levels (pEC50 in control cells expressing CB2 7.4 ± 0.2, to a minimum of 39 ± 7%; in brodifacoum pEC50 of 7.5 ± 0.1; to a minimum of 45 ± 4%).
We also examined the possibility that brodifacoum could affect the sustained responses to CP55940 or THC. As previously described (Cawston et al., 2013), prolonged application of cannabinoids in AtT20-CB1 cells produces a response that wanes over time, reflecting desensitization of receptor signaling. The degree to which this desensitization reflects changes in signaling specific to cannabinoid receptors is tested by application of somatostatin, which activates receptors native to AtT20 cells (Günther, Culler & Schulz, 2016; Heblinski, Bladen & Connor, 2019). In these experiments, CP55940 (100 nM) or THC (10 µM) were applied 2 min after addition of brodifacoum (1 µM), and the fluorescence monitored for 30 min before the addition of somatostatin (100 nM) (Fig. 4). Desensitization was quantified after 30 min of agonist application, and was expressed as the % decline from the peak response. We did not observe any significant difference in the desensitization of CB1 signaling mediated by CP55940 (100 nM) when co-applied with brodifacoum (Control, 71 ± 4%; brodifacoum treated, 66 ± 7%, P = 0.55). The presence of brodifacoum had no effect on the somatostatin (100 nM) induced hyperpolarization alone, or after 30 mins of CP55940 treatment (P = 0.75) (Fig. S2). The desensitization produced by THC (10 µM, 30 mins) in AtT20-CB1 cells was not different when co-applied with brodifacoum, (Control, 65 ± 6%; brodifacoum treated, 53 ± 8%, P = 0.3) (Fig. 4). A similar reversal of the hyperpolarization produced by CP55940 (100 nM) in AtT20-CB2 cells was also observed. Treatment with brodifacoum did not significantly affect the desensitization produced by CP55940 compared to control cells (Control, 77 ± 6%; brodifacoum treated, 63 ± 8%, P = 0.2). THC (10 µM, 30 mins) signaling at CB2, although modest, also declined during continuous drug exposure, and this was also not affected by co-application of brodifacoum (37 ± 14% in control, 20 ± 7% in brodifacoum treated, P = 0.3) (Fig. 4). The hyperpolarization induced by somatostatin after prolonged application of CP55940 (P = 0.56) or THC (P = 0.87) to AtT20-CB2 cells was also not significantly different in the presence of brodifacoum (Fig. S2).
The principal finding of this study is that brodifacoum does not affect CB1 or CB2 signaling, either to K channels in AtT20 cells or adenylyl cyclase in HEK 293 cells. In the assay of K channel activation, there was no effect on the concentration response relationship for CP55940 or THC, and brodifacoum did not affect the desensitization of signaling produced by prolonged application of either drug. Brodifacoum had no effect on the potency, maximum effect or time-dependence of the actions of the high efficacy synthetic cannabinoid CP55940 or the lower efficacy phytocannabinoid THC, indicating that it is unlikely to act as modulator of the pharmacodynamic effects of cannabinoids.
Activation of GIRK is mediated by the Gβγ subunits of G protein heterotrimers, and many Gi/Go coupled receptors effectively signal through this pathway in AtT20 cells (e.g., Mackie et al., 1995; Günther, Culler & Schulz, 2016; Knapman et al., 2013; Heblinski, Bladen & Connor, 2019). We have previously used the fluorescent measurement of membrane potential to study CB1 and CB2 agonists, antagonists, and allosteric modulators of CB1 (Cawston et al., 2013). Inhibition of adenylyl cyclase activity by CB receptors is mediated via the Gα subunits of G protein heterotrimers, and brodifacoum also failed to affect this signal transduction pathway. The precise cellular signaling mechanisms responsible for the subjective effects of Cannabis and synthetic cannabinoid agonists are not established, although the signal transduction of cannabinoid receptors has been extensively studied (Howlett & Abood, 2017; Ibsen, Connor & Glass, 2017) and it is unlikely that any one pathway is responsible. It remains formally possible that brodifacoum could selectively modulate pathways other than Gβγ-mediated activation of GIRK or Gα-mediated inhibition of cAMP accumulation, but the lack of any effect whatsoever on the effects of CP55940 or THC suggests that ligand interactions with cannabinoid receptors are unaffected by brodifacoum.
The concentration of brodifacoum in blood or brain after co-ingestion with synthetic cannabinoids is unknown. However, concentrations of up to 3 µM have been reported in the serum of people who have deliberately ingested large quantities of rat poison (Weitzel et al., 1990; Hollinger & Pastoor, 1993), and inhalation of BFC via smoked synthetic cannabinoids may produce higher serum concentrations of BFC than ingestion. Brodifacoum at 1 µM failed to affect CB1 or CB2 receptor signaling when measured continuously over a period of 30 min, and 10 µM brodifacoum failed to mimic or affect the acute response to a maximally effective concentration of CP 55940, although at this concentration prolonged application of brodifacoum produced a decrease in the fluorescence of wild type AtT20 cells, as well as those expressing CB1 and CB2 receptors. This effect at higher concentrations may reflect direct interactions of brodifacoum with cell membranes (Marangoni et al., 2016). Concentrations of brodifacoum in the upper range of what we tested are achieved only after ingestion of large amounts of rat bait, it is possible that they could be achieved while ingesting contaminated synthetic cannabinoids, but this remains unreported.
Several case reports suggest an interaction between therapeutic warfarin and cannabis or cannabidiol (Grayson et al., 2018; Yamreudeewong et al., 2009; Damkier et al., 2019). It has been suggested that cannabinoid inhibition of enzymes responsible for the metabolism of warfarin can increase blood levels of the drug, and while these studies have focussed on potentially dangerous changes in warfarin concentration, levels of cannabinoids could also be reciprocally elevated. Such interactions may inform a decision to deliberately combine “superwarfarin” with SCRA, as has been previously suggested for cannabis (La Rosa, Clarke & Lefkowitz, 1997; Spahr, Maul & Rodgers, 2007), although whether brodifacoum is metabolized by pathways shared with SCRA in humans is unknown. Information about how or even whether BFC is metabolized in humans is very sparse, although available evidence suggests metabolism is very limited or absent (Hauck, Feinstein & Van Breeman, 2016). Apart from the obvious danger of ingesting brodifacoum, altering the metabolism of SCRA is likely to have unpredictable consequences, as some metabolites of SCRA retain cannabinoid receptor activity (e.g., Brents et al., 2011; Chimalakonda et al., 2012; Longworth et al., 2017; Cannaert et al., 2016), and may contribute to the overall SCRA experience.
Ingestion of brodifacoum is relatively common, while death from exposure is rare, owing to ready treatment with vitamin K (King & Tran, 2015; Gummin et al., 2018). The high number of deaths associated with the combination of SCRA and anticoagulants in 2018 (at least eight; Connors, 2018) may point to an interaction between the drugs. It may also reflect the identity and dose of the synthetic cannabinoid(s) consumed, as well as the general health status of the drug users. Deaths from synthetic cannabinoid exposure are uncommon, but well documented (e.g., Kasper et al., 2015; Trecki, Gerona & Schwartz, 2015). While there is a general acceptance that brodifacoum or a similar agent is responsible for the coagulopathies associated with synthetic cannabinoid ingestion, identification of the synthetic cannabinoid has not been reported in most cases, but a recent report identified a metabolite of AB-FUBINACA in one patient following ingestion of “King Kong”, a brodifacoum laced SCRA (Riley et al., 2019). It seems unlikely, though, that brodifacoum would interact with higher efficacy or potency SCRAs at cannabinoid receptors when it clearly does not interact with CP55940 or THC signaling (Noble et al., 2019; Sachdev et al., in press). Intriguingly, several groups have reported cannabinoid receptor ligands based on a coumarin scaffold (Behrenswerth et al., 2009; Han et al., 2015). While these drugs have been reported to be either antagonists/inverse agonists (Behrenswerth et al., 2009) or CB2-selective agonists (Han et al., 2015), they remain largely uncharacterized. Given the propensity of chemists producing and, in some cases, designing cannabinoids for the recreational market, it cannot be ruled out that coagulopathy may be an unanticipated adverse effect of a synthetic cannabinoid, which may have arisen from a novel, coumarin-based cannabinoid that retains some of the vitamin K epoxide reductase inhibitory of warfarin and brodifacoum.
In conclusion, we report that brodifacoum does not appear to be an agonist or antagonist of human cannabinoid receptors, and it also does not appear to be an allosteric modulator of CB1 or CB2 activation of K channels or inhibition of adenylyl cyclase. Why brodifacoum has been mixed with synthetic cannabinoid receptor agonists remains a matter for speculation, although an intended effect on synthetic cannabinoid drug pharmacokinetics cannot entirely be ruled out.
Effects of brodifacoum (BFC) and CP55940 in wild type AtT20 cells
Scatter dot plot representing the percentage change in fluorescence for BFC (30 µM), BFC (10 µM), CP55940 (10 µM), and Vehicle (0.1% DMSO) alone (blue dots), and the response to the subsequent addition of SOMATOSTATIN (100 nM) to AtT20-WT cells (black dots). Data represents the mean ± SEM of five independent experiments performed in duplicate (p ¿0.05).
Effect of brodifacoum (BFC) on somatostatin (SRIF) challenge after 30 minutes of drugs on AtT20-CB1 and -CB2 cells
Comparison of percentage change in fluorescence after SRIF (100 nM) challenge on AtT20-CB1, and AtT20-CB2 in the continuous presence of (A), (C) CP55940 or (B), (D) Δ9-THC added with either HBS or BFC (1 µM). BFC did not affect the hyperpolarization induced by SRIF after prolonged application of CP55940 or Δ9-THC. Data represents the mean ± SEM of five independent experiments performed in duplicate ( p > 0.05).