In vitro determination of the CB1 efficacy of illicit synthetic cannabinoids

Abstract

BACKGROUND AND PURPOSEThe morbidity and mortality associated with recreational use of synthetic cannabinoid receptor agonists (SCRAs) is a major health concern, and may involve over-activation of CB1 receptors.Thus, we sought to determine the efficacy of 13 SCRAs at CB1 using receptor depletion with the irreversible CB1 antagonist AM6544 followed by fitting the curve with the Black and Leff operational model to calculate efficacy. EXPERIMENTAL APPROACHReceptor depletion in mouse AtT-20 neuroblastoma cells stably expressing human CB1 was achieved by pre-treatment of cells with AM6544 (10 µM, 60 mins). The CB1-mediated hyperpolarisation of AtT20 cells was measured using membrane potential dye. From data fit to the operational model, the efficacy (tau) and affinity (KA) parameters were obtained for each drug. KEY RESULTSAM6544 did not affect the potency or maximal effect of native somatostatin receptor-induced hyperpolarisation (Control, pEC50 9.13 ± 0.05, Emax 38 ± 1%; AM6544 treated pEC50 9.18 ± 0.04, Emax 39 ± 0.7%). The tau value of ∆ 9 -THC was 70-fold less than the reference CB-agonist CP55940, and 240-fold less than the highest efficacy SCRA, 5F-MDMB-PICA. Most of the SCRAs had about 50% of the efficacy of CP55940. There was no correlation between the tau and KA values for any SCRA. CONCLUSION AND IMPLICATIONSAll the SCRA tested showed substantially higher agonist activity at CB1 than ∆ 9 -THC, which may contribute to the adverse effects seen with these drugs but not ∆ 9 -THC, although the mechanisms underlying SCRA toxicity are still poorly defined.

Detection and quantitation of synthetic cannabinoid receptor agonists in infused papers from prisons in a constantly evolving illicit market

“…As might be expected from available pharmacological data for structurally similar compounds, 4F‐MDMB‐BINACA has been shown by Jawalosky (as referenced in a recent World Health Organization Critical Review) to bind to the CB 1 receptor (4F‐MDMB‐BINACA at CB 1 : Ki = 14.3 nM; (R)‐(+)‐WIN‐55,212–2: Ki = 172 nM; Δ 9 ‐THC: Ki =22.5 nM using HEK cells and [3H]CP‐55,940 (~1.3 nM) as a radioligand) and to have functional activity as assessed using an adenylate cyclase assay using a cyclic AMP ELISA kit (4F‐MDMB‐BINACA, EC 50 = 0.20 nM (E max = 67.7%); (−)CP‐55,940, EC 50 = 0.40 nM (E max = 95.6%); Δ 9 ‐THC, EC 50 = 14.2 nM (E max = 82.9%)). To the best of the author's knowledge there is currently no publicly available data on the pharmacology of MDMB‐4en‐PINACA; however, based on existing structural–activity relationships it is highly likely to be a potent CB 1 and CB 2 receptor agonist …”

“…This, as well as the enactment of other national and international legislative controls, has led to a proliferation of new SCRA compounds, with 260 SCRAs being reported to the United Nations Office for Drugs and Crime (UNODC) by December 2018 and over 180 reported to the EU Early Warning System. The rate of the emergence of new compounds may be slowing, but there has been a general trend of increasing potency as the understanding of SCRA structure–activity relationships has improved …”

Adding more “spice” to the pot: A review of the chemistry and pharmacology of newly emerging heterocyclic synthetic cannabinoid receptor agonists

“…At present, it remains unclear if this presence of 9 in certain batches of SCRA‐containing products is deliberate or accidental. However, 9 does not appear to modulate in vitro cannabinoid (CB) receptor subtype‐1 (CB 1 ) or ‐2 (CB 2 ) signaling in the presence of known CB ligands; CP‐55,940 ( 10 ) or (−)‐ trans ‐Δ 9 ‐tetrahydrocannabinol (Δ 9 ‐THC, 11 ) …”

“…Importantly, functional assays, such as FLIPR®, can be significantly influenced by the level of receptor expression within the system and their propensity to couple with G‐proteins. These factors are often collectively referred to as “receptor reserve” and are a well‐documented limitation associated with such in vitro functional assays . Pharmacological investigation of the effects of 5F‐CUMYL‐P7AICA ( 19 ) in mice has demonstrated its ability to induce hypothermia when injected intraperitoneally (≥ 3 mg/kg).…”

Differential activation of G-protein-mediated signalling by synthetic cannabinoid receptor agonists

“…Many SCRAs are agonists at cannabinoid type-1 and type-2 receptors (CB1 and CB2, respectively [10]; with the psychoactive effects attributed to the activation of CB1 [11]. We have previously described the in vitro quantitative measurement of SCRA efficacy at CB1, where all SCRAs tested showed between 20-300 fold greater agonist activity at CB1 compared to THC [12]. Cannabinoid receptors mediate downstream signalling predominantly through the Gαi/o protein family [13]; however, under some circumstances, CB1 can also stimulate adenylyl cyclase (AC) through Gαs-proteins [14,15,16].…”