The pharmacology of valinate and tert-leucinate synthetic cannabinoids

Synthetic cannabinoids (SCs) with indole or indazole cores and featuring L-valinate or L-tert leucinate groups are epidemic recreational drugs in many parts of the world, and are reported to be associated with severe toxicity. We evaluated the cannabimimetic activity of these compounds on human cannabinoid type 1 (CB1) and type 2 (CB2) receptors. We also examined desensitization of CB1 receptor signaling on continued exposure to SCs. We used a fluorescence-based membrane potential assay to measure the potassium channel-mediated cellular hyperpolarization of AtT20 cells expressing CB1 or CB2. Compound 101, Trametinib and SCH772984 were used to study the involvement of G Protein-Coupled Receptor Kinase2/3 (GRK), Mitogen activated protein kinase enzyme (MEK) and extracellular signal regulated kinase (ERK) in CB1 receptor desensitization. All 16 indole and indazole SCs tested activated CB1 and CB2, with a modest preference for CB1. The most potent was 5F-MDMB-PICA, with an EC₅₀ of 0.45 nM at CB1 receptor. The desensitization of the CB1 mediated hyperpolarization produced by EC₅₀ and EC₉₀ concentrations of 5F-MDMB-PICA was 65±5% and 78±2% after 30 min (n=6). Like CB1 receptor, CB2 receptor also shows a decline in signaling on continuous exposure to EC₅₀ and EC₉₀ of 5F-MDMB-PICA. A significant change in fluorescence was observed for somatostatin after 30 mins of EC₉₀ 5F-MDMB-PICA to that of SST alone at CB2 receptor (P<0.05). Although, the hyperpolarization to a subsequent application of SST (100 nM) after SCs to SST alone was unchanged at CB1 receptor. In cells treated with Cmpd101 (10 μM), we did not observe any difference in the desensitization of CB1 receptor evoked by the EC₉₀ concentration of 5F-MDMB-PICA, but at a concentration of 10 μM 5F-MDMB-PICA, Cmpd 101 reduced desensitization from 97±3.9% to 77±3.5%. Inhibition of MEK or ERK had no effect on CB1 receptor desensitization. All SCs tested in this study have greater potency and maximum effect than Δ⁹-THC. CB1 receptor desensitization was largely homologous, with little effect on the native SST receptor responses, whereas desensitization at CB2 receptors was found to be both homologous and heterologous. Our data demonstrate a role of GRK2/3 in CB1 receptor desensitization to high concentrations of agonist in AtT20 cells, but suggest that other mechanisms may be recruited by lower concentrations of drug.