Functional Characterization of CB1 Agonists

Each compound was pharmacologically characterized using a functional fluorescent CB1 activated Gαq₁₆-coupled intracellular calcium mobilization assay in CHO-K1 cells, as has been described in our previous publications and apparent affinity (K_e) values were determined.^{13 (link)} Briefly, CHO-K1 cells were engineered to coexpress human CB1 and Gαq₁₆. Activation of CB1 by an agonist then leads to generation of inositol phospahatase 3 (IP3) and activation of IP3 receptors, which leads to mobilization of intracellular calcium. Calcium flux was monitored in a 96-well format using the fluorescent dye Calcein-4 a.m. in an automated platereader (Flexstation, Molecular Devices). The antagonism of a test compound was measured by its ability to shift the concentration response curve of the synthetic CB1 agonist CP55940 rightwards using the equation:
K_e = [Ligand]/[DR-1] where DR is the EC₅₀ ratio of CP55940 in the presence or absence of a test agent. Standard errors were between 5 and 25% of mean in most cases and have been left out for clarity.
Further characterization of select compounds was performed using radioligand displacement of [³H]SR141716 and equilibrium dissociation constant (K_i) values were determined as described previously.^{13 (link)} Selectivity of these compounds at CB1 versus CB2 was also determined by obtaining K_i values at either receptor using displacement of [³H]CP55940 in membranes of CHO-K1 cells overexpressing either receptor. Data reported are average values from 3 to 6 measurements. The standard errors for most measurements were between 5 and 25% of mean and have been left out from the tables and figures for clarity.