Radioligand Binding Assay for hCB1R and hCB2R

The binding affinities toward
hCB1R and hCB2R were determined according
to a previously published protocol.²² In
brief, membrane preparations obtained from CHO cell lines stably transfected
with either human CB1R (hCB1R-CHO; obtained from Euroscreen, Gosselies,
Belgium) or human CB2R (hCB2R-CHO; obtained from Paul L. Prather,
Department of Pharmacology and Toxicology, College of Medicine, University
of Arkansas for Medical Sciences, USA) were incubated with [³H]SR141716A (1,554 GBq/mmol; PerkinElmer Life and Analytical Sciences,
Rodgau, Germany; final concentration ∼2 nM) or [³H]WIN55212-2 (6,438 GBq/mmol; PerkinElmer Life and Analytical Sciences,
Rodgau, Germany; final concentration ∼3 nM) and the respective
test compounds at different concentrations (final concentration 10^–5–10^–11 M) diluted from DMSO
stock solutions (1% DMSO final concentration) in incubation buffer
(50 mM TRIS-HCl, pH 7.4, supplemented with 0.1% bovine serum albumin,
5 mM MgCl₂, and 1 mM EDTA) at rt for 90 min.
Homologous
radioligand displacement studies investigating the potential of RM365
to displace [¹⁸F]RM365 from binding sites in membrane homogenates
of rat spleen or hCB2R-CHO cells were performed according to the same
protocol.
The nonspecific binding of the respective radioligand
was determined
by coincubation with CP55,940 (final concentration 10^–5 M). The normalized values of bound activity (% specific binding)
were calculated and plotted vs the logarithm of the concentration
of the respective test compound. The IC₅₀ values of the
resulting inhibition curves were estimated by nonlinear regression
analysis (GraphPad Prism 2.01). To calculate the K_i, the equation of Cheng and Prusoff was used. For homologous
competition experiments, K_i = K_D.^{38 (link)}