3.3. Biology—Opioid Binding Assay

Ligand binding assays were determined for compounds under study, at μ-, δ-, and κ-opioid receptors, as described in detail elsewhere [30,31]. Binding affinities for μ-, δ-, and κ-opioid receptors were determined by displacing, respectively, [³H]DAMGO (1 nM), [³H]DADLE (1 nM) and [³H]{"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 (1 nM) from mouse brain membrane binding sites. Brain membranes were incubated with the appropriate [³H]-ligand in 50 mM Tris–HCl buffer, pH 7.4, at 25 °C for 60 min in the absence or presence of 10 μM naloxone. IC₅₀ values were determined from log dose displacement curves, and K_i values were calculated from the obtained IC₅₀ values by means of the equation of Cheng and Prusoff [32].