AM1241

La unión a los receptores cannabinoides se prueba utilizando la unión de equilibrio competitivo vs. [³H]CP55,940. AM-1241 se diluye en 25 mM Tris base (pH 7,4)/5 mM MgCl₂/1 mM EDTA/0,1% BSA esencialmente libre de ácidos grasos y se transfiere a placas de 96 pocillos tratadas con Regisil. Se añade [³H]CP55,940 (DuPont_NEN; actividad específica 100–180 Ci/mmol; 1 Ci =37 GBq) a una concentración de 0,8 nM. Se añaden membranas preparadas a partir de cerebro de rata (que contienen receptores CB1) o bazo de ratón (que contienen receptores CB2) (≈50 μg de proteína de membrana por pocillo), las placas se incuban a 30 °C durante 1 hora y el contenido se filtra a través de filtros Packard Unifilter GF/B de 96 pocillos utilizando un recolector de células Packard Filtermate 196. Los filtros se lavan con 50 mM Tris base/5 mM MgCl₂/0,5% BSA helado y se secan. La radioactividad unida se cuantifica y se corrige para la unión no específica, y los resultados se normalizan entre 0% y 100% de [³H]CP-55,940 específicamente unido. La IC50 se determina mediante análisis de regresión no lineal utilizando GraphPad PRISM y se transforma a un valor de Ki. Todos los datos se recopilan por duplicado. Los valores de IC50 y Ki se determinan a partir de tres experimentos independientes.

HEK cells and CHO cell lines stably express human CB2 receptor and CB1 receptor.

12 concentrations between 0.1 nM–10 μM

90 minutes

Membrane samples are prepared from HEK cells stably expressing the human CB2 receptors previously generated (Mukherjee et al., 2004), or the CHO cell line that stably expresses the human CB1 receptor. Radioligand binding assays are performed as following. Briefly, the cells are harvested and homogenized using a Polytron for 2 × 10 s bursts in a buffer containing 50 mM Tris-HCl, pH 7.4, 1 mM MgCl₂, and 1mM EDTA in the presence of protease inhibitors followed by centrifugation at 45 000 g for 20 minutes. The membrane pellets are washed and frozen at -80 °C in aliquots until use. Saturation binding reactions are performed at 30 °C for 90 minutes using [³H]CP 55,940 (0.01–8 nM) in an assay buffer containing 50 mM Tris-HCl, pH 7.4, 2.5 mM EDTA, 5mM MgCl₂, and 0.05% fatty acid free bovine serum albumin (BSA) and the reactions are terminated by rapid vacuum filtration through UniFilter-96 GF/C filter plates and four washes with cold assay buffer. Competition experiments are conducted using 0.5 nM [³H]CP 55,940 in the presence of test compounds (0.1 nM–10 μM). Nonspecific binding is defined by 10 mM unlabeled CP 55,940. K_D values from saturation binding assays and Ki values from competition binding assays are determined with one site binding or one site competition curve fitting using the Prism software.

250-350 g male Sprague-Dawley rats

0, 100, 300, 1000, 3000 μg/kg

Administered via i.p.