Original Article Optimum Conditions of Radioligand Receptor Binding Assay of

To obtain drugs which are more selective at benzodiazepine (BZD) receptors, design and synthesis of functionally selective ligands for BZD receptors is the current strategy of our pharmaceutical chemistry department. The affinity of newly synthesized ligands is assessed by radioligand receptor binding assays. Based on our previous studies, 2-phenyl-5-oxo-7-methyl-1,3, 4-oxadiazolo[a,2,3]-pyrimidine (compound A) was chosen for design and synthesis of new triazole derivatives as GABAA BZD receptor agonist. The cortical membrane of male Sprague-Dawley rats was prepared as the source of the BZD receptors. Different concentrations of membrane protein and [3H]-flumazenil were incubated at room temperature at different time periods to reach the steady-state. To saturate the receptors, increased amounts of radioligand were incubated with membrane protein. The bound and un-bound ligands were separated by centrifugation. The affinity of compound A was measured in competition studies at optimum conditions by displacement of [3H]-Flumazenil from rat cortical membrane. Based on results, the optimum conditions of radioligand receptor binding assay of benzodiazepines were 35 min incubation of ligands with 100 µg cortical membrane protein and 8.6 × 10-5 nmole 3H-flumazenil in a final volume of 0.