Title Page Title: A New Radioligand Binding Assay to Measure the Concentration of Drugs in Rodent Brain Ex Vivo Running Title Page Running Title: A New Competitive-Saturation Binding Assay

Abstract We have developed a new radioligand binding assay method to measure the concentration of non-radiolabeled drugs in the brain ex vivo. This new method fuses the concepts of standard competition and saturation binding assays, and utilizes a transformed version of the Cheng-Prusoff equation to calculate the drug concentration. After testing the validity of this method, we demonstrated its utility by measuring the brain concentration of sazetidine-A, a newly developed nicotinic receptor ligand, and its elimination rate after a single subcutaneous administration. Our results indicate that sazetidine-A reaches brain concentrations that are known to occupy and desensitize the majority of nAChR binding sites. Furthermore, using this method, we estimated the half-life of sazetidine-A in the rat brain to be ~65 min. It is important to note that the method described here to measure sazetidine-A in brain should be generalizable to other drugs acting at any receptor that can be reliably measured with a radiolabeled ligand