Detection of adenosine receptor antagonists in rat brain using a modified radioreceptor assay

The present study describes a modified radioreceptor binding assay using brain homogenate or serum from drug treated animals as the 'competing drug' in a conventional in vitro radioligand binding assay. Method validation involved measurement of the brain and serum concentration of three adenosine receptor antagonists following systemic administration, using a [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) binding assay. The intrinsic [3H]DPCPX binding capacity of test samples was abolished by protein denaturation (80 degrees C, 15 min) and, endogenous ligand was depleted enzymatically, prior to determination of drug concentration. Brain and serum concentrations of the adenosine A1 receptor antagonist, DPCPX increased in a dose related manner when measured 20 min after intraperitoneal injection. Estimated brain concentrations were 13.8, 87.7 and 288 nM following injection of 0.01, 0.1 and 1.0 mg/kg DPCPX, and serum concentrations were 26.5, 195 and 1370 nM respectively. A time dependent decrease in both brain and serum concentration was noted 20-180 min following injection of 1.0 mg/kg DPCPX. The peripheral adenosine receptor antagonists, 1,3-dipropyl-8-p-sulphophenylxanthine (DPSPX; 5.6 mg/kg) and 8-(p-sulphophenyl)theophylline (8-PST; 20 mg/kg), were not detected in brain tissue 20 min after intraperitoneal injection, despite serum concentrations of 56 and 52 microM respectively. This assay provides a useful and versatile method for determining the central penetration of neuroactive drugs.