Development of methodology for analysis of brain function using radioisotope labeled compounds.

Abstract

To evaluate the relationship between the pharmacological effect of benzodiazepine (BZP) and BZP receptor binding in the conscious mouse brain, a response of the glucose utilization (GU) to clonazepam (CNZ) was measured as an index for the pharmacological effect. GU was measured by the simultaneous use of [14C] 2-deoxyglucose (2DG), the glucose analogue which can be phosphorylated in the brain, and [3H] 3-O-methylglucose (3MG), the nonmetabolizable glucose analogue. The distribution volume of unphosphorylated 2DG in the brain was not significantly different from that of 3MG (VM), indicating that the phosphorylation rate of 2DG can be estimated by subtracting VM from apparent volume of distribution of 2DG. By this double tracer technique, it is possible to determine GU within 10min after administration of both tracers. Pharmacological and pathophysiological changes of the isotope correction factor (lumped constant) can also be estimated by this technique. In the cerebral cortex, GU decreased to 70 ?? 80% at 60 min after i.v. administration of CNZ (0.005 ?? 1.0mg/kg), and this effect was completely diminished by the administration of a benzodiazepine antagonist, Ro-15-1788 (5mg/kg). The maximum effect of CNZ on GU (about 30% decrease) was found at 0.1mg/kg of CNZ, but increasing the dose to 1mg/kg had very little additional effect. In vivo BZP receptor occupancy, measured using [3H] Ro-15-1788, increased from less than 10% at a dose of 0.005mg/kg up to essentially 100% at doses of 1mg/kg or greater. ID50 in dose response curve of the receptor occupancy for CNZ and ED50 in that of decrease in GU were 0.3mg/kg and 0.007mg/kg, respectively. A nonlinear and hyperbolic relationship was observed between the receptor occupancy and the response for the glucose metabolic rate, indicating that BZP exerts the maximum glucose metabolic change at a low fractional receptor occupancy (30 ?? 40%). By using positron emission tomography, these techniques can be applied to living human brain, which makes it possible to determine the optimal doses of BZP in the effective therapeutic drug monitoring. Furthermore, a mathematical allosteric coupling model has been proposed to des cribe the process by which binding to the GABA/benzodiazepine receptor complex initiates a biological response. The modeling exercise shows that the benzodiazepine concentration required for half-maximal biological response is lower than that required for half-maximal receptor binding. The degree of discrepancy between the two profiles (receptor occupancy and biological response) concerning benzodiazepine concentration dependency increased with the decrease in the dissociation constants based on the GABA receptor-benzod iazepine receptor interaction. This model offers a simple explanation for discrepancies between receptor occupancy-concentration profile of benzodiazepine and biological response-concentration curve that are often observed in vivo and/or in vitro.