The improved EC 50 accuracy with unbound concentrations was 10–250 folds in plasma and 10–170 folds in the brain. The correlation between the unbound EC 50 of the antagonists and corresponding Ki values was good ( r 2=0.828). The test drug quetiapine, eplivanserin, and clozapine showed high free fractions in plasma, but for ketanserin and olanzapine, the brain free fraction was higher. Except for quetiapine, the ED 50 values in the dose-occupancy curves of antagonists are close and ranged from 1 to 3 mg/kg. The corresponding binding affinities (Ki) correlated with the unbound concentrations.

Drug-free fractions derived the unbound concentration (EC 50) in plasma and brain at test doses. Equilibrium dialysis method determines the in vitro free fraction of the test antagonist in untreated rat brain homogenates and plasma. The 5-HT 2A occupancy of antagonist in the rat brain has determined with non-radiolabeled tracer MDL-100,907 at an optimized dose (3 µg/kg) and treatment time (30 min). With the application of mass spectrometry, the current work proposes a definite measure of test drug exposures at serotonin-2A occupancy. Drug exposures from preclinical are total concentrations of those over/underestimate the clinical dose projection. Unbound drug concentration in the brain would be the true exposure responsible for specific target occupancy.

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