Comparison of in Vivo Receptor Occupancy Studies using a Radioligand-Based Approach or a Liquid Chromatography/Mass Spectrometry - Based Approach

Biomedical Research Models, Inc. In vivo receptor occupancy (RO) is a valuable tool used in preclinical drug development to confirm target engagement and faci litate selection of compounds for further development. We have been using radioligand-based RO as means of measuring target engagement in the rat brain. In this approach, compounds of interest are peripherally injected prior to intravenous injection of a selective radioligand. If the compounds of interest occupy the target, binding sites are no longer available for the radioligand. Therefore, binding of the radioligand, which is evaluated by imaging brain sections, is inversely correlated to target occupancy. This approach is very sensitive, has a fast turnaround (2-3 days) and provides the ability to analyze multiple brain areas independently of their size. However, many targets have no validated radioligand avai lable, and the cost of radioligand-based RO can be prohibitive. These limitations prompted us to evaluate liquid chromatography/mass spectrometry (LCMS)-based RO as an alternative method. In this method, levels of the cold ligand are measured in brain homogenates. LCMSbased RO is an inexpensive and rapid method for determining RO of compounds in small animals as well as screening for potential new ligands. In order to compare radioligand-based RO with LCMS-based RO, two different ligands were chosen: raclopride for D2 RO and MDL100907 for 5-HT2A RO. Dose-response curves were generated using both methods for haloperidol and quinpirole (D2 RO), and olanzapine (5-HT2A RO). We found an excellent correlation between the RO50 (in terms of dose and plasma levels) measured using radioligands and cold ligands. Binding of MDL100907 was not affected by citalopram and baclofen (negative controls) which substantiate the selectivity of the tracer and validate the LCMS detection method. In conclusion, LCMS-based RO is a good alternative method compared with radioligand-based RO and will allow us to screen potential ligands for new projects.