In Vivo Receptor Occupancy in Rodents by LC-MS/MS

Receptor (or enzyme) Occupancy (RO) is a quantitative measure of what percentage of the total number of available target receptors is engaged by or bound to a particular ligand. In drug discovery, it is important to determine the relationship of a receptor’s occupancy to its final observed pharmacological efficacy and/or toxicological readout. RO is related to but distinct from a ligand’s potency and maximal efficacy. Different compounds may achieve different percentages and durations of occupancy. Receptor occupancy/exposure relationships as well as the duration of occupancy have been used to project clinical doses.The drug discovery lead optimization flow scheme can be improved by inclusion of a receptor occupancy assay that determines the level of target engagement to allow rational triage and selection of compounds to advance into more labor intensive and longer duration efficacy studies.In the clinic, RO is measured using radioisotopically labeled tracer ligands whose distributions can be determined by non-invasive imaging methods, most commonly positron emission tomography (PET) and single photon emission computed tomography (SPECT) (1). Recent advances in the sensitivity of LC-MS/MS detectors have enabled determination of RO in preclinical studies without the need for a radiotracer (2, 3). This chapter will cover the details of performing an in vivo receptor occupancy assay using an LC-MS/MS methodology. This chapter assumes that a tracer exists for the target for which occupancy measures are being made, and will not detail the process of identifying a novel tracer (4, 5). A suitable LC-MS/MS tracer should be selective and have high affinity for the pharmacologic target, be readily tissue-penetrant, have low nonspecific binding, and exhibit suitable kinetics.