Receptor Occupancy


Receptor OccupancyA receptor occupancy assay measures the degree to which the test drug occupies its target receptor in the tissue or animal. Receptor occupancy is determined by measuring the ability of the unlabeled drug to competitively compete with binding of a radiotracer to the receptor. The test is useful to determine the dose or plasma concentration of drug needed to reach a therapeutically-effective receptor occupancy level.

Ex vivo receptor occupancy protocol

The test drug is administered to the animal and the level of receptor occupancy by the drug subsequently measured ex vivo.  This is acheived by harvesting the tissue from the drug-treated animal, sectioning the tissue on a cryostat and determining the level to which the binding of a radiotracer applied briefly to the sections is inhibited by receptor-bound drug in the sections.  Radioactivity is quantified using autoradiographic imaging.

In vivo receptor occupancy protocol

The animal is given the test drug followed a short time later by a radiotracer for the targeted receptor, the latter given intravenously.  At the time of peak tissue levels of the test drug, the percentage to which the in vivo radiotracer binding to receptors in the tissue has been inhibited by the presence of the test drug is determined.  Radiotracer levels are measured either ex vivo by euthanizing the animal and directly counting tissue radioactivity in organs using liquid scintillation or gamma counting or in vivo in the anesthetised animal by PET imaging.


Fig 1

ex vivo occupancy

Ex vivo occupancy study: Autoradiographic binding of [3H]DAMGO to striatal sections from a rat that had been given naloxone (3 mg/kg, i.p.) 20 min prior to sacrifice. Autoradiographic images are shown on the left together and the corresponding ROI analysis on the right. Incubation time of the sections in [3H]DAMGO in vitro was between 1 to 45 min, as indicated. Binding is reduced at all incubation time points in the sections from the naloxone-treated animal due to occupancy of opiate receptors in the tissue by the drug.

Fig 2

CB1 cannabinoid receptor occupancy by a cannabinoid drug, as determined by inhibition of the corresponding radioiodinated tracer. Values are ratios of radioactivity in cerebellum (i.e. a CB1 receptor-rich region) to brain stem (a reference region) and are the means of 5 - 6 animals per dose. Unlabeled drug reduced specific radiotracer binding by 50 % (~ 50 % receptor occupancy) at 0.3 mg/kg. Dotted line indicates the level of non-specific binding.

Fig 3

Dopamine transporter occupancy of cocaine and methylphenidate as determined by inhibition of [3H]cocaine binding in the striatum. Values are ratios of radioactivity in striatum (receptor-rich region) to cerebellum (reference region) and are the means of 5 - 6 animals per dose. Both cocaine and methylphenidate reduced specific [3H]cocaine binding with 50 % inhibition at about 0.25 mg/kg for both drugs. Dotted line indicates the level of non-specific binding, as determined by administration of a blocking dose of a high-affinity cocaine analogue.