Identification of dopamine D3 receptors (D3R) in vivo is important to understand several brain functions related to addiction. The goal of this work was to identify D3R binding of the dopamine D2 receptor (D2R)/D3R imaging agent, 18F-fallypride. Brain slices from male Sprague-Dawley rats (n=6) and New Zealand White rabbits (n=6) were incubated with 18F-fallypride and D3R selective agonist (R)-7-OH-DPAT (98-fold D3R selective). Rat slices were also treated with BP 897 (68-fold D3R selective partial agonist) and NGB 2904 (56-fold D3R selective antagonist). In vivo rat studies (n=6) were done on Inveon PET using 18–37 MBq 18F-fallypride and drug-induced displacement by (R)-7-OH-DPAT, BP 897 and NGB 2904. PET/CT imaging of wild type (WT, n=2) and D2R knock-out (KO, n=2) mice were carried out with 18F-fallypride. (R)-7-OH-DPAT displaced binding of 18F-fallypride, both in vitro and in vivo. In vitro, at 10 nM (R)-7-OH-DPAT, 18F-fallypride binding in the rat ventral striatum (VST) and dorsal striatum (DST) and rabbit nucleus accumbens were reduced by ~10–15%. At 10 µM (R)-7-OH-DPAT all regions in rat and rabbit were reduced by ≥85%. In vivo reductions for DST and VST before and after (R)-7-OH-DPAT were: low-dose (0.015mg/kg) DST −22%, VST −29%; high-dose (1.88 mg/kg) DST −58%, VST −77%, suggesting D3R/D2R displacement. BP 897 and NGB 2904 competed with 18F-fallypride in vitro, but unlike BP 897, NGB2904 did not displace 18F-fallypride in vivo. The D2R KO mice lacked 18F-fallypride binding in the DST. In summary, our findings suggest that up to 20% of 18F-fallypride may be bound to D3R sites in vivo.

Imaging the high-affinity, functional state (HA) of dopamine D2 and D3 receptors has been pursued in PET imaging studies of various brain functions. We report further evaluation of 18F-5-OH-FPPAT, and the newer 18F-5-OH-FHXPAT and 18F-7-OH-FHXPAT. Syntheses of 18F-5-OH-FHXPAT and 18F-7-OH-FHXPAT were improved by modifications of our previously reported procedures. Brain slices and brain homogenates from male Sprague-Dawley rats were used with the 3 radiotracers (74–111 kBq/cc). Competition with dopamine (1–100 nM) and Gpp(NH)p (10–50 μM) were carried out to demonstrate binding to dopamine D2 and D3 HA-states and binding kinetics of 18F-5-OH-FPPAT measured. Ex vivo brain slice autoradiography was carried out on rats administered with 18F-5-OH-FHXPAT to ascertain HA-state binding. PET/CT imaging in rats and wild type (WT) and D2 knock-out mice were carried out using 18F-7-OH-FHXPAT (2–37 MBq). Striatum was clearly visualized by the three radiotracers in brain slices and dopamine displaced more than 80% of binding, with dissociation rate in homogenates of 2.2 × 10−2 min−1 for 18F-5-OH-FPPAT. Treatment with Gpp(NH)p significantly reduced 50–80% striatal binding with faster dissociation rates (5.0 × 10−2 min−1), suggesting HA-state binding of 18F-5-OH-FPPAT and 18F-5-OH-FHXPAT. Striatal binding of 18F-5-OH-FHXPAT in ex vivo brain slices were sensitive to Gpp(NH)p, suggesting HA-state binding in vivo. PET binding ratios of 18F-7-OH-FHXPAT in rat brain were ventral striatum/cerebellum=2.09 and dorsal striatum/cerebellum=1.65; similar binding ratios were found in the D2 WT mice. These results suggest that in vivo PET measures of agonists in the brain at least in part reflect binding to the membrane-bound HA-state of the dopamine receptor.