NGD-941

The suitability of an (123)I-labeled form of the putative D(4) receptor ligand L750,667 as a radiotracer for single photon emission computed tomography imaging was assessed in nonhuman primates. [(123)I]L750,667, labeled by iododestannylation, was administered to baboons in bolus and bolus plus constant infusion paradigms and imaged for 6 h. Total [(123)I]L750,667 brain uptake peaked (2.3% injected dose) at 15 min postinjection. [(123)I]L750,667 uptake was observed in all brain regions measured including diencephalon, brainstem, basal ganglia, cingulate cortex, and cerebellum, and slightly lower levels were noted in the frontal, parietal, temporoinsular, and occipital cortices. Administration of the D(4) receptor antagonist NGD 94-1 (2 mg/kg) did not displace radioactivity from any of the brain regions examined. Thus, while L750,667 is selective for the D(4) receptor in vitro, because brain [(123)I]L750,667 uptake was not displaced by NGD 94-1 at receptor saturating doses, [(123)I]L750,667 does not appear to be a suitable radiotracer for in vivo imaging of the D(4) receptor.

It has been difficult to observe functional coupling of the D4 receptor to second messenger systems and a robust functional assay system for this receptor is still lacking. In the present study, the rat dopamine D4 receptor was cloned from rat retina. Sequence comparison revealed identity with the published sequence of Ashgari and co-workers, including the two amino acid insertions (V-Q) at position 92 which are not present in the published sequence of O'Malley and coworkers. The rat dopamine D4 receptor was stably expressed in Chinese hamster lung fibroblast CCL39 cells. [3H]spiperone saturation binding yielded a Bmax of 2,370+/-546 fmol/mg protein and a pKD of 8.74+/-0.14 (n=4). Forskolin-stimulated cAMP accumulation was inhibited by dopamine (Emax 61+/-1% inhibition of forskolin-stimulated levels, pEC50 7.33+/-0.06, n=23). A similar concentration-dependent inhibition was observed with the dopamine D2-like receptor agonists quinpirole and 7-OH-DPAT which elicited nearly the same Emax as dopamine. By contrast, apomorphine and a number of compounds with reported affinity for human dopamine D4 receptors (PD168077, U-101958, SDZ GLC 756, L-745,870 and NGD 94-1) behaved as partial agonists (Emax ranging between 26% and 56% of that of dopamine). The agonist effect of dopamine was completely blocked by preincubation with pertussis toxin, no further accumulation of cAMP above the forskolin-stimulated levels being observed. Antagonist pKB-values obtained against dopamine in this system were: 8.55+/-0.19 (n=3) for the partial agonist L-745,870, 8.38+/-0.23 (n=5) for spiperone, 7.18+/-0.17 (n=4) for haloperidol, 7.04+/-0.13 (n=4) for clozapine and <6 for raclopride. Other functional assays applicable were stimulation of [35S]GTPgammaS binding, extracellular acidification rate and a serum-responsive element using luciferase expression as a reporter gene. However, the receptor did not couple to phosphatidylinositol turnover or to intracellular Ca2+. Thus, expression of the rat dopamine D4 receptor in CCL39 cells provided several functional assay systems, of which inhibition of cAMP appeared to be the most robust one. These functional models can be used to evaluate the activity of compounds at the rat dopamine D4 receptor.