Development of a pet agonist radioligand for imaging the high-affinity state of the dopamine D2/3 receptor in vivo: the road from bench to bedside

Positron emission tomography (PET) is a molecular imaging technique that allows for non-invasive examination of the distribution and density of biochemical markers such as receptors, in the living brain. Another important application of PET is to measure endogenous levels of neurotransmitters such as dopamine (DA) in vivo in both man and animals. Fundamental information has been obtained using this approach, notably with the observations that schizophrenia is associated with an enhanced amphetamine-induced DA release whereas the opposite is observed in drug abuse. Changes in DA levels have been extensively studied using antagonist PET radioligand for DA D2/3 receptors such as [11C]-raclopride. The technique however suffers from the major limitation that a substantial portion of antagonist radiotracer binding is impervious to DA manipulations. This limitation has been related to the inability of antagonist PET radiotracers to distinguish the low-affinity (D2/3Low) from the high-affinity states (D2/3High) of D2/3 receptors. Only agonist radiotracers can distinguish between the D2/3High and D2/3Low, binding preferentially to the D2/3High, and only agonist radiotracers are thought to be fully sensitive to the effects of endogenous DA. Despite the critical importance of imaging the D2/3High, which is the functionally active receptor state, most existing PET D2/3 radiotracers are antagonists, and the state of development and validation of agonist D2/3 radiotracers is currently in its beginnings. Therefore, the aims of the present thesis were to develop and validate a D2/3 agonist radioligand for in vivo measurement of the D2/3High using PET and to evaluate the sensitivity of the radiolabeled D2/3 agonist to pharmacologically-induced changes in endogenous DA in the living brain of animals and humans. In studies I and 2, the potent D2/3 receptor agonist (+)-PHNO, namely [(+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol], was radiolabeled with carbon-11 and evaluated in rodents. [11C]-(+)-PHNO exhibited high brain penetration, favorable in vivo kinetics and metabolite profile, and had high specificity for the D2/3 receptors both ex vivo and in vivo. Comparison of [11C]-(+)-PHNO ex vivo binding in DA-depleted rats and in control rats suggested that about 35% of the D2/3 receptors were occupied by DA at baseline. Moreover, in vivo comparison of [11C]-(+)-PHNO with two other D2/3-agonist radiotracers, namely [11C]-(-)-NPA and [11C]-(+)-MHNO, the methyl analog of [11C]-(+)-PHNO, demonstrated the superiority of [11C]-(+)-PHNO over the two other agonist radioligands for imaging the D2/3High. PET imaging studies in cats (Study 3) revealed that [11C]-(+)-PHNO was more sensitive than the antagonist radioligand [11C]-raclopride to amphetamine-induced changes in endogenous DA levels. The improved sensitivity was at least ~1.5 fold across three doses. Importantly, and in contrast to [11C]-raclopride, amphetamine-released DA had virtually access to the entire population of D2/3 receptors labelled by the agonist radioligand, with no ceiling effect, thus suggesting that [11C]-(+)-PHNO indeed preferentially binds the D2/3High. It was estimated that about 79% of the total pool of D2/3 receptors was in the high affinity state. Scatchard analyses of [11C]-(+)-PHNO and [11C]-raclopride binding indicated that striatal D2/3 receptors densities measured with the two radioligands were identical, a finding that questioned the existence of two affinity states for D2/3 receptors unless it is speculated that all receptors are configured in the high affinity state at in vivo conditions. [11C]-(+)-PHNO was then successfully introduced as the first agonist radioligand for studying the D2/3 receptors in human (study 4). [11C]-(+)-PHNO enabled an exquisite delineation of D2/3 receptors in the striatal complex but also in extrastriatal regions such as the substantia nigra. The prominent binding of [11C]-(+)-PHNO in D3-rich regions suggested a significant contribution of the D3 receptor subtype to the radioligand binding in vivo. Full kinetic modeling analysis of [11C]-(+)-PHNO binding in the human brain indicated that simplified quantitative methods, using the cerebellum as reference region, provided meaningful indexes of regional D2/3High receptor density and thus could be used in routine clinical studies (Study 5). Finally, [11C]-(+)-PHNO was also found to be highly sensitive for detecting changes in extracellular DA levels in the living human brain (Study 6). Thus, in addition to providing an access to the functional state of the DA D2/3 receptor, our studies showed that [11C]-(+)-PHNO also provides a more refined and sensitive index of endogenous DA levels and enables for the first time an opportunity to explore the D3 receptor in D3-rich areas in the brain. Since the DA system has been implicated in learning and reward, as well as disorders such as schizophrenia, movement disorders, mood disorders, and addiction - [11C]-(+)-PHNO thus had the prospect for future explorations of the pathophysiology and therapeutics of these disorders.