A preliminary PET evaluation of the new dopamine D2 receptor agonist [11C]MNPA in cynomolgus monkey

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Abstract

This study describes the preliminary positron emission tomography (PET) evaluation of a dopamine D2-like receptor agonist, (R)-2-11CH3O-N-n-propylnorapomorphine ([11C]MNPA), as a potential new radioligand for in vivo imaging of the high-affinity state of the dopamine D2 receptor (D2R). MNPA is a selective D2-like receptor agonist with a high affinity (Ki=0.17 nM). [11C]MNPA was successfully synthesized by direct O-methylation of (R)-2-hydroxy-NPA using [11C]methyl iodide and was evaluated in cynomolgus monkeys. This study included baseline PET experiments and a pretreatment study using unlabeled raclopride (1 mg/kg). High uptake of radioactivity was seen in regions known to contain high D2R, with a maximum striatum-to-cerebellum ratio of 2.23±0.21 at 78 min and a maximum thalamus-to-cerebellum ratio of 1.37±0.06 at 72 min. The pretreatment study demonstrated high specific binding to D2R by reducing the striatum-to-cerebellum ratio to 1.26 at 78 min. This preliminary study indicates that the dopamine agonist [11C]MNPA has potential as an agonist radioligand for the D2-like receptor and has potential for examination of the high-affinity state of the D2R in human subjects and patients with neuropsychiatric disorders.

Introduction

Studies of the dopaminergic system utilizing positron emission tomography (PET) have primarily focused on the use of antagonists, such as [11C]raclopride [1]. High signal relies mainly on the fact that antagonists have equal binding affinity for receptors in both the high- and low-affinity states [2], [3], [4], [5], [6]. The use of antagonists to quantify receptor levels within the brain has been applied to examine the dopamine (DA) system in various psychiatric disorders. In addition to these studies, drug-induced receptor occupancy analysis has been used in drug development of antipsychotic drugs, themselves being antagonists, in order to demonstrate therapeutic efficacy.

A limitation with DA antagonists, however, is that they have no inherent potential to distinguish receptors in high- vs. low-affinity states as proportion varies with conditions. The study of neurotransmission is therefore very difficult because endogenous DA binds preferentially to receptors in the high-affinity state. An agonist radioligand may thus be more sensitive to endogenous DA levels when compared with an antagonist radioligand.

To overcome this limitation, studies aiming at the development of agonist radioligands have recently been undertaken [7], [8], [9], [10], [11]. Some of the most potent DA agonists identified thus far are apomorphine and its analogues [12], [13]. One of the apomorphine analogues, (R)-N-11C-propyl-norapomorphine ([11C]NPA; Fig. 1), has been evaluated in baboon and showed a striatum-to-cerebellum ratio of 2.8 [8]. These developments of agonist radioligands are of particular interest since they may have increased sensitivity for endogenous DA levels. Immediate support for this hypothesis is found in an agonist–antagonist comparison, where an induced increase in synaptic DA due to acute amphetamine exposure resulted in a 42% higher decrease in [11C]NPA binding, as compared to [11C]raclopride binding [14].

Among the apomorphine analogues, (R)-2-CH3O-N-n-propylnorapomorphine (MNPA; Fig. 1) has a very high D2-like receptor binding affinity [1.02 nM (IC50) or 0.17 nM (Ki)], as compared to NPA [4.80 nM (IC50) or 0.8 nM (Ki)] [12], [13]. A preliminary study in a cynomolgus monkey and with an old PET system has demonstrated a marked uptake in the striatum after iv injection of [11C]MNPA. The striatum-to-cerebellum ratio was approximately 2, with accumulation of radioactivity in regions known to contain high dopamine D2 receptor (D2R) [15].

The aim of the present study was to optimize the radiosynthesis of [11C]MNPA by direct methylation and to examine its potential as a radioligand. The specificity and selectivity of binding were examined by baseline and pretreatment experiments in cynomolgus monkeys. [11C]MNPA metabolism was measured in monkey plasma by gradient HPLC.

Section snippets

General procedures

Dimethyl sulfoxide (DMSO) and 5 M NaOH were obtained from Fluka Sweden and the Swedish Pharmacy, respectively. Additional chemicals were obtained from various commercial sources and were, whenever possible, of analytical grade. [11C]Methane was produced with a GEMS PETtrace cyclotron at the Karolinska University Hospital, Stockholm, Sweden, using 16.4-MeV protons in the 14N(p,α)11C reaction on nitrogen gas containing 10% H2. The target gas was irradiated for 20 min with a beam intensity of 35

Radiochemistry

The incorporation yield of [11C]methyl iodide to [11C]MNPA was approximately 75%, and total synthesis time was 30–35 min. [11C]MNPA was purified by semipreparative reversed-phase HPLC with a retention time of 6.5–8 min (Fig. 3). The retention time on the analytical reversed-phase HPLC system was 3–4 min, indicating a radiochemical purity better than 98%. The specific radioactivity obtained at the time of administration of [11C]MNPA was approximately 350 Ci/mmol (13 GBq/μmol), and the

Radiochemistry

Direct alkylation with [11C]methyl iodide of 2-OH-NPA is selective to the 2-OH position, which was confirmed by derivatization. Derivatization with acetic anhydride/pyridine and dibromomethane resulted in products that coeluted on HPLC with their reference compounds. Protection of the catechol group was therefore not needed. A plausible explanation for the selective labeling of the 2-OH position could be less sterical hindrance as compared to the 10- and 11-OH positions.

The synthesis gave a

Conclusion

[11C]MNPA was successfully synthesized by a direct methylation approach. Preliminary PET studies showed uptake of radioactivity in brain regions known to contain D2R. The specificity of [11C]MNPA binding to D2R was demonstrated by a pretreatment measurement which showed a marked reduction of binding after iv injection of unlabeled raclopride. [11C]MNPA has potential as an agonist radioligand for the D2-like receptor and has potential for examination of the high-affinity state of the D2R in

Acknowledgments

The authors would like to thank the members of the Stockholm PET group for their assistance in the PET experiments. A grant from the Swedish Medical Research Council (41205-7) was used to support this work. Nick Seneca was supported by a grant from the NIH-KI joint PhD program. We are also grateful to Dr Cyrill Burger for providing the PMOD (version 2.5) software.

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