Synthesis and evaluation in rats of homologous series of [(18)F]-labeled dopamine D 2/3 receptor agonists based on the 2-aminomethylchroman scaffold as potential PET tracers.

BACKGROUND: Agonist positron emission tomography (PET) tracers for dopamine D2/3 receptors (D2/3Rs) offer greater sensitivity to changes in endogenous dopamine levels than D2/3R antagonist tracers. D2/3R agonist tracers currently available for clinical research are labeled with the short-lived isotope carbon-11, which limits their use. We aimed to develop high-affinity D2R agonists amenable for labeling with the longer-living fluorine-18. Here, we report the evaluation as potential PET tracers of two homologous series of [(18)F]fluorinated tracers based on the 2-aminomethylchroman-7-ol (AMC) scaffold: (R)-2-((4-(2-fluoroalkoxy)benzylamino)methyl)chroman-7-ols (AMC13 homologues) and (R)-2-((2-(4-(4-(fluoroalkoxy)phenyl)piperazin-1-yl)ethylamino)methyl)chroman-7-ols (AMC15 homologues). We varied the length of the (18)F-fluoroalkyl chain in these structures to balance brain penetration and non-specific binding of the radioligands by adjusting their lipophilicity. METHODS: The tracers were evaluated in brain slices of Sprague-Dawley rats by in vitro autoradiography and in living rats by microPET imaging and ex vivo autoradiography. PET data were analyzed with one- and two-tissue compartmental models (1TCM/2TCM), simplified reference tissue model (SRTM), and Logan graphical analysis. Specificity of binding was tested by blocking D2/3R with raclopride. RESULTS: Homologues with a shorter fluoroalkyl chain consistently showed greater D2/3R-specific-to-total binding ratios in the striatum than those with longer chains. The fluoroethoxy homologue of AMC13 ([(18)F]FEt-AMC13) demonstrated the highest degree of D2/3R-specific binding among the evaluated tracers: mean striatum-to-cerebellum uptake ratio reached 4.4 in vitro and 2.1/2.8 in vivo/ex vivo (PET/autoradiography). Striatal binding potential (BPND) relative to cerebellum was 0.51-0.63 depending on the estimation method. Radiometabolites of [(18)F]FEt-AMC13 did not enter the brain. In vitro, application of 10 µmol/L raclopride reduced D2/3R-specific binding of [(18)F]FEt-AMC13 in the striatum by 81 %. In vivo, pre-treatment with 1 mg/kg (2.9 µmol/kg) raclopride led to 17-39 % decrease in D2/3R-specific binding in the striatum. CONCLUSIONS: Varying the length of the [(18)F]fluoroalkyl chain helped improve the characteristics of the original candidate tracers. Further modifications of the current lead [(18)F]FEt-AMC13 can provide an agonist radiopharmaceutical suitable for D2/3R imaging by PET.

123I-iododexetimide preferentially binds to the muscarinic receptor subtype M1 in vivo.

UNLABELLED: The muscarinic M1 receptor (M1R) is highly involved in cognition, and selective M1 agonists have procognitive properties. Loss of M1R has been found in postmortem brain tissue for several neuropsychiatric disorders and may be related to symptoms of cognitive dysfunction. (123)I-iododexetimide is used for imaging muscarinic acetylcholine receptors (mAchRs). Considering its high brain uptake and intense binding in M1R-rich brain areas, (123)I-iododexetimide may be an attractive radiopharmaceutical to image M1R. To date, the binding affinity and selectivity of (123)I-iododexetimide for the mAchR subtypes has not been characterized, nor has its brain distribution been studied intensively. Therefore, this study aimed to address these topics. METHODS: The in vitro affinity and selectivity of (127)I-iododexetimide (cold-labeled iododexetimide), as well as its functional antagonist properties (guanosine 5'-[γ-(35)S-thio]triphosphate [GTPγ(35)S] assay), were assessed on recombinant human M1R-M5R. Distributions of (127)I-iododexetimide and (123)I-iododexetimide in the brain were evaluated using liquid chromatography-mass spectrometry and storage phosphor imaging, respectively, ex vivo in rats, wild-type mice, and M1-M5 knock-out (KO) mice. Inhibition of (127)I-iododexetimide and (123)I-iododexetimide binding in M1R-rich brain areas by the M1R/M4R agonist xanomeline, or the antipsychotics olanzapine (M1R antagonist) and haloperidol (low M1R affinity), was assessed in rats ex vivo. RESULTS: In vitro, (127)I-iododexetimide displayed high affinity for M1R (pM range), with modest selectivity over other mAchRs. In biodistribution studies on rats, ex vivo (127)I-iododexetimide binding was much higher in M1R-rich brain areas, such as the cortex and striatum, than in cerebellum (devoid of M1Rs). In M1 KO mice, but not M2-M5 KO mice, (127)I-iododexetimide binding was strongly reduced in the frontal cortex compared with wild-type mice. Finally, acute administration of both an M1R/M4R agonist xanomeline and the M1R antagonist olanzapine was able to inhibit (123)I-iododexetimide ex vivo, and (123)I-iododexetimide binding in M1-rich brain areas in rats, whereas administration of haloperidol had no effect. CONCLUSION: The current results suggest that (123)I-iododexetimide preferentially binds to M1R in vivo and can be displaced by M1R ligands. (123)I-iododexetimide may therefore be a useful imaging tool as a way to further evaluate M1R changes in neuropsychiatric disorders, as a potential stratifying biomarker, or as a clinical target engagement biomarker to assess M1R.

Synthesis and evaluation in rats of the dopamine D2/3 receptor agonist 18F-AMC20 as a potential radioligand for PET.

UNLABELLED: Dopamine D(2/3) receptor (D(2/3)R) agonist PET tracers are better suited for the imaging of synaptic dopaminergic neurotransmission than D(2/3)R antagonists and may also offer the opportunity to study in vivo the high-affinity state of D(2/3)R (D(2/3)RHigh). With the aim to develop (18)F-labeled D2/3R agonists suitable for widespread clinical application, we report here on the synthesis and in vitro and in vivo evaluation of a D(2/3)R agonist ligand from the aminomethyl chromane (AMC) class-(R)-2-[(4-(18)F-fluorobenzylamino)methyl]chroman-7-ol ((18)F- AMC20: ). METHODS: In vitro affinities of AMC20: toward dopaminergic receptor subtypes were measured in membrane homogenates prepared from HEK293 cells expressing human dopamine receptors. Agonism of AMC20: was assessed in the arrestin recruitment assay in Chinese hamster ovary-K(1) cells expressing the long isoform of D(2)R (D(2)RLong). D(2/3)R-specific binding of (18)F- AMC20: was evaluated in brain slices of Sprague-Dawley rats by in vitro autoradiography and in living rats by in vivo small-animal PET imaging and ex vivo autoradiography. PET data were analyzed with 1- and 2-tissue compartmental models, the simplified reference tissue model, and Logan graphical analysis. Specificity of binding was tested by blocking D(2/3)R with raclopride (coincubation with 10 µM in vitro, administration of 1.0 mg/kg in vivo). RESULTS: In membrane homogenates, AMC20: demonstrated picomolar affinity at D(2)RHigh (mean inhibition constant [K(i)] = 85 pM) and excellent selectivity against the low-affinity state of D(2)R (D(2)RLow) (mean K(i) = 84 nM, 988-fold selectivity) and D(1)-like receptors (mean K(i) = 5,062 nM at D1R). The efficacy of AMC20: was 90% of that of dopamine in the arrestin recruitment assay. Up to 70% of total binding of (18)F- AMC20: in the D2/3R-rich striatum in rat brain slices was D(2/3)R-specific; in living rats, the uptake ratio between the striatum and the D(2/3)R-poor cerebellum reached 2.0-2.5, depending on the measurement method. Radiometabolites of (18)F- AMC20: did not enter the brain. Striatal binding potential of (18)F- AMC20: varied between 0.49 and 0.59 depending on the estimation method. Pretreatment with 1 mg of raclopride per kilogram reduced the apparent specific binding of (18)F- AMC20: in the striatum. CONCLUSION: (18)F- AMC20: shows specific binding to D(2/3)R in the striatum of living rats. Further optimization of the chemical structure of (18)F- AMC20: can lead to (18)F-labeled D(2/3) agonist PET tracers more suitable for in vivo clinical application.

Synthesis and characterization of a novel series of agonist compounds as potential radiopharmaceuticals for imaging dopamine D2/3 receptors in their high-affinity state.

Imaging of dopamine D2/3 receptors (D2/3R) can shed light on the nature of several neuropsychiatric disorders in which dysregulation of D2/3R signaling is involved. Agonist D2/3 tracers for PET/SPECT imaging are considered to be superior to antagonists because they are more sensitive to dopamine concentrations and may selectively label the high-affinity receptor state. Carbon-11-labeled D2/3R agonists have been developed, but these short-lived tracers can be used only in centers with a cyclotron. Here, we report the development of a series of novel D2R agonist compounds based on the 2-aminomethylchromane (AMC) scaffold that provides ample opportunities for the introduction of longer-lived [(18)F] or [(123)I]. Binding experiments showed that several AMC compounds have a high affinity and selectivity for D2/3R and act as agonists. Two fluorine-containing compounds were [(18)F]-labeled, and both displayed specific binding to striatal D2/3R in rat brain slices in vitro. These findings encourage further in vivo evaluations.

Ex Vivo Characterization of a Novel Iodine-123-Labelled Aminomethylchroman as a Potential Agonist Ligand for SPECT Imaging of Dopamine D2/3 Receptors.

For imaging of dopamine D2/3 receptors, agonist tracers are favoured over antagonists because they are more sensitive to detection of dopamine release and because they may selectively label the high-affinity receptor state. We have developed novel D2/3 receptor selective agonists that can be radiolabelled with [(123)I], which label is advantageous over most other labels, such as carbon-11, as it has a longer half-life. Particularly, we considered (R) N-[7-hydroxychroman-2-yl]-methyl 4-iodobenzyl amine (compound 1) as an attractive candidate for development as it shows high binding affinity to D2/3 receptors in vitro, and here we report on the characterization of this first [(123)I]-labelled D2/3 receptor agonist radiopharmaceutical intended for SPECT imaging. The appropriate tin precursor for [(123)I]-1 was developed and was successfully radiolabelled with iodine-123 giving a moderate yield (30-35%) and a good purity (>95%) for [(123)I]-1. In biodistribution experiments in Wistar rats intravenous injection of [(123)I]-1 resulted in a fast brain uptake, where the observed binding in the D2/3 receptor-rich striatum was slightly higher than that in the cerebellum 30 min to 4 h p.i. Storage phosphor imaging experiments, however, did not show specific D2/3 receptor binding. In conclusion, despite promising in vitro data for 1, neither specific ex vivo binding nor high signal-to-noise ratios were found in rodents for [(123)I]-1.

Agonist signalling properties of radiotracers used for imaging of dopamine D2/3 receptors.

BACKGROUND: Dopamine D2/3 receptor (D2/3R) agonist radiopharmaceuticals are considered superior to antagonists to detect dopamine release, e.g. induced by amphetamines. Agonists bind preferentially to the high-affinity state of the dopamine D2R, which has been proposed as the reason why agonists are more sensitive to detect dopamine release than antagonist radiopharmaceuticals, but this theory has been challenged. Interestingly, not all agonists similarly activate the classic cyclic adenosine mono phosphate (cAMP) and the ?-arrestin-2 pathway, some stimulate preferentially one of these pathways; a phenomenon called biased agonism. Because these pathways can be affected separately by pathologies or drugs (including dopamine releasers), it is important to know how agonist radiotracers act on these pathways. Therefore, we characterized the intracellular signalling of the well-known D2/3R agonist radiopharmaceuticals NPA and PHNO and of several novel D2/3R agonists. METHODS: cAMP accumulation and ?-arrestin-2 recruitment were measured on cells expressing human D2R. RESULTS: All tested agonists showed (almost) full agonism in both pathways. CONCLUSIONS: The tested D2/3R agonist radiopharmaceuticals did not exhibit biased agonism in vitro. Consequently, it is likely that drugs (including psychostimulants like amphetamines) and/or pathologies that influence the cAMP and/or the ?-arrestin-2 pathway may influence the binding of these radiopharmaceuticals.

The new radioligand [(3)H]-L 748,337 differentially labels human and rat ß3-adrenoceptors.

As no suitable radioligand exists for the detection of ß3-adrenoceptors, we have explored the radioligand binding properties of a tritiated version of the selective ß3-adrenoceptor antagonist L 748,337. Kinetic and equilibrium saturation and competition binding experiments were performed with [(3)H]-L 748,337 on membrane fractions of HEK and CHO cells stably transfected with human and rat ß-adrenoceptor subtypes. Based on both association/dissociation kinetic and equilibrium saturation binding studies in transfected HEK cells, [(3)H]-L 748,337 exhibited an affinity of approximately 2 nM for human ß3-adrenoceptors. Competition studies with agonists and subtype-selective antagonists validated its binding to ß3-adrenoceptors. In CHO cells transfected with human ß3-adrenoceptors similar saturable high-affinity of [(3)H]-L 748,337 was observed. While some isoprenaline-sensitive [(3)H]-L 748,337 binding was also observed in CHO cells transfected with human ß1- or ß2-adrenoceptors, this was not saturable in a similar concentration range and/or not sensitive to the antagonists propranolol and SR 59,230, indicating that it did not primarily involve ß-adrenoceptors. In CHO cells transfected with rat ß3-adrenoceptors [(3)H]-L 748,337 exhibited a considerably lower affinity than with the human subtype (12-95 nM). Low affinity for the rat ß3-adrenoceptor was also found with unlabelled L 748,337 in rat bladder strip relaxation experiments. We conclude that L 748,337 apparently has lower affinity for the rat than the human ß3-adrenoceptors and that [(3)H]-L 748,337 can bind to a low-affinity site distinct from the orthosteric pocket of ß-adrenoceptors. Nevertheless, [(3)H]-L 748,337 appears to be the most promising radioligand for the selective labelling of human ß3-adrenoceptors reported to date.

Agonist high- and low-affinity states of dopamine D2 receptors: methods of detection and clinical implications.

Dopamine D(2) receptors, similar to other G-protein-coupled receptors, exist in a high- and low-affinity state for agonists. Based upon a review of the methods for detecting D(2) receptor agonist high-affinity states, we discuss alterations of such states in animal models of disease and the implications of such alterations for their labelling with positron emission tomography (PET) and single-photon emission computed tomography (SPECT) tracers. The classic approach of detecting agonist high-affinity states compares agonist competition for antagonist radioligands, in most cases using [(3)H]-spiperone as the radioligand; alternative approaches and radioligands have been proposed, but their claimed advantages have not been substantiated by other investigators. In view of the advantages and disadvantages of various techniques, we critically have reviewed reported findings on the detection of D(2) receptor agonist high-affinity states in a variety of animal models. These data are compared to the less numerous findings from human in vivo studies based on PET and SPECT tracers; they are interpreted in light of the finding that D(2) receptor agonist high-affinity states under control conditions may differ between rodent and human brain. The potential advantages of agonist ligands in studies of pathophysiology and as diagnostics are being discussed.

Novice drivers' performance after different alcohol dosages and placebo in the divided-attention steering simulator (DASS).

RATIONALE: The divided-attention steering simulator (DASS) is designed to measure lane-keeping (i.e., a tracking task using a steering wheel) while performing a secondary visual task (responding to digits that appear in the corners of the computer screen). Some studies have already used the DASS, but the magnitude of impairment is difficult to interpret because reference values are lacking. OBJECTIVE: To examine the magnitude of impairment after administration of four different dosages of alcohol and placebo. MATERIALS AND METHODS: Thirty-two healthy young adults participated in this randomized, single-blind crossover trial. Subjects received alcohol to gain a blood alcohol concentration (BAC) of 0.02%, 0.05%, 0.08%, and 0.10% or alcohol-placebo. Sixteen subjects performed a 30-min test in DASS (dual-task condition). Outcome measures were steering error, reaction time, and percentage of errors. Sixteen other subjects performed the test without performing the secondary peripheral task (single-task condition). RESULTS: Twenty-eight subjects (novice drivers; drivers' license up to 5 years) were included in the analyses. Dose-dependent impairment was found in both the single-task condition (F ((4,11)) = 10.86, p < 0.001) and the dual-task condition (F ((4,9)) = 5.58, p < 0.015). Performance at all BAC levels differed significantly (p < 0.05) from alcohol-placebo, except BAC 0.02%. With increasing BAC levels, subjects made more errors and reacted slower on the peripheral visual search task, but these effects did not reach significance. CONCLUSION: With increasing BAC, dose-dependent impairment was found. The DASS seems to be a suitable divided-attention task that is useful in psychopharmacological research and training of novice drivers.