Leonhard-Key Mien

Reduced serotonin-1A receptor binding in social anxiety disorder.

BACKGROUND Results from studies in serotonin-1A (5-HT1A) knockout mice and previous positron emission tomography (PET) studies in humans imply a role for 5-HT1A receptors in normal state anxiety as well as in certain anxiety disorders. The objective of this study was to investigate 5-HT1A receptor binding potential (BP) in social anxiety disorder (SAD). METHODS Using PET and [carbonyl-11C]WAY-100635, we compared a homogeneous group of 12 unmedicated, male SAD patients with 18 healthy control subjects (HC). A multivariate ANOVA with all regional BP values as dependent variables, age and four radiochemical variables as covariates was performed. RESULTS We found a significantly lower 5-HT1A BP in several limbic and paralimbic areas but not in the hippocampus (p = .234) of SAD patients. The difference in 5-HT1A binding was most significant in the amygdala (-21.4%; p = .003). There was also a more than 20% lower 5-HT(1A) BP of SAD patients in the anterior cingulate cortex (p = .004), insula (p = .003), and dorsal raphe nuclei (p = .030). CONCLUSIONS The lower 5-HT1A binding in the amygdala and mesiofrontal areas of SAD patients is consistent with 1) preclinical findings of elevated anxiety in 5-HT1A knockout mice, 2) a previous PET study in healthy volunteers showing an inverse correlation between 5-HT1A BP and state anxiety, and 3) another human PET study in patients with panic disorder showing reduced 5-HT1A binding, thus corroborating the potential validity of 5-HT1A receptors as targets in the treatment of human anxiety disorders.

Influence of escitalopram treatment on 5-HT 1A receptor binding in limbic regions in patients with anxiety disorders.

There is an increasing interest in the underlying mechanisms of the antidepressant and anxiolytic treatment effect associated with changes in serotonergic neurotransmission after treatment with selective serotonin (5-HT) reuptake inhibitors (SSRIs) in humans. The 5-HT1A receptor is known to play a crucial role in the pathophysiology of affective disorders, and altered 5-HT1A receptor binding has been found in anxiety patients. SSRI treatment raises the 5-HT level in the synaptic cleft and might change postsynaptic receptor densities. Therefore, our study in patients suffering from anxiety disorders investigated the effects of long-term treatment with escitalopram on the 5-HT1A receptor. A longitudinal positrone emission tomography (PET) study in 12 patients suffering from anxiety disorders was conducted. Two dynamic PET scans were performed applying the selective 5-HT1A receptor antagonist [carbonyl-11C]WAY-100635. Eight regions of interest were defined a priori (orbitofrontal cortex, amygdala, hippocampus, subgenual cortex, anterior and posterior cingulate cortex, dorsal raphe nucleus and cerebellum as reference). After the baseline PET scan, patients were administered escitalopram (average dose of 11.2±6.0 mg day−1) for a minimum of 12 weeks. A second PET scan was conducted after 109±27 days. 5-HT1A receptor binding potentials in 12 patients were assessed by PET applying the Simplified Reference Tissue Model.There was a significant reduction in the 5-HT1A receptor binding potential after a minimum of 12 weeks of escitalopram treatment in the hippocampus (P=0.006), subgenual cortex (P=0.017) and posterior cingulate cortex (P=0.034). The significance of the hippocampus region survived the Bonferroni-adjusted threshold for multiple comparisons. These PET data in humans in vivo demonstrate a reduction of the 5-HT1A binding potential after SSRI treatment.

The serotonin-1A receptor distribution in healthy men and women measured by PET and [carbonyl-11C]WAY-100635

PurposeThe higher prevalence rates of depression and anxiety disorders in women compared to men have been associated with sexual dimorphisms in the serotonergic system. The present positron emission tomography (PET) study investigated the influence of sex on the major inhibitory serotonergic receptor subtype, the serotonin-1A (5-HT1A) receptor.MethodsSixteen healthy women and 16 healthy men were measured using PET and the highly specific radioligand [carbonyl-11C]WAY-100635. Effects of age or gonadal hormones were excluded by restricting the inclusion criteria to young adults and by controlling for menstrual cycle phase. The 5-HT1A receptor BPND was quantified using (1) the ‘gold standard’ manual delineation approach with ten regions of interest (ROIs) and (2) a newly developed delineation method using a PET template normalized to the Montreal Neurologic Institute space with 45 ROIs based on automated anatomical labeling.ResultsThe 5-HT1A receptor BPND was found equally distributed in men and women applying both the manual delineation method and the automated delineation approach. Women had lower mean BPND values in every region investigated, with a borderline significant sex difference in the hypothalamus (p = 0.012, uncorrected). There was a high intersubject variability of the 5-HT1A receptor BPND within both sexes compared to the small mean differences between men and women.ConclusionsTo conclude, when measured in the follicular phase, women do not differ from men in the 5-HT1A receptor binding. To explain the higher prevalence of affective disorders in women, further studies are needed to evaluate the relationship between hormonal status and the 5-HT1A receptor expression.

Escitalopram Enhances the Association of Serotonin-1A Autoreceptors to Heteroreceptors in Anxiety Disorders

Selective serotonin reuptake inhibitors (SSRIs) represent one of the most common treatment options in major depression and anxiety disorders. By blocking the serotonin transporter, SSRIs modulate serotonergic neurotransmission as well as the function of autoreceptors and heteroreceptors. However, treatment-induced changes on a network level primarily remain unknown. Thus, we evaluated the association between serotonin-1A (5-HT1A) autoreceptors and heteroreceptors before and after SSRIs. Twenty-one patients with anxiety disorders underwent positron emission tomography using [carbonyl-11C]WAY-100635 before and after 12 weeks of escitalopram treatment; 15 of them completed the study protocol. Additionally, 36 drug-naive healthy controls were measured once. The 5-HT1A receptor binding potential (BPND) was quantified for the dorsal raphe nucleus (DRN) using a region-of-interest approach and for the entire brain by calculating parametric maps. Voxel-wise linear regression was applied between DRN autoreceptor and whole-brain heteroreceptor 5-HT1A BPND. Consistent with previous observations, healthy subjects showed widespread positive correlations of 5-HT1A BPND between autoreceptors and heteroreceptors. Comparing patients before versus after escitalopram treatment revealed enhanced associations of autoreceptor-to-heteroreceptor 5-HT1A BPND within the amygdala and hippocampus (R2 = 0.21–0.28 vs 0.49–0.81; p < 0.05–0.001). In contrast, no significant SSRI-induced changes were found for correlations of heteroreceptor-to-heteroreceptor 5-HT1A BPND between several limbic regions. This interregional approach suggests a treatment-induced reinforcement of the association of 5-HT1A binding between autoreceptors and heteroreceptors specifically in areas involved in anxiety disorders. These findings provide complementary information about treatment effects on a network level and confirm the central role of the DRN as a prime regulatory area.

Simple and fully automated preparation of [carbonyl-11C]WAY-100635

So far, [carbonyl-11C]WAY-100635 is the PET-tracer of choice for 5HT1A-receptor-imaging. Since the preparation is still a challenge, we aimed at (1) the evaluation of various essential parameters for the successful preparation, (2) the simplification of the radiosynthesis and (3) the establishment of a safe and fully automated system. The preparation is based on a commercial synthesizer and all chemicals are used without further processing. We found a low failure rate (7.7%), high average yield (4.0 ± 1.0 GBq) and a specific radioactivity of 292 ± 168 GBq/μmol (both at the end of synthesis, EOS).

[18F]FE@SUPPY and [18F]FE@SUPPY:2 — metabolic considerations

INTRODUCTION Recently, [(18)F]FE@SUPPY and [(18)F]FE@SUPPY:2 were introduced as the first positron emission tomography (PET) tracers for the adenosine A(3) receptor. Thus, aim of the present study was the metabolic characterization of the two adenosine A(3) receptor PET tracers. METHODS In vitro carboxylesterase (CES) experiments were conducted using incubation mixtures containing different concentrations of the two substrates, porcine CES and phosphate-buffered saline. Enzymatic reactions were stopped by adding acetonitrile/methanol (10:1) after various time points and analyzed by a high-performance liquid chromatography (HPLC) standard protocol. In vivo experiments were conducted in male wild-type rats; tracers were injected through a tail vein. Rats were sacrificed after various time points (n=3), and blood and brain samples were collected. Sample cleanup was performed by an HPLC standard protocol. RESULTS The rate of enzymatic hydrolysis by CES demonstrated Michaelis-Menten constants in a micromolar range (FE@SUPPY, 20.15 microM, and FE@SUPPY:2, 13.11 microM) and limiting velocities of 0.035 and 0.015 microM/min for FE@SUPPY and FE@SUPPY:2, respectively. Degree of metabolism in blood showed the following: 15 min pi 47.7% of [(18)F]FE@SUPPY was intact compared to 33.1% of [(18)F]FE@SUPPY:2; 30 min pi 30.3% intact [(18)F]FE@SUPPY was found compared to 15.6% [(18)F]FE@SUPPY:2. In brain, [(18)F]FE@SUPPY:2 formed an early hydrophilic metabolite, whereas metabolism of [(18)F]FE@SUPPY was not observed before 30 min pi CONCLUSION Knowing that metabolism in rats is several times faster than in human, we conclude that [(18)F]FE@SUPPY should be stable for the typical time span of a clinical investigation. As a consequence, from a metabolic point of view, one would tend to decide in favor of [(18)F]FE@SUPPY.

Automatisation and First Evaluation of [18F]FE@SUPPY:2, an AlternativePET-Tracer for the Adenosine A3 Receptor: A Comparison with[18F]FE@SUPPY

Introduction: Since the Adenosine-A3-receptor was identified in the late 1990´s, there is little data available de- scribing its distribution in vivo. Recently, we introduced ( 18 F)FE@SUPPY as the first PET-tracer for this receptor. In the present investigation we translated this fluoroethyl-ester into the fluoroethyl-thioester ( 18 F)FE@SUPPY:2 (5-ethyl 2,4- diethyl-3-((2-( 18 F)fluoroethyl) sulfanylcarbonyl)-6-phenylpyridine-5-carboxylate). Aims of the present study were the evaluation of (1) the automatized preparation of both ( 18 F)FE@SUPPY-derivatives, (2) the biodistribution of ( 18 F)FE@SUPPY:2, (3) the lipophilicity and (4) the comparison of the findings of ( 18 F)FE@SUPPY and ( 18 F)FE@SUPPY:2. Methods: The automated preparations of both ( 18 F)FE@SUPPY-analogs were performed on a GE TRACERlab FxFN syn- thesizer using suitable precursors. Biodistribution experiments were performed using Sprague-Dawley rats/Him:OFA. Lipophilicity of the compounds was determined using an HPLC assay. Results: 22 automated radiosyntheses were performed for both radiotracers. Specific radioactivity was 70 ± 26GBq/� mol for ( 18 F)FE@SUPPY and 340 ± 140GBq/� mol for ( 18 F)FE@SUPPY:2. Biodistribution experiments evinced bowels and liver as organs with highest uptake and intermediate uptake in kidney, lung and heart. LogP values of both molecules ranged from 3.99 to 4.12 at different pH. Conclusion: From a radiopharmaceutical perspective, drastically better specific radioactivities would militate in favour of ( 18 F)FE@SUPPY:2; preclinical evaluations, so far, do not permit the decision upon the selection of the optimum ( 18 F)FE@SUPPY-derivative. With ( 18 F)FE@SUPPY:2, we are able to provide a second potential tracer that could help to further characterize the still quite unexplored Adenosine-A3-receptor.

Radiosynthesis of the adenosine A3 receptor ligand 5-(2-[18F]fluoroethyl) 2,4-diethyl-3-(ethylsulfanylcarbonyl)- 6-phenylpyridine-5-carboxylate ([18F]FE@SUPPY)

Summary Since to date very limited information on the distribution and function of the adenosine A3 receptor is available, the development of a suitable radioligand is needed. Such a selective radioligand can then be used for quantitative autoradiography, preclinical studies in animals and subsequent human PET applications. Recently, a promising candidate compound, 5-(2-fluoroethyl) 2,4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate (FE@SUPPY), has been presented. The successful preparation of a suitable labelling precursor and the evaluation and optimization of the radiosynthesis of [18F] FE@SUPPY is presented herewith. For satisfactory yields, a reaction temperature of 75 °C has to be applied for at least 20 min using 8–10 mg of precursor. Until now, 15 complete high-scale radiosyntheses were performed. Starting from an average of 51±12 GBq (average ±SD; range: 30–67 GBq) [18F]fluoride, 9.4–3.6 GBq of formulated [18F]FE@SUPPY (32.3±12.4%, based on [18F]fluoride, corrected for decay) were prepared in <105 min.

Radiosynthesis of a novel potential adenosine A3 receptor ligand, 5-ethyl 2,4-diethyl-3-((2-[18F]fluoroethyl)sulfanylcarbonyl)-6-phenylpyridine-5-carboxylate ([18F]FE@SUPPY:2)

Abstract Since, to date very limited information on the distribution and function of the adenosine A3 receptor is available, the development of suitable radioligands is needed. Recently, we introduced [ 18F]FE@SUPPY (5-(2-[ 18F]fluoroethyl) 2,4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate) as the first PET-ligand for the A3R. Regarding the metabolic profile – this class of dialkylpyridines comprises two ester functions within one molecule, one carboxylic and one thiocarboxylic – one could expect carboxylesterases significantly contributing to cleavage and degradation. Therefore, our aim was the development of [ 18F]FE@SUPPY:2 (5-ethyl 2,4-diethyl-3-((2-[ 18F]fluoroethyl)sulfanylcarbonyl)-6-phenylpyridine-5-carboxylate), the functional isomer containing the label at the thiocarboxylic moiety. For satisfactory yields in high scale radiosyntheses, a reaction temperature of 75 °C has to be applied for at least 20 min using 20 mg/mL of precursor. So far, 6 complete high-scale radiosyntheses were performed. Starting from an average of 51.2±21.8 GBq (mean±SD) [ 18F]fluoride, 5.8±4.1 GBq of formulated [ 18F]FE@SUPPY:2 (12.0±5.4%, based on [ 18F]fluoride, not corrected for decay) were prepared in 75±8 min.

Preparation and radiosynthesis of [18F]FE@CFN (2-[18F]fluoroethyl 4-[N-(1-oxopropyl)-N-phenylamino]-1-(2-phenylethyl)-4-piperidinecarboxylate): a potential μ-opioid receptor imaging agent

PET imaging of the μ-opioid receptor (OR) is still restricted to [11C]carfentanil ([11C]CFN) but its use is limited due to its short half-life and high agonistic potency. Recently, the radiosynthesis of [18F]fluoroalkyl esters of CFN was proposed, unfortunately yielding products not suitable for human PET due to their low specific activities. Therefore, our rationale was to develop a reliable radiosynthesis of a [18F]fluoroethylated CFN derivative overcoming these drawbacks. The [18F]fluoroethyl ester of carfentanil, [18F]FE@CFN (2-[18F]fluoroethyl 4-[N-(1-oxopropyl)-N-phenylamino]-1-(2-phenylethyl)-4-piperidinecarboxylate), and its corresponding inactive standard compound were prepared. Purification of [18F]FE@CFN was achieved via a simple solid phase extraction method. [18F]FE@CFN was prepared with excellent purity (> 98%) and sufficient yields. Specific activity surpassed the level required for safe administration. We therefore conclude that our simplified synthesis of [18F]FE@CFN, for the first time, overcomes the shortcomings of [11C]CFN and the previously suggested alternatives, namely, (1) longer half-life; (2) easy production and (3) adequate specific activity, should make a wider application possible. Hence, [18F]FE@CFN may become a valuable PET tracer for the imaging of the μ-OR in human brain and heart.