[18F]DPA-714: Effect of co-medications, age, sex, BMI and TSPO polymorphism on the human plasma input function.

PURPOSE: We aimed to assess the effect of concomitant medication, age, sex, body mass index and 18-kDa translocator protein (TSPO) binding affinity status on the metabolism and plasma pharmacokinetics of [18F]DPA-714 and their influence on the plasma input function in a large cohort of 201 subjects who underwent brain and whole-body PET imaging to investigate the role of neuroinflammation in neurological diseases. METHODS: The non-metabolized fraction of [18F]DPA-714 was estimated in venous plasma of 138 patients and 63 healthy controls (HCs; including additional arterial sampling in 16 subjects) during the 90 min brain PET acquisition using a direct solid-phase extraction method. The mean fraction between 70 and 90 min post-injection ([18F]DPA-71470-90) and corresponding normalized plasma concentration (SUV70-90) were correlated with all factors using a multiple linear regression model. Differences between groups (arterial vs venous measurements; HCs vs patients; high- (HAB), mixed- (MAB) and low-affinity binders (LAB); subjects with vs without co-medications, females vs males were also assessed using the non-parametric Mann-Whitney or Kruskal-Wallis ANOVA tests. Finally, the impact of co-medications on the brain uptake of [18F]DPA-714 at equilibrium was investigated. RESULTS: As no significant differences were observed between arterial and venous [18F]DPA-71470-90 and SUV70-90, venous plasma was used for correlations. [18F]DPA-71470-90 was not significantly different between patients and HCS (59.7 ± 12.3% vs 60.2 ± 12.9%) despite high interindividual variability. However, 47 subjects exhibiting a huge increase or decrease of [18F]DPA-71470-90 (up to 88% or down to 23%) and SUV70-90 values (2-threefold) were found to receive co-medications identified as inhibitors or inducers of CYP3A4, known to catalyse [18F]DPA-714 metabolism. Comparison between cortex-to-plasma ratios using individual input function (VTIND) or population-based input function derived from untreated HCs (VTPBIF) indicated that non-considering the individual metabolism rate led to a bias of about 30% in VT values. Multiple linear regression model analysis of subjects free of these co-medications suggested significant correlations between [18F]DPA-71470-90 and age, BMI and sex while TSPO polymorphism did not influence the metabolism of the radiotracer. [18F]DPA-714 metabolism fell with age and BMI and was significantly faster in females than in males. Whole-body PET/CT exhibited a high uptake of the tracer in TSPO-rich organs (heart wall, spleen, kidneys…) and those involved in metabolism and excretion pathways (liver, gallbladder) in HAB and MAB with a strong decrease in LAB (-89% and -85%) resulting in tracer accumulation in plasma (4.5 and 3.3-fold increase). CONCLUSION: Any co-medication that inhibits or induces CYP3A4 as well as TSPO genetic status, age, BMI and sex mostly contribute to interindividual variations of the radiotracer metabolism and/or concentration that may affect the input function of [18F]DPA-714 and consequently its human brain and peripheral uptake. TRIAL REGISTRATION: INFLAPARK, NCT02319382, registered December 18, 2014, retrospectively registered; IMABIO 3, NCT01775696, registered January 25, 2013, retrospectively registered; INFLASEP, NCT02305264, registered December 2, 2014, retrospectively registered; EPI-TEP, EudraCT 2017-003381-27, registered September 24, 2018.

The SIGMA rat brain templates and atlases for multimodal MRI data analysis and visualization.

Preclinical imaging studies offer a unique access to the rat brain, allowing investigations that go beyond what is possible in human studies. Unfortunately, these techniques still suffer from a lack of dedicated and standardized neuroimaging tools, namely brain templates and descriptive atlases. Here, we present two rat brain MRI templates and their associated gray matter, white matter and cerebrospinal fluid probability maps, generated from ex vivo [Formula: see text]-weighted images (90 µm isotropic resolution) and in vivo T2-weighted images (150 µm isotropic resolution). In association with these templates, we also provide both anatomical and functional 3D brain atlases, respectively derived from the merging of the Waxholm and Tohoku atlases, and analysis of resting-state functional MRI data. Finally, we propose a complete set of preclinical MRI reference resources, compatible with common neuroimaging software, for the investigation of rat brain structures and functions.

The dynamics of stress: a longitudinal MRI study of rat brain structure and connectome.

Stress is a well-established trigger for a number of neuropsychiatric disorders, as it alters both structure and function of several brain regions and its networks. Herein, we conduct a longitudinal neuroimaging study to assess how a chronic unpredictable stress protocol impacts the structure of the rat brain and its functional connectome in both high and low responders to stress. Our results reveal the changes that stress triggers in the brain, with structural atrophy affecting key regions such as the prelimbic, cingulate, insular and retrosplenial, somatosensory, motor, auditory and perirhinal/entorhinal cortices, the hippocampus, the dorsomedial striatum, nucleus accumbens, the septum, the bed nucleus of the stria terminalis, the thalamus and several brain stem nuclei. These structural changes are associated with increasing functional connectivity within a network composed by these regions. Moreover, using a clustering based on endocrine and behavioural outcomes, animals were classified as high and low responders to stress. We reveal that susceptible animals (high responders) develop local atrophy of the ventral tegmental area and an increase in functional connectivity between this area and the thalamus, further spreading to other areas that link the cognitive system with the fight-or-flight system. Through a longitudinal approach we were able to establish two distinct patterns, with functional changes occurring during the exposure to stress, but with an inflection point after the first week of stress when more prominent changes were seen. Finally, our study revealed differences in functional connectivity in a brainstem-limbic network that distinguishes resistant and susceptible responders before any exposure to stress, providing the first potential imaging-based predictive biomarkers of an individual's resilience/vulnerability to stressful conditions.

White matter changes in microstructure associated with a maladaptive response to stress in rats.

In today's society, every individual is subjected to stressful stimuli with different intensities and duration. This exposure can be a key trigger in several mental illnesses greatly affecting one's quality of life. Yet not all subjects respond equally to the same stimulus and some are able to better adapt to them delaying the onset of its negative consequences. The neural specificities of this adaptation can be essential to understand the true dynamics of stress as well as to design new approaches to reduce its consequences. In the current work, we employed ex vivo high field diffusion magnetic resonance imaging (MRI) to uncover the differences in white matter properties in the entire brain between Fisher 344 (F344) and Sprague-Dawley (SD) rats, known to present different responses to stress, and to examine the effects of a 2-week repeated inescapable stress paradigm. We applied a tract-based spatial statistics (TBSS) analysis approach to a total of 25 animals. After exposure to stress, SD rats were found to have lower values of corticosterone when compared with F344 rats. Overall, stress was found to lead to an overall increase in fractional anisotropy (FA), on top of a reduction in mean and radial diffusivity (MD and RD) in several white matter bundles of the brain. No effect of strain on the white matter diffusion properties was observed. The strain-by-stress interaction revealed an effect on SD rats in MD, RD and axial diffusivity (AD), with lower diffusion metric levels on stressed animals. These effects were localized on the left side of the brain on the external capsule, corpus callosum, deep cerebral white matter, anterior commissure, endopiriform nucleus, dorsal hippocampus and amygdala fibers. The results possibly reveal an adaptation of the SD strain to the stressful stimuli through synaptic and structural plasticity processes, possibly reflecting learning processes.

Modifications of the endosomal compartment in peripheral blood mononuclear cells and fibroblasts from Alzheimer's disease patients.

Identification of blood-based biomarkers of Alzheimer's disease (AD) remains a challenge. Neuropathological studies have identified enlarged endosomes in post-mortem brains as the earliest cellular change associated to AD. Here the presence of enlarged endosomes was investigated in peripheral blood mononuclear cells from 48 biologically defined AD patients (25 with mild cognitive impairment and 23 with dementia (AD-D)), and 23 age-matched healthy controls using immunocytochemistry and confocal microscopy. The volume and number of endosomes were not significantly different between AD and controls. However, the percentage of cells containing enlarged endosomes was significantly higher in the AD-D group as compared with controls. Furthermore, endosomal volumes significantly correlated to [C(11)]PiB cortical index measured by positron emission tomography in the AD group, independently of the APOE genotype, but not to the levels of amyloid-beta, tau and phosphorylated tau measured in the cerebrospinal fluid. Importantly, we confirmed the presence of enlarged endosomes in fibroblasts from six unrelated AD-D patients as compared with five cognitively normal controls. This study is the first, to our knowledge, to report morphological alterations of the endosomal compartment in peripheral cells from AD patients correlated to amyloid load that will now be evaluated as a possible biomarker.

Hyper-responsivity to stress in rats is associated with a large increase in amygdala volume. A 7T MRI study.

Stress is known to precipitate psychiatric disorders in vulnerable people. Individual differences in the stress responsivity can dramatically affect the onset of these illnesses. Animal models of repeated stress represent valuable tools to identify region-specific volumetric changes in the brain. Here, using high resolution 7T MRI, we found that amygdala is the most significant parameter for distinction between F344 and SD rats known to have differential response to stress. A significant substantial increase (45%) was found in the amygdala volume of rats that do not habituate to the repeated stress procedure (F344 rats) compared to SD rats. This strain-specific effect of stress was evidenced by a significant strain-by-stress interaction. There were no significant strain differences in the volumes of hippocampi and prefrontal cortices though stress produces significant reductions of smaller amplitude in the medial prefrontal cortex (mPFC) (9% and 12%) and dorsal hippocampus (5% and 6%) in both strains. Our data further demonstrate the feasibility and relevance of high isotropic resolution structural ex vivo 7T MRI in the study of the brain effects of stress in small animals. Neuroimaging is a valuable tool to follow up brain volumetric reorganization during the stress response and could also be easily used to test pharmacological interventions to prevent the deleterious effects of stress.

Plasma DYRK1A as a novel risk factor for Alzheimer's disease.

To determine whether apparent involvement of DYRK1A in Alzheimer's disease (AD) pathology makes it a candidate plasma biomarker for diagnosis, we developed a method to quantify plasma DYRK1A by immunoblot in transgenic mouse models having different gene dosages of Dyrk1a, and, consequently, different relative protein expression. Then, we measured plasma DYRK1A levels in 26 patients with biologically confirmed AD and 25 controls (negative amyloid imaging available on 13). DYRK1A was detected in transgenic mouse brain and plasma samples, and relative levels of DYRK1A correlated with the gene copy number. In plasma from AD patients, DYRK1A levels were significantly lower compared with controls (P<0.0001). Results were similar when we compared AD patients with the subgroup of controls confirmed by negative amyloid imaging. In a subgroup of patients with early AD (CDR=0.5), lower DYRK1A expression was confirmed. In contrast, no difference was found in levels of DYRK1B, the closest relative of DYRK1A, between AD patients and controls. Further, AD patients exhibited a positive correlation between plasma DYRK1A levels and cerebrospinal fluid tau and phosphorylated-tau proteins, but no correlation with amyloid-ß42 levels and Pittsburgh compound B cortical binding. DYRK1A levels detected in lymphoblastoid cell lines from AD patients were also lower when compared with cells from age-matched controls. These findings suggest that reduced DYRK1A expression might be a novel plasma risk factor for AD.

Alteration of the in vivo nicotinic receptor density in ADNFLE patients: a PET study.

Nicotinic acetylcholine receptors (nAChRs) are involved in a familial form of frontal lobe epilepsy, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). In several ADNFLE families, mutations were identified in the nAChR alpha4 or beta2 subunit, which together compose the main cerebral nAChR. Electrophysiological assessment using in vitro expression systems indicated a gain of function of the mutant receptors. However the precise mechanisms by which they contribute to the pathogenesis of a focal epilepsy remain obscure, especially since alpha4beta2 nAChRs are known to be widely distributed within the entire brain. PET study using [18F]-F-A-85380, a high affinity agonist at the alpha4beta2 nAChRs, allows the determination of the regional distribution and density of the nAChRs in healthy volunteers and in ADNFLE patients, thus offering a unique opportunity to investigate some in vivo consequences of the molecular defect. We have assessed nAChR distribution in eight non-smoking ADNFLE patients (from five families) bearing an identified mutation in nAChRs and in seven age-matched non-smoking healthy volunteers using PET and [(18)F]-F-A-85380. Parametric images of volume of distribution (Vd) were generated as the ratio of tissue to plasma radioactivities. The images showed a clear difference in the pattern of the nAChR density in the brains of the patients compared to the healthy volunteers. Vd values revealed a significant increase (between 12 and 21%, P < 0.05) in the ADNFLE patients in the mesencephalon, the pons and the cerebellum when compared to control subjects. Statistical parametric mapping (SPM) was then used to better analyse subtle regional differences. This analysis confirmed clear regional differences between patients and controls: patients had increased nAChR density in the epithalamus, ventral mesencephalon and cerebellum, but decreased nAChR density in the right dorsolateral prefrontal region. In five patients who underwent an additional [(18)F]-fluorodeoxyglucose (FDG) PET experiment, hypometabolism was observed in the neighbouring area of the right orbitofrontal cortex. The demonstration of a regional nAChR density decrease in the prefrontal cortex, despite the known distribution of these receptors throughout the cerebral cortex, is consistent with a focal epilepsy involving the frontal lobe. We also propose that the nAChR density increase in mesencephalon is involved in the pathophysiology of ADNFLE through the role of brainstem ascending cholinergic systems in arousal.

Improved synthesis of the peripheral benzodiazepine receptor ligand [11C]DPA-713 using [11C]methyl triflate.

Recently, the pyrazolopyrimidine, [11C] N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide (DPA-713) has been reported as a new promising marker for the study of peripheral benzodiazepine receptors with positron emission tomography. In the present study, DPA-713 has been labelled from the corresponding nor-analogue using [11C]methyl triflate (CH3OTf). Conditions for HPLC were also modified to include physiological saline (aq. 0.9% NaCl)/ethanol:60/40 as mobile phase making it suitable for injection. The total time of radiosynthesis, including HPLC purification, was 18-20 min. This reported synthesis of [11C]DPA-713, using [11C]CH3OTf, resulted in an improved radiochemical yield (30-38%) compared to [11C]methyl iodide (CH3I) (9) with a simpler purification method. This ultimately enhances the potential of [11C]DPA-713 for further pharmacological and clinical evaluation. These improvements make this radioligand more suitable for automated synthesis which is of benefit where multi-dose preparations and repeated syntheses of radioligand are required.

Radiosynthesis and pharmacological evaluation of [11C]EMD-95885: a high affinity ligand for NR2B-containing NMDA receptors.

EMD-95885, 6-[3-[4-(4-fluorobenzyl)piperidino]propionyl]-3H-benzoxazol-2-one (1) has been described as a selective antagonist for the NMDA receptors containing NR2B subunits, displaying an IC50 of 3.9 nM for this subtype. EMD-95885 (1) has been synthesized in good overall yield and labelled with carbon-11 ( T1/2 : 20.4 min) at its benzoxazolinone moiety using [11C]phosgene. The pharmacological profile of [11C]EMD-95885 ([11C]-1) was evaluated in vivo in rats with biodistribution studies and brain radioactivity monitored with intracerebral radiosensitive beta-microprobes. The brain uptake of [11C]-1 was homogeneous (0.4-0.6%ID/mL) across the different brain structures studied. This in vivo brain regional distribution of [11C]-1 was not consistent with the known distribution of NR2B subunits. Also as a measure of specificity the hippocampus/cerebellum ratio reached 0.8 throughout the time course of the experiment supporting the lack of specificity. Competition studies with the NR2B prototypic ligand ifenprodil and EMD-95885 (1), 30 min before the radioligand injection, displayed homogeneous reduction of [11C]-1 uptake of 40-60%. Pre-treatment of rats with DTG (sigma ligand), MDL105519 (glycine site antagonist) and MK801 (ion channel blocker) had no inhibitory effect on [11C]-1 uptake. Use of haloperidol as a blocking drug also resulted in a homogeneous inhibition of [11C]-1 uptake by 66-60%, which does not reflect binding to dopamine or sigma receptors. Due to the homogeneous radioligand uptake and inhibition and no measure of cerebral blood flow effects during these blocking studies it is uncertain whether any specific binding is observed. In view of these results, [11C]EMD-95885 ([11C]-1) does not have the required properties for imaging NR2B containing NMDA receptors using positron emission tomography.

In vivo detection of striatal dopamine release during reward: a PET study with [(11)C]raclopride and a single dynamic scan approach.

A new simple method is proposed to detect, using PET and [(11)C]raclopride, changes in striatal extracellular dopamine concentration during a rewarded effortful task. This approach aimed to increase the sensitivity in detection of these effects. It requires a single-dynamic PET study and combines the classic kinetic compartmental model with the general linear model of SPM to provide statistical inference on changes in [(11)C]raclopride time-activity curve due to endogenous dopamine release during two short periods of activation. Kinetic simulations predicted that 100% dopamine increase during two 5-min periods starting at 30 and 60 min after the injection can be detected. Moreover the effects of dopamine release on the [(11)C]raclopride time-activity-curve are different from those induced by CBF increase. These simulated curves were used to construct the statistical linear model and to test voxel-by-voxel in healthy subjects the hypothesis that dopamine is released in the ventral striatum during periods of unexpected monetary gains, but not during periods of unexpected monetary loss. The experimental results are in line with the expected results although the amplitude of the effects due to dopamine release is moderate. The advantages and the limits of this method as well as the relevance of the results for dopamine involvement in reward processing are discussed.

Parametric images of the extrastriatal D2 receptor density obtained using a high-affinity ligand (FLB 457) and a double-saturation method.

The potential of positron emission tomography for the quantitative estimation of receptor concentration in extrastriatal regions has been limited in the past because of the low density of the D2 receptor sites in these regions and the insufficient affinity of the most widely used radioligands for dopamine receptors. The new method described in this paper permits the estimate of the D2 receptor concentration in the extrastriatal regions using a two-injection protocol and FLB 457, a ligand with a high affinity (20 pmol/L in vitro ) with D2 dopamine receptors. This approach is not valid for the striatal regions because some hypotheses cannot be verified (because of the high receptor concentration in these regions). The experimental protocol includes two injections with ligand doses designed to significantly occupy the extrastriatal receptor sites (approximately 90%), while leaving less than 60% of the receptor sites occupied by the ligand in the striatal regions. The results obtained using this double-saturation method are in line with the concentration estimates previously obtained using the multiinjection approach. The receptor concentration is 2.9 +/- 0.5 pmol/mL in the thalamus, 1.0 +/- 0.2 pmol/mL in the temporal cortex, and 0.35 +/- 0.13 pmol/mL in the occipital cortex. This study provides new arguments supporting the presence of a small receptor-site concentration in the cerebellum, estimated at 0.35 +/- 0.16 pmol/mL The simplicity of the calculation used to estimate the receptor concentration lends itself easily to parametric imaging. The receptor concentration is estimated pixel by pixel, without filtering. This method permits estimation of the extrastriatal D2 receptor concentration using an experimental protocol that can easily be used in patient studies (i.e., single experiment, no blood sampling, short experiment duration).

Anatomic and biochemical correlates of the dopamine transporter ligand 11C-PE2I in normal and parkinsonian primates: comparison with 6-[18F]fluoro-L-dopa.

Positron emission tomography (PET) coupled to 6-[18F]Fluoro-L-Dopa (18F-Dopa) remains the gold standard for assessing dysfunctionality concerning the dopaminergic nigrostriatal pathway in Parkinson's disease and related disorders. The use of ligands of the dopamine transporters (DAT) is an attractive alternative target; consequently, the current aim was to validate one of them, 11C-PE2I, using a multiinjection modeling approach allowing accurate quantitation of DAT densities in the striatum. Experiments were performed in three controls, three MPTP-treated (parkinsonian) baboons, and one reserpine-treated baboon. 11C-PE2I B'max values obtained with this approach were compared with 18F-Dopa input rate constant values (Ki), in vitro Bmax binding of 125I-PE2I, and the number of dopaminergic neurons in the substantia nigra estimated postmortem by stereology. In the caudate nucleus and putamen, control values for 11C-PE2I B'max were 673 and 658 pmol/mL, respectively, whereas it was strongly reduced in the MPTP-treated (B'max = 26 and 36 pmol/mL) and reserpine-treated animals (B'max = 338 and 483 pmol/mL). In vivo 11C-PE2I B'max values correlated with 18F-Dopa Ki values and in vitro 125I-PE2I Bmax values in the striatum and with the number of nigral dopaminergic neurons. Altogether, these data support the use of 11C-PE2I for monitoring striatal dopaminergic disorders and the effect of potential neuroprotective strategies.

Absolute quantification by positron emission tomography of the endogenous ligand.

The results of several recent papers have shown a significant influence of the endogenous neurotransmitters on the exogenous ligand kinetics measured by positron emission tomography. For example, several groups found that the percentage of D2 receptor sites occupied by the endogenous dopamine ranged from 25% to 40% at basal level. An obvious consequence of this significant occupancy is that the ligand-receptor model parameters, usually estimated by a model that does not take into account the endogenous ligand (EL) kinetics, can be significantly biased. In the current work, the authors studied the biases obtained by using the multiinjection approach. The results showed that in the classical ligand-receptor model, the receptor concentration is correctly estimated and that only the apparent affinity is biased by not taking the EL into account. At present, all absolute quantifications of the EL have been obtained through pharmacologic manipulation of the endogenous transmitter concentration, which is often too invasive a method to be used in patients. A theoretical reasoning showed that a noninvasive approach is necessarily based on both the apparent affinity measurement and on a multiregion approach. The correlation between the receptor concentration and the apparent affinity, previously observed with some ligands, verifies these two conditions; thus, the authors suggest that this correlation could be the result of the EL effect. To test this assumption experimentally, the effect of reserpine-induced dopamine depletion on the interactions between the D2 receptor sites and the FLB 457 is studied. With untreated baboons, the apparent FLB 457 affinity was smaller in the receptor-rich regions (striatum) than in the receptor-poor regions. This discrepancy disappeared after dopamine depletion, strongly suggesting that this affinity difference was related to the EL effect. Therefore, the purpose of the current study was to test the ability to quantify the EL based on the observed correlation between the receptor concentration and the apparent affinity. This approach offers a method for estimating the percentage of receptor sites occupied by the EL and, if its affinity is known, the free EL concentration. From the data obtained using FLB 457 with baboons, the authors found that approximately 53% of the D2 receptor sites are occupied by dopamine in the striatum and that the free dopamine concentration is approximately 120 nmol/L at basal level. This approach is transferable to patients, because the experimental data are obtained without pharmacologically induced modification of the EL.

Synthesis of high-specific-radioactivity 4- and 6-[18F]fluorometaraminol-PET tracers for the adrenergic nervous system of the heart.

Fluorine-18- (t(1/2) 109.8 min) and carbon-11 (t(1/2) 20.4 min)-labeled norepinephrine analogues have been found previously to be useful positron-emission-tomography (PET) radioligands to map adrenergic nerve terminals of the heart. Metaraminol ((1R,2S)-2-amino-1-(3-hydroxyphenyl)-1-propanol) is a metabolically stable structural analogue of norepinephrine and possesses high affinity towards the norepinephrine transporter and the vesicular monoamine transporter. This paper presents the radiosynthesis of new positron-emission-tomography halogeno analogues of metaraminol labeled with high specific radioactivity. Firstly, fluorine-18-labeled 4-fluorometaraminol (4-[18F]FMR or (1R,2S)-2-amino-1-(4-[18F]fluoro-3-hydroxyphenyl)-1-propanol) and its three other stereoisomers were prepared based on the following key steps: (a) condensation of the corresponding no-carrier-added labeled fluorobenzaldehyde with nitroethane, and (b) HPLC (C18 and chiral) resolution of the diastereomeric product mixture into the four individual enantiomers. Secondly, the corresponding 6-fluoro analogues, fluorine-18-labeled 6-fluorometaraminol (6-[18F]FMR or (1R,2S)-2-amino-1-(2-[18F]fluoro-5-hydroxyphenyl)-1-propanol) and its three other enantiomers, were prepared in an analogous way. Typically, 0.48-0.55 GBq of 4-[18F]FMR and 0.14-0.15 GBq of 6-[18F]FMR could be obtained after 120-160 min total synthesis time, with a specific radioactivity of 56-106 GBq/micromol. Furthermore, the synthesis of racemic 4-fluorometaraminol and 6-fluorometaraminol as reference compounds was performed. as well as independent chiral syntheses of the optically active (1R,2S) enantiomers. For the chiral syntheses, the key step was an electrophilic fluorination with acetyl hypofluorite of (1R,2S)-configurated organometallic derivatives of metaraminol. Tissue distribution studies in rats suggested that both 4-[18F]FMR and 6-[18F]FMR display similar affinity towards the presynaptic adrenergic nerve terminal in the heart. From a practical point of view, 4-[18F]FMR appeared to be the more attractive candidate for future PET investigations, due to higher radiochemical yields.

High specific radioactivity (1R,2S)-4-[(18)F]fluorometaraminol: a PET radiotracer for mapping sympathetic nerves of the heart.

The radiolabeled catecholamine analogue (1R, 2S)-6-[(18)F]fluorometaraminol (6-[(18)F]FMR) is a substrate for the neuronal norepinephrine transporter. It has been used as a positron emission tomography (PET) ligand to map sympathetic nerves in dog heart. 6-[(18)F]FMR could be only synthesized with low specific radioactivity, which precluded its use in human subjects. We have recently prepared (1R,2S)-4-[(18)F]fluorometaraminol (4-[(18)F]FMR), a new fluoro-analogue of metaraminol, with high specific radioactivity (56-106 GBq/micromol). In the present study, we demonstrate in rats that 4-[(18)F]FMR possesses similar affinity toward myocardial norepinephrine transport mechanisms as 6-[(18)F]FMR. When compared with control animals, an 80% and 76% reduction in myocardial uptake was observed in animals pretreated with desipramine (an inhibitor of the neuronal norepinephrine transporter) and with reserpine (a blocker of the vesicular storage of monoamines), respectively. The entire radioactivity in rat myocardium represented unmetabolized parent tracer as determined by high performance liquid chromatography analysis of tissue extracts. In dogs, myocardial kinetics of 4-[(18)F]FMR were assessed using PET. A rapid and high uptake was observed, followed by prolonged cardiac retention. A heart-to-lung ratio of 15 was reached 10 min after injection of the radiotracer. Pretreatment with desipramine reduced the heart half-life of 4-[(18)F]FMR by 90% compared with control. Moreover, an infusion of tyramine caused a rapid decline of radioactivity in the heart. This demonstrates that 4-[(18)F]FMR specifically visualizes sympathetic neurons in dog heart. High specific radioactivity 4-[(18)F]FMR is a promising alternative to 6-[(18)F]FMR for myocardial neuronal mapping with PET in humans.

Characterization of bromine-76-labelled 5-bromo-6-nitroquipazine for PET studies of the serotonin transporter.

The development of suitable radioligands for brain imaging of the serotonin transporter is of great importance for the study of depression and other affective disorders. The potent and selective serotonin transporter ligand, 5-iodo-6-nitro-2-piperazinylquinoline, has been labelled with iodine-123 and used as a radioligand for single photon emission computerized tomography. To evaluate the potential of the bromine-76-labelled analogue, 5-bromo-6-nitroquipazine, as a radioligand for positron emission tomography (PET), its brain distribution and binding characteristics were examined in rats. In vivo brain distribution and ex vivo autoradiography demonstrated that [76Br]5-bromo-6-nitroquipazine enters the brain rapidly. The regional brain distribution of [76Br]5-bromo-6-nitroquipazine was consistent with the known distribution of serotonin transporters in the midbrain, pons, thalamus, striatum, and neocortex. Specific binding was inhibited by the selective serotonin reuptake inhibitor citalopram. The peripheral metabolism in plasma was rapid, but more than 90% of the radioactivity in brain represented unchanged radioligand 2 h postinjection (p.i.). A preliminary PET study was also performed in a baboon. Following the intravenous injection of [76Br]5-bromo-6-nitroquipazine in a baboon, there was a conspicuous accumulation of radioactivity in thalamus, striatum, and pons. The radioactivity in these brain regions was 1.5 times higher than in the cerebellum at 3 h and 2.5-4 times higher at 24 h. A rapid metabolism of the radioligand in plasma was observed (38% unchanged after 5 min). The results indicate that [76Br]5-bromo-6-nitroquipazine has potential for PET imaging of the serotonin transporter.

Imaging central nicotinic acetylcholine receptors in baboons with [18F]fluoro-A-85380.

UNLABELLED: Central nicotinic acetylcholine receptors (nAChRs) have been implicated in learning-memory processes. Postmortem brain tissue of patients who suffered senile dementia or Parkinson's disease shows low density of nAChRs. In this study, we used PET to evaluate the distribution and kinetics of the fluoro derivative of the high-affinity and alpha4beta2-subtype-selective, nicotinic ligand 3-[2(S)-2-azetidinylmethoxy]pyridine (A-85380) in baboons. METHODS: After intravenous injection of 37 MBq (1 mCi, 1-1.5 nmol) [18F]fluoro-A-85380 into isoflurane-anesthetized baboons, dynamic PET data were acquired for 180 min. Time-activity curves were generated from regions of interest. Displacement experiments (80 min after injection of the radiotracer) were performed using cytisine (1 mg/kg subcutaneously) and unlabeled fluoro-A-85380 (0.1 and 0.3 mg/kg intravenously). Toxicological studies were performed in mice. RESULTS: Brain radioactivity reached a plateau within 40-50 min of injection of the tracer. In the thalamic area, radioactivity remained constant for 180 min, while clearance from the cerebellum was slow (t1/2 = 145-190 min). Cytisine and unlabeled fluoro-A-85380 reduced brain radioactivity at 180 min by 50%-60%, 30%-35% and 20%-35% of control values in the thalamus, cerebellum and frontal cortex, respectively. A slight, transient increase (20 mm Hg) in blood pressure was observed with the highest displacing dose of unlabeled fluoro-A-85380. Lethal dose in mice was found to be 2.2 mg/kg intravenously. CONCLUSION: These results demonstrate the feasibility and the safety of imaging nAChRs in vivo using labeled or unlabeled fluoro-A-85380.

Synthesis and nicotinic acetylcholine receptor in vivo binding properties of 2-fluoro-3-[2(S)-2-azetidinylmethoxy]pyridine: a new positron emission tomography ligand for nicotinic receptors.

The lead compound of a new series of 3-pyridyl ethers, the azetidine derivative A-85380 (3-[(S)-2-azetidinylmethoxy]pyridine), is a potent and selective ligand for the human alpha4beta2 nicotinic acetylcholine receptor (nAChR) subtype. In vitro, the fluoro derivative of A-85380 (2-fluoro-3-[(S)-2-azetidinylmethoxy]pyridine or F-A-85380) competitively displaced [3H]cytisine or [3H]epibatidine with Ki values of 48 and 46 pM, respectively. F-A-85380 has been labeled with the positron emitter fluorine-18 (t1/2 (half-life) = 110 min) by no-carrier-added nucleophilic aromatic substitution by K[18F]F-K222 complex with (3-[2(S)-N-(tert-butoxycarbonyl)-2-azetidinylmethoxy]pyridin-2-yl) tri methylammonium trifluoromethanesulfonate as a highly efficient labeling precursor, followed by TFA removal of the Boc protective group. The total synthesis time was 50-53 min from the end of cyclotron fluorine-18 production (EOB). Radiochemical yields, with respect to initial [18F]fluoride ion radioactivity, were 68-72% (decay-corrected) and 49-52% (non-decay-corrected), and the specific radioactivities at EOB were 4-7 Ci/micromol (148-259 GBq/micromol). In vivo characterization of [18F]F-A-85380 showed promising properties for PET imaging of central nAChRs. This compound does not bind in vivo to alpha7 nicotinic or 5HT3 receptors. Moreover, its cerebral uptake can be modulated by the synaptic concentration of the endogenous ligand acetylcholine. The preliminary PET experiments in baboons with [18F]F-A-85380 show an accumulation of the radiotracer in the brain within 60 min. In the thalamus, a nAChR-rich area, uptake of radioactivity reached a maximum at 60 min (4% I.D./100 mL of tissue). [18F]F-A-85380 appears to be a suitable radioligand for brain imaging nAChRs with PET.

Quantitation of extrastriatal D2 receptors using a very high-affinity ligand (FLB 457) and the multi-injection approach.

The multi-injection approach has been used to study in baboon the in vivo interactions between the D2 receptor sites and FLB 457, a ligand with a very high affinity for these receptors. The model structure was composed of four compartments (plasma, free ligand, and specifically and unspecifically bound ligands) and seven parameters (including the D2 receptor site density). The arterial plasma concentration, after correction for metabolites, was used as the input function. The experimental protocol, which consisted of three injections of labeled and/or unlabeled ligand, allowed the evaluation of all model parameters from a single positron emission tomography experiment. In particular, the concentration of receptor sites available for binding (B'max) and the apparent in vivo FLB 457 affinity were estimated in seven brain regions, including the cerebellum and several cortex regions, in which these parameters are estimated in vivo for the first time (B'max is estimated to be 4.0+/-1.3 pmol/mL in the thalamus and from 0.32 to 1.90 pmol/mL in the cortex). A low receptor density was found in the cerebellum (B'max = 0.39+/-0.17 pmol/mL), whereas the cerebellum is usually used as a reference region assumed to be devoid of D2 receptor sites. In spite of this very small concentration (1% of the striatal concentration), and because of the high affinity of the ligand, we demonstrated that after a tracer injection, most of the PET-measured radioactivity in the cerebellum results from the labeled ligand bound to receptor sites. The estimation of all the model parameters allowed simulations that led to a precise knowledge of the FLB 457 kinetics in all brain regions and gave the possibility of testing the equilibrium hypotheses and estimating the biases introduced by the usual simplified approaches.