[18F]F13640, a 5-HT1A Receptor Radiopharmaceutical Sensitive to Brain Serotonin Fluctuations.

INTRODUCTION: Serotonin is involved in a variety of physiological functions and brain disorders. In this context, efforts have been made to investigate the in vivo fluctuations of this neurotransmitter using positron emission tomography (PET) imaging paradigms. Since serotonin is a full agonist, it binds preferentially to G-protein coupled receptors. In contrast, antagonist PET ligands additionally interact with uncoupled receptors. This could explain the lack of sensitivity to serotonin fluctuations of current 5-HT1A radiopharmaceuticals which are mainly antagonists and suggests that agonist radiotracers would be more appropriate to measure changes in neurotransmitter release. The present study evaluated the sensitivity to endogenous serotonin release of a recently developed, selective 5-HT1A receptor PET radiopharmaceutical, the agonist [18F]F13640 (a.k.a. befiradol or NLX-112). MATERIALS AND METHODS: Four cats each underwent three PET scans with [18F]F13640, i.e., a control PET scan of 90 min, a PET scan preceded 30 min before by an intravenous injection 1 mg/kg of d-fenfluramine, a serotonin releaser (blocking challenge), and a PET scan comprising the intravenous injection of 1 mg/kg of d-fenfluramine 30 min after the radiotracer injection (displacement challenge). Data were analyzed with regions of interest and voxel-based approaches. A lp-ntPET model approach was implemented to determine the dynamic of serotonin release during the challenge study. RESULTS: D-fenfluramine pretreatment elicited a massive inhibition of [18F]F13640 labeling in regions known to express 5-HT1A receptors, e.g., raphe nuclei, hippocampus, thalamus, anterior cingulate cortex, caudate putamen, occipital, frontal and parietal cortices, and gray matter of cerebellum. Administration of d-fenfluramine during PET acquisition indicates changes in occupancy from 10% (thalamus) to 31% (gray matter of cerebellum) even though the dissociation rate of [18F]F13640 over the 90 min acquisition time was modest. The lp-ntPET simulation succeeded in differentiating the control and challenge conditions. CONCLUSION: The present findings demonstrate that labeling of 5-HT1A receptors with [18F]F13640 is sensitive to serotonin concentration fluctuations in vivo. Although the data underline the need to perform longer PET scan to ensure accurate measure of displacement, they support clinical development of [18F]F13640 as a tool to explore experimental paradigms involving physiological or pathological (neurological or neuropsychiatric pathologies) fluctuations of extracellular serotonin.

Preclinical validation of [18F]2FNQ1P as a specific PET radiotracer of 5-HT6 receptors in rat, pig, non-human primate and human brain tissue.

INTRODUCTION: The aim of this study was to perform in-vitro and in-vivo radiopharmacological characterizations of [18F]2FNQ1P, a new PET radiotracer of 5-HT6 receptors, in rat, pig, non-human primate and human tissues. The 5-HT6 receptor is one of the more recently identified serotonin receptors in central nervous system and, because of its role in memory and cognitive processes, is considered as a promising therapeutic target. METHODS: In-vitro autoradiography and saturation binding assays were performed in postmortem brain tissues from rat, pig, non-human primate and human caudate nucleus, completed by serum stability assessment in all species and cerebral radiometabolite and biodistribution studies in rat. RESULTS: In all species, autoradiography data revealed high binding levels of [18F]2FNQ1P in cerebral regions with high 5-HT6 receptor density. Binding was blocked by addition of SB258585 as a specific antagonist. Binding assays provided KD and Bmax values of respectively 1.34 nM and 0.03 pmol·mg-1 in rat, 0.60 nM and 0.04 pmol·mg-1 in pig, 1.38 nM and 0.07 pmol·mg-1 in non-human primate, and 1.39 nM and 0.15 pmol·mg-1 in human caudate nucleus. In rat brain, the proportion of unmetabolized [18F]2FNQ1P was >99% 5 min after iv injection and 89% at 40 min. The biodistribution studies found maximal radioactivity in lungs and kidneys (3.5 ± 1.2% ID/g and 2.0 ± 0.7% ID/g, respectively, 15 min post-injection). CONCLUSION: These radiopharmacological data confirm that [18F]2FNQ1P is a specific radiotracer for molecular imaging of 5-HT6 receptors and suggest that it could be used as a radiopharmaceutical in humans.

Serotonin 5-HT1A Receptor Biased Agonists Induce Different Cerebral Metabolic Responses: A [18F]-Fluorodesoxyglucose Positron Emission Tomography Study in Conscious and Anesthetized Rats.

Serotonin 5-HT1A receptors constitute an attractive therapeutic target for various psychiatric or neurodegenerative disorders. These receptors are expressed in multiple brain regions on different neuronal populations and can be coupled with distinct G-protein subtypes; such functional diversity complicates the use of 5-HT1A ligands in several pathologies where it would be desirable to stimulate the receptors in a precise region. Therefore, using "biased agonists" able to target specifically certain subpopulations of 5-HT1A receptors would enable achievement of better therapeutic benefit. Several 5-HT1A receptor biased agonists are currently in development, including NLX-101 (aka F15599) and NLX-112 (aka F13640, befiradol), with preclinical data suggesting that they preferentially target different populations of 5-HT1A receptors. However, most previous studies used invasive and regionally limited approaches. In this context, [18F]-fluorodesoxyglucose (FDG)-positron emission tomography (PET) imaging constitutes an interesting technique as it enables noninvasive mapping of the regional brain activity changes following a pharmacological challenge in conscious animals. We report here the evaluation of cerebral glucose metabolism following intraperitoneal injection of different doses of NLX-112 or NLX-101 in conscious or isoflurane-anesthetized rats. The biased agonists produced different metabolic "fingerprints" with distinct regional preferences, consistent with previous studies. At equal doses, the effect of NLX-101 was less marked than NLX-112 in the piriform cortex, in the striatum (in terms of inhibition), and in the pontine nuclei and the cerebellum (in terms of activation); furthermore, only NLX-112 increased the glucose metabolism in the parietal cortex, whereas only NLX-101 induced a clear activation in the colliculi and the frontal cortex, which may be related to its distinctive procognitive profile. Both agonist effects were almost completely unapparent in anesthetized animals, underlining the importance of studying serotonergic neurotransmission in the conscious state. In this regard, [18F]FDG-PET imaging seems very complementary with other functional imaging techniques such as pharmacological MRI.

In vivo biased agonism at 5-HT1A receptors: characterisation by simultaneous PET/MR imaging.

In neuropharmacology, the recent concept of 'biased agonism' denotes the capacity of certain agonists to target-specific intracellular pathways of a given receptor in specific brain areas. In the context of serotonin pharmacotherapy, 5-HT1A receptor-biased agonists could be of great interest in several neuropsychiatric disorders. The aim of this study was to determine whether biased agonists could be differentiated in terms of regional targeting by use of simultaneous functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) brain imaging. We compared two 5-HT1A-biased agonists, NLX-112 and NLX-101, injected at three different doses in anaesthetised cats (n = 4). PET imaging was acquired for 90 min after bolus administration followed by constant infusion of the 5-HT1A radiotracer, [18F]MPPF. Drug occupancy was evaluated after injection at 50 min and BOLD fMRI was simultaneously acquired to evaluate subsequent brain activation patterns. 5-HT1A receptor occupancy was found to be dose-dependent for both agonists, but differed in magnitude and spatial distribution at equal doses with distinct BOLD patterns. Functional connectivity, as measured by BOLD signal temporal correlations between regions, was also differently modified by NLX-112 or NLX-101. Voxel-based correlation analyses between PET and fMRI suggested that NLX-112 stimulates both 5-HT1A autoreceptors and post-synaptic receptors, whereas NLX-101 preferentially stimulates post-synaptic cortical receptors. In cingulate cortex, the agonists induced opposite BOLD signal changes in response to receptor occupancy. These data constitute the first simultaneous exploration of 5-HT1A occupancy and its consequences in terms of brain activation, and demonstrates differential signalling by two 5-HT1A-biased agonists. Combined PET/fMRI represents a powerful tool in neuropharmacology, and opens new ways to address the concept of biased agonism by translational approaches.

18F-F13640 preclinical evaluation in rodent, cat and primate as a 5-HT1A receptor agonist for PET neuroimaging.

Serotonin 1A receptors are known to play an important role in many psychiatric and neurodegenerative disorders. Currently, all available 5-HT1A receptor PET radiopharmaceuticals that are radiolabeled with fluorine-18 are antagonists. As agonists bind preferentially to the high-affinity state of receptors, it would be of great interest to develop agonist radioligands which could provide a measure of the functional 5-HT1A receptors in pathophysiological processes. The 5-HT1A receptor agonist candidates we recently proposed had promising in vitro properties but were not optimal in terms of PET imaging. F13640, a.k.a befiradol or NLX-112, is a 5-HT1A receptor agonist with a high affinity (Ki = 1 nM) and a high selectivity that would be suitable for a potential PET radiopharmaceutical. With propose here the first preclinical evaluation of 18F-F13640. 18F-F13640's nitro-precursor was synthesized and radiolabeled via a fluoro-nucleophilic substitution. Its radiopharmacological characterization included autoradiographic studies, metabolic studies, and in vivo PET scans in rat, cat and non-human primate. Some of the results were compared with the radiotracer 18F-MPPF, a 5-HT1A receptor antagonist. The radiochemical purity of 18F-F13640 was > 98%. In vitro binding pattern was consistent with the 5-HT1A receptor distribution. Metabolic studies revealed that the radiotracer rapidly entered the brain and led to few brain radiometabolites. Although 18F-F13640 in vivo binding was blocked by the 5-HT1A antagonist WAY-100635 and the 5-HT1A agonist 8-OH-DPAT, the distribution pattern was markedly different from antagonist radiotracers in the three species, suggesting it provides novel information on 5-HT1A receptors. Preliminary studies also suggest a high sensitivity of 18F-F13640 to endogenous serotonin release. 18F-F13640 has suitable characteristics for probing in vitro and in vivo the 5-HT1A receptors in high-affinity state. Quantification analyses with kinetic modeling are in progress to prepare the first-in-man study of 18F-F13640.

[11C]PF-3274167 as a PET radiotracer of oxytocin receptors: Radiosynthesis and evaluation in rat brain.

INTRODUCTION: Oxytocin plays a major role in the regulation of social interactions in mammals by interacting with the oxytocin receptor (OTR) expressed in the brain. Furthermore, the oxytocin system appears as a possible therapeutic target in autism spectrum disorders and other psychiatric troubles, justifying current pharmacological researches. Since no specific PET radioligand is currently available to image OTR in the brain, the aim of this study was to radiolabel the specific OTR antagonist PF-3274167 and to evaluate [11C]PF-3274167 as a potential PET tracer for OTR in rat brains. METHODS: [11C]PF-3274167 was prepared via the O-methylation of its desmethyl precursor with [11C]methyl iodide. The lipophilicity of the radioactive compound was evaluated by measuring the n-octanol-buffer partition coefficient (logD). Autoradiography experiments were performed on rat brain tissue to evaluate the in vitro distribution of the [11C]PF-3274167. MicroPET experiments were conducted with and without pre-injection of ciclosporin in order to evaluate the influence of the P-glycoprotein (P-gp) on the brain uptake. RESULTS: [11C]PF-3274167 was synthesized with high radiochemical and chemical purities (>95%) and good specific activity. The measured logD was 1.93. In vitro, [11C]PF-3274167 did not show any evidence of specific binding to OTR. PET imaging showed that [11C]PF-3274167 uptake in rat brain was very low in basal conditions but increased significantly after the administration of ciclosporin, suggesting that it is a substrate of the P-gp. In the ciclosporin-pre-injected rat, however, [11C]PF-3274167 distribution did not match with the known distribution of OTR in rats. CONCLUSION: [11C]PF-3274167 is not a suitable tracer for imaging of OTR in rat brain, probably because of a too low affinity for this receptor in addition to a poor brain penetration.

Characterization and Reliability of [18F]2FNQ1P in Cynomolgus Monkeys as a PET Radiotracer for Serotonin 5-HT6 Receptors.

Brain serotonin-6 receptor (5-HT6R) is the one of the most recently identified serotonin receptors. Accumulating evidence suggests that it is a potent therapeutic target for psychiatric and neurological diseases. Since [18F]2FNQ1P was recently proposed as the first fluorinated positron emission tomography (PET) radioligand for this receptor, the objective of the present study was to demonstrate its suitability for 5-HT6R neuroimaging in primates. [18F]2FNQ1P was characterized by in vitro autoradiography and in vivo PET imaging in cynomolgus monkeys. Following in vivo PET imaging, tracer binding indices were computed using the simplified reference tissue model and Logan graphical model, with cerebellum as reference region. The tracer binding reproducibility was assessed by test-retest in five animals. Finally, specificity was assessed by pre-injection of a 5-HT6R antagonist, SB258585. In vitro, results showed wide cerebral distribution of the tracer with specificity toward 5-HT6Rs as binding was effectively displaced by SB258585. In vivo brain penetration was good with reproducible distribution at cortical and subcortical levels. The automated method gave the best spatial normalization. The Logan graphical model showed the best tracer binding indices, giving the highest magnitude, lowest standard deviation and best reproducibility and robustness. Finally, 5-HT6R antagonist pre-injection significantly decreased [18F]2FNQ1P binding mainly in the striatum and sensorimotor cortex. Taken together, these preclinical results show that [18F]2FNQ1P is a good candidate to address 5-HT6 receptors in clinical studies.

Oxytocin and Serotonin Brain Mechanisms in the Nonhuman Primate.

Oxytocin (OT) is increasingly studied for its therapeutic potential in psychiatric disorders, which are associated with the deregulation of several neurotransmission systems. Studies in rodents demonstrated that the interaction between OT and serotonin (5-HT) is critical for several aspects of social behavior. Using PET scan in humans, we have recently found that 5-HT 1A receptor (5-HT1AR) function is modified after intranasal oxytocin intake. However, the underlying mechanism between OT and 5-HT remains unclear. To understand this interaction, we tested 3 male macaque monkeys using both [11C]DASB and [18F]MPPF, two PET radiotracers, marking the serotonin transporter and the 5-HT1AR, respectively. Oxytocin (1 IU in 20 µl of ACSF) or placebo was injected into the brain lateral ventricle 45 min before scans. Additionally, we performed postmortem autoradiography. Compared with placebo, OT significantly reduced [11C]DASB binding potential in right amygdala, insula, and hippocampus, whereas [18F]MPPF binding potential increased in right amygdala and insula. Autoradiography revealed that [11C]DASB was sensitive to physiological levels of 5-HT modification, and that OT does not act directly on the 5-HT1AR. Our results show that oxytocin administration in nonhuman primates influences serotoninergic neurotransmission via at least two ways: (1) by provoking a release of serotonin in key limbic regions; and (2) by increasing the availability of 5-HT1AR receptors in the same limbic areas. Because these two molecules are important for social behavior, our study sheds light on the specific nature of their interaction, therefore helping to develop new mechanisms-based therapies for psychiatric disorders.SIGNIFICANCE STATEMENT Social behavior is largely controlled by brain neuromodulators, such as oxytocin and serotonin. While these are currently targeted in the context of psychiatric disorders such as autism and schizophrenia, a new promising pharmaceutical strategy is to study the interaction between these systems. Here we depict the interplay between oxytocin and serotonin in the nonhuman primate brain. We found that oxytocin provokes the release of serotonin, which in turn impacts on the serotonin 1A receptor system, by modulating its availability. This happens in several key brain regions for social behavior, such as the amygdala and insula. This novel finding can open ways to advance treatments where drugs are combined to influence several neurotransmission networks.

Agonist and antagonist bind differently to 5-HT1A receptors during Alzheimer's disease: A post-mortem study with PET radiopharmaceuticals.

PET imaging studies using 5-HT1A receptor radiotracers show a decreased density of this receptor in hippocampi of patients with Alzheimer's disease (AD) at advanced stages. However, current 5-HT1A receptor radiopharmaceuticals used in neuroimaging are antagonists, thought to bind to 5-HT1A receptors in different functional states (i.e., both the one which displays high affinity for agonists and is thought to mediate receptor activation, as well as the state which has low affinity for agonists). Comparing the PET imaging obtained using an agonist radiotracer, which binds selectively to functional receptors, with the PET imaging obtained using an antagonist radiotracer would therefore provide original information on 5-HT1A receptor impairment during AD. Quantitative autoradiography using [(18)F]F13640 and [(18)F]MPPF, a 5-HT1A agonist and antagonist, respectively, was measured in hippocampi of patients with AD (n = 25, at different Braak stages) and control subjects (n = 9). The neuronal density was measured in the same tissues by NeuN immunohistochemistry. The specific binding of both radiotracers was determined by addition of WAY-100635, a selective 5-HT1A receptor antagonist. The autoradiography distribution of both 5-HT1A PET radiotracers varied across hippocampus regions. The highest binding density was in the pyramidal layer of CA1. Incubation with Gpp(NH)p, a non-hydrolysable analogue of GTP, reduced significantly [(18)F]F13640 binding in hippocampal regions, confirming its preferential interaction with G-coupled receptors, and slightly increased [(18)F]MPPF binding. In the CA1 subfield, [(18)F]F13640 binding was significantly decreased at Braak stages I/II (-19%), Braak stages III/IV (-23%), and Braak stages V/VI (-36%) versus control. In contrast, [(18)F]MPPF binding was statistically reduced only at the most advanced Braak stages V/VI compared to control (-33%). Since [(18)F]F13640 and [(18)F]MPPF can be used in vivo in humans, this neuropharmacological paradigm supports testing the concept of functional imaging using agonist radiopharmaceuticals in future clinical studies.

PIXSIC: A Wireless Intracerebral Radiosensitive Probe in Freely Moving Rats.

The aim of this study was to demonstrate the potential of a wireless pixelated ß+-sensitive intracerebral probe (PIXSIC) for in vivo positron emission tomographic (PET) radiopharmacology in awake and freely moving rodents. The binding of [(11)C]raclopride to D2 dopamine receptors was measured in anesthetized and awake rats following injection of the radiotracer. Competitive binding was assessed with a cold raclopride injection 20 minutes later. The device can accurately monitor binding of PET ligands in freely moving rodents with a high spatiotemporal resolution. Reproducible time-activity curves were obtained for pixels throughout the striatum and cerebellum. A significantly lower [(11)C]raclopride tracer-specific binding was observed in awake animals. These first results pave the way for PET tracer pharmacokinetics measurements in freely moving rodents.

Syntheses, radiolabelings, and in vitro evaluations of fluorinated PET radioligands of 5-HT6 serotoninergic receptors.

The 5-HT6 receptors are potent therapeutic targets for psychiatric and neurological diseases (schizophrenia, Alzheimer's disease, etc.). However, with lack of specific radiopharmaceuticals, their pharmacology is still incomplete and their exploration is limited to animal models. In this context, we have designed a fluorinated PET radiotracer, [(18)F]2FNQ1P, that possesses a high affinity and selectivity for 5-HT6. In vitro PET autoradiographies in rat brain sections with this radiotracer were in accordance with the 5-HT6 distribution pattern.