Guillaume Becker

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, Alzheimers 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.

Radiosynthesis and Preclinical Evaluation of 18F-F13714 as a Fluorinated 5-HT1A Receptor Agonist Radioligand for PET Neuroimaging

PET brain imaging of the serotonin 1A (5-hydroxytryptamine 1A [5-HT1A]) receptor has been widely used in clinical studies. Currently, only a few well-validated radiolabeled antagonist tracers are available for in vivo imaging of this central receptor. 5-HT1A receptors exist in high- and low-affinity states, depending on their coupling to G proteins. Agonists bind preferentially to receptors in the high-affinity state and thereby could provide a measure of functional 5-HT1A receptors. Therefore, it is of great interest to develop an 18F-labeled full agonist 5-HT1A receptor radiotracer. In this study, we radiolabeled the high-affinity 5-HT1A receptor agonist 18F-F13714 and investigated its potential as a PET tracer. Methods: F13714 nitro precursor was synthesized and radiolabeled via a fluoronucleophilic substitution. In vitro binding assays were performed using established protocols. Radiopharmacologic evaluations included in vitro autoradiography in rat brain and PET scans on anesthetized cats. Results: The chemical and radiochemical purities of 18F-F13714 were greater than 98%. F13714 has a high affinity (0.1 nM) and selectivity for 5-HT1A receptors. In vitro 18F-F13714 binding in rats was consistent with the known 5-HT1A receptors distribution (hippocampus and cortical areas) and was particularly high in the dorsal raphe. In vitro binding of 18F-F13714 was blocked in a dose-dependent fashion by WAY100635, the prototypical 5-HT1A antagonist, and by the endogenous agonist, serotonin (5-HT). Addition of Gpp(NH)p also inhibited in vitro 18F-F13714 binding, consistent with a preferential binding of the compound to G-protein–coupled receptors. Ex vivo tissue measurements in rat revealed an absence of brain radioactive metabolites. In vivo studies showed that the radiotracer entered the cat brain readily and displayed a preferential labeling of 5-HT1A receptors located in cingulate cortex. In vivo labeling was prevented by preinjection of WAY100635. Conclusion: 18F-F13714 is a radiofluorinated agonist that presents suitable characteristics for probing the high-affinity states of the 5-HT1A receptors in vitro and in vivo. Thus, it is a promising tool for investigation of 5-HT1A agonist binding in the living human brain.

Pharmacokinetic Characterization of [18F]UCB-H PET Radiopharmaceutical in the Rat Brain

The synaptic vesicle glycoprotein 2A (SV2A), a protein essential to the proper nervous system function, is found in presynaptic vesicles. Thus, SV2A targeting, using dedicated radiotracers combined with positron emission tomography (PET), allows the assessment of synaptic density in the living brain. The first-in-class fluorinated SV2A specific radioligand, [18F]UCB-H, is now available at high activity through an efficient radiosynthesis compliant with current good manufacturing practices (cGMP). We report here a noninvasive method to quantify [18F]UCB-H binding in rat brain with microPET. Validation study in rats confirmed the need of high enantiomeric purity to target SV2A in vivo. We demonstrated the reliability of a population-based input function to quantify SV2A in preclinical microPET setting. Finally, we investigated the in vivo metabolism of [18F]UCB-H and confirmed the negligible amount of radiometabolites in the rat brain. Hence, the in vivo quantification of SV2A using [18F]UCB-H microPET seems a promising tool for the assessment of the synaptic density in the rat brain, and opens the way for longitudinal follow-up in neurodegenerative disease rodent models.

Measuring brain synaptic vesicle protein 2A with positron emission tomography and [18F]UCB-H.

Brain distribution of synaptic vesicle protein 2A was measured with fluorine‐18 UCB‐H ([18F]UCB‐H) and positron emission tomography (PET).

Nanofitin as a New Molecular-Imaging Agent for the Diagnosis of Epidermal Growth Factor Receptor Over-Expressing Tumors

Epidermal growth-factor receptor (EGFR) is involved in cell growth and proliferation and is over-expressed in malignant tissues. Although anti-EGFR-based immunotherapy became a standard of care for patients with EGFR-positive tumors, this strategy of addressing cancer tumors by targeting EGFR with monoclonal antibodies is less-developed for patient diagnostic and monitoring. Indeed, antibodies exhibit a slow blood clearance, which is detrimental for positron emission tomography (PET) imaging. New molecular probes are proposed to overcome such limitations for patient monitoring, making use of low-molecular-weight protein scaffolds as alternatives to antibodies, such as Nanofitins with better pharmacokinetic profiles. Anti-EGFR Nanofitin B10 was reformatted by genetic engineering to exhibit a unique cysteine moiety at its C-terminus, which allows the development of a fast and site-specific radiolabeling procedure with 18F-4-fluorobenzamido-N-ethylamino-maleimide (18F-FBEM). The in vivo tumor targeting and imaging profile of the anti-EGFR Cys-B10 Nanofitin was investigated in a double-tumor xenograft model by static small-animal PET at 2 h after tail-vein injection of the radiolabeled Nanofitin 18F-FBEM-Cys-B10. The image showed that the EGFR-positive tumor (A431) is clearly delineated in comparison to the EGFR-negative tumor (H520) with a significant tumor-to-background contrast. 18F-FBEM-Cys-B10 demonstrated a significantly higher retention in A431 tumors than in H520 tumors at 2.5 h post-injection with a A431-to-H520 uptake ratio of 2.53 ± 0.18 and a tumor-to-blood ratio of 4.55 ± 0.63. This study provides the first report of Nanofitin scaffold used as a targeted PET radiotracer for in vivo imaging of EGFR-positive tumor, with the anti-EGFR B10 Nanofitin used as proof-of-concept. The fast generation of specific Nanofitins via a fully in vitro selection process, together with the excellent imaging features of the Nanofitin scaffold, could facilitate the development of valuable PET-based companion diagnostics.

Regiospecific radiolabelling of Nanofitin on Ni magnetic beads with [18F]FBEM and in vivo PET studies

INTRODUCTION Nanofitins are low molecular weight, single chain and cysteine-free protein scaffolds able to selectively bind a defined biological target. They derive from Sac7d bacterial protein family and are highly stable over a wide range of pH (0-13) and temperature (Tm ~80°C). Their extreme stability, low cost of production and high tolerability for chemical coupling make Nanofitins a very interesting alternative to antibodies and their fragments. Here, a hexahistidine tagged model Nanofitin (H4) directed against hen egg white lysozyme was radiolabelled and injected in mice to provide a baseline biodistribution and pharmacokinetic profiles to support future Nanofitin development programs. METHOD A single cysteine residue has been genetically inserted in a model Nanofitin and its regioselective radiolabelling has been performed with 4-[18F]fluorobenzamido-N-ethylamino-maleimide ([18F]FBEM). The synthesis of [18F]FBEM has been completely implemented on a radiosynthesis unit (FastLab) including HPLC purification and formulation. Coupling with the [18F]FBEM has been achieved on a solid support (Ni magnetic beads) allowing rapid purification at room temperature without organic solvent. PET-MRI studies on C57BL/6 mice were conducted after injection of [18F]FBEM-Cys-H4 in order to access the biodistribution of this Nanofitin model. RESULTS Radiochemical yield (decay corrected) of 54±7% (n=4) was obtained after optimization for coupling the [18F]FBEM to Nanofitin. Pharmacokinetics results of [18F]FBEM-Cys-H4 revealed a fast clearance through the liver and the kidneys. CONCLUSION An efficient new method on Ni magnetic beads was developed to radiolabelled his-tagged biomolecules with [18F]FBEM. This procedure was applied on a Nanofitin model Cys-H4 and biodistribution kinetic studies were achieved to evaluate the potential use of Nanofitin for diagnostic imaging. Fast clearance indicates that Nanofitins represent very interesting tools for diagnostic imaging.

The severe deficiency of the somatotrope GH-releasing hormone/growth hormone/insulin-like growth factor 1 axis of Ghrh-/- mice is associated with an important splenic atrophy and relative B lymphopenia

A debate is still open about the precise control exerted by the somatotrope GH-releasing hormone (GHRH)/growth hormone (GH)/insulin-like growth factor 1 axis on the immune system. The objective of this study was to directly address this question through the use of Ghrh−/− mice that exhibit a severe deficiency of their somatotrope axis. After control backcross studies and normalization for the reduced global weight of transgenic mice, no difference in weight and cellularity of the thymus was observed in Ghrh−/− mice when compared with C57BL/6 wild-type (WT) control mice. Similarly, no significant change was observed in frequency and number of thymic T cell subsets. In the periphery, Ghrh−/− mice exhibited an increase in T cell proportion associated with a higher frequency of sjTREC and naïve T cells. However, all Ghrh−/− mice displayed an absolute and relative splenic atrophy, in parallel with a decrease in B cell percentage. GH supplementation of transgenic mice for 6 weeks induced a significant increase in their global as well as absolute and relative splenic weight. Interestingly, the classical thymus involution following dexamethasone administration was shown to recover in WT mice more quickly than in mutant mice. Altogether, these data show that the severe somatotrope deficiency of Ghrh−/− mice essentially impacts the spleen and B compartment of the adaptive immune system, while it only marginally affects thymic function and T cell development.

Development and validation of a new mouse model to investigate the role of sv2a in epilepsy

SV2A is a glycoprotein present in the membranes of most synaptic vesicles. Although it has been highly conserved throughout evolution, its physiological role remains largely unknown. Nevertheless, Levetiracetam, a very effective anti-epileptic drug, has been recently demonstrated to bind to SV2A. At present, our understanding of the normal function of SV2A and its possible involvement in diseases like epilepsy is limited. With this study, we sought to develop a relevant model enabling analysis of SV2A’s role in the occurrence or progression of epilepsy. For this purpose, we generated a floxed SV2A mouse model with conditional alleles carrying LoxP sites around exon 3 by means of a gene-targeting strategy. The SV2A lox/lox mouse line is indistinguishable from wild-type mice. When the recombination was observed in all cells, a model of mice with both SV2A alleles floxed around exon 3 recapitulated the phenotype of SV2A KO mice, including seizures. However, the specific invalidation of SV2A in the CA3 hippocampal region was not followed by epileptic seizures or decrease in the epileptic threshold on pentylenetetrazol (PTZ) test. These results demonstrate that the floxed SV2A mouse line has been successfully established. This transgenic mouse model will be useful for investigating SV2A functions related to cell types and developmental stages.

Biodistribution of Novel 68Ga-Radiolabelled HER2 Aptamers in Mice

Background: Two novel HER2 aptamers were recently selected with great potential for the in vitro diagnosis of HER2-positive cancer. The goal of this study was to examine the in vivo diagnostic potential of these HER2 aptamers. Methods: Both HER2 aptamers were radiolabelled with 68Ga, injected in mice bearing a HER2-positive and HER2-negative tumour and evaluated by PET/MRI. Results: Ex vivo bio distribution analysis revealed high uptake in the blood, tissues and organs, except the brain. Interestingly, this high uptake was explained by the slow blood clearance due to non-specific aptamer binding to blood proteins. We observed accumulation of radioactivity in both tumours in time. Although higher uptake in the HER2-positive tumour compared to the HER2-negative tumour was observed, this was accompanied with more necrosis in the HER2-negative tumour, which was observed by 18FDG PET/CT. Conclusion: This work presents a first step towards the development of 68Ga-labelled aptamers for molecular cancer imaging.

Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-L-dopa to evaluate dopaminergic presynaptic integrity in a Parkinson’s disease rat model.

Because of the progressive loss of nigro‐striatal dopaminergic terminals in Parkinsons disease (PD), in vivo quantitative imaging of dopamine (DA) containing neurons in animal models of PD is of critical importance in the preclinical evaluation of highly awaited disease‐modifying therapies. Among existing methods, the high sensitivity of positron emission tomography (PET) is attractive to achieve that goal. The aim of this study was to perform a quantitative comparison of brain images obtained in 6‐hydroxydopamine (6‐OHDA) lesioned rats using two dopaminergic PET radiotracers, namely [18F]fluoro‐3,4‐dihydroxyphenyl‐L‐alanine ([18F]FDOPA) and 6‐[18F]fluoro‐L‐m‐tyrosine ([18F]FMT). Because the imaging signal is theoretically less contaminated by metabolites, we hypothesized that the latter would show stronger relationship with behavioural and post‐mortem measures of striatal dopaminergic deficiency. We used a within‐subject design to measure striatal [18F]FMT and [18F]FDOPA uptake in eight partially lesioned, eight fully lesioned and ten sham‐treated rats. Animals were pretreated with an L‐aromatic amino acid decarboxylase inhibitor. A catechol‐O‐methyl transferase inhibitor was also given before [18F]FDOPA PET. Quantitative estimates of striatal uptake were computed using conventional graphical Patlak method. Striatal dopaminergic deficiencies were measured with apomorphine‐induced rotations and post‐mortem striatal DA content. We observed a strong relationship between [18F]FMT and [18F]FDOPA estimates of decreased uptake in the denervated striatum using the tissue‐derived uptake rate constant Kc. However, only [18F]FMT Kc succeeded to discriminate between the partial and the full 6‐OHDA lesion and correlated well with the post‐mortem striatal DA content. This study indicates that the [18F]FMT could be more sensitive, with respect of [18F]FDOPA, to investigate DA terminals loss in 6‐OHDA rats, and open the way to in vivo L‐aromatic amino acid decarboxylase activity targeting in future investigations on progressive PD models.