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See detailDesign and Synthesis of [18F]BPAM121, a PET-Probe Targeting AMPA-subtype Glutamatergic Receptors.
Manos-Turvey, Alexandra ULg; Lemaire, Christian ULg; Becker, Guillaume ULg et al

Poster (2017, August)

AMPA receptors (AMPARs), one of three sub-groups of ionotropic glutamate receptors present in the central nervous system, are recognised for their involvement in long-term potentiation (LTP), and learning ... [more ▼]

AMPA receptors (AMPARs), one of three sub-groups of ionotropic glutamate receptors present in the central nervous system, are recognised for their involvement in long-term potentiation (LTP), and learning and memory processes. [1] They represent a valid cognitive enhancer target, particularly in the fight against Alzheimer’s disease (AD). [2,3] Benzothiadiazine 1,1-dioxides, such as BPAM121, have emerged as important allosteric modulators of AMPARs, working solely in the presence of the endogenous transmitter. [4] Synthesis of BPAM121 labelled with fluorine-18 was proposed, to investigate the utility of this molecule as a PET probe in vivo, and evaluate its potential as an AD diagnostic tool (Figure 1). [Figure 1. a) Structure of BPAM121, b) Established radiochemical synthesis of [18F]BPAM121.] This work documents the successful optimization of synthesis, purification and formulation of [18F]BPAM121 using an automated FASTlab (GE Healthcare) synthesizer. In particular, the influence of higher-level [18F]fluoride ion starting concentrations on final product formulation requirements is discussed. Initial results revealed [18F]BPAM121 successfully passes the blood brain barrier, and further biological studies are currently underway. References [1] S. F. Traynelis et al. Pharmacol. Rev. 2010, 62, 405-496. 
 [2] J. Keifer, Z. Zheng, Eur. J. Neurosci. 2010, 32, 269-277. 
 [3] L. Gao et al. J. Neurochem. 2016, 136, 620-636. 
 [4] P. Francotte et al. J. Med. Chem. 2010, 53, 1700-1711. 
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See detailRegiospecific radiolabelling of Nanofitin on Ni Magnetic Beads with [18F]FBEM and in vivo PET studies
Dammicco, Sylvestre ULg; Goux, Marine; Lemaire, Christian ULg et al

in Nuclear Medicine & Biology (2017)

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 ... [more ▼]

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. [less ▲]

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See detailPharmacokinetic characterization of [18F]UCB-H PET radiopharmaceutical in the rat brain.
Becker, Guillaume ULg; Warnier, Corentin; Serrano Navacerrada, Maria Elisa ULg et al

in Molecular Pharmaceutics (2017), 14(8), 2719-2725

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 ... [more ▼]

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 the current good manufacturing practices (cGMP). We report here a non-invasive 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 diseases rodents’ models. [less ▲]

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See detail[18F]BPAM121: An AMPAR Modulator with Potential as a PET Probe
Manos-Turvey, Alexandra ULg; Lemaire, Christian ULg; Deverdenne, François et al

Poster (2017, June)

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See detailComparative 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.
Becker, Guillaume ULg; Bahri, Mohamed Ali ULg; Michel, Anne et al

in Journal of Neurochemistry (2017), 141

Because of the progressive loss of nigro-striatal dopaminergic terminals in Parkinson’s disease (PD), in vivo quantitative imaging of dopamine (DA) containing neurons in animal models of PD is of critical ... [more ▼]

Because of the progressive loss of nigro-striatal dopaminergic terminals in Parkinson’s disease (PD), in vivo quantitative imaging of dopamine (DA) containing neurons in animal models of PD is of critical importance in the pre-clinical 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 (AADC) 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 AADC activity targeting in future investigations on progressive PD models. [less ▲]

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See detailEvaluating the specificity of [18F] UCB-H for the isoform SV2A, compared with isoforms SV2B and SV2C
Serrano Navacerrada, Maria Elisa ULg; Becker, Guillaume ULg; Bahri, Mohamed Ali ULg et al

Poster (2017, February 01)

Background: SV2A is the most studied isoform of the Synaptic Vesicle 2 proteins, which are involved in the synaptic vesicle trafficking, being important in normal and pathological process, like the ... [more ▼]

Background: SV2A is the most studied isoform of the Synaptic Vesicle 2 proteins, which are involved in the synaptic vesicle trafficking, being important in normal and pathological process, like the epilepsy (1, 2). [18F]UCB-H was developed like a tool to study the role of this isoform with neuroimaging techniques (3, 4). The objective of this study was to evaluate its specificity to this isoform comparing with the others, through a competition assay in rats with ex-vivo autoradiography and mPET imaging. Methods: Forty male Sprague-Dawley were used in ex-vivo autoradiography experiments (N=20) and in microPET imaging (N=20). Animals were pre-treated 30 minutes before the injection of [18F]UCB-H with a dose IP either of vehicle, Keppra (SV2A ligand), UCB068 (SV2B ligand) or UCB054 (SV2C ligand). Ex-vivo autoradiography was carried out 5 minutes after radiotracer injection while mPET images were acquiring with a dynamic scanner of 1 hour. Data were expressed in Standard Uptake Value and then, the area under the curve was calculated for the total process. Results: In ex-vivo autoradiography, ANOVA of two-ways showed statistical significant differences in brain uptake of [18F]UCB-H among the groups pretreated with Keppra or the ligand for SV2B and the control group. Regarding mPET data, statistical significant differences were found between the group injected with keppra and the rest of groups. Conclusion: Even if a considerable affinity between the ligands UCB068 and UCB054, and the receptor for the isoform SV2A exists, it is only detected during the first 5 minutes (ex-vivo technique), being certainly due to a nonspecific binding. This binding is not strong enough to show a direct competition with the radiotracer during a mPET acquisition. These results allow us to conclude that [18F]UCB-H is a suitable radiotracer for the imaging of the isoform SV2A in vivo, allowing us the clinical study about the molecular base of a disease with a high population impact, like the epilepsy. [less ▲]

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See detailIN VIVO STUDY OF THE SV2A PROTEIN IN THE KAINIC ACID EPILEPSY RAT MODEL
Serrano Navacerrada, Maria Elisa ULg; Becker, Guillaume ULg; Bahri, Mohamed Ali ULg et al

Poster (2017)

Introduction Epilepsy is one of the commonest neurological disorders [1]. Antiepileptic drugs mainly target the SV2A protein [2] but its actual role is still largely unknown. [18F]UCB-H was developed to ... [more ▼]

Introduction Epilepsy is one of the commonest neurological disorders [1]. Antiepileptic drugs mainly target the SV2A protein [2] but its actual role is still largely unknown. [18F]UCB-H was developed to study in vivo SV2A brain proteins [3, 4]. The present pilot study was undertaken to evaluate for the first time in vivo in rats SV2A expression in the Kaïnic Acid (KA) epilepsy model [5]. Although this model is well studied in mice, few reports were devoted to rats. Imaging-wise, rats are very interesting thanks to a bigger brain size (reduction of the partial volume effect). Methods Three male Sprague-Dawley were used, one injected with saline and two with multiple KA injections (3 x 5mg/kg) [6]. 75 days later, when spontaneous seizures started to appear, microPET (Focus 120 ) was performed under isoflurane anesthesia (2.5-3 % in air) for 1 hour with [18F]UCB-H (41 ± 5 MBq IV tail vein) followed by MRI (9.4T Agilent, anatomical T2). Coregistration was done with PMOD 3.6 software. Data were expressed as SUV and areas under the curve were calculated for the different regions. Results [18F]UCB-H microPET images showed an important reduction (20-30%) for SV2A after KA injections mainly localized in amygdala, hippocampus, lateral parietal association cortex and cingulate cortex. The rest of the brain was globally unchanged. MRI revealed atrophy and inflammation in amygdala and hippocampus. Conclusions These preliminary results obtained in KA treated rats showed that [18F]UCB-H was able to detect important modifications for SV2A in relevant regions for epilepsy and appears as a valuable tool to follow in vivo SV2A through longitudinal studies. KA model in rats deserves for further development and validation as a tool for the study of epilepsy. [less ▲]

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See detailIN VIVO STUDY OF THE SV2A PROTEIN IN AN EPILEPTIC RAT MODEL
Serrano Navacerrada, Maria Elisa ULg; Becker, Guillaume ULg; Bahri, Mohamed Ali ULg et al

Poster (2017)

Introduction Epilepsy is one of the commonest neurological disorders, affecting more than 60 million people worldwide [1]. New and effective antiepileptic drugs mainly target the SV2A protein [2] but its ... [more ▼]

Introduction Epilepsy is one of the commonest neurological disorders, affecting more than 60 million people worldwide [1]. New and effective antiepileptic drugs mainly target the SV2A protein [2] but its actual role is still largely unknown. [18F]UCB-H was developed as a tool to study in vivo the brain expression of this isoform [3, 4]. Due to the fact that only post-mortem studies were reported so far [5] the present pilot study was undertaken in order to evaluate for the first time in vivo in rats the SV2A expression in the validated Kaïnic Acid (KA) epilepsy model [6]. Methods Three male Sprague-Dawley were used, one injected with saline (Sham) and two with multiple KA systemic injections (5mg/kg x 3) [9]. SV2A brain levels were estimated at day 75, when spontaneous seizures started to appear. Animals were anesthetized (2.5 to 3 % isoflurane), and scanned for 1 hour with [18F]UCB-H (41 ± 5 MBq IV tail vein) in a Focus 120 microPET system and with MRI (9.4T Agilent, anatomical T2). Coregistration was done with PMOD 3.6 software. Data were expressed in SUV and areas under the curve were calculated for the different regions. Results [18F]UCB-H microPET images showed an important reduction (20-30%) for SV2A after KA injections mainly localized in amygdala, hippocampus, lateral parietal association cortex and cingulate cortex. The rest of the brain was globally unchanged. MRI revealed atrophy and inflammation in amygdala and hippocampus. Conclusions These preliminary results in KA treated rats presenting spontaneous seizures showed that [18F]UCB-H microPET was able to detect important reductions for the SV2A proteins in relevant regions for epilepsy [5]. Accordingly to this, we can infer that the KA model in rats deserves for further development and validation as a tool for the study of epilepsy. [18F]UCB-H appears as a valuable tool to follow in vivo SV2A proteins through longitudinal protocols and in turn to better understand its actual role in epilepsy. References/acknowledgements This work was funded by University of Liège, F.R.S.-FNRS, Walloon Region and UCB Pharma. Alain Plenevaux is research director from F.R.S.-FNRS. [1] Alexopoulos, Epileptology, 2004 [2] Hamann et al., Eur J Pharmacol, 2008 [3] Bretin et al., Molecular Imaging and Biology, 2015 [4] Warnock et al., J Nucl Med., 2014 [5] Wang et al., J Mol Neurosci., 2014 [6] Hellier et al., Epilepsy Res., 1998 [less ▲]

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See detail[18F]UCB-H RADIOTRACER AS A TOOL TO UNDERSTAND THE ROLE OF THE SV2A PROTEIN
Serrano Navacerrada, Maria Elisa ULg; Becker, Guillaume ULg; Bahri, Mohamed Ali ULg et al

Poster (2017)

Background: SV2A is the most studied isoform of the Synaptic Vesicle 2 proteins, which are involved in the synaptic vesicle trafficking, being important both in normal as in pathological process (1, 2 ... [more ▼]

Background: SV2A is the most studied isoform of the Synaptic Vesicle 2 proteins, which are involved in the synaptic vesicle trafficking, being important both in normal as in pathological process (1, 2). Until now, only one study in vivo has been reported, showing a reduction of SV2A levels in the epilepsy (3). [18F]UCB-H was developed like a current tool to study the role of SV2A with in vivo techniques (4, 5), and as a tool in clinical investigations. The objective of this research was to evaluate the radiotracer specificity to this isoform comparing with the others, through a competition assay in rats with ex-vivo autoradiography and mPET imaging. Methods: Forty male Sprague-Dawley were used in ex-vivo autoradiography experiments (N=20) and in microPET imaging (N=20). Animals were pre-treated 30 minutes before the injection of [18F]UCB-H with a dose IP either of vehicle, Keppra (SV2A ligand), UCB068 (SV2B ligand) or UCB054 (SV2C ligand). Ex-vivo autoradiography was carried out 5 minutes after radiotracer injection while mPET images were acquiring with a dynamic scanner of 1 hour. Standard Uptake Value (SUV) and Distribution Volume (VT) were calculated and the correlation between both parameters was determined. Results: In ex-vivo autoradiography, ANOVA of two-ways showed statistical significant differences in brain uptake of [18F]UCB-H among the groups pretreated with Keppra or the ligand for SV2B and the control group. Regarding mPET data, statistical significant differences were found between the group injected with keppra and the rest of groups. Pearson Correlation between SUV and VT was strong, with a value of 0.955. Conclusion: Even if a considerable affinity between the ligands UCB068 and UCB054, and the receptor for the isoform SV2A exists, it is only detected during the first 5 minutes (ex-vivo technique), being certainly due to a nonspecific binding. This binding is not strong enough to show a direct competition with the radiotracer during a mPET acquisition. These results allow us to conclude that [18F]UCB-H is a suitable radiotracer for the imaging of the isoform SV2A in vivo, allowing us the clinical study about the molecular base of a disease with a high population impact, like the epilepsy. 1) Van Vliet et al., 2009. Epilepsia 2) Crèvecœur et al., 2013. BMC Neurosci. 3) Finnema et al., 2016; Sci Transl Med. 4) Bretin et al., 2013.EJNMMI Res 5) Bretin et al., 2015.Mol Imaging Biol [less ▲]

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See detailEVALUATING THE SPECIFICITY OF [18F]UCB-H FOR THE ISOFORM SV2A, COMPARED WITH ISOFORMS SV2B AND SV2C
Serrano Navacerrada, Maria Elisa ULg; Aerts, Joël ULg; Bahri, Mohamed Ali ULg et al

Poster (2016, November 18)

Background: SV2A is the most studied isoform of the Synaptic Vesicle 2 proteins, which are involved in the synaptic vesicle trafficking, being important in normal and pathological process, like the ... [more ▼]

Background: SV2A is the most studied isoform of the Synaptic Vesicle 2 proteins, which are involved in the synaptic vesicle trafficking, being important in normal and pathological process, like the epilepsy (1, 2). [18F]UCB-H was developed like a tool to study the role of this isoform with neuroimaging techniques (3, 4). The objective of this study was to evaluate its specificity to this isoform comparing with the others, through a competition assay in rats with ex-vivo autoradiography and mPET imaging. Methods: Forty male Sprague-Dawley were used in ex-vivo autoradiography experiments (N=20) and in microPET imaging (N=20). Animals were pre-treated 30 minutes before the injection of [18F]UCB-H with a dose IP either of vehicle, Keppra (SV2A ligand), UCB068 (SV2B ligand) or UCB054 (SV2C ligand). Ex-vivo autoradiography was carried out 5 minutes after radiotracer injection while mPET images were acquiring with a dynamic scanner of 1 hour. Data were expressed in Standard Uptake Value and then, the area under the curve was calculated for the total process. Results: In ex-vivo autoradiography, ANOVA of two-ways showed statistical significant differences in brain uptake of [18F]UCB-H among the groups pretreated with Keppra or the ligand for SV2B and the control group. Regarding mPET data, statistical significant differences were found between the group injected with keppra and the rest of groups. Conclusion: Even if a considerable affinity between the ligands UCB068 and UCB054, and the receptor for the isoform SV2A exists, it is only detected during the first 5 minutes (ex-vivo technique), being certainly due to a nonspecific binding. This binding is not strong enough to show a direct competition with the radiotracer during a mPET acquisition. These results allow us to conclude that [18F]UCB-H is a suitable radiotracer for the imaging of the isoform SV2A in vivo, allowing us the clinical study about the molecular base of a disease with a high population impact, like the epilepsy. [less ▲]

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See detailEnabling efficient PET imaging of Synaptic Vesicle glycoprotein 2A (SV2A) with a robust and one-step radiosynthesis of a highly potent 18F-labelled ligand ([18F]UCB-H)
Warnier, Corentin ULg; Lemaire, Christian ULg; Becker, Guillaume ULg et al

in Journal of Medicinal Chemistry (2016), 59

We herein describe the straightforward synthesis of a stable pyridyl(4- methoxyphenyl)iodonium salt and its [18F]radiolabelling within a one-step, fully automated and cGMP compliant radiosynthesis of [18F ... [more ▼]

We herein describe the straightforward synthesis of a stable pyridyl(4- methoxyphenyl)iodonium salt and its [18F]radiolabelling within a one-step, fully automated and cGMP compliant radiosynthesis of [18F]UCB-H ([18F]7), a PET tracer for the imaging of Synaptic Vesicle glycoprotein 2A (SV2A). Over the course of one year, 50 automated productions provided 34±2% of injectable [18F]7 from up to 285 GBq (7.7 Ci) of [18F]fluoride in 50 minutes (uncorrected radiochemical yield. Specific Activity = 815±185 GBq/μmol). The successful implementation of our synthetic strategy within routine, high-activity and cGMP productions attests to its practicality and reliability for the production of large doses of [18F]7. In addition to enabling efficient and cost-effective clinical research on a range of neurological pathologies through the imaging of SV2A, this work further demonstrates the real value of iodonium salts for the cGMP 18F-PET tracer manufacturing industry, and their ability to fulfill practical and regulatory requirements in that field. [less ▲]

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See detailFully automated radiosynthesis of N1-[18F]fluoroethyl-tryptophan and study of its biological activity as a new potential substrate for indoleamine 2,3-dioxygenase PET imaging
Henrottin, Jean ULg; Lemaire, Christian ULg; Egrise, Dominique et al

in Nuclear Medicine & Biology (2016), 43(6), 379-389

Introduction: Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial step in the catabolism of L-tryptophan along the kynurenine pathway and exerts immunosuppressive properties in inflammatory and tumor ... [more ▼]

Introduction: Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial step in the catabolism of L-tryptophan along the kynurenine pathway and exerts immunosuppressive properties in inflammatory and tumor tissues by blocking locally T-lymphocyte proliferation. Recently, 1-(2-[19F]fluoroethyl)-DL-tryptophan (1-[19F]FE-DL-Trp) was reported as a good and specific substrate of this enzyme. Herein, the radiosynthesis of its radioactive isotopomer (1-[18F]FE-DL-Trp, DL-[18F]5) is presented along with in vitro enzymatic and cellular uptake studies. Methods: The one-pot n.c.a. radiosynthesis of this novel potential PET imaging tracer, including HPLC purification and formulation, has been fully automated on a FASTlabTM synthesizer. Chiral separation of both isomers and their formulation were implemented on a second cassette. In vitro enzymatic and cellular uptake studies were then conducted with the D-, L- and DL-radiotracers. Results: The radiolabeling of the tosylate precursor was performed in DMF (in 5 min; RCY: 57% (d.c.), n=3). After hydrolysis, HPLC purification and formulation, DL-[18F]5 was obtained with a global radiochemical yield of 18±3% (not decay corrected, n=7, in 80 min) and a specific activity of 600±180 GBq/µmol (n=5). The subsequent separation of L- and D-enantiomers was performed by chiral HPLC and both were obtained after formulation with a RCY (d.c.) of 6.1% and 5.8%, respectively. In vitro enzymatic assays reveal that L-[18F]5 is a better substrate than D-[18F]5 for human IDO. In vitro cellular assays show an IDO-specific uptake of the racemate varying from 30% to 50% of that of L-[18F]5, and a negligible uptake of D-[18F]5. Conclusion: In vitro studies show that L-[18F]5 is a good and specific substrate of hIDO, while presenting a very low efflux. These results confirm that L-[18F]5 could be a very useful PET radiotracer for IDO expressing cells in cancer imaging. [less ▲]

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See detailDevelopment of solid-supported methodology for the preparation of peptidoglycan fragments containing (2S,6R)-diaminopimelic acid
Simon, Justine ULg; Lamborelle, Nicolas ULg; Zervosen, Astrid et al

in Tetrahedron Letters (2016)

Herein, we describe the development of an efficient solid-supported methodology for the stereoselective synthesis of two peptides containing (2S,6R)-diaminopimelic acid, (S)-Ala-γ-(R)-Glu-(2S,6R)-A2pm-(R ... [more ▼]

Herein, we describe the development of an efficient solid-supported methodology for the stereoselective synthesis of two peptides containing (2S,6R)-diaminopimelic acid, (S)-Ala-γ-(R)-Glu-(2S,6R)-A2pm-(R)-Ala 1 and γ-(R)-Glu-(2S,6R)-A2pm 2. The platform consists of a Wang resin anchored by an amino acid chain including allylglycine. By olefin cross metathesis with vinylglycine, unsaturated protected (2S,6R)-A2pm was fixed on solid support. Peptides were achieved by cleavage of cross metathesis products from resin, followed by reduction of double bonds along removing of protecting groups. Furthermore, this efficient solid phase approach will lead to peptide and muropeptide libraries. [less ▲]

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See detailEVALUATION OF SV2Alox/Cre TRANSGENIC MICE USING [18F]UCB-H IN VITRO AUTORADIOGRAPHY
Serrano Navacerrada, Maria Elisa ULg; Becker, Guillaume ULg; MENTEN, Catherine ULg et al

Poster (2016, March 09)

Introduction Epilepsy is one of the commonest neurological disorders [1]. Antiepileptic drugs mainly target the SV2A protein [2] but its actual role is still largely unknown. [18F]UCB-H was developed to ... [more ▼]

Introduction Epilepsy is one of the commonest neurological disorders [1]. Antiepileptic drugs mainly target the SV2A protein [2] but its actual role is still largely unknown. [18F]UCB-H was developed to study in vivo SV2A brain proteins [3, 4]. The present pilot study was undertaken to evaluate for the first time in vivo in rats SV2A expression in the Kaïnic Acid (KA) epilepsy model [5]. Although this model is well studied in mice, few reports were devoted to rats. Imaging-wise, rats are very interesting thanks to a bigger brain size (reduction of the partial volume effect). Methods Three male Sprague-Dawley were used, one injected with saline and two with multiple KA injections (3 x 5mg/kg) [6]. 75 days later, when spontaneous seizures started to appear, microPET (Focus 120 ) was performed under isoflurane anesthesia (2.5-3 % in air) for 1 hour with [18F]UCB-H (41 ± 5 MBq IV tail vein) followed by MRI (9.4T Agilent, anatomical T2). Coregistration was done with PMOD 3.6 software. Data were expressed as SUV and areas under the curve were calculated for the different regions. Results [18F]UCB-H microPET images showed an important reduction (20-30%) for SV2A after KA injections mainly localized in amygdala, hippocampus, lateral parietal association cortex and cingulate cortex. The rest of the brain was globally unchanged. MRI revealed atrophy and inflammation in amygdala and hippocampus. Conclusions These preliminary results obtained in KA treated rats showed that [18F]UCB-H was able to detect important modifications for SV2A in relevant regions for epilepsy and appears as a valuable tool to follow in vivo SV2A through longitudinal studies. KA model in rats deserves for further development and validation as a tool for the study of epilepsy. [less ▲]

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See detailEVALUATION OF SV2Alox/Cre TRANSGENIC MOUSE USING [18F]UCB-H IN IN VITRO AUTORADIOGRAPHY
Serrano Navacerrada, Maria Elisa ULg; Becker, Guillaume ULg; MENTEN, Catherine ULg et al

Poster (2015, September 04)

Background: SV2A is the most studied isoform of the Synaptic Vesicle 2 proteins, which are involved in the synaptic vesicle trafficking. Interestingly, the SV2A has been identify as the binding site for ... [more ▼]

Background: SV2A is the most studied isoform of the Synaptic Vesicle 2 proteins, which are involved in the synaptic vesicle trafficking. Interestingly, the SV2A has been identify as the binding site for the antiepileptic drug levetiracetam, showing a close relation between the epilepsy, the dysregulation of the SV2A levels and the response to antiepileptic medications. SV2A floxed-mice were developed using a cre-lox technique, leading to a strong decrease of SV2A expression in the CA3 field of the hippocampus. We aim here to validate this model using [18F]UCB-H, a novel PET imaging radiotracer with a nanomolar affinity for human SV2A. Methods: In vitro autoradiography were performed on SV2Alox/Cre+ transgenic mouse brain slices. SV2Alox/Cre- mouse was used as control. To obtain a structural reference, brain slices underwent eosin-haematoxylin staining. Images of both procedures were coregistered using π-PMOD software. Regions of interest (Dentate Gyrus, CA1, CA2 and CA3) were drawn according to a stereotaxic atlas of the mouse brain. Results: Analyses showed significant differences in radiotracer binding (p<0.001) between SV2Alox/Cre+ mouse and SV2Alox/Cre- mouse highlighting an important reduction for the labelling density in Ammon's horn, particularly in CA1, compared to Dentate Gyrus where the diminution was less marked. Conclusions: Here, we used the radiotracer [18F]UCB-H to probe the decreased expression of SV2A protein in the hippocampus of SV2Alox/Cre+ mouse versus SV2Alox/Cre- control mouse. Our results contribute to the validation of the model, and encourage us to proceed with further longitudinal and behavioural studies. [less ▲]

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See detailIn vivo quantification of dopaminergic terminals loss in Parkinson’s Disease rat model: comparison between [18F]FMT and [18F]FDOPA.
Becker, Guillaume ULg; Bahri, Mohamed Ali ULg; Michel, Anne et al

Poster (2015, September 02)

Objectives: Rat models of Parkinson’s disease (PD), such as unilaterally lesioned rats with 6-hydroxydopamine (6-OHDA), are useful to evaluate novel antiparkinsonian therapies. MicroPET imaging, using L-3 ... [more ▼]

Objectives: Rat models of Parkinson’s disease (PD), such as unilaterally lesioned rats with 6-hydroxydopamine (6-OHDA), are useful to evaluate novel antiparkinsonian therapies. MicroPET imaging, using L-3,4-dihydroxy-6-[18F]-fluoro-phenylalanine ([18F]FDOPA) allows longitudinal evaluations of DA terminals loss. However, chemical structure of [18F]FDOPA leads to suboptimal PET imaging. 18F-fluoro-m-tyrosine ([18F]FMT) is an effective PET tracer to evaluate DA terminals integrity and L-aromatic amino acid decarboxylase (AAAD) metabolic pathway. So far, there are no available quantitative PET studies comparing the two methods in hemiparkinsonian rats. In this study, we compare imaging data provided by [18F]FMT PET and [18F]FDOPA PET in 6-OHDA-lesioned rats. Methods: 10 µg of 6-OHDA were injected into the right medial forebrain bundle (MFB) of male Sprague-Dawley rats (n=8). As control, sham-treated rats (n=8) were injected with vehicle only but otherwise treated identically. Striatal DA presynaptic activity was assessed by dynamic PET with both [18F]FMT and [18F]FDOPA. Structural T2-weighted brain images were acquired on a 9.4T MRI and were used for co-registration. After normalization on a MRI template, kinetic analysis was performed by “Patlak Reference” model, using PMOD software. Six days after the last PET scan, rats were sacrificed, and striatum were rapidly removed for striatal DA and metabolites quantification. Results: Striatal accumulation was observed for both tracers. However, while the administration of [18F]FDOPA required two peripheral inhibitors (benserazide and entacapone), only benserazide is needed with [18F]FMT. As consequence of the 6-OHDA-lesion, significant decrease of both [18F]FMT and [18F]DOPA accumulation was recorded in the striatum ipsilateral to the lesion. Lesioned rats had dramatically reduced uptake constant Ki in the ipsilateral striatum compared to the contralateral striatum (p<0.001 for [18F]FMT and p<0.05 for [18F]DOPA) and to the ipsilateral striatum of sham-treated rats (p<0.001 for both tracers). The DA content in the ipsilateral striatum was significantly lower (p<0.001) than in the contralateral striatum in the 6-OHDA-injected group, whereas such difference was not measured with the sham group. This indicate that [18F]FMT PET is as effective as [18F]DOPA PET to quantify loss of DA presynaptic function in unilaterally 6-OHDA lesioned rats. Conclusions: Our results are in agreement with data reporting correlation between these two tracers in a Non-human primate model of PD. The sensitivity of the data quantification obtained in this study, confirms the interest to pursue longitudinal investigations with [18F]FMT to monitor dopaminergic dysfunction in a more progressive preclinical model of PD. [less ▲]

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See detailAutomated production at the curie level of no-carrier-added 6-[18F]fluoro-L-dopa and 2-[18F]fluoro-L-tyrosine on a FASTlab synthesizer
Lemaire, Christian ULg; Libert, Lionel; Franci, Xavier et al

in Journal of Labelled Compounds (2015), 58

An efficient, fully automated, enantioselective multi-step synthesis of no-carrier-added (nca) 6-[18F]fluoro-L-dopa ([18F]FDOPA) and 2-[18F]fluoro-L-tyrosine ([18F]FTYR) on a GE FASTlab synthesizer in ... [more ▼]

An efficient, fully automated, enantioselective multi-step synthesis of no-carrier-added (nca) 6-[18F]fluoro-L-dopa ([18F]FDOPA) and 2-[18F]fluoro-L-tyrosine ([18F]FTYR) on a GE FASTlab synthesizer in conjunction with an additional high-performance liquid chromatography (HPLC) purification has been developed. A PTC (phase-transfer catalyst) strategy wasused to synthesize these two important radiopharmaceuticals. According to recent chemistry improvements, automationof the whole process was implemented in a commercially available GE FASTlab module, with slight hardware modificationusing single use cassettes and stand-alone HPLC. [18F]FDOPA and [18F]FTYR were produced in 36.3 ± 3.0 % (n = 8) and50.5 ± 2.7 % (n = 10) FASTlab radiochemical yield (decay corrected). The automated radiosynthesis on the FASTlab modulerequires about 52 min. Total synthesis time including HPLC purification and formulation was about 62 min. Enantiomericexcesses for these two aromatic amino acids were always >95 %, and the specific activity of was >740 GBq/μmol. Thisautomated synthesis provides high amount of [18F]FDOPA and [18F]FTYR (>37 GBq end of synthesis (EOS)). The process, fullyadaptable for reliable production across multiple PET sites, could be readily implemented into a clinical good manufacturingprocess (GMP) environment. [less ▲]

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See detail[18F]UCB-H as a new PET radiotracer for Synaptic vesicle protein 2A: A first clinical trial.
Bahri, Mohamed Ali ULg; Stifkens, M; Bastin, Christine ULg et al

in Tijdschrift voor Nucleaire Geneeskunde (2015, May 09), 37(3), 1457-1458

The synaptic vesicle protein 2A (SV2A) is widely distributed in the brain and has been demonstrated to be involved in vesicle trafficking. The critical role of SV2A in proper nervous system function is ... [more ▼]

The synaptic vesicle protein 2A (SV2A) is widely distributed in the brain and has been demonstrated to be involved in vesicle trafficking. The critical role of SV2A in proper nervous system function is shown, e.g., by the fact that it is a binding site and the primary mechanism of the antiepileptic drug levetiracetam. This drug has recently been suggested to reduce synaptic deficits in a mouse model for Alzheimer’s disease. We here aimed to investigate the cerebral distribution of [18F]UCB-H, which has a high affinity with the SV2A. Dynamic PET data of the head of 4 healthy volunteers were acquired over 100 minutes after injection of 170.4 ± 24.9 MBq of GMP produced [18F]UCB-H. The arterial input function (IF) was obtained by blood sampling but also derived from the dynamic data using the correlation coefficient method. Blood data revealed a consistent amount of [18F]UCB-H in whole blood and plasma indicating a very low degree of binding of the tracer to the red blood cells. The unchanged fraction of [18F]UCB-H in plasma showed a bi-exponential behavioral decrease with a starting fraction of 92% of the injected amount of the tracer, measured at 3 min post injection. This fraction decreased to about 50% at 10 min post injection. The image-derived arterial IFs showed to be very similar to the measured ones with a peak-ratio around 0.91 and an area-under-curve ratio about 0.98. The PET images showed a high and rapid uptake of [18F]UCB-H in the grey matter structures, matching the known ubiquitous distribution of the SV2A in the brain. The kinetics of the tracer in the brain was characterized by an initial high uptake phase followed by rapid washout. For the three standard compartmental models (1-tissue, 2-tissue, and Logan Plot), similar results were obtained with both the measured and image-derived IFs. Nevertheless the two-tissue compartment model fitted the experimental data best and provided a total distribution volume of the [18F]UCB-H in the brain greater than 7 mL/cm3 and a specific distribution volume around 3 mL/cm3. Our results suggest that [18F]UCB-H is a good candidate as radiotracer for brain SV2A proteins and could be used for human studies (dosimetry has already been reported elsewhere). Image-derived IF showed to be useful for quantitative studies without the need to the arterial blood sampling. This new tracer could help to assess SV2A modifications in neurological pathologies such as Alzheimer’s disease. [less ▲]

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See detail[18F]FMT: a reliable PET tracer for in vivo evaluation of dopaminergic dysfunction in Parkinson’s Disease rat model.
Seret, Alain ULg; Becker, Guillaume ULg; Bahri, Mohamed Ali ULg et al

Poster (2015, May 09)

Background: Rat models of Parkinson’s disease (PD), such as lesioned rats with 6-hydroxydopamine (6-OHDA), are useful for studying dopamine (DA)-related functions. 6-[18F]fluoro-m-tyrosine (6-[18F]FMT) is ... [more ▼]

Background: Rat models of Parkinson’s disease (PD), such as lesioned rats with 6-hydroxydopamine (6-OHDA), are useful for studying dopamine (DA)-related functions. 6-[18F]fluoro-m-tyrosine (6-[18F]FMT) is an effective PET tracer to evaluate of DA terminals integrity and L-aromatic amino acid decarboxylase (AAAD) metabolic pathway. However, there are currently no available quantitative PET studies using [18F]FMT in 6-OHDA lesioned rats. In this context, we investigated the feasibility of in vivo PET study using [18F]FMT on 6-OHDA PD’s model. Methods: 10 µg of 6-OHDA were injected into the right medial forebrain bundle (MFB) of male Sprague-Dawley rats (n=8). As control, sham-treated rats (n=8) were injected with vehicle only but otherwise treated identically. Striatal DA presynaptic activity was assessed by dynamic [18F]FMT PET, 30 min after benserazide pretreatment. Structural T2-weighted brain images were acquired on a 9.4T MRI and were used for co-registration. After normalization on a MRI template, kinetic analysis was performed by “Patlak Reference” model, using PMOD software. Results: Striatal accumulation of [18F]FMT was observed in rats pretreated with benserazide, a peripheral AAAD inhibitor. As consequence of the 6-OHDA-lesion, significant decrease of [18F]FMT accumulation was recorded in the striatum ipsilateral to the lesion. Lesioned rats had dramatically reduced uptake constant Ki in the ipsilateral striatum compared to the contralateral striatum (p<0.001) and to the ipsilateral striatum of sham-treated rats (p<0.005). The Ki ratio (Ipsi./Contra.) was equivalent to 94% in the sham group and dropped to 41% in the lesioned group. Conclusions: [18F]FMT PET enables us to quantify loss of DA presynaptic function in unilaterally 6-OHDA lesioned rats. These results encourage us to pursue further investigations in a longitudinal way and to monitor the progression of the dopaminergic dysfunction in more moderate and gradual preclinical PD models. [less ▲]

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