multi‐patient dose synthesis of [18F]Flumazenil via a copper‐mediated 18F‐fluorination

Background Flumazenil (FMZ) is a functionally silent imidazobenzodiazepine which binds to the benzodiazepine binding site of approximately 75% of the brain γ-aminobutyric acid-A receptors (GABAARs). Positron Emission Tomography (PET) imaging of the GABAARs with [11C]FMZ has been used to evidence alterations in neuronal density, to assess target engagement of novel pharmacological agents, and to study disorders such as epilepsy and Huntington’s disease. Despite the potential of FMZ PET imaging the short half-life (t1/2) of carbon-11 (20 min) has limited the more widespread clinical use of [11C]FMZ. The fluorine-18 (18F) isotopologue with a longer t1/2 (110 min) is ideally suited to address this drawback. However, the majority of current radiochemical methods for the synthesis of [18F]FMZ are non-trivial and low yielding. We report a robust, automated protocol that is good manufacturing practice (GMP) compatible, and yields multi-patient doses of [18F]FMZ. Results The fully automated synthesis was developed on the Trasis AllinOne (AIO) platform using a single-use cassette. [18F]FMZ was synthesized in a one-step procedure from [18F]fluoride, via a copper-mediated 18F-fluorination of a boronate ester precursor. Purification was performed by semi-preparative radio-HPLC and the collected fraction formulated directly into the final product vial. The overall process from start of synthesis to delivery of product is approximately 55 min. Starting with an initial activity of 23.6 ± 5.8 GBq (n = 3) activity yields of [18F]FMZ were 8.0 ± 1 GBq (n = 3). The synthesis was successfully reproduced at two independent sites, where the product passed quality control release criteria in line with the European Pharmacopoeia standards and ICH Q3D(R1) guidelines to be suitable for human use. Conclusion Reported is a fully automated cassette-based synthesis of [18F]FMZ that is Good Manufacturing Practice (GMP) compatible and produces multi-patient doses of [18F]FMZ

On the GMP production of [18]FUCB-H : Imaging neurotransmission

We herein describe the straightforward synthesis of a stable pyridyl(aryl)iodonium salt and its [18F]radiolabelling within a one-step, fully automated and cGMP compliant radi-osynthesis of [18F]UCB-H, a PET tracer for the imaging of Synaptic Vesicle glycoprotein 2A (SV2A). Over the course of one year, 40 automated productions provided 34±2% of injectable [18F]UCB-H from up to 7.7 Ci of [18F]fluoride in 50 minutes (uncorrected radiochemical yield. Specific Activity = 22±5 Ci/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]UCB-H. 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 in the cGMP 18F-PET tracer manufacturing industry and their ability to fulfill practical and regulatory requirements in that field

Synthèse du [18F]4-(4-fluorophenyl)-1,2,4-triazole-3,5-dione: un agent pour le radiomarquage spécifique de la tyrosine.

peer reviewedObjectives: Metal-free and mild tyrosine modification reactions are an attractive alternative to the commonly used lysine and cysteine modification protocols for peptide and proteins labelling. Recently, Ban and co-workers have reported a tyrosine bioconjugation through ene-type reactions. Cyclic diazodicarboxamides, which are electrophilic compounds, react selectively in o-position on the phenol side chain of tyrosine in mild aqueous conditions and the 1,2,4-triazolidine-3,5-dione linkage is hydrolytically and thermally stable. We herein present the synthesis of [18F]4-(4-fluorophenyl)-1,2,4-triazole-3,5-dione and the coupling with N-acyl tyrosine methylamide. Methods: The N,N,N-trimethyl-4-nitrobenzeneammonium trifluoromethanesulfonate 1 was prepared following a procedure previously reported [2]. The [18F]prosthetic group 6, [18F]4-(4-fluorophenyl)-1,2,4-triazole-3,5-dione, was synthesized in five steps. Results: The synthesis of the [18F]prosthetic group has been realized with a decay-corrected radiochemical yield of 20% in 90 minutes. The radiochemical yield of the coupling with N-acyl tyrosine methylamide is 40% (DC). This presented synthetic pathway should be easily automated: particulary because the purifications between the different steps are exclusively done on SPE cartridges. Conclusions: We successfully developed an efficient bioconjugation method for fluorine-18 labelling of tyrosine without prior modifications of the peptide in very mild conditions

Light-induced Hetero-Diels Alder cycloaddition as a new coupling method to biomolecule radiolabeling

The formation of a C-18F bond requires hard conditions which is problematic for the biomolecule radiolabelling. The alternative method which has been developed since a few decades consists in incorporating the 18F on a prosthetic group and coupling it to the biomolecule. The copper (I)-catalysed 1,2,3-triazole formation involving azides and terminal alkynes is a powerful and rapid method of coupling but present the inconvenient of the employment of cytotoxic reagents. The photoclick conjugation is a promising alternative with no need of catalyst[1]. Recently, a light-induced hetero-Diels Alder cycloaddition involving a 3-(hydroxymethyl)-2-naphthol derivative and an electron-rich olefin has been developed[2]. This reaction seems well adapted for the fast conjugation of radionuclides to biomolecules. Herein we report the synthesis of a [18F]fluoronaphtoquinone derivative as prosthetic group and its reaction with vinyl ethers

[18F]UCB-H BINDING QUANTIFICATION IN RAT BRAIN: FROM MODELLING TO SUV

Introduction Image quantification in Positron Emission Tomography (PET) is usually achieved through the invasive and sometimes infeasible arterial blood sampling [1, 2]. Alternative methods have been proposed, but a validation of their results is necessary [3, 4]. In the scope of improving the use of [18F]UCB-H, a specific biomarker for the Synaptic Vesicle protein 2A (SV2A) [5, 6, 7, 8], we have compared the distribution volume (VT) obtained through full kinetic modelling using a Population Based Input Function (PBIF) [9], and the Standardized Uptake Value (SUV). Methods Twelve Sprague Dawley male rats were pre-treated with vehicle (saline), 1 or 10 mg/kg of SV2A ligand (Keppra®, IP). Thirty minutes later, [18F]UCB-H was injected (IV) and a 90 min microPET dynamic acquisition was started followed by a T2 structural MRI. Primary image analysis was focused in examining tracer measurement stability through 10 min time windows. Subsequently, we calculated the correlation between VT (90 minutes) and SUV values over consecutive 20-minute time frames searching for the optimal frame to perform a static acquisition [10]. Finally, we did a supplementary test-retest static acquisition, from 60 to 80 minutes, in order to test group differences in SUV. Results/Discussion Evaluation of ten minutes time windows showed more stability in VT than in SUV measures, for all the groups. This change in signal seems to decrease in late time frames. We found also a strong correlation (R2>0.6) between dynamic VT and twenty minutes frame SUV, especially between 20 min and 60 min. From this, we can infer that an optimal frame to perform a static acquisition with [18F]UCB-H would be between 50 and 80 minutes. Using a static acquisition from 60 to 80 minutes, the SUV highlighted statistically significant differences between the group injected with vehicle and the other groups (p<0.01), but not between groups pre-treated with 1mg/kg and 10mg/kg of Keppra®. Conclusions Our work shows that a strong correlation between the SUV and the VT parameter based on a PBIF does exist. This opens the way to a possible simplification for SV2A in vivo imaging with [18F]UCB-H. Despite the fact that SUV is affected by many factors [11] and that it can overestimate results relative to VT [10], it is able to detect important differences in SV2A expression. Based on these results, SUV could become an interesting and easy to obtain parameter to study group differences in the context of several diseases. References 1. Acton PD et al. Radiologic Clinics of North America. 2004; 42(6):1055. 2. Kinahan PE & Fletcher JW. Seminars in Ultrasound, CT and MRI 2010; 31(6): 496. 3. Heurling K et al. Brain Res. 2017; 1670:220. 4. Tomasi G et al. Molecular Imaging and Biology. 2012; 14(2):131. 5. Bretin F et al. EJNMMI res. 2013; 3(1):35. 6. Warnock GI et al. J Nucl Med. 2014; 55(8):1336. 7. Bretin F et al. Molecular Imaging and Biology. 2015; 17(4):557. 8. Salmon E et al. Alzheimer's & Dementia. 2017; 13(7):781. 9. Becker G et al. Molecular Pharmaceutics. 2017; 14(8):2719. 10. Lockhart SN et al. PLoS One. 2016; 11(6):e0158460. 11. Boellaard R. J Nucl Med. 2009; 50(Suppl 1):11S-20S. Acknowledgement 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.SV2A Projec

Gallium-68-labelled NOTA-oligonucleotides: An optimized method for their preparation

One of the most essential aspects to the success of radiopharmaceuticals is an easy and reliable radiolabelling protocol to obtain pure and stable products. In this study, we optimized the bioconjugation and gallium-68 ((68) Ga) radiolabelling conditions for a single-stranded 40-mer DNA oligonucleotide, in order to obtain highly pure and stable radiolabelled oligonucleotides. Quantitative bioconjugation was obtained for a disulfide-functionalized oligonucleotide conjugated to the macrocylic bifunctional chelator MMA-NOTA (maleimido-mono-amide (1,4,7-triazanonane-1,4,7-triyl)triacetic acid). Next, this NOTA-oligonucleotide bioconjugate was radiolabelled at room temperature with purified and pre-concentrated (68) Ga with quantitative levels of radioactive incorporation and high radiochemical and chemical purity. In addition, high chelate stability was observed in physiological-like conditions (37 °C, PBS and serum), in the presence of a transchelator (EDTA) and transferrin. A specific activity of 51.1 MBq/nmol was reached using a 1470-fold molar excess bioconjugate over (68) Ga. This study presents a fast, straightforward and reliable protocol for the preparation of (68) Ga-radiolabelled DNA oligonucleotides under mild reaction conditions and without the use of organic solvents. The methodology herein developed will be applied to the preparation of oligonucleotidic sequences (aptamers) targeting the human epidermal growth factor receptor 2 (HER2) for cancer imaging