Optimised GMP-compliant production of [18F]DPA-714 on the Trasis AllinOne module

Abstract Background The translocator protein 18 kDa is recognised as an important biomarker for neuroinflammation due to its soaring expression in microglia. This process is common for various neurological disorders. DPA-714 is a potent TSPO-specific ligand which found its use in Positron Emission Tomography following substitution of fluorine-19 with fluorine-18, a positron-emitting radionuclide. [18F]DPA-714 enables visualisation of inflammatory processes in vivo non-invasively. Radiolabelling of this tracer is well described in literature, including validation for clinical use. Here, we report significant enhancements to the process which resulted in the design of a fully GMP-compliant robust synthesis of [18F]DPA-714 on a popular cassette-based system, Trasis AllinOne, boosting reliability, throughput, and introducing a significant degree of simplicity. Results [18F]DPA-714 was synthesised using the classic nucleophilic aliphatic substitution on a good leaving group, tosylate, with [18F]fluoride using tetraethylammonium bicarbonate in acetonitrile at 100∘C. The process was fully automated on a Trasis AllinOne synthesiser using an in-house designed cassette and sequence. With a relatively small precursor load of 4 mg, [18F]DPA-714 was obtained with consistently high radiochemical yields of 55-71% (n=6) and molar activities of 117-350 GBq/µmol at end of synthesis. With a single production batch, starting with 31-42 GBq of [18F]fluoride, between 13-20 GBq of the tracer can be produced, enabling multi-centre studies. Conclusion To the best of our knowledge, the process presented herein is the most efficient [18F]DPA-714 synthesis, with advantageous GMP compliance. The use of a Trasis AllinOne synthesiser increases reliability and allows rapid training of production staff.

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Translocator Protein (18 kDa) Polymorphism (rs6971) Explains in-vivo Brain Binding Affinity of the PET Radioligand [18F]-FEPPA

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2012.46 ◽

2012 ◽

Vol 32 (6) ◽

pp. 968-972 ◽

Cited By ~ 79

Author(s):

Romina Mizrahi ◽

Pablo M Rusjan ◽

James Kennedy ◽

Bruce Pollock ◽

Benoit Mulsant ◽

...

Keyword(s):

Binding Affinity ◽

Microglial Activation ◽

Translocator Protein ◽

Time Activity ◽

Genetic Groups ◽

Positron Emission ◽

Translocator Protein 18 Kda ◽

Human Brains ◽

New Generation

[18F]-FEPPA binds to the 18-kDa translocator protein (TSPO) and is used in positron emission tomography (PET) to detect microglial activation. However, quantitative interpretations of the PET signal with new generation TSPO PET radioligands are confounded by large interindividual variability in binding affinity. This presents as a trimodal distribution, reflecting high-affinity binders (HABs), low-affinity binder (LAB), and mixed-affinity binders (MABs). Here, we show that one polymorphism (rs6971) located in exon 4 of the TSPO gene, which results in a nonconservative amino-acid substitution from alanine to threonine (Ala147Thr) in the TSPO protein, predicts [18F]-FEPPA total distribution volume in human brains. In addition, [18F]-FEPPA exhibits clearly different features in the shape of the time activity curves between genetic groups. Testing for the rs6971 polymorphism may allow quantitative interpretation of TSPO PET studies with new generation of TSPO PET radioligands.

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Cellular sources of TSPO expression in healthy and diseased brain

European Journal of Nuclear Medicine and Molecular Imaging ◽

10.1007/s00259-020-05166-2 ◽

2021 ◽

Author(s):

Erik Nutma ◽

Kelly Ceyzériat ◽

Sandra Amor ◽

Stergios Tsartsalis ◽

Philippe Millet ◽

...

Keyword(s):

Cell Activity ◽

Brain Regions ◽

Cellular Level ◽

Translocator Protein ◽

Disease Stage ◽

Animal Models Of Disease ◽

Positron Emission ◽

Neuropsychiatric Diseases ◽

Tspo Expression

AbstractThe 18 kDa translocator protein (TSPO) is a highly conserved protein located in the outer mitochondrial membrane. TSPO binding, as measured with positron emission tomography (PET), is considered an in vivo marker of neuroinflammation. Indeed, TSPO expression is altered in neurodegenerative, neuroinflammatory, and neuropsychiatric diseases. In PET studies, the TSPO signal is often viewed as a marker of microglial cell activity. However, there is little evidence in support of a microglia-specific TSPO expression. This review describes the cellular sources and functions of TSPO in animal models of disease and human studies, in health, and in central nervous system diseases. A discussion of methods of analysis and of quantification of TSPO is also presented. Overall, it appears that the alterations of TSPO binding, their cellular underpinnings, and the functional significance of such alterations depend on many factors, notably the pathology or the animal model under study, the disease stage, and the involved brain regions. Thus, further studies are needed to fully determine how changes in TSPO binding occur at the cellular level with the ultimate goal of revealing potential therapeutic pathways.

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Simplified 89Zr-Labeling Protocol of Oxine (8-Hydroxyquinoline) Enabling Prolonged Tracking of Liposome-Based Nanomedicines and Cells

Pharmaceutics ◽

10.3390/pharmaceutics13071097 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1097

Author(s):

Andras Polyak ◽

Jens P. Bankstahl ◽

Karen F. W. Besecke ◽

Constantin Hozsa ◽

Wiebke Triebert ◽

...

Keyword(s):

Reaction Times ◽

Extraction Methods ◽

Cell Labeling ◽

Size Exclusion ◽

Cell Therapies ◽

Biodistribution Studies ◽

Positron Emission ◽

Human Ipscs ◽

Radiochemical Yields

In this work, a method for the preparation of the highly lipophilic labeling synthon [89Zr]Zr(oxinate)4 was optimized for the radiolabeling of liposomes and human induced pluripotent stem cells (hiPSCs). The aim was to establish a robust and reliable labeling protocol for enabling up to one week positron emission tomography (PET) tracing of lipid-based nanomedicines and transplanted or injected cells, respectively. [89Zr]Zr(oxinate)4 was prepared from oxine (8-hydroxyquinoline) and [89Zr]Zr(OH)2(C2O4). Earlier introduced liquid–liquid extraction methods were simplified by the optimization of buffering, pH, temperature and reaction times. For quality control, thin-layer chromatography (TLC), size-exclusion chromatography (SEC) and centrifugation were employed. Subsequently, the 89Zr-complex was incorporated into liposome formulations. PET/CT imaging of 89Zr-labeled liposomes was performed in healthy mice. Cell labeling was accomplished in PBS using suspensions of 3 × 106 hiPSCs, each. [89Zr]Zr(oxinate)4 was synthesized in very high radiochemical yields of 98.7% (96.8% ± 2.8%). Similarly, high internalization rates (≥90%) of [89Zr]Zr(oxinate)4 into liposomes were obtained over an 18 h incubation period. MicroPET and biodistribution studies confirmed the labeled nanocarriers’ in vivo stability. Human iPSCs incorporated the labeling agent within 30 min with ~50% efficiency. Prolonged PET imaging is an ideal tool in the development of lipid-based nanocarriers for drug delivery and cell therapies. To this end, a reliable and reproducible 89Zr radiolabeling method was developed and tested successfully in a model liposome system and in hiPSCs alike.

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Positron Emission Tomography in the Inflamed Cerebellum: Addressing Novel Targets among G Protein-Coupled Receptors and Immune Receptors

Pharmaceutics ◽

10.3390/pharmaceutics12100925 ◽

2020 ◽

Vol 12 (10) ◽

pp. 925

Author(s):

Margit Pissarek

Keyword(s):

G Protein ◽

Cannabinoid Receptors ◽

G Protein Coupled Receptors ◽

Translocator Protein ◽

Brain Diseases ◽

Present Contribution ◽

Binding Behavior ◽

Positron Emission ◽

Inflammatory Processes ◽

G Protein Coupled

Inflammatory processes preceding clinical manifestation of brain diseases are moving increasingly into the focus of positron emission tomographic (PET) investigations. A key role in inflammation and as a target of PET imaging efforts is attributed to microglia. Cerebellar microglia, with a predominant ameboid and activated subtype, is of special interest also regarding improved and changing knowledge on functional involvement of the cerebellum in mental activities in addition to its regulatory role in motor function. The present contribution considers small molecule ligands as potential PET tools for the visualization of several receptors recognized to be overexpressed in microglia and which can potentially serve as indicators of inflammatory processes in the cerebellum. The sphingosine 1 phosphate receptor 1 (S1P1), neuropeptide Y receptor 2 (NPY2) and purinoceptor Y12 (P2Y12) cannabinoid receptors and the chemokine receptor CX3CR1 as G-protein-coupled receptors and the ionotropic purinoceptor P2X7 provide structures with rather classical binding behavior, while the immune receptor for advanced glycation end products (RAGE) and the triggering receptor expressed on myeloid cells 2 (TREM2) might depend for instance on further accessory proteins. Improvement in differentiation between microglial functional subtypes in comparison to the presently used 18 kDa translocator protein ligands as well as of the knowledge on the role of polymorphisms are special challenges in such developments.

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Development of a 18F-Labeled Radiotracer with Improved Brain Kinetics for Positron Emission Tomography Imaging of Translocator Protein (18 kDa) in Ischemic Brain and Glioma

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.7b00374 ◽

2017 ◽

Vol 60 (9) ◽

pp. 4047-4061 ◽

Cited By ~ 13

Author(s):

Masayuki Fujinaga ◽

Rui Luo ◽

Katsushi Kumata ◽

Yiding Zhang ◽

Akiko Hatori ◽

...

Keyword(s):

Positron Emission Tomography ◽

Positron Emission Tomography Imaging ◽

Translocator Protein ◽

Emission Tomography ◽

Ischemic Brain ◽

Tomography Imaging ◽

Positron Emission ◽

Translocator Protein 18 Kda

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Noninvasive quantification of macrophagic lung recruitment during experimental ventilation-induced lung injury

Journal of Applied Physiology ◽

10.1152/japplphysiol.00825.2018 ◽

2019 ◽

Vol 127 (2) ◽

pp. 546-558

Author(s):

Laurent Bitker ◽

Nicolas Costes ◽

Didier Le Bars ◽

Franck Lavenne ◽

Maciej Orkisz ◽

...

Keyword(s):

Lung Injury ◽

Protective Ventilation ◽

Lung Recruitment ◽

Translocator Protein ◽

Emission Tomography ◽

Dynamic Strain ◽

Lung Uptake ◽

Positron Emission ◽

Ventilation Induced Lung Injury

Macrophagic lung infiltration is pivotal in the development of lung biotrauma because of ventilation-induced lung injury (VILI). We assessed the performance of [11C](R)-PK11195, a positron emission tomography (PET) radiotracer binding the translocator protein, to quantify macrophage lung recruitment during experimental VILI. Pigs ( n = 6) were mechanically ventilated under general anesthesia, using protective ventilation settings (baseline). Experimental VILI was performed by titrating tidal volume to reach a transpulmonary end-inspiratory pressure (∆PL) of 35–40 cmH2O. We acquired PET/computed tomography (CT) lung images at baseline and after 4 h of VILI. Lung macrophages were quantified in vivo by the standardized uptake value (SUV) of [11C](R)-PK11195 measured in PET on the whole lung and in six lung regions and ex vivo on lung pathology at the end of experiment. Lung mechanics were extracted from CT images to assess their association with the PET signal. ∆PL increased from 9 ± 1 cmH2O under protective ventilation, to 36 ± 6 cmH2O during experimental VILI. Compared with baseline, whole-lung [11C](R)-PK11195 SUV significantly increased from 1.8 ± 0.5 to 2.9 ± 0.5 after experimental VILI. Regional [11C](R)-PK11195 SUV was positively associated with the magnitude of macrophage recruitment in pathology ( P = 0.03). Compared with baseline, whole-lung CT-derived dynamic strain and tidal hyperinflation increased significantly after experimental VILI, from 0.6 ± 0 to 2.0 ± 0.4, and 1 ± 1 to 43 ± 19%, respectively. On multivariate analysis, both were significantly associated with regional [11C](R)-PK11195 SUV. [11C](R)-PK11195 lung uptake (a proxy of lung inflammation) was increased by experimental VILI and was associated with the magnitude of dynamic strain and tidal hyperinflation. NEW & NOTEWORTHY We assessed the performance of [11C](R)-PK11195, a translocator protein-specific positron emission tomography (PET) radiotracer, to quantify macrophage lung recruitment during experimental ventilation-induced lung injury (VILI). In this proof-of-concept study, we showed that the in vivo quantification of [11C](R)-PK11195 lung uptake in PET reflected the magnitude of macrophage lung recruitment after VILI. Furthermore, increased [11C](R)-PK11195 lung uptake was associated with harmful levels of dynamic strain and tidal hyperinflation applied to the lungs.

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Radiosynthesis of [18F]-Labelled Pro-Nucleotides (ProTides)

Molecules ◽

10.3390/molecules25030704 ◽

2020 ◽

Vol 25 (3) ◽

pp. 704

Author(s):

Alessandra Cavaliere ◽

Katrin C. Probst ◽

Stephen J. Paisey ◽

Christopher Marshall ◽

Abdul K. H. Dheere ◽

...

Keyword(s):

Total Synthesis ◽

Pet Imaging ◽

Early Stage ◽

Proof Of Concept ◽

Synthesis Time ◽

Positron Emission ◽

Anti Cancer ◽

Radiochemical Yields ◽

Radiochemical Synthesis

Phosphoramidate pro-nucleotides (ProTides) have revolutionized the field of anti-viral and anti-cancer nucleoside therapy, overcoming the major limitations of nucleoside therapies and achieving clinical and commercial success. Despite the translation of ProTide technology into the clinic, there remain unresolved in vivo pharmacokinetic and pharmacodynamic questions. Positron Emission Tomography (PET) imaging using [18F]-labelled model ProTides could directly address key mechanistic questions and predict response to ProTide therapy. Here we report the first radiochemical synthesis of [18F]ProTides as novel probes for PET imaging. As a proof of concept, two chemically distinct radiolabelled ProTides have been synthesized as models of 3′- and 2′-fluorinated ProTides following different radiosynthetic approaches. The 3′-[18F]FLT ProTide was obtained via a late stage [18F]fluorination in radiochemical yields (RCY) of 15–30% (n = 5, decay-corrected from end of bombardment (EoB)), with high radiochemical purities (97%) and molar activities of 56 GBq/μmol (total synthesis time of 130 min.). The 2′-[18F]FIAU ProTide was obtained via an early stage [18F]fluorination approach with an RCY of 1–5% (n = 7, decay-corrected from EoB), with high radiochemical purities (98%) and molar activities of 53 GBq/μmol (total synthesis time of 240 min).

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Automated Synthesis of Fluorine-18 Labeled CXCR4 Ligand via the Conjugation with Nicotinic Acid N-Hydroxysuccinimide Ester (6-[18F]SFPy)

Molecules ◽

10.3390/molecules25173924 ◽

2020 ◽

Vol 25 (17) ◽

pp. 3924

Author(s):

Falguni Basuli ◽

Xiang Zhang ◽

Tim E. Phelps ◽

Elaine M. Jagoda ◽

Peter L. Choyke ◽

...

Keyword(s):

Nicotinic Acid ◽

Specific Binding ◽

Automated Synthesis ◽

Bone Marrow Niche ◽

Positron Emission ◽

Straightforward Method ◽

Radiochemical Yields ◽

Chemokine Receptor 4

The C-X-C motif chemokine receptor 4 (CXCR4) is a seven-transmembrane G protein-coupled receptor that is overexpressed in numerous diseases, particularly in various cancers and is a powerful chemokine, attracting cells to the bone marrow niche. Therefore, CXCR4 is an attractive target for imaging and therapeutic purposes. The goal of this study is to develop an efficient, reproducible, and straightforward method to prepare a fluorine-18 labeled CXCR4 ligand. 6-[18F]Fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester (6-[18F]FPy-TFP) and nicotinic acid N-hydroxysuccinimide ester (6-[18F]SFPy) have been prepared using ‘fluorination on the Sep-Pak’ method. Conjugation of 6-[18F]SFPy or 6-[18F]FPy-TFP with the alpha-amino group at the N terminus of the protected T140 precursor followed by deprotection, yielded the final product 6-[18F]FPy-T140. The overall radiochemical yields were 6–17% (n = 15, decay-corrected) in a 90-min radiolabeling time with a radiochemical purity >99%. 6-[18F]FPy-T140 exhibited high specific binding and nanomolar affinity for CXCR4 in vitro, indicating that the biological activity of the peptide was preserved. For the first time, [18F]SFPy has been prepared using ‘fluorination on the Sep-Pak’ method that allows rapid automated synthesis of 6-[18F]FPy-T140. In addition to increased synthetic efficiency, this construct binds with CXCR4 in high affinity and may have potential as an in vivo positron emission tomography (PET) imaging agent. This radiosynthesis method should encourage wider use of this PET agent to quantify CXCR4 in both research and clinical settings.

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