Design, synthesis, conjugation and reactivity of novel trans,trans-1,5-cyclooctadiene-derived bioorthogonal linkers

Funding Information: The authors thank Irene Feiner, Marion van Leeuwen-Chomet, and Joey Muns for their helpful insight. We acknowledge financial support from the University of Aberdeen and European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 675417.Peer reviewedPostprin

The binding characteristics and orientation of a novel radioligand with distinct properties at 5-HT3A and 5-HT3AB receptors.

VUF10166 (2-chloro-3-(4-methyl piperazin-1-yl)quinoxaline) is a ligand that binds with high affinity to 5-HT3 receptors. Here we synthesise [(3)H]VUF10166 and characterise its binding properties at 5-HT3A and 5-HT3AB receptors. At 5-HT3A receptors [(3)H]VUF10166 displayed saturable binding with a Kd of 0.18 nM. Kinetic measurements gave monophasic association (6.25 × 10(7) M(-1) min(-1)) and dissociation (0.01 min(-1)) rates that yielded a similar Kd value (0.16 nM). At 5-HT3AB receptors two association (6.15 × 10(-7), 7.23 M(-1) min(-1)) and dissociation (0.024, 0.162 min(-1)) rates were seen, yielding Kd values (0.38 nM and 22 nM) that were consistent with values obtained in saturation (Kd = 0.74 nM) and competition (Ki = 37 nM) binding experiments respectively. At both receptor types, specific binding was inhibited by classical 5-HT3 receptor-selective orthosteric ligands (5-HT, allosetron, d-tubocurarine, granisetron, mCPBG, MDL72222, quipazine), but not by non-competitive antagonists (bilobalide, ginkgolide B, picrotoxin) or competitive ligands of other Cys-loop receptors (ACh, bicuculline, glycine, gabazine). To explore VUF10166 ligand-receptor interactions we used in silico modelling and docking, and tested the predictions using site directed mutagenesis. The data suggest that VUF10166 adopts a similar orientation to 5-HT3 receptor agonists bound in AChBP (varenicline) and 5HTBP (5-HT) crystal structures.This is the final version. It was first published by Elsevier at http://www.sciencedirect.com/science/article/pii/S002839081400291

Design, synthesis, radiosynthesis and biological evaluation of Fenretinide analogues as anticancer and metabolic syndromepreventive agents

We thank the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement N° 675417 (PET3D project) for financial support of the project and the studentship of I.P. We also thank the British Heart Foundation for the project grant PG/16/90/32518.Peer reviewedPostprin

Monitoring Response to Radiotherapy in Human Squamous Cell Cancer Bearing Nude Mice: Comparison of 2′-deoxy-2′-[18F]fluoro-d-glucose (FDG) and 3′-[18F]fluoro-3′-deoxythymidine (FLT)

Objective: The uptake of 3′-[18F]fluoro-3′- deoxythymidine (FLT), a proliferation marker, was measured before and during fractionated radiotherapy to evaluate the potential of FLT-positron emission tomography (PET) imaging as an indicator of tumor response compared to 2′-deoxy-2′-[18F]fluoro-d-glucose (FDG). Materials and Methods: Nude mice bearing established human head and neck xenografts (HNX-OE; nu/nu mice) were locally irradiated (three fractions/week; 22 Gy) using a 150-kVp unit. Multiple FDG- and FLT-PET scans were acquired during treatment. Tumor volume was determined regularly, and tissue was analyzed for biomarkers involved in tracer uptake. Results: Both groups revealed a significant decline in tumor volume (P∈<∈0.01) compared to untreated tumors. For FDG as well as for FLT, a significant decline in retention was observed at day 4. For FLT, most significant decline in retention was observed at day 12; whereas, for FDG, this was already noted at day 4. Maximum decline in tumor-to-nontumor ratios (T/NT) for FDG and FLT was 42∈±∈18% and 49∈±∈16% (mean∈± ∈SD), respectively. FLT uptake was higher then that of FDG. For FLT, statistical significant correlations were found for both tumor volume at baseline and at day 29 with T/NT and ΔT/NT. All tumors demonstrated expression of glucose transporter-1, thymidine kinase-1, and hexokinase II. No differences were found for amount of tumor cells and necrosis at the end of treatment. Conclusion: This new experimental in vivo model supports the promise of using FLT-PET, as with FDG-PET, to monitor response to external radiotherapy. This warrants further clinical studies to compare these two tracers especially in cancers treated with radiotherapy

Reproducibility of quantitative (R)-[11C]verapamil studies

Background P-glycoprotein [Pgp] dysfunction may be involved in neurodegenerative diseases, such as Alzheimer's disease, and in drug resistant epilepsy. Positron emission tomography using the Pgp substrate tracer (R)-[11C]verapamil enables in vivo quantification of Pgp function at the human blood-brain barrier. Knowledge of test-retest variability is important for assessing changes over time or after treatment with disease-modifying drugs. The purpose of this study was to assess reproducibility of several tracer kinetic models used for analysis of (R)-[11C]verapamil data. Methods Dynamic (R)-[11C]verapamil scans with arterial sampling were performed twice on the same day in 13 healthy controls. Data were reconstructed using both filtered back projection [FBP] and partial volume corrected ordered subset expectation maximization [PVC OSEM]. All data were analysed using single-tissue and two-tissue compartment models. Global and regional test-retest variability was determined for various outcome measures. Results Analysis using the Akaike information criterion showed that a constrained two-tissue compartment model provided the best fits to the data. Global test-retest variability of the volume of distribution was comparable for single-tissue (6%) and constrained two-tissue (9%) compartment models. Using a single-tissue compartment model covering the first 10 min of data yielded acceptable global test-retest variability (9%) for the outcome measure K1. Test-retest variability of binding potential derived from the constrained two-tissue compartment model was less robust, but still acceptable (22%). Test-retest variability was comparable for PVC OSEM and FBP reconstructed data. Conclusion The model of choice for analysing (R)-[11C]verapamil data is a constrained two-tissue compartment model

Quantification of Dynamic 11C-Phenytoin PET Studies

The overexpression of P-glycoprotein (Pgp) is thought to be an important mechanism of pharmacoresistance in epilepsy. Recently, 11C-phenytoin has been evaluated preclinically as a tracer for Pgp. The aim of the present study was to assess the optimal plasma kinetic model for quantification of 11C-phenytoin studies in humans. Methods: Dynamic 11C-phenytoin PET scans of 6 healthy volunteers with arterial sampling were acquired twice on the same day and analyzed using single- and 2-tissue-compartment models with and without a blood volume parameter. Global and regional test– retest (TRT) variability was determined for both plasma to tissue rate constant (K1) and volume of distribution (VT). Results: According to the Akaike information criterion, the reversible single-tissue-compartment model with blood volume parameter was the preferred plasma input model. Mean TRT variability ranged from 1.5% to 16.9% for K1 and from 0.5% to 5.8% for VT. Larger volumes of interest showed better repeatabilities than smaller regions. A 45-min scan provided essentially the same K1 and VT values as a 60-min scan. Conclusion: A reversible single-tissue-compartment model with blood volume seems to be a good candidate model for quantification of dynamic 11C-phenytoin studies. Scan duration may be reduced to 45 min without notable loss of accuracy and precision of both K1 and VT, although this still needs to be confirmed under pathologic conditions

Impact of New Scatter Correction Strategies on High-Resolution Research Tomograph Brain PET Studies

The aim of this study is to evaluate the impact of different scatter correction strategies on quantification of high-resolution research tomograph (HRRT) data for three tracers covering a wide range in kinetic profiles. Healthy subjects received dynamic HRRT scans using either (R)-[C-11]verapamil (n = 5), [C-11]raclopride (n = 5) or [C-11]flumazenil (n = 5). To reduce the effects of patient motion on scatter scaling factors, a margin in the attenuation correction factor (ACF) sinogram was applied prior to 2D or 3D single scatter simulation (SSS). Some (R)-[C-11]verapamil studies showed prominent artefacts that disappeared with an ACF-margin of 10 mm or more. Use of 3D SSS for (R)-[C-11]verapamil showed a statistically significant increase in volume of distribution compared with 2D SSS (p 0.05). When there is a patient motion-induced mismatch between transmission and emission scans, applying an ACF-margin resulted in more reliable scatter scaling factors but did not change (and/or deteriorate) quantification

Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [Zr-89]Zr-DFO-PEG(5)-Tz

The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-ofconcept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new Zr-89-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels-Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 (t(1/2) = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [Zr-89]Zr-DFO-PEG S -Tz ([Zr-89]Zr-3) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans-cyclooctene (TCO)-functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 +/- 0.2 vs 17.1 +/- 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for Zr-89-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics.Peer reviewe

Evaluation of the Novel Folate Receptor Ligand [18F] Fluoro-PEG-Folate for Macrophage Targeting in a Rat Model of Arthritis.

Introduction Detection of (subclinical) synovitis is relevant for both early diagnosis and monitoring of therapy of rheumatoid arthritis (RA). Previously, the potential of imaging (sub)clinical arthritis was demonstrated by targeting the translocator protein in activated macrophages using (R)-[11C]PK11195 and positron emission tomography (PET). Images, however, also showed significant peri-articular background activity. The folate receptor (FR)-β is a potential alternative target for imaging activated macrophages. Therefore, the PET tracer [18F]fluoro-PEG-folate was synthesized and evaluated in both in vitro and ex vivo studies using a methylated BSA induced arthritis model. Methods [18F]fluoro-PEG-folate was synthesized in a two-step procedure. Relative binding affinities of non-radioactive fluoro-PEG-folate, folic acid and naturally circulating 5-methyltetrahydrofolate (5-Me-THF) to FR were determined using KB cells with high expression of FR. Both in vivo [18F]fluoro-PEG-folate PET and ex vivo tissue distribution studies were performed in arthritic and normal rats and results were compared with those of the established macrophage tracer (R)-[11C]PK11195. Results [18F]fluoro-PEG-folate was synthesized with a purity \u3e97%, a yield of 300 to 1,700 MBq and a specific activity between 40 and 70 GBq/µmol. Relative in vitro binding affinity for FR of F-PEG-folate was 1.8-fold lower than that of folic acid, but 3-fold higher than that of 5-Me-THF. In the rat model, [18F]fluoro-PEG-folate uptake in arthritic knees was increased compared with both contralateral knees and knees of normal rats. Uptake in arthritic knees could be blocked by an excess of glucosamine-folate, consistent with [18F]fluoro-PEG-folate being specifically bound to FR. Arthritic knee-to-bone and arthritic knee-to-blood ratios of [18F]fluoro-PEG-folate were increased compared with those of (R)-[11C]PK11195. Reduction of 5-Me-THF levels in rat plasma to those mimicking human levels increased absolute [18F]fluoro-PEG-folate uptake in arthritic joints, but without improving target-to-background ratios. Conclusions The novel PET tracer [18F]fluoro-PEG-folate, designed to target FR on activated macrophages provided improved contrast in a rat model of arthritis compared with the accepted macrophage tracer (R)-[11C]PK11195. These results warrant further exploration of [18F]fluoro-PEG-folate as a putative PET tracer for imaging (sub)clinical arthritis in RA patients