In vitro evaluation of PET radiotracers for imaging synaptic density, the acetylcholine transporter, AMPA-tarp-γ8 and muscarinic M4 receptors in Alzheimer's disease.

Several therapeutics and biomarkers that target Alzheimer's disease (AD) are under development. Our clinical positron emission tomography (PET) research programs are interested in six radiopharmaceuticals to image patients with AD and related dementias, specifically [11C]UCB-J and [18F]SynVesT-1 for synaptic vesicle glycoprotein 2A as a marker of synaptic density, two vesicular acetylcholine transporter PET radiotracers: [18F]FEOBV and [18F]VAT, as well as the transmembrane AMPA receptor regulatory protein (TARP)-γ8 tracer, [18F]JNJ-64511070, and the muscarinic acetylcholine receptor (mAChR) M4 tracer [11C]MK-6884. The goal of this study was to compare all six radiotracers (labeled with tritium or 18F) by measuring their density variability in pathologically diagnosed cases of AD, mild cognitive impairment (MCI) and normal healthy volunteer (NHV) human brains, using thin-section in vitro autoradiography (ARG). Region of interest analysis was used to quantify radioligand binding density and determine whether the radioligands provide a signal-to-noise ratio optimal for showing changes in binding. Our preliminary study confirmed that all six radiotracers show specific binding in MCI and AD. An expected decrease in their respective target density in human AD hippocampus tissues compared to NHV was observed with [3H]UCB-J, [3H]SynVesT-1, [3H]JNJ-64511070, and [3H]MK-6884. This preliminary study will be used to guide human PET imaging of SV2A, TARP-γ8 and the mAChR M4 subtype for imaging in AD and related dementias.

Impact of Surgical Lights on the Performance of Fluorescence-Guided Surgery Systems: A Pilot Study.

Fluorescence-guided surgery can aid in the intraoperative visualization of target tissues, with promising applications in human and veterinary surgical oncology. The aim of this study was to evaluate the performances of two fluoresce camera systems, IC-FlowTM and VisionsenseTM VS3 Iridum, for the detection of two non-targeted (ICG and IRDye-800) and two targeted fluorophores (AngiostampTM and FAP-Cyan) under different room light conditions, including ambient light, new generation LED, and halogen artificial light sources, which are commonly used in operating theaters. Six dilutions of the fluorophores were imaged in phantom kits using the two camera systems. The limit of detection (LOD) and mean signal-to-background ratio (mSBR) were determined. The highest values of mSBR and a lower LOD were obtained in dark conditions for both systems. Under room lights, the capabilities decreased, but the mSBR remained greater than 3 (=clearly detectable signal). LOD and mSBR worsened under surgical lights for both camera systems, with a greater impact from halogen bulbs on VisionsenseTM VS3 Iridium and of the LED lights on IC-Flow due to a contribution of these lights in the near-infrared spectrum. When considering implementing FGS into the clinical routine, surgeons should cautiously evaluate the spectral contribution of the lights in the operating theater.

Cooperative Capture Synthesis of Functionalized Heterorotaxanes─Chemical Scope, Kinetics, and Mechanistic Studies.

The self-assembly of molecularly interlocked molecules offers new opportunities for creating bioactive molecules for applications in medicine. Cooperative capture synthesis of heterorotaxanes in water is an attractive methodology for developing multifunctional supramolecular imaging agents or drugs, but derivatizing the rotaxane scaffold with biologically active vectors like peptides and proteins, or reporter probers like radioactive metal ion complexes and fluorophores, requires the installation of reactive functional groups. Here, we explored the chemical scope of ß-cyclodextrin (ß-CD) derivatization on the cucurbit[6]uril (CB[6])-mediated cooperative capture synthesis of hetero[4]rotaxanes with the objective of identifying which reactive groups can be used for further functionalization without compromising the efficiency of rotaxane synthesis. Nine ß-CD derivatives featuring an electrophilic leaving group (tosylate), aliphatic amines, a carboxylic acid, aliphatic azides, anilines, and aryl isothiocyanate were evaluated in the synthesis of hetero[4]rotaxanes. Experimental measurements on the kinetics of rotaxane synthesis were combined with detailed computational studies using the density functional theory to elucidate the mechanistic pathways and rate determining step in the cooperative capture process. Computational studies on the structure and bonding also revealed why intermolecular interactions between the ß-CD and CB[6] macrocycles improve the rate and efficiency of rotaxane formation through cooperative capture. Understanding the mechanistic details and synthetic scope will facilitate broader access to functionalized hetero[4]rotaxanes for applications in biomedicine and beyond.

Asymmetric rotaxanes as dual-modality supramolecular imaging agents for targeting cancer biomarkers.

Dual-modality imaging agents featuring both a radioactive complex for positron emission tomography (PET) and a fluorophore for optical fluorescence imaging (OFI) are crucial tools for reinforcing clinical diagnosis and intraoperative surgeries. We report the synthesis and characterisation of bimodal mechanically interlocked rotaxane-based imaging agents, constructed via the cucurbit[6]uril CB[6]-mediated alkyne-azide 'click' reaction. Two synthetic routes involving four- or six-component reactions are developed to access asymmetric rotaxanes. Furthermore, by using this rapid and versatile approach, a peptide-based rotaxane targeted toward the clinical prostate cancer biomarker, prostate-specific membrane antigen (PSMA), and bearing a 68Ga-radiometal ion complex for positron emission tomography and fluorescein as an optically active imaging agent, was synthesised. The chemical and radiochemical stability, and the cellular uptake profile of the radiolabelled and fluorescent rotaxane was evaluated in vitro where the experimental data demonstrate the viability of using an asymmetric rotaxane platform to produce dual-modality imaging agents that specifically target prostate cancer cells.

Performance of two clinical fluorescence imaging systems with different targeted and non-targeted near-infrared fluorophores: a cadaveric explorative study.

Introduction: Near-infrared (NIR) fluorescence-guided surgery is increasingly utilized in humans and pets. As clinical imaging systems are optimized for Indocyanine green (ICG) detection, the usage of targeted dyes necessitates the validation of these systems for each dye. We investigated the impact of skin pigmentation and tissue overlay on the sensitivity of two NIR cameras (IC-FlowTM, VisionsenseTM VS3 Iridum) for the detection of non-targeted (ICG, IRDye800) and targeted (AngiostampTM, FAP-Cyan) NIR fluorophores in an ex vivo big animal model. Methods: We quantitatively measured the limit of detection (LOD) and signal-to-background ratio (SBR) and implemented a semi-quantitative visual score to account for subjective interpretation of images by the surgeon. Results: VisionsenseTM VS3 Iridum outperformed IC-FlowTM in terms of LOD and SBR for the detection of all dyes except FAP-Cyan. Median SBR was negatively affected by skin pigmentation and tissue overlay with both camera systems. Level of agreement between quantitative and semi-quantitative visual score and interobserver agreement were better with VisionsenseTM VS3 Iridum. Conclusion: The overlay of different tissue types and skin pigmentation may negatively affect the ability of the two tested camera systems to identify nanomolar concentrations of targeted-fluorescent dyes and should be considered when planning surgical applications.

A rotaxane-based platform for tailoring the pharmacokinetics of cancer-targeted radiotracers.

Radiolabelled monoclonal antibodies (mAbs) are a cornerstone of molecular diagnostic imaging and targeted radioimmunotherapy in nuclear medicine, but one of the major challenges in the field is to identify ways of reducing the radiation burden to patients. We reasoned that a rotaxane-based platform featuring a non-covalent mechanical bond between the radionuclide complex and the biologically active mAb could offer new ways of controlling the biophysical properties of cancer-specific radiotracers for positron emission tomography (PET). Herein, we present the photoradiosynthesis and characterisation of [89Zr]ZrFe-[4]rotaxane-azepin-onartuzumab ([89Zr]ZrFe-2), a unique rotaxane-antibody conjugate for PET imaging and quantification of the human hepatocyte growth factor receptor (c-MET). Multiple component self-assembly reactions were combined with simultaneous 89Zr-radiolabelling and light-induced bioconjugation methods to give [89Zr]ZrFe-2 in 15 ± 1% (n = 3) decay-corrected radiochemical yield, with >90% radiochemical purity, and molar activities suitable for PET imaging studies (>6.1 MBq mg-1 of protein). Cellular assays confirmed the specificity of [89Zr]ZrFe-2 binding to the c-MET receptor. Temporal PET imaging in athymic nude mice bearing subcutaneous MKN-45 gastric adenocarcinoma xenografts demonstrated specific binding of [89Zr]ZrFe-2 toward c-MET in vivo, where tumour uptake reached 9.8 ± 1.3 %ID g-1 (72 h, n = 5) in a normal group and was reduced by ∼56% in a control (blocking) group. Head-to-head comparison of the biodistribution and excretion profile of [89Zr]ZrFe-2versus two control compounds, alongside characterisation of two potential metabolites, showed that the rotaxane-radiotracer has an improved clearance profile with higher tumour-to-tissue contrast ratios and reduced radiation exposure to critical (dose-limiting) organs including liver, spleen, and kidneys. Collectively, the experimental results suggested that non-covalent mechanical bonds between the radionuclide and mAb can be used to fine-tune the pharmacokinetic profile of supramolecular radiopharmaceuticals in ways that are simply not accessible when using traditional covalent design.

Supramolecular Rotaxane-Based Multi-Modal Probes for Cancer Biomarker Imaging.

Mechanically interlocked molecules present opportunities to construct therapeutic drugs and diagnostic imaging agents but harnessing supramolecular chemistry to make biologically active probes in water is a challenge. Here, we describe a rotaxane-based approach to synthesise radiolabelled proteins and peptides for molecular imaging of cancer biomarkers in vivo. Host-guest chemistry using ß-cyclodextrin- and cucurbit[6]uril-catalysed cooperative capture synthesis produced gallium-68 or zirconium-89 radiolabelled metallo[4]rotaxanes. Photochemical conjugation to trastuzumab led to a viable positron emission tomography (PET) radiotracer. The rotaxane architecture can be tuned to accommodate different radiometal ion complexes, other protein- or peptide-based drugs, and fluorophores for optical detection. This technology provides a platform to explore how mechanical bonding can improve drug delivery, enhance tumour specificity, control radiotracer pharmacokinetics, and reduce dosimetry.

Charting the Chemical and Mechanistic Scope of Light-Triggered Protein Ligation.

The creation of discrete, covalent bonds between a protein and a functional molecule like a drug, fluorophore, or radiolabeled complex is essential for making state-of-the-art tools that find applications in basic science and clinical medicine. Photochemistry offers a unique set of reactive groups that hold potential for the synthesis of protein conjugates. Previous studies have demonstrated that photoactivatable desferrioxamine B (DFO) derivatives featuring a para-substituted aryl azide (ArN3) can be used to produce viable zirconium-89-radiolabeled monoclonal antibodies (89Zr-mAbs) for applications in noninvasive diagnostic positron emission tomography (PET) imaging of cancers. Here, we report on the synthesis, 89Zr-radiochemistry, and light-triggered photoradiosynthesis of 89Zr-labeled human serum albumin (HSA) using a series of 14 different photoactivatable DFO derivatives. The photoactive groups explore a range of substituted, and isomeric ArN3 reagents, as well as derivatives of benzophenone, a para-substituted trifluoromethyl phenyl diazirine, and a tetrazole species. For the compounds studied, efficient photochemical activation occurs inside the UVA-to-visible region of the electromagnetic spectrum (∼365-450 nm) and the photochemical reactions with HSA in water were complete within 15 min under ambient conditions. Under standardized experimental conditions, photoradiosynthesis with compounds 1-14 produced the corresponding 89ZrDFO-PEG3-HSA conjugates with decay-corrected isolated radiochemical yields between 18.1 ± 1.8% and 62.3 ± 3.6%. Extensive density functional theory (DFT) calculations were used to explore the reaction mechanisms and chemoselectivity of the light-induced bimolecular conjugation of compounds 1-14 to protein. The photoactivatable DFO-derivatives operate by at least five distinct mechanisms, each producing a different type of bioconjugate bond. Overall, the experimental and computational work presented here confirms that photochemistry is a viable option for making diverse, functionalized protein conjugates.

Expanding the Chemistry Palette for Radiotracer Synthesis.

The synthesis, characterisation and application of radiolabelled compounds for use in diagnostic and therapeutic medicine requires a diverse skill set. This article highlights a selection of our ongoing projects that aim to provide new synthetic methods and radiochemical tools for building molecular imaging agents with various radionuclides.

Synthesis, characterization and biological activity of organometallic derivatives of the antimalarial drug mefloquine as new antischistosomal drug candidates.

We present the design, synthesis, characterization and biological evaluation of new ferrocenyl and ruthenocenyl derivatives of the organic antimalarial mefloquine, a drug also known for its antischistosomal activity. The two metallocenyl derivatives prepared (3 and 4) demonstrated comparable activity to mefloquine against adult-stage Schistosoma mansoni in vitro. Importantly, both compounds were found to have lower toxicity in all cell lines than mefloquine itself. Administration of a 200 mg kg-1 oral dose of 3 and 4 to S. mansoni-infected mice did not significantly reduce worm burden, contrary to mefloquine.