Development of new strategies for the synthesis of radiotracers labeled with short-lived isotopes: application to 11C and 13N

S'ha desenvolupat una nova estratègia per la síntesi ràpida i eficient de L-[metil-11C]metionina basada en el captive solvent method. La reacció de L-homocisteína (dissolució bàsica en aigua/etanol 1:1) amb [11C]CH3I en un loop de HPLC va permetre la formació del radiotraçador desitjat amb elevat rendiment radioquímic (38.4 ± 4.1%) en un temps curt (90%) és el propi procés de bombardeig, probablement degut a la combustió (causada per les altes temperatures i pressions assolides durant la irradiació) dels compostos que contenen carboni i que es troben al gas irradiat (o a l'interior del blanc). Es van establir procediments generals per realitzar abans, durant i després de la radiosíntesi per prevenir la contaminació exterior i, d'aquesta manera, augmentar l'activitat específica dels radiotraçadors sintetitzats.En quant al marcatge amb nitrogen-13, s'ha desenvolupat un procés totalment automàtic per a la producció de [13N]NO2- a partir de [13N]NO3- generat en el ciclotró. El precursor radioactiu [13N]NO2- s'ha utilitzat per la radiosíntesi de compostos amb interès biològic com ara S-nitrosotiols (donadors de NO.), N-nitrosamines (molècules amb potencials efectes carcinogènics) i azo compostos (amb possible aplicació com a radiotraçadors per a la detecció in vivo de plaques de β-amiloide). En tots els casos es van obtenir excel·lents conversions radioquímiques (48.7% - 74.5% per S-[13N]nitrosotiols, 45.6% - 53.4% per N-[13N]nitrosamines i 40.0% - 58.3% per 13N-azo compostos) i bons rendiments radioquímics (33.8% - 60.6% per S-[13N]nitrosotiols, 34.0% - 37.8% per N-[13N]nitrosamines i 20.4% - 47.2% per 13N-azo compostos). A més a més, s'ha dissenyat i implementat un mòdul automàtic amb control remot pel marcatge de molècules amb 13N.Se ha desarrollado una nueva estrategia para la síntesis rápida y eficiente de L-[metil-11C]metionina basada en el captive solvent method. La reacción de L-homocisteína (disolución básica en agua/etanol 1:1) con [11C]CH3I en un loop de HPLC permitió la formación del radiotrazador deseado con elevado rendimiento radioquímico (38.4 ± 4.1%) en un tiempo corto (90%) es el propio proceso de bombardeo, probablemente debido a la combustión (causada por las altas temperaturas y presiones alcanzadas durante la irradiación) de los compuestos que contienen carbono y que se encuentran presentes en el gas irradiado (o en el mismo cuerpo del blanco). Se establecieron procedimientos generales para realizar antes, durante y con posterioridad a la radiosíntesis para prevenir la contaminación exterior y, de esta manera, aumentar la actividad específica de los radiotrazadores sintetizados.Respecto al marcaje con nitrógeno-13, se ha desarrollado un proceso totalmente automático para la producción de [13N]NO2- a partir del [13N]NO3- generado en el ciclotrón. El precursor radiactivo [13N]NO2- se ha utilizado para la radiosíntesis de compuestos con interés biológico tales como S-nitrosotioles (donadores de NO.), N-nitrosaminas (moléculas con potenciales efectos carcinogénicos) y azo compuestos (con posible aplicación como radiotrazadores para la detección in vivo de placas de β-amiloide). En todos los casos se obtuvieron excelentes conversiones radioquímicas (48.7% - 74.5% para S-[13N]nitrosotioles, 45.6% - 53.4% para N-[13N]nitrosaminas y 40.0% - 58.3% para 13N-azo compuestos) y buenos rendimientos radioquímicos (33.8% - 60.6% para S-[13N]nitrosotioles, 34.0% - 37.8% para N-[13N]nitrosaminas y 20.4% - 47.2% para 13N-azo compuestos). Además, se ha diseñado e implementado un módulo automático con control remoto para el marcaje de moléculas con 13N.A new strategy for the fast and efficient synthesis of L-[methyl-11C]methionine based on the captive solvent method has been developed. The in loop reaction of a basic water/ethanol 1:1 solution of L-homocysteine with [11C]CH3I led to the formation of the desired radiotracer with high radiochemical yield (38.4 ± 4.1%) in short production time (90%) was shown to be generated during the bombardment process, probably due to the combustion (caused by high temperature and pressure during irradiation) of carbon carrier compounds present in the irradiated gas (or target body). General procedures to be performed before, during and after the radiosynthesis were established to prevent external contamination and to improve the specific radioactivity of 11C-labeled radiotracers synthesized from [11C]CH3I produced via the "wet" method. Concerning 13N-labeling, a fully automatic process for the production of [13N]NO2- from cyclotron generated [13N]NO3- has been developed. The radioactive precursor [13N]NO2- has been used for the synthesis of biologically interesting 13N-labeled compounds such as S-nitrosothiols (well-known NO. donors), N-nitrosamines (molecules with potent carcinogenic effects) and azo compounds (with putative application as imaging probes for in vivo detection of β-amyloid plaques). In all cases, excellent radiochemical conversion (48.7% - 74.5% for S-[13N]nitrosothiols, 45.6% - 53.4% for N-[13N]nitrosamines and 40.0% - 58.3% for 13N-labeled azo compounds) and good radiochemical yields (33.8% - 60.6% for S-[13N]nitrosothiols, 34.0% - 37.8% for N-[13N]nitrosamines and 20.4% - 47.2% for 13N-labeled azo compounds) were achieved. An automatic remote controlled synthesis module for the preparation of 13N-labeled structures has been designed and implemented

Medical imaging for the tracking of micromotors

Micro/nanomotors are useful tools for several biomedical applications, including targeted drug delivery and minimally invasive microsurgeries. However, major challenges such as in vivo imaging need to be addressed before they can be safely applied on a living body. Here, we show that positron emission tomography (PET), a molecular imaging technique widely used in medical imaging, can also be used to track a large population of tubular Au/PEDOT/Pt micromotors. Chemisorption of an iodine isotope onto the micromotor’s Au surface rendered them detectable by PET, and we could track their movements in a tubular phantom over time frames of up to 15 min. In a second set of experiments, micromotors and the bubbles released during self-propulsion were optically tracked by video imaging and bright-field microscopy. The results from direct optical tracking agreed with those from PET tracking, demonstrating that PET is a suitable technique for the imaging of large populations of active micromotors in opaque environments, thus opening opportunities for the use of this mature imaging technology for the in vivo localization of artificial swimmers

Gold Nanoparticles as Boron Carriers for Boron Neutron Capture Therapy: Synthesis, Radiolabelling and In Vivo Evaluation

Background: Boron Neutron Capture Therapy (BNCT) is a binary approach to cancer therapy that requires accumulation of boron atoms preferentially in tumour cells. This can be achieved by using nanoparticles as boron carriers and taking advantage of the enhanced permeability and retention (EPR) effect. Here, we present the preparation and characterization of size and shape-tuned gold NPs (AuNPs) stabilised with polyethylene glycol (PEG) and functionalized with the boron-rich anion cobalt bis(dicarbollide), commonly known as COSAN. The resulting NPs were radiolabelled with 124I both at the core and the shell, and were evaluated in vivo in a mouse model of human fibrosarcoma (HT1080 cells) using positron emission tomography (PET). Methods: The thiolated COSAN derivatives for subsequent attachment to the gold surface were synthesized by reaction of COSAN with tetrahydropyran (THP) followed by ring opening using potassium thioacetate (KSAc). Iodination on one of the boron atoms of the cluster was also carried out to enable subsequent radiolabelling of the boron cage. AuNPs grafted with mPEG-SH (5 Kda) and thiolated COSAN were prepared by ligand displacement. Radiolabelling was carried out both at the shell (isotopic exchange) and at the core (anionic absorption) of the NPs using 124I to enable PET imaging. Results: Stable gold nanoparticles simultaneously functionalised with PEG and COSAN (PEG-AuNPs@[4]) with hydrodynamic diameter of 37.8 0.5 nm, core diameter of 19.2 1.4 nm and -potential of 18.0 0.7 mV were obtained. The presence of the COSAN on the surface of the NPs was confirmed by Raman Spectroscopy and UV-Vis spectrophotometry. PEG-AuNPs@[4] could be efficiently labelled with 124I both at the core and the shell. Biodistribution studies in a xenograft mouse model of human fibrosarcoma showed major accumulation in liver, lungs and spleen, and poor accumulation in the tumour. The dual labelling approach confirmed the in vivo stability of the PEG-AuNPs@[4]. Conclusions: PEG stabilized, COSAN-functionalised AuNPs could be synthesized, radiolabelled and evaluated in vivo using PET. The low tumour accumulation in the animal model assayed points to the need of tuning the size and geometry of the gold core for future studies

Oral Treatment with Iododiflunisal Delays Hippocampal Amyloid-β Formation in a Transgenic Mouse Model of Alzheimer's Disease: A Longitudinal in vivo Molecular Imaging Study

Transthyretin (TTR) is a tetrameric, amyloid-β (Aβ)-binding protein, which reduces Aβ toxicity. The TTR/Aβ interaction can be enhanced by a series of small molecules that stabilize its tetrameric form. Hence, TTR stabilizers might act as disease-modifying drugs in Alzheimer's disease. Objective: We monitored the therapeutic efficacy of two TTR stabilizers, iododiflunisal (IDIF), which acts as small-molecule chaperone of the TTR/Aβ interaction, and tolcapone, which does not behave as a small-molecule chaperone, in an animal model of Alzheimer's disease using positron emission tomography (PET). Methods: Female mice (AβPPswe/PS1A246E/TTR+/-) were divided into 3 groups (n=7 per group): IDIF-treated, tolcapone-treated, and non-treated. The oral treatment (100mg/Kg/day) was started at 5 months of age. Treatment efficacy assessment was based on changes in longitudinal deposition of Aβ in the hippocampus (HIP) and the cortex (CTX) and determined using PET-[18F]florbetaben. Immunohistochemical analysis was performed at age=14 months. Results: Standard uptake values relative to the cerebellum (SUVr) of [18F]florbetaben in CTX and HIP of non-treated animals progressively increased from age=5 to 11 months and stabilized afterwards. In contrast, [18F]florbetaben uptake in HIP of IDIF-treated animals remained constant between ages=5 and 11 months and significantly increased at 14 months. In the tolcapone-treated group, SUVr progressively increased with time, but at lower rate than in the non-treated group. No significant treatment effect was observed in CTX. Results from immunohistochemistry matched the in vivo data at age=14 months. Conclusion: Our work provides encouraging preliminary results on the ability of small-molecule chaperones to ameliorate Aβ deposition in certain brain regions

Opposite Alterations of 5¬HT2A Receptor Brain Density in Subjects with Schizophrenia: Relevance of Radiotracers Pharmacological Profile

The status of serotonin 5HT2A receptors (5HT2ARs) in schizophrenia has been controversial. In vivo positron emission tomography neuroimaging and in vitro post-mortem binding studies have reported conflicting results about 5HT2AR density. Radiotracers bind different receptor conformations depending on their agonist, antagonist or inverse agonist properties. This study investigates 5HT2AR density in the post-mortem prefrontal cortex from subjects with schizophrenia and controls using three radiotracers with a different pharmacological profile. The specific binding parameters of the inverse agonist [18F]altanserin, the agonist [3 H]lysergic acid diethylamide (LSD) and the antagonist [ 3 H]MDL100907 to brain cortex membranes from 20 subjects with schizophrenia and 20 individually matched controls were evaluated under similar methodological conditions. Ten schizophrenia subjects were antipsychotic-free at death. Saturation curve analyses were performed by non-linear regression to obtain a maximal density of binding sites (Bmax) and the affinity of the respective radiotracers (Kd). In schizophrenia subjects, 5-HT2AR density was decreased when quantified by [18F]altanserin binding, whereas increased when evaluated by [3 H]LSD binding. However, [3 H] MDL100907 binding was unaltered. A slight loss of affinity (higher Kd) was observed exclusively in [3 H]LSD binding. The findings were more evident in antipsychotic-free subjects than in antipsychotic-treated subjects. In conclusion, a higher proportion of the 5-HT2AR-active functional conformation, which is rather identified by agonist radiotracers, was observed in schizophrenia patients. A consequent reduction of the inactive 5-HT2AR conformation, which is preferentially identified by inverse agonist radiotracers, was also obtained. Antagonist radiotracers do not distinguish between molecular conformations of the receptor, and accordingly, the absence of changes was shown. These results are compatible with the proposed increased functional activity of brain cortical 5-HT2ARs in schizophrenia.This study was supported by the Spanish State Research Agency, Ministry of Science and ERD Funds (SAF-2009-08460, SAF-2017-88126-R, RYC-2017-22412 and CTQ-2017-87637-R), and the Basque Government (SAIOTEK S-PE13UN019 and IT-1211-19). Part of this work was conducted under the Maria de Maeztu Units of Excellence Programme (Grant MDM-2017-0720). C.M. and A.G.-B. were recipients of fellowships from the Marie Slodowska-Curie Programme (European Union’s Horizon 2020, Grant 747487) and the Basque Government predoctoral training Programme, respectivel

In vivo PET Imaging of Gliogenesis After Cerebral Ischemia in Rats

In vivopositron emission tomography of neuroinflammation has mainly focused on the evaluation of glial cell activation using radiolabeled ligands. However, the non-invasive imaging of neuroinflammatory cell proliferation has been scarcely evaluated so far.In vivoandex vivoassessment of gliogenesis after transient middle cerebral artery occlusion (MCAO) in rats was carried out using PET imaging with the marker of cell proliferation 3 '-Deoxy-3 '-[18F] fluorothymidine ([F-18]FLT), magnetic resonance imaging (MRI) and fluorescence immunohistochemistry. MRI-T2W studies showed the presence of the brain infarction at 24 h after MCAO affecting cerebral cortex and striatum.In vivoPET imaging showed a significant increase in [F-18]FLT uptake in the ischemic territory at day 7 followed by a progressive decline from day 14 to day 28 after ischemia onset. In addition, immunohistochemistry studies using Ki67, CD11b, and GFAP to evaluate proliferation of microglia and astrocytes confirmed the PET findings showing the increase of glial proliferation at day 7 after ischemia followed by decrease later on. Hence, these results show that [F-18]FLT provides accurate quantitative information on the time course of glial proliferation in experimental stroke. Finally, this novel brain imaging method might guide on the imaging evaluation of the role of gliogenesis after stroke.The authors would like to thank A. Leukona, X. Rios-Anglada, and V. Salinas for technical support in the radiosynthesis. This study was funded by grants from the Spanish Ministry of Education and Science/FEDER RYC-2017-22412, SAF2016-75292-R, PID2019-107989RB-I00, the Basque Government (IT1203/19, BIO18/IC/006) and CIBERNED. Maria Ardaya holds a fellowship from the University of Pais Vasco. Ana Joya acknowledges funding from Fundacio La Marato de TV3 (17/C/2017). Part of the work has been performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (Grant No. MDM-2017-0720)

Longitudinal evaluation of neuroinflammation and oxidative stress in a mouse model of Alzheimer disease using positron emission tomography

[EN] Background: Validation of new biomarkers of Alzheimer disease (AD) is crucial for the successful development and implementation of treatment strategies. Additional to traditional AT(N) biomarkers, neuroinflammation biomarkers, such as translocator protein (TSPO) and cystine/glutamine antiporter system (x(c)(-)), could be considered when assessing AD progression. Herein, we report the longitudinal investigation of [F-18]DPA-714 and [F-18]FSPG for their ability to detect TSPO and x(c)(-) biomarkers, respectively, in the 5xFAD mouse model for AD. Methods: Expression of TSPO and x(c)(-) system was assessed longitudinally (2-12 months of age) on 5xFAD mice and their respective controls by positron emission tomography (PET) imaging using radioligands [F-18]DPA-714 and [F-18]FSPG. In parallel, in the same mice, amyloid-beta plaque deposition was assessed with the amyloid PET radiotracer [F-18]florbetaben. In vivo findings were correlated to ex vivo immunofluorescence staining of TSPO and x(c)(-) in microglia/macrophages and astrocytes on brain slices. Physiological changes of the brain tissue were assessed by magnetic resonance imaging (MRI) in 12-month-old mice. Results: PET studies showed a significant increase in the uptake of [F-18]DPA-714 and [F-18]FSPG in the cortex, hippocampus, and thalamus in 5xFAD but not in WT mice over time. The results correlate with A beta plaque deposition. Ex vivo staining confirmed higher TSPO overexpression in both, microglia/macrophages and astrocytes, and overexpression of x(c)(-) in non-glial cells of 5xFAD mice. Additionally, the results show that A beta plaques were surrounded by microglia/macrophages overexpressing TSPO. MRI studies showed significant tissue shrinkage and microstructural alterations in 5xFAD mice compared to controls. Conclusions: TSPO and x(c)(-) overexpression can be assessed by [F-18]DPA-714 and [F-18]FSPG, respectively, and correlate with the level of A beta plaque deposition obtained with a PET amyloid tracer. These results position the two tracers as promising imaging tools for the evaluation of disease progression.J.L. and P.R. thank the Spanish Ministry of Science and Innovation MCIN/AEI/10.13039/501100011033 (PID2020-117656RB-100 and PID2020-118546RBI00, respectively) and the Interreg Atlantic Area Programme (EAPA_791/2018). Abraham Martin acknowledges funding from the Spanish Ministry of Education and Science (RYC-2017-22412, PID2019-107989RB-I00), the Basque Government (BIO18/IC/006), and Fundacio La Marato de TV3 (17/C/2017). Estibaliz Capetillo-Zarate acknowledges funding from the Basque Government (IT120319; ELKARTEK KK-2020/00034) and CIBERNED (CB06/0005/0076). The work was performed under the Maria de Maeztu Units of Excellence Programme -Grant MDM-2017-0720 funded by MCIN/AEI/10.13039/50110001103

Longitudinal evaluation of a novel BChE PET tracer as an early in vivo biomarker in the brain of a mouse model for Alzheimer disease

Purpose: The increase in butyrylcholinesterase (BChE) activity in the brain of Alzheimer disease (AD) patients and animal models of AD position this enzyme as a potential biomarker of the disease. However, the information on the ability of BChE to serve as AD biomarker is contradicting, also due to scarce longitudinal studies of BChE activity abundance. Here, we report 11C-labeling, in vivo stability, biodistribution, and longitudinal study on BChE abundance in the brains of control and 5xFAD (AD model) animals, using a potent BChE selective inhibitor, [11C]4, and positron emission tomography (PET) in combination with computerised tomography (CT). We correlate the results with in vivo amyloid beta (Aβ) deposition, longitudinally assessed by [18F]florbetaben-PET imaging. Methods: [11C]4 was radiolabelled through 11C-methylation. Metabolism studies were performed on blood and brain samples of female wild type (WT) mice. Biodistribution studies were performed in female WT mice using dynamic PET-CT imaging. Specific binding was demonstrated by ex vivo and in vivo PET imaging blocking studies in female WT and 5xFAD mice at the age of 7 months. Longitudinal PET imaging of BChE was conducted in female 5xFAD mice at 4, 6, 8, 10 and 12 months of age and compared to age-matched control animals. Additionally, Aβ plaque distribution was assessed in the same mice using [18F]florbetaben at the ages of 2, 5, 7 and 11 months. The results were validated by ex vivo staining of BChE at 4, 8, and 12 months and Aβ at 12 months on brain samples. Results: [11C]4 was produced in sufficient radiochemical yield and molar activity for the use in PET imaging. Metabolism and biodistribution studies confirmed sufficient stability in vivo, the ability of [11C]4 to cross the blood brain barrier (BBB) and rapid washout from the brain. Blocking studies confirmed specificity of the binding. Longitudinal PET studies showed increased levels of BChE in the cerebral cortex, hippocampus, striatum, thalamus, cerebellum and brain stem in aged AD mice compared to WT littermates. [18F]Florbetaben-PET imaging showed similar trend of Aβ plaques accumulation in the cerebral cortex and the hippocampus of AD animals as the one observed for BChE at ages 4 to 8 months. Contrarily to the results obtained by ex vivo staining, lower abundance of BChE was observed in vivo at 10 and 12 months than at 8 months of age. Conclusions: The BChE inhibitor [11C]4 crosses the BBB and is quickly washed out of the brain of WT mice. Comparison between AD and WT mice shows accumulation of the radiotracer in the AD-affected areas of the brain over time during the early disease progression. The results correspond well with Aβ accumulation, suggesting that BChE is a promising early biomarker for incipient AD

PEG-copolymer-coated iron oxide nanoparticles that avoid the reticuloendothelial system and act as kidney MRI contrast agents

In vitro experiments have shown the great potential of magnetic nanocarriers for multimodal imaging diagnosis and non-invasive therapies. However, their extensive clinical application is still jeopardized by a fast retention in the reticuloendothelial system (RES). The other issue that restrains their potential performance is slow degradation and excretion, which increases their risks of toxicity. We report a promising case in which multicore iron oxide nanoparticles coated with a poly(4-vinylpyridine) polyethylene glycol copolymer show low RES retention and high urinary excretion, as confirmed by single photon emission computerized tomography (SPECT), gamma counting, magnetic resonance imaging (MRI) and electron microscopy (EM) biodistribution studies. These iron oxide-copolymer nanoparticles have a high PEG density in their coating which may be responsible for this effect. Moreover, they show a clear negative contrast in the MR imaging of the kidneys. These nanoparticles with an average hydrodynamic diameter of approximately 20 nm were nevertheless able to cross the glomerulus wall which has an effective pore size of approximately 6 nm. A transmission electron microscopy inspection of kidney tissue revealed the presence of iron containing nanoparticle clusters in proximal tubule cells. This therefore makes them exceptionally useful as magnetic nanocarriers and as new MRI contrast agents for the kidneys.Financial support by the Spanish Ministry of Science and Innovation (MAT2014-52069-R) (SAF2014-53413-R) (PC2015-1-05 (53-80)) is gratefully acknowledged.Peer reviewe