Synthesis of positron emitter-labelled amino acids: Application to the investigation of metabolic preferences and vulnerabilities in a mouse prostate cancer model.

152 p.El carcinoma de próstata es una de las principales causas de muerte en todo el mundo. Por ello, su estudio en modelos animales que mimeticen la enfermedad en humanos se convierte en un tema de interés científico. El objetivo de esta tesis es investigar los marcadores moleculares relacionados con la enfermedad. Con este fin, se eligieron los aminoácidos radiomarcados, para ser utilizados como sondas PET (Positron Emission Tomography) en un modelo animal de cáncer de próstata. Se han desarrollado estrategias sintéticas para la preparación de L- y D-[metil-11C]alanina y L-[13N]alanina, basadas en la catálisis por transferencia de fase y la biocatálisis, respectivamente. Se ha llevado a acabo su estudio de imagen PET, que mostró diferente biodistribución in vivo y una elevada acumulación en las próstatas de los animales inyectados con D-[metil-11C]alanina. Asimismo, se ha abierto una nueva ruta biosintética para la producción de L-[13N]glutamina, cuyo refinamiento hará posible su uso en estudios de imagen

Fluorine Labeling of Nanoparticles and In Vivo 19F Magnetic Resonance Imaging

Fluorinated nanoparticles have increasing applications, but they are still challenging to prepare, especially in the case of water-soluble fluorinated nanoparticles. Herein, a fluorine labeling strategy is presented that is based on the conjugation of custom-made small fluorinated building blocks, obtained by simple synthetic transformations, with carboxylated gold nanoparticles through a convenient phase-transfer process. The synthesis of four fluorinated building blocks with different chemical shifts in 19F nuclear magnetic resonance and varied functionalities is reported, along with their conjugation onto nanoparticles. Fluorinated nanoparticles of small core size obtained by this conjugation methodology and by direct synthesis presented high transverse relaxation times (T2) ranging from 518 to 1030 ms, and a large number of equivalent fluorine atoms per nanoparticle (340-1260 fluorine atoms), which made them potential candidates for 19F magnetic resonance related applications. Finally, nontargeted fluorinated nanoparticles were probed by performing in vivo 19F magnetic resonance spectroscopy (19F MRS) in mice. Nanoparticles were detected at both 1 and 2 h after being injected. 19F MRI images were also acquired after either intravenous or subcutaneous injection. Their fate was studied by analyzing the gold content in tissues by ICP-MS. Thus, the present work provides a general fluorination strategy for nanoparticles and shows the potential use of small fluorinated nanoparticles in magnetic-resonance-related applicationsThis work was supported in part by grants from the Spanish State Research Agency (MCI/PID2019-107449RB-I00/AEI/10.13039/501100011033, RYC-2017-22412, and SAF2017-84494-C2-R), from the Fundación Biofísica Bizkaia, the Basque Excellence Research Centre (BERC) program, and projects IT1196-19 and Grant KK-2019/bmG19 of the Basque Government. We also thank the UE for funding (Nova MRI, H2020-MSCA-ITN-2018-811382). This work was performed under the Maria de Maeztu Units of Excellence Programme Grant MDM-2017-0720 ministry of Science, Innovation and Universities. The authors thank for technical and human support the Materials and Surfaces Service and the Central Analysis Service of Bizkaia provided by SGIker (UPV/EHU) and European funding (ERDF and ESF) for the XPS, TEM and ICP-AES analyses. The MRI service members of CIC biomaGUNE are also acknowledged for their support of the in vivo experiment

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

Alkynyl Gold(I) complexes derived from 3-hydroxyflavones as multi-targeted drugs against colon cancer

The design of multi-targeted drugs has gained considerable interest in the last decade thanks to their advantages in the treatment of different diseases, including cancer. The simultaneous inhibition of selected targets from cancerous cells to induce their death represents an attractive objective for the medicinal chemist in order to enhance the efficiency of chemotherapy. In the present work, several alkynyl gold(I) phosphane complexes derived from 3-hydroxyflavones active against three human cancer cell lines, colorectal adenocarcinoma Caco-2/TC7, breast adenocarcinoma MCF-7 and hepatocellular carcinoma HepG2, have been synthesized and characterized. Moreover, these compounds display high selective index values towards differentiated Caco-2 cells, which are considered as a model of non-cancerous cells. The antiproliferative effect of the most active complexes [Au(L2b)PPh] (3b) and [Au(L2c)PTA] (4c) on Caco-2 cells, seems to be mediated by the inhibition of the enzyme cyclooxygenase-1/2 and alteration of the activities of the redox enzymes thioredoxin reductase and glutathione reductase. Both complexes triggered cell death by apoptosis, alterations in cell cycle progression and increased of ROS production. These results provide support for the suggestion that multi-targeting approach involving the interaction with cyclooxygenase-1/2 and the redox enzymes that increases ROS production, enhances cell death in vitro. All these results indicate that complexes [Au(L2b)PPh] and [Au(L2c)PTA] are promising antiproliferative agents for further anticancer drug development.We thank The Ministerio de Economía y Competitividad (CTQ2016-75816-C2-1-P and SAF2016-75441-R), CIBEROBN under Grant (CB06/03/1012) of the Instituto Carlos III, European Grant Interreg/SUDOE (Redvalue, SOE1/PI/E0123) and Aragon Regional Government (B16-R17 and E07-17R) for financial support