Complexos de Ga (III) de ligandos macrocíclicos anfifílicos com relevância para medicina nuclear

Dissertação de mestrado em Química MedicinalNeste trabalho foram sintetizados, purificados e caracterizados três ligandos macrocíclicos anfifílicos, o DOTAC6 (ácido 1,4,7,10-tetraazaciclododecano-1-octanóico-4,7,10- triacético), o DOTAC8 (ácido 1,4,7,10-Tetraazaciclododecano-1-decanóico-4,7,10-triacético) e o DOTAC14 (ácido 1,4,7,10-Tetraazaciclododecano-1-hexadecanóico-4,7,10-triacético). O péptido Gly-Arg-Gly-Asp-Gly (GRGDG) foi sintetizado em fase sólida e conjugado com os referidos ligandos, bem como com o ligando DOTAC4 (ácido 1,4,7,10-tetraazaciclododecano-1-hexanóico- 4,7,10-triacético), originando os bioconjugados DOTAC4-GRGDG, DOTAC6-GRGDG, DOTAC8- GRGDG e DOTAC14-GRGDG. A determinação dos valores de LogP (1-octanol/água), permitiu verificar que o complexo Ga(DOTAC14)- possui características acentuadamente hidrofóbicas, ao passo que os complexos Ga(DOTAC6)- e Ga(DOTAC8)- possuem características acentuadamente hidrofílicas. As características hidrofóbicas do complexo Ga(DOTAC14)- são igualmente evidenciadas pelo seu baixo valor de concentração micelar crítica (cmc), valor esse que vai aumentando com a diminuição do número de carbonos da cadeia alquílica dos restantes quelatos (cmc do Ga(DOTAC14)- < cmc do Ga(DOTAC8)- < cmc do Ga(DOTAC6)-). Os estudos efectuados em soro sanguíneo com o quelato Ga(DOTAC8)- permitiram verificar que este tipo de quelatos é cineticamente bastante estável, não se dissociando em condições fisiológicas. Nos ensaios realizados em ratos Wistar com quelatos marcados com 67Ga verificou-se que o carácter apolar do quelato Ga(DOTAC14)- leva a uma captação preferencial no fígado, enquanto que o carácter polar do quelato Ga(DOTAC8)- conduz a uma captação preferencial nos rins, demonstrando uma rápida eliminação renal. Ambos os quelatos mostram captações significativas noutros tecidos, devido à elevada irrigação sanguínea dos mesmos e são completamente eliminados após 24 horas.In this work three amphiphilic macrocyclic ligands, DOTAC6 (1,4,7,10- tetraazacyclododecane-1-octanoic-4,7,10-triacetic acid), DOTAC8 (1,4,7,10- tetraazacyclododecane-1-decanoic-4,7,10-triacetic acid), DOTAC14 (1,4,7,10- tetraazacyclododecane-1-hexadecanoic-4,7,10-triacetic acid), were synthesized, purified and characterized. The bioconjugates of those ligands with a Gly-Arg-Gly-Asp-Gly (GRGRG) peptide were also synthesized, as well as the DOTAC4 (1,4,7,10-tetraazacyclododecane-1-hexanoic- 4,7,10-triacetic acid)-GRGDG bioconjugate. The LogP (1-octanol/water) values, allowed verifying that the Ga(DOTAC14)- complex possesses hydrophobic characteristics, and on the other hand the Ga(DOTAC6)- and Ga(DOTAC8)- complexes possess hydrophilic characteristics. The hydrophobic characteristics of the Ga(DOTAC14)- chelate are also shown in its low critic micellar concentration (cmc) value. The cmc value increases with the decrease of the number of carbon atoms of the alkylic chain (cmc of Ga(DOTAC14)- < cmc of Ga(DOTAC8)- < cmc of Ga(DOTAC6)-). Kinetic stability studies in serum blood with the complex Ga(DOTAC8)- showed that these kind of chelates are very stable, and do not dissociate under physiological conditions. The studies with the chelates labeled with 67Ga in Wistar rats showed that the hydrophobic nature of Ga(DOTAC14)- leads to a preferential uptake in the liver, and on the other hand the hydrophilic nature of Ga(DOTAC8)- leads to a preferential uptake in the kidneys, consistent with a fast renal clearance. Both quelates show significant uptake in other tissues, due to the high irrigation of those tissues, and they are completely removed from the system 24 hours after injection

Ga(III) chelates of amphiphilic DOTA-based ligands : synthetic route and in vitro and in vivo studies

In this work we report a synthetic strategy of amphiphilic DOTA-based chelators bearing a variable size α-alkyl chain at one of the pendant acetate arms (from six to fourteen carbon atoms), compatible with their covalent coupling to amine-bearing biomolecules. The amphiphilic behavior of the micelles-forming Ga(III) chelates (critical micelle concentration), their stability in blood serum and their lipophilicity (logP) were investigated. Biodistribution studies with the 67Ga-labeled chelates were performed in Wistar rats showing a predominant liver uptake with almost no traces of the radiochelates in the body after 24 hours.Fundação para a Ciência e a Tecnologia (FCT

Quelatos do tipo DOTA de Ga(III) e Gd(III) para imagem médica (TEP, cintigrafia gama e IRM)

PhD Thesis in Sciences Specialization in ChemistryThe work developed aimed at the design, synthesis and characterization of new Gd(III) and Ga(III) chelates with potential application as imaging probes. The initial part of the work is focused on the synthesis of new DOTA-based bifunctional ligands. The chelator DOTA-AHA (1,4,7,10-tetraazacyclododecane-1-[(6-amino)hexanoic]- 4,7,10-triacetic acid) was successfully synthesized and characterized. This ligand was the starting point for the development of three sets of molecular constructs, which include dimeric ligands, PEGylated chelators and c(RGDWK) peptide bioconjugates. The Gd(III) chelates of DOTA-AHA, dimeric ligands and DOTA-AHA PEGylated ligands were obtained. All Gd(III) chelates were studied by variable temperature 1H NMRD (nuclear magnetic relaxation dispersion) and 17O NMR (nuclear magnetic resonance) spectroscopy in order to measure the relaxivity and the parameters that govern it. Gd(DOTA-AHA) and the binuclear chelates form weakly bound aggregates and even if the aggregates contain only 10 to 15% of the total amount of Gd(III) ions a marked increase in relaxivity between 30 and 100 MHz is observed. PEGylation did not show to be a very efficient process for relaxivity improvement. Despite the moderate water exchange rates of the PEGylated Gd(III) chelates and the high global rotational correlation times, these chelates present lower relaxivity values than the binuclear chelates. The distance between the two Gd(III) centers in the binuclear compounds has been determined by double electron-electron resonance (DEER) experiments and by molecular modelling studies giving comparable distances. 1H NMR spectra of paramagnetic lanthanide chelates of DOTA-A(PEG750)HA were recorded at different temperatures. The data obtained gave information on the structure and dynamics of the chelates in solution. In vitro studies with 67Ga-radiolabeled DOTA-AHA and DOTA-A(PEG750)HA showed that both chelates are extremely hydrophilic. The lack of biospecificity of 67Ga(DOTA-AHA) and [67Ga(DOTA-A(PEG750)HA)]- is revealed by their biodistribution profiles, which show that both radiolabeled chelates have significant uptake in major tissues.O trabalho desenvolvido teve como objectivo o desenho, síntese e caracterização de novos quelatos de Gd(III) e Ga(III) com potencial aplicação como agentes de imagem. A parte inicial do trabalho focou-se na síntese de novos ligandos funcionais do tipo DOTA. O ligando DOTA-AHA (ácido 1,4,7,10-tetraazaciclododecano-1-[(6- amino)hexanóico]-4,7,10-triacético) foi sintetizado e caracterizado com sucesso. Este ligando foi o ponto de partida para o desenvolvimento de ligandos diméricos, ligandos PEGuilados e bioconjugados com o péptido c(RGDWK). Preparam-se os quelatos de Gd(III) do ligando DOTA-AHA, dos ligandos diméricos e dos ligandos PEGuilados. Todos os quelatos de Gd(III) foram estudados por DRMN (dispersão de relaxação magnética nuclear) e RMN (ressonância magnética nuclear) de 17O, de modo a obter os valores de relaxividade e os parâmetros que a afectam. O quelato Gd(DOTA-AHA) e os quelatos binucleares formam agregados fracamente ligados e apesar de os agregados só conterem 10 a 15% da quantidade total de iões Gd(III) verifica-se um aumento acentuado na relaxividade para frequências entre 30 e 100 MHz. A PEGuilação mostrou ser um processo pouco eficiente para melhorar a relaxividade. Apesar das constantes de troca de água dos respectivos complexos de Gd(III) se mostrarem consideráveis e dos seus elevados tempos de correlação rotacional globais, estes quelatos apresentam valores de relaxividade inferiores à dos quelatos binucleares. A distância entre os dois centros de Gd(III) nos compostos binucleares foi determinada por ressonância dupla electrão-electrão (RDEE) e por estudos de modelação molecular, tendo sido obtidos resultados comparáveis. Estudos de 1H RMN dos quelatos de DOTA-A(PEG750)HA com lantanídeos paramagnéticos foram efectuados a diferentes temperaturas. Os dados recolhidos forneceram informação sobre a estrutura e dinâmica destes quelatos em solução. Estudos in vitro com os ligandos DOTA-AHA e DOTA-A(PEG750)HA marcados com 67Ga mostraram que ambos os quelatos são extremamente hidrofílicos. A falta de bioespecificidade de 67Ga(DOTA-AHA) e [67Ga(DOTA-A(PEG750)HA)]- é evidenciada através dos perfis de biodistribuição destes radiocomplexos.Fundação para a Ciência e Tecnologia PhD grant SFRH /BD/63676/2009

Assessment of calcinosis in Portuguese patients with systemic sclerosis: a multicenter study

© The Author(s), under exclusive licence to International League of Associations for Rheumatology (ILAR) 2023Introduction/objectives: The study aims to define the clinical and subclinical calcinosis prevalence, the sensitivity of radiographed site and clinical method for its diagnosis, and the phenotype of Portuguese systemic sclerosis (SSc) patients with calcinosis. Method: A cross-sectional multicenter study was conducted with SSc patients fulfilling Leroy/Medsger 2001 or ACR/EULAR 2013 classification criteria, registered in the Reuma.pt. Calcinosis was assessed through clinical examination and radiographs of hands, elbows, knees, and feet. Independent parametric or non-parametric tests, multivariate logistic regression, and sensitivity calculation of radiographed site and clinical method for calcinosis detection were performed. Results: We included 226 patients. Clinical calcinosis was described in 63 (28.1%) and radiological calcinosis in 91 (40.3%) patients, of which 37 (40.7%) were subclinical. The most sensitive location to detect calcinosis was the hand (74.7%). Sensitivity of the clinical method was 58.2%. Calcinosis patients were more often female (p = 0.008) and older (p < 0.001) and had more frequently longer disease duration (p < 0.001), limited SSc (p = 0.017), telangiectasia (p = 0.039), digital ulcers (p = 0.001), esophageal (p < 0.001) and intestinal (p = 0.003) involvements, osteoporosis (p = 0.028), and late capillaroscopic pattern (p < 0.001). In multivariate analysis, digital ulcers (OR 2.63, 95% CI 1.02-6.78, p = 0.045) predicted overall calcinosis, esophageal involvement (OR 3.52, 95% CI 1.28-9.67, p = 0.015) and osteoporosis (OR 4.1, 95% CI 1.2-14.2, p = 0.027) predicted hand calcinosis, and late capillaroscopic pattern (OR 7.6, 95% CI 1.7-34.9, p = 0.009) predicted knee calcinosis. Anti-nuclear antibody positivity was associated with less knee calcinosis (OR 0.021, 95% CI 0.001-0477, p = 0.015). Conclusions: Subclinical calcinosis high prevalence suggests that calcinosis is underdiagnosed and radiographic screening might be relevant. Multifactorial pathogenesis may explain calcinosis predictors' variability. Key Points • Prevalence of subclinical calcinosis in SSc patients is substantial. • Hand radiographs are more sensitive to detect calcinosis than other locations or clinical method. • Digital ulcers were associated with overall calcinosis, esophageal involvement and osteoporosis were associated with hand calcinosis, and late sclerodermic pattern in nailfold capillaroscopy was associated with knee calcinosis. • Anti-nuclear antibody positivity may be a protective factor for knee calcinosis.info:eu-repo/semantics/publishedVersio

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved