abstract: A tumor is a heterogeneous combination of proliferating tumor cells, infiltrating immune cells and stromal components along with a variety of associated host tissue cells, collectively termed the tumor microenvironment (TME). The constituents of the TME and their interaction with the host organ shape and define the properties of tumors and contribute towards the acquisition of hallmark traits such as hypoxia. Hypoxia imparts resistance to cancer from chemotherapy and radiotherapy due to the decreased production of reactive oxygen species and also promotes angiogenesis, malignant progression and metastasis. It also provides a powerful physiological stimulus that can be exploited as a tumor-specific condition, allowing for the rational design of anticancer hypoxia-activated pro-drugs (HAP). Accurate evaluation of tumor oxygenation in response to therapeutics interventions at various stages of growth should provide a better understanding of tumor response to therapy, potentially allowing therapy to be tailored to individual characteristics. The primary goal of this research was to investigate the utility of prospective identification of hypoxic tumors, by two different Magnetic Resonance Imaging (MRI) based oximetry approaches, in successful treatment with hypoxia activated therapy. In the present study, I report the utility of these two techniques 1) PISTOL (Proton Imaging of Siloxanes to map Tissue Oxygenation Levels) and 2) use of a hypoxia binding T1 contrast agent GdDO3NI in reporting the modulations of hypoxia pre and post hypoxia activated therapies in pre-clinical models of cancer. I have performed these studies in non-small cell lung cancer (NSCLC) and epidermoid carcinoma (NCI-H1975 and A431 cell lines, respectively) as well as in patient derived xenograft models of NSCLC. Both the oximetry techniques have the potential to differentiate between normoxic and hypoxic regions of the tumor and reveal both baseline heterogeneity and differential response to therapeutic intervention. The response of the tumor models to therapeutic interventions indicates that, in conjunction with pO2, other factors such as tumor perfusion (essential for delivering HAPs) and relative expression of nitroreductases (essential for activating HAPs) may play an important role. The long term goal of the proposed research is the clinical translation of both the MRI techniques and aiding the design and development of personalized therapy (e.g. patient stratification for novel hypoxia activated pro-drugs) particularly for cancer. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2017

Indiana University-Purdue University Indianapolis (IUPUI)

Cystinosis is a metabolic disorder characterised by the abnormal accumulation of the amino acid cystine in cells leading to a slow destruction of all major organs. If patients diagnosed with cystinosis are untreated, death due to kidney failure ensues in the second decade of life. A number of studies have shown the ability of the drug cysteamine (Cystagon®) to lower cystine accumulation within cells resulting in reduced organ and tissue damage. Cysteamine therapy however, is associated with a number of side effects involving the gastrointestinal tract and the central nervous system. Most of these arise due to the large amount of cysteamine present in the stomach and gut following administration. In addition, cysteamine possesses an unpleasant taste and smell, resulting in poor patient compliance. In an attempt to overcome these problems, a number of pro-drug derivatives of cysteamine and cystamine, the disulfide analogue of cysteamine, have been synthesised and evaluated. Pro-drugs were synthesised using a route established in our laboratories. Briefly, cystamine dihydrochloride was basified and allowed to react with a number of cyclic anhydrides under basic conditions. The resulting di-acids were reacted with carbonyldiimidazole and monoBoc-cystamine to yield the desired pro-drugs. Removal of the tBoc-protecting group was achieved in a facile manner by use of trifluoroacetic acid to yield product. The efficacy of the synthesised pro-drugs was determined by incubation of 50μM compound in a suspension of cultured cystinotic fibroblasts, with 50μM cysteamine as control. Cell growth was measured at 72 h and the level of thiol determined. All except one of the pro-drugs tested were significantly more effective than the control at lowering the cystine burden of the cells. Further work will concentrate on repeating these studies and evaluating a more robust Structure Activity Relationship for these compounds. The overall aim of all this work remains the production of an odourless, tasteless and orally active treatment for cystinosis and, if possible, improve on the current dosing regimen of every 6h. By using pro-drugs, cysteamine will be chemically camouflaged and hence, the side effects associated with its administration will be minimised or even entirely abolished.

Malária, doença de Chagas e leishmaniose, consideradas doenças negligenciadas, e tuberculose, infecção bacteriana reemergente, que grassa em diversas regiões do mundo, constituem-se grandes desafios médico-sociais para os países acometidos. Nitroderivados são substâncias utilizadas na terapêutica como antimicrobianos de amplo espectro. O mecanismo de ação proposto para estes compostos engloba a redução do grupo nitro por nitrorredutases inespecíficas, levando à produção de radicais livres. Estes, altamente reativos, reagiriam, por sua vez, com macromoléculas, organelas, membranas e mesmo ácidos nucléicos danificando-os irreversivelmente e levando o microrganismo - bactérias e parasitos -- à morte. No caso particular da doença de Chagas, a redutase envolvida na redução seria a tripanotiona redutase, que poderia ser inibida por estes compostos. Compostos 5-nitro-2-heterocíclicos benzidrazídicos são derivados estudados como antibacterianos e antiparasitários. Sabe-se que alterações na posição 2 dos anéis 5-nitrofurânicos interferem com o potencial redox do grupo nitro e, assim, com a atividade biológica destes compostos. Com base nesta informação e supondo que a estrutura destas moléculas seja completamente ressonante, propôs-se a síntese de série de derivados 5-nitro-heterocíclicos benzidrazídicos variando-se a estrutura dos análogos em dois pontos principais: substituições no anel benzênico da estrutura e o tipo de anel heterocíclico ligado diretamente ao grupo nitro. Com relação ao tipo de anel ligado ao grupo nitro foram propostos derivados 5-nitrofurânicos e 5-nitrotiofênicos. Quanto aos substituintes no anel benzênico propuseram-se derivados mono e dissubstituídos nas posições meta e para de acordo com suas contribuições eletrônicas e hidrofóbicas para o sistema. Para tanto foram utilizados os parâmetros físico-químicos π e σ. Para analisar o efeito das modificações na atividade biológica destes compostos aplicou-se a metodologia de Topliss como ponto de partida para a análise quantitativa, QSAR-2D, pelo método misto de Hansch-Free Wilson. A biblioteca planejada foi obtida por síntese paralela em solução, empregando-se sintetizadores paralelos automatizados. Foram obtidos 56 derivados, que foram purificados, caracterizados estruturalmente e, então, analisados frente a quatro microrganismos: Plasmodium falciparum, Leishmania donovani, Trypanosoma cruz e Mycobacterium tuberculosis. Os estudos de QSAR-2D revelaram importância significativa do tipo de anel heterocíclico ligado ao grupo nitro para a atividade destes compostos. Não foi possível, entretanto, obter correlação das atividades com as substituições realizadas no anel aromático, o que levou ao arrolamento de diferentes hipóteses, incluindo aquelas relativas à deslocalização eletrônica nas estruturas estudadas. Estas hipóteses, bem como os resultados sobre a influência do tipo de anel heterocíclico ligado ao grupo nitro nas atividades biológicas estudadas fornecem subsídios para novos projetos de pesquisa dedicados à exploração das informações obtidas neste trabalho. / Malaria, Chagas\' disease and leishmaniasis, considered as neglected diseases, and tuberculosis, reemerging bacterial infection disseminated worldwide, are medical-social challenges for the countries involved. Nitroderivatives are therapeutic compounds used as broad spectrum antimicrobial drugs. The most accepted mechanism of action of these compounds is based on nitro group reduction by unspecific nitroreductases or, in case of Chagas\' disease, inhibition of tripanotiona redutase. Free radical species are formed from this reduction, which can react with macromolecules, organelles, membranes and nucleic acids of microorganisms leading them to death. The 5-nitro-2-heterocyclic benzhydrazide derivatives are nitrofuran analogs that have been tested as antibacterial and antiprotozoal drugs. As already known, modifications in position 2 of 5-nitroheterociclic rings can interfere with nitro group redox potential and then with their biological activities. Based on this information and considering these compound structures are completely conjugated structures, we suggested the synthesis of a library of 5-nitro-2-heterocyclic benzhydrazide derivatives with two main points of structural modification: the substituents in benzenic ring and the kind of heterocyclic ring directly linked to the nitro group. The former was achieved by suggesting mono and dissubstituted analogs based on their hydrophobic and electronic contributions whose values were obtained from physico-chemical parameters π and σ. Nitrofurans and nitrothiophenes were elected to study the influence of the heterocyclic ring directly linked to the nitro group. Topliss methodology was used as a starting point to 2D-QSAR mixed quantitative method through Hansch-Free Wilson analysis. The designed library was synthesized by solution phase parallel synthesis using automated parallel synthesizers. Fifty-six analogs were synthesized, purified, characterized and biologically analyzed against four microorganisms: Plasmodium falciparum, Leishmania donovani, Trypanosoma cruzi e Mycobacterium tuberculosis. The 2D-QSAR studies provided information about the significative influence of the kind of heterocyclic ring on the biological activity of the series. Nevertheless, it was not possible to obtain information about the influence of different substitutions on benzene ring and different hypothesis were advanced to explain the electronic distribution in the structures herein studied. These hypotheses as well as the data about the influence of the kind of heterocyclic ring directly linked to the nitro group on the biological activities studied deserve to be tested and explored in future researches.

Malária, doença de Chagas e leishmaniose, consideradas doenças negligenciadas, e tuberculose, infecção bacteriana reemergente, que grassa em diversas regiões do mundo, constituem-se grandes desafios médico-sociais para os países acometidos. Nitroderivados são substâncias utilizadas na terapêutica como antimicrobianos de amplo espectro. O mecanismo de ação proposto para estes compostos engloba a redução do grupo nitro por nitrorredutases inespecíficas, levando à produção de radicais livres. Estes, altamente reativos, reagiriam, por sua vez, com macromoléculas, organelas, membranas e mesmo ácidos nucléicos danificando-os irreversivelmente e levando o microrganismo - bactérias e parasitos -- à morte. No caso particular da doença de Chagas, a redutase envolvida na redução seria a tripanotiona redutase, que poderia ser inibida por estes compostos. Compostos 5-nitro-2-heterocíclicos benzidrazídicos são derivados estudados como antibacterianos e antiparasitários. Sabe-se que alterações na posição 2 dos anéis 5-nitrofurânicos interferem com o potencial redox do grupo nitro e, assim, com a atividade biológica destes compostos. Com base nesta informação e supondo que a estrutura destas moléculas seja completamente ressonante, propôs-se a síntese de série de derivados 5-nitro-heterocíclicos benzidrazídicos variando-se a estrutura dos análogos em dois pontos principais: substituições no anel benzênico da estrutura e o tipo de anel heterocíclico ligado diretamente ao grupo nitro. Com relação ao tipo de anel ligado ao grupo nitro foram propostos derivados 5-nitrofurânicos e 5-nitrotiofênicos. Quanto aos substituintes no anel benzênico propuseram-se derivados mono e dissubstituídos nas posições meta e para de acordo com suas contribuições eletrônicas e hidrofóbicas para o sistema. Para tanto foram utilizados os parâmetros físico-químicos π e σ. Para analisar o efeito das modificações na atividade biológica destes compostos aplicou-se a metodologia de Topliss como ponto de partida para a análise quantitativa, QSAR-2D, pelo método misto de Hansch-Free Wilson. A biblioteca planejada foi obtida por síntese paralela em solução, empregando-se sintetizadores paralelos automatizados. Foram obtidos 56 derivados, que foram purificados, caracterizados estruturalmente e, então, analisados frente a quatro microrganismos: Plasmodium falciparum, Leishmania donovani, Trypanosoma cruz e Mycobacterium tuberculosis. Os estudos de QSAR-2D revelaram importância significativa do tipo de anel heterocíclico ligado ao grupo nitro para a atividade destes compostos. Não foi possível, entretanto, obter correlação das atividades com as substituições realizadas no anel aromático, o que levou ao arrolamento de diferentes hipóteses, incluindo aquelas relativas à deslocalização eletrônica nas estruturas estudadas. Estas hipóteses, bem como os resultados sobre a influência do tipo de anel heterocíclico ligado ao grupo nitro nas atividades biológicas estudadas fornecem subsídios para novos projetos de pesquisa dedicados à exploração das informações obtidas neste trabalho. / Malaria, Chagas\' disease and leishmaniasis, considered as neglected diseases, and tuberculosis, reemerging bacterial infection disseminated worldwide, are medical-social challenges for the countries involved. Nitroderivatives are therapeutic compounds used as broad spectrum antimicrobial drugs. The most accepted mechanism of action of these compounds is based on nitro group reduction by unspecific nitroreductases or, in case of Chagas\' disease, inhibition of tripanotiona redutase. Free radical species are formed from this reduction, which can react with macromolecules, organelles, membranes and nucleic acids of microorganisms leading them to death. The 5-nitro-2-heterocyclic benzhydrazide derivatives are nitrofuran analogs that have been tested as antibacterial and antiprotozoal drugs. As already known, modifications in position 2 of 5-nitroheterociclic rings can interfere with nitro group redox potential and then with their biological activities. Based on this information and considering these compound structures are completely conjugated structures, we suggested the synthesis of a library of 5-nitro-2-heterocyclic benzhydrazide derivatives with two main points of structural modification: the substituents in benzenic ring and the kind of heterocyclic ring directly linked to the nitro group. The former was achieved by suggesting mono and dissubstituted analogs based on their hydrophobic and electronic contributions whose values were obtained from physico-chemical parameters π and σ. Nitrofurans and nitrothiophenes were elected to study the influence of the heterocyclic ring directly linked to the nitro group. Topliss methodology was used as a starting point to 2D-QSAR mixed quantitative method through Hansch-Free Wilson analysis. The designed library was synthesized by solution phase parallel synthesis using automated parallel synthesizers. Fifty-six analogs were synthesized, purified, characterized and biologically analyzed against four microorganisms: Plasmodium falciparum, Leishmania donovani, Trypanosoma cruzi e Mycobacterium tuberculosis. The 2D-QSAR studies provided information about the significative influence of the kind of heterocyclic ring on the biological activity of the series. Nevertheless, it was not possible to obtain information about the influence of different substitutions on benzene ring and different hypothesis were advanced to explain the electronic distribution in the structures herein studied. These hypotheses as well as the data about the influence of the kind of heterocyclic ring directly linked to the nitro group on the biological activities studied deserve to be tested and explored in future researches.

[ES] La presente tesis doctoral titulada "Nuevas sondas, portadores y pro-fármacos dirigidos a células senescentes in vivo" se centra en el diseño, preparación, caracterización y evaluación de sondas basadas en fluoróforos funcionalizados o en nanopartículas, así como en el desarrollo de profármacos, aplicadas al campo de la senescencia celular. En el primer capítulo se introducen, a nivel general, los diferentes conceptos relacionados con el reconocimiento molecular y la detección de biomarcadores. Seguidamente se introducen conceptos básicos de senescencia celular y envejecimiento, así como el papel que juegan las células senescentes a nivel fisiológico, en enfermedades asociadas al envejecimiento celular y en el cáncer. Por último, se aborda también la creciente necesidad de desarrollar nuevas herramientas de diagnóstico y terapias centradas en la detección y tratamiento de dichas enfermedades. En el segundo capítulo se exponen los objetivos generales de la presente tesis doctoral, así como los objetivos concretos que son abordados en los diferentes capítulos experimentales. En el tercer capítulo se describe una nueva sonda molecular para la detección de senescencia in vivo. En concreto, se describe el diseño de una sonda de dos fotones basada en un fluoróforo de naftalimida conectada, mediante un éster metílico de L-histidina, con una galactosa acetilada que esta unida a uno de los átomos de nitrógeno aromático de la L-histidina a través de un enlace N-glicosídico hidrolizable (AHGa). La sonda inicial presenta una baja emsión pero en células senescentes se transforma en un fluoróforo con alto rendimiento cuantico que permite visualizar estas células debido a la hidrólisis del enlace N-glicosidico por la enzima ¿-galactosidasa sobreexpresada en este tipo de células. La sonda es capaz de detectar in vitro células de melanoma humano SK-Mel-103 tratadas con quimioterapia inductora de senescencia frente a células control. Adicionalmente, se validó la detección in vivo de senescencia en ratones con xenoinjertos tumorales tratados con quimioterapia inductora de senescencia. Basándonos en los resultados obtenidos en el capítulo tres, en el capítulo cuatro se describe un sistema similar cuyas características confieren al fluoróforo una mayor longitud de onda de excitación (HeckGal). La nueva sonda es capaz de detectar células senescentes debido a la sobreexpresión de la enzima ¿-galactosidasa asociada a senescencia en gran variedad de líneas celulares con diversos métodos de inducción de senescencia. Esta sonda se validó también in vivo en un modelo ortotópico de cáncer de mama de ratón tratado con quimioterapia inductora de senescencia, y en un modelo de ratón de fibrosis renal. Seguidamente, el capítulo cinco, se centra en un nuevo concepto de sondas moleculares no invasivas, que proporcionan una señal fácilmente legible a través de simples medidas de fluorescencia de la orina. Esta idea se llevó a cabo mediante la funcionalización de una sonda con grupos que le confieren características diuréticas (rápida eliminación renal) y el concepto se aplicó al diseño de una sonda para a la detección en orina de la carga senescente en diversos modelos. La sonda (Cy7Gal) está basada en la cianina-7 que es hidrolizada por la enzima ¿-galactosidasa en un fluoróforo altamente emisivo Cy7. Cy7Gal y Cy7 contienen restos de ácido sulfónico que aumentan su solubilidad en agua y desencadenan su rápida excreción por el sistema urinario. El grado de senescencia se cuantificó por medición directa de fluorescencia en orina en tres modelos de senescencia en ratones: (i) un modelo ortotópico de cáncer de mama en ratones BALB/cByJ con senescencia inducida por quimioterapia; (ii) ratones BALB / cByJ envejecidos de forma natural; y (iii) ratones con senescencia acelerada (SAMP8, del inglés Senescence Accelerated Mouse-Prone 8). Además, las imágenes IVIS ex vivo permite / [CA] La tesi doctoral titulada "Noves sondes, portadors i profàrmacs dirigits a cèl·lules senescents in vivo" se centra en el desenvolupament i avaluació de sondes basades en fluoròfors funcionalitzats o en nanopartícules, així com en el desenvolupament de profàrmacs, aplicades a el camp de la senescència cel·lular. Primer s'introdueixen els diferents conceptes relacionats amb el sensing i amb la sescencia cel·lular, abordant la necessitat de noves eines diagnòstiques i terapèutiques d'aquestes malalties. En els capítols 3:04 es descriuen una nova sonda molecular dos fotons per a la detecció de senescència in vivo en ratolins amb xenoempelts tumorals tractats amb quimioteràpia inductora de senescència, una altra sonda per a la detecció in vivo en un model ortotòpic de càncer de mama de ratolí tractat amb quimioteràpia inductora de senescència, i en un model de ratolí de fibrosi renal. El capítol 5, se centra en un nou concepte de sondes moleculars no invasives, que proporcionen un senyal fàcilment llegible a través de simples mesures de fluorescència de l'orina. Finalment, pel que fa a detecció de senescència cel·lular es refereix, es presenta en el capítol sis la detecció in vivo de senescència cel·lular mitjançant l'alliberament controlat de Nile Blue en nanopartícules de sílice mesoporoses funcionalitzades amb un galactohexasacárido. Un cop assolits els objectius planificats pel que fa a la diagnosi, en el capítol set es va abordar la millora d'un dels senolíticos més potents i àmpliament conegut en el camp de la senescència cel·lular que existeix a dia d'avui al mercat: el Navitoclax. / [EN] The doctoral thesis entitled "New probes, carriers and pro-drugs directed to senescent cells in vivo" focuses on the development and evaluation of probes based on functionalized fluorophores or nanoparticles, as well as on the development of prodrugs, applied to the field of cellular senescence. First, the different concepts related to sensing and cell sescence are introduced, addressing the need for new diagnostic and therapeutic tools for these diseases. Chapters three and four describe a new two-photon molecular probe for in vivo senescence detection in mice with tumor xenografts treated with senescence-inducing chemotherapy, another probe for in vivo detection in an orthotopic mouse breast cancer model. treated with senescence-inducing chemotherapy, and in a mouse model of renal fibrosis. Chapter five focuses on a new concept of non-invasive molecular probes, which provide an easily readable signal through simple measurements of urine fluorescence. Finally, regarding the detection of cellular senescence, chapter six presents the in vivo detection of cellular senescence through the controlled release of Nile Blue in mesoporous silica nanoparticles functionalized with a galactohexasaccharide. Once the planned objectives with regard to diagnosis had been achieved, chapter seven addressed the improvement of one of the most powerful and widely known senolytics in the field of cellular senescence that exists on the market today: Navitoclax. / The authors thank the financial support from the Spanish Government (projects MAT2015-64139-C4-1-R and AGL2015-70235-C2-2-R) and the Generalitat Valenciana (project PROMETEOII/2014/047). R.M laboratory members thank the financial support from the Spanish Government (project RTI2018-100910-B-C41) and the Generalitat Valenciana (project PROMETEO 2018/024). B.L-T. is grateful to the Spanish Ministry of Economy for their PhD grants (FPU15/02707). Work in the laboratory of MS was funded by the IRB and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (ERDF) (SAF2013-48256-R), the European Research Council (ERC-2014-AdG/669622), and the “laCaixa” Foundation. D.M.-E. laboratory is supported by the Cancer Research UK (CRUK) Cambridge Centre Early Detection Programme, by a CRUK Early Detection OHSU Project Award (C62187/A26989), by a Medical Research Council (MRC) New Investigators Research Grant (NIRG, MR/R000530/1). Work in the laboratory of I.F. is supported by grants from the Spanish Government (SAF2017-86690-R), Instituto de Salud Carlos III (CIBERNED and RETIC Tercel), and Generalitat Valenciana (Prometeo 2017/030). / Lozano Torres, B. (2021). Novel probes, carriers and prodrugs to target senescent cells in vivo [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172175