Transport into and across the cells of the human body is a prerequisite for the pharmacological action of drugs. Passive membrane permeability and active transport mechanisms are major determinants of the intestinal absorption of drugs, as well as of the distribution to target tissues and the subsequent metabolism and excretion from the body. In this thesis, the role of ATP-binding cassette (ABC) transporters and passive permeability on drug absorption and disposition was investigated. Particular emphasis was placed on defining the molecular properties important for these transport mechanisms. The influence of different transport pathways on predictions of intestinal drug absorption was investigated using experimental models of different complexity. Experimental models that include the paracellular pathway gave improved predictions of intestinal drug absorption, especially for incompletely absorbed drugs. Further, the inhibition of the ABC transporters breast cancer resistance protein (BCRP/ABCG2) and multidrug-resistance associated protein 2 (MRP2/ABCC2) was experimentally investigated using structurally diverse datasets that were representative of orally administered drugs. A large number of previously unknown inhibitors were identified among registered drugs, but their clinical relevance for drug-drug interactions and drug-induced toxicity remains to be determined. The majority of the inhibitors affected all three major ABC transporters BCRP, MRP2 and P-glycoprotein (P gp/ABCB1), and these multi-specific inhibitors were found to be enriched in highly lipophilic weak bases. To summarize, the present work has led to an increased knowledge of the molecular features of importance for ABC transporter inhibition and passive membrane permeability. Previously unknown ABC transporter inhibitors were identified and predictive computational models were developed for the different drug transport mechanisms. These could be valuable tools to assist in the prioritization of experimental efforts in early drug discovery.

L'enveloppe cellulaire des bactéries à Gram négatif constitue la première ligne de défense contre les antibiotiques. Sous l’effet, d’une part, de la faible perméabilité de la membrane externe qui s'oppose à la pénétration des agents antibactériens, d’autre part des pompes d'efflux qui favorisent leur expulsion, elle empêche nombre de composés potentiellement actifs in vitro d'atteindre leur cible, limitant l’effet antibactérien. Un enjeu important pour restaurer l’activité de ces molécules est de trouver une stratégie pour en augmenter la concentration intracellulaire. L'objectif de cette thèse est de développer des métallodrogues comme nouvelle stratégie de vectorisation de drogues dans les cellules. Cette stratégie repose sur l’association d'une drogue active in vitro, et d’un ligand auxiliaire ayant des propriétés perméabilisantes ou inhibitrices de pompe d’efflux, dans un complexe qui jouera le rôle de chaperone. Les agents antibactériens utilisés sont des inhibiteurs (dérivés d’acides hydroxamiques) de peptide déformylase (PDF) et de méthionine aminopeptidase (MetAP) développés au laboratoire. Tout d’abord, une étude globale de la stratégie de vectorisation a été réalisée (i) étude de stabilité de métallodrogues modèles : en utilisant un acide hydroxamique fluorescent, nous avons montré que, seules, des métallodrogues à Co(III), à la différence de celles à Cu(II) et Fe(III), satisfaisaient aux conditions de stabilité compatibles avec les conditions de tests biologiques. (ii) Etude de la libération de la drogue : nous avons établi par une étude RMN 1H et UV-vis qu’en milieu tampon pH = 7,4, la libération de la drogue se faisait par échange de ligand avec un thiol exogène. Récemment, une nouvelle série d’inhibiteurs de PDF a été synthétisée au laboratoire. Elle est basée sur un squelette hétérocyclique à 5 chaînons fonctionnalisé par une chaîne en C4, puis via un espaceur monocarboné, à un acide hydroxamique. Les meilleurs résultats ont été obtenus avec un oxadiazole (AT002 16 µg/ml sur E. coli en présence de perméabilisant PMBN). Au cours de cette thèse, pour améliorer la lipophilie, des groupements aromatiques ont été fixés sur cet hétérocycle. Les MICs sur la souche d’E. coli sauvage n’ont pas été améliorées mais en présence de PMBN, le dérivé présentant la meilleure activité est le composé AT015 (2 µg/ml sur E. coli en présence de PMBN) qui a donc été choisi pour concevoir des métallodrogues. La métallodrogue réunit autour d’un métal deux parties: (i) un ligand auxiliaire fonctionnalisé via un espaceur par un perméabilisant peptidique analogue de peptide antimicrobien ou par un modulateur de l’efflux (ii) un acide hydroxamique inhibiteur de PDF. Au cours de la SAR réalisée en faisant varier la drogue, le ligand auxiliaire et le métal, nous avons montré que les meilleures métallodrogues permettent d’améliorer l’activité de la drogue sur la souche d’E. coli sauvage d’un facteur 16. Un des ligands auxiliaires fonctionnalisé par un tétrapeptide présente, seul, une activité sur une souche d’E. aerogenes résistante aux fluoroquinolones. Sur ce cas, l’activité biologique a été reliée, par des expériences de mapping par fluorescence, à son accumulation intracellulaire, en utilisant un analogue fluorescent de ce composé. / The gram negative bacterias’ cell envelopes are the first line of defense against antibiotics. First thanks to the low permeability of the external membrane that prevents the penetration of the antibiotics, but also thanks to the efflux pumps that help expelling the antibiotics from the cell. These mechanisms prevent many compounds, potentially active in vitro, from reaching their targets, thus limiting the antimicrobial effect. To increase the molecules’ intracellular concentration is one of the means to restore their activity. This thesis’ objective is to develop metallodrugs as a new drug vectorization strategy in cells. We here associate an active drug in vitro and an auxiliary ligand with permeabilization or efflux pumps inhibition abilities in a complex playing the role of a chaperone. We used peptide deformylase (PDF) and methionine aminopeptidase (MetAP) inhibitors (derived from hydroxamic acids) developed at the laboratory as antimicrobial agents. I’ll begin with a global study of the vectorization strategy we’ve adopted (i) Stability study of the metallodrugs models: using a fluorescent hydroxamic acid, we showed that only Co(III) metallodrugs are in agreement with the stability conditions compatible with the biological tests, in opposition with the Cu(II) and Fe(III) ones. (ii) Drug release study: we showed in 1H NMR and UV-vis studies that in a buffer solution pH 7.4, a ligand exchange with an exogenous thiol is responsible for the drug release. Recently, a new series of PDF inhibitors was synthesized at the laboratory. It is composed of a 5 membered heterocyclic skeleton functionalized by a chain in C4 followed by an hydroxamic acid via a monocarbonated spacer. The best results were obtained with an oxadiazole (AT002 16 µg/ml with E. coli and PMBN as permeabilizing agent). During this thesis, to enhance lipophilicity, we attached aromatic groups on the heterocycle. CMIs on the E. coli strain have not been increased but the compounds displaying the best activity in presence of PMBN (AT015, 2 µg/ml with E. coli and PMBN) was chosen to conceive metallodrugs. The metallodrug is composed of a metal center and two other parts: (i) an auxiliary ligand functionalized via a spacer by a permeabilizing peptide, an antimicrobial peptide analogue, or by an efflux modulator. (ii) An hydroxamic acid PDF inhibitor. We showed that the best metallodrugs enhance the drug activity on the wild E.coli strain by a 16 factor, with the SAR we realized, changing the drug, the auxiliary ligand and the metal. One of the auxiliary ligands functionalized by a tetrapeptide show an activity on a fluoroquinolone-resistant E. aerogenes strain while alone. Utilizing a fluorescent analogous of this compound, we linked the biological activity to its intracellular accumulation with fluorescence mapping experiments.

La perméabilité membranaire joue un rôle important dans la résistance aux antibiotiques chez lesbactéries à Gram négatif.L’objectif de notre travail était de caractériser la fonction tenue par les deux régulateurs globaux dela perméabilité membranaire chez Enterobacer aerogenes: mar et ram. L’objectif initial futd’identifier le répresseur spécifique de RamA qui manquait en tant qu’élément de la cascade derégulation actuellement définie. La sélection de 60 souches nous a permis de confirmer le rôlecentral joué par RamA dans la régulation, ainsi qu’identifier des mutations pouvant être critiques, auniveau de RamR. Ainsi, l’absence variations observées dans le régulon marRAB et l’expressionmodérée des transcrits montrée par qRT-PCR laisse penser, que RamA a un rôle clef dans larégulation de l’expression des porines et des pompes d’efflux chez E. aerogenes.L’autre partie de notre travail reposait sur l’étude de la translocation des antibiotiques au traversdes porines. L’étude des interactions porine-carbapénèmes s’est faite sur la porine sauvage OmpFd'Escherichia coli et deux mutations. Les résultats indiquent également l'importance de l'aspartateen position 113 dans la sélectivité de translocation des carbapénèmes au sein de la porine OmpF.Ce travail montre ainsi que la translocation des pénicillines est aussi sous la dépendance desinteractions qui se créent entre le substrat et le résidu en position 113 de OmpF et limitent alorsleur passage au niveau du canal porine. Nous avons recherché la contribution attribuée à la porineOmp36 d'E. aerogenes dans la translocation de certaines béta-lactamines. Les mesures ont permisde conclure que les deux beta-lactamin / Genetic permeability plays an important role in antibiotic resistance of Gram-negative bacteria.Our work was to characterize and better understand of the genetic regulation of membranepermeability in E. aerogenes. We focused on two global regulators, mar and ram, in about 60clinical isolates. Alterations in the upstream region of ramA and in ramR but no mutations in marAnor marR were observed. Overexpression of ramA or ramR led to an altered antibiotic susceptibilityassociated to decrease of porins expression and over-expression of efflux-pumps. qRT-PCR pointedout the estimated importance of the ram-regulon in the regulation cascade.Another part of this work was to characterize the translocation of compounds through porins andthe role of porins in drug uptake in general. Measurement of the rate of antibiotic action of threecarbapenems in an E. coli strain solely expressing OmpF as porin clearly indicated the importanceof the aspartate at position 113 in antibiotic translocation. A multi-disciplinary three way approachof computer modeling, black-lipid-bilayer assays and measurement of antibiotic action, suggestedthat interactions with residue D113 of E. coli porin OmpF are rate-limiting for transport throughthe porin channel. Combination of biological and biophysical measurements with E. aerogenesporin Omp36 denoted that interactions between the porin channel and the antibiotic facilitate andaccelerate transport.

Gallic acid is a known antioxidant and has anti-inflammatory activity in addition to other biological activities, but GA efficiency is restricted due to low permeability and low oral bioavailability. This study was designed to investigate the solubility, permeability, oral bioavailability, enzymatic stability with cytochrome CYP2D6, antioxidant and anti-inflammatory activity of novel gallic acid sulfonamide derivatives; TMBS, and THBS. In addition, a novel in silico permeability model was designed to predict the permeability and bioavailability of eighty derivatives of GA. In sillico prediction of intestinal permeability of GA derivative indicated an increase in permeability with increased lipophilicity and decreased aqueous solubility, replacing the carboxylic group with sulfonamide group has increased intestinal permeability. A significant (P <0.01) increase was observed in the permeability of TMBS and THBS over GA, in both gastric fluids and HIEC cells. TMBS was O-demethylated by CYP2D6. TMBS had greater ROS scavenging activity than GA in HIEC-6 cells. There was a significant (P< 0.05) increase in anti-inflammatory activity of THBS, and TMBS compared to ibuprofen. TMBS, and THBS had better oral bioavailability than GA. This data suggests that the in silico permeability model can be used in the future to study new candidate of gallic acid, and further in vivo and clinical investigations are required to introduce TMBS and THBS as a new antioxidant and anti-inflammatory drugs.

El reactor anaerobio de membranas sumergidas (SAnMBR) está considerado como tecnología candidata para mejorar la sostenibilidad en el sector de la depuración de aguas residuales, ampliando la aplicabilidad de la biotecnología anaerobia al tratamiento de aguas residuales de baja carga (v.g. agua residual urbana) o a condiciones medioambientales extremas (v.g. bajas temperaturas de operación). Esta tecnología alternativa de tratamiento de aguas residuales es más sostenible que las tecnologías aerobias actuales ya que el agua residual se transforma en una fuente renovable de energía y nutrientes, proporcionando además un recurso de agua reutilizable. SAnMBR no sólo presenta las principales ventajas de los reactores de membranas (i.e. efluente de alta calidad, y pocas necesidades de espacio), sino que también presenta las principales ventajas de los procesos anaerobios. En este sentido, la tecnología SAnMBR presenta una baja producción de fangos debido a la baja tasa de crecimiento de los microorganismos implicados en la degradación de la materia orgánica, presenta una baja demanda energética debido a la ausencia de aireación, y permite la generación de metano, el cual representa una fuente de energía renovable que mejora el balance energético neto del sistema. Cabe destacar el potencial de recuperación de nutrientes del agua residual bien cuando el efluente es destinado a irrigación directamente, o bien cuando debe ser tratado previamente mediante tecnologías de recuperación de nutrientes. El objetivo principal de esta tesis doctoral es evaluar la viabilidad de la tecnología SAnMBR como núcleo en el tratamiento de aguas residuales urbanas a temperatura ambiente. Por lo tanto, esta tesis se centra en las siguientes tareas: (1) implementación, calibración y puesta en marcha del sistema de instrumentación, control y automatización requerido; (2) identificación de los parámetros de operación clave que afectan al proceso de filtración; (3) modelación y simulación del proceso de filtración; y (4) desarrollo de estrategias de control para la optimización del proceso de filtración minimizando los costes de operación. En este trabajo de investigación se propone un sistema de instrumentación, control y automatización para SAnMBR, el cual fue esencial para alcanzar un comportamiento adecuado y estable del sistema frente a posibles perturbaciones. El comportamiento de las membranas fue comparable a sistemas MBR aerobios a escala industrial. Tras más de dos años de operación ininterrumpida, no se detectaron problemas significativos asociados al ensuciamiento irreversible de las membranas, incluso operando a elevadas concentraciones de sólidos en el licor mezcla (valores de hasta 25 g·L-1 ). En este trabajo se presenta un modelo de filtración (basado en el modelo de resistencias en serie) que permitió simular de forma adecuada el proceso de filtración. Por otra parte, se propone un control supervisor basado en un sistema experto que consiguió reducir el consumo energético asociado a la limpieza física de las membranas, un bajo porcentaje de tiempo destinado a la limpieza física respecto al total de operación, y, en general, un menor coste operacional del proceso de filtración. Esta tesis doctoral está integrada en un proyecto nacional de investigación, subvencionado por el Ministerio de Ciencia e Innovación (MICINN), con título ¿Modelación de la aplicación de la tecnología de membranas para la valorización energética de la materia orgánica del agua residual y la minimización de los fangos producidos¿ (MICINN, proyecto CTM2008-06809- C02-01/02). Para obtener resultados representativos que puedan ser extrapolados a plantas reales, esta tesis doctoral se ha llevado a cabo utilizando un sistema SAnMBR que incorpora módulos comerciales de membrana de fibra hueca. Además, esta planta es alimentada con el efluente del pre-tratamiento de la EDAR del Barranco del Carraixet (Valencia, España). / Robles Martínez, Á. (2013). Modelling, simulation and control of the filtration process in a submerged anaerobic membrane bioreactor treating urban wastewater [Tesis doctoral]. Editorial Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34102 / TESIS / Premios Extraordinarios de tesis doctorales

Membrane filtration processes are widely utilized across different industrial sectors for biological and environmental separations. Examples of the former are sterile filtration and protein fractionation via microfiltration (MF) and ultrafiltration (UF) while drinking water treatment, tertiary treatment of wastewater, water reuse and desalination via MF, UF, nanofiltration (NF) and reverse-osmosis (RO) are examples of the latter. A common misconception is that the performance of membrane separation is solely dependent on the membrane pore size, whereas a multitude of parameters including solution conditions, solute concentration, presence of specific ions, hydrodynamic conditions, membrane structure and surface properties can significantly influence the separation performance and the membrane’s fouling propensity. The conventional approach for studying filtration performance is to use a single lab- or pilot-scale module and perform numerous experiments in a sequential manner which is both time-consuming and requires large amounts of material. Alternatively, high-throughput (HT) techniques, defined as the miniaturized version of conventional unit operations which allow for multiple experiments to be run in parallel and require a small amount of sample, can be employed. There is a growing interest in the use of HT techniques to speed up the testing and optimization of membrane-based separations. In this work, different HT screening approaches are developed and utilized for the evaluation and optimization of filtration performance using flat-sheet and hollow-fiber (HF) membranes used in biological and environmental separations. The effects of various process factors were evaluated on the separation of different biomolecules by combining a HT filtration method using flat-sheet UF membranes and design-of-experiments methods. Additionally, a novel HT platform was introduced for multi-modal (constant transmembrane pressure vs. constant flux) testing of flat-sheet membranes used in bio-separations. Furthermore, the first-ever HT modules for parallel testing of HF membranes were developed for rapid fouling tests as well as extended filtration evaluation experiments. The usefulness of the modules was demonstrated by evaluating the filtration performance of different foulants under various operating conditions as well as running surface modification experiments. The techniques described herein can be employed for rapid determination of the optimal combination of conditions that result in the best filtration performance for different membrane separation applications and thus eliminate the need to perform numerous conventional lab-scale tests. Overall, more than 250 filtration tests and 350 hydraulic permeability measurements were performed and analyzed using the HT platforms developed in this thesis. / Thesis / Doctor of Philosophy (PhD) / Membrane filtration is widely used as a key separation process in different industries. For example, microfiltration (MF) and ultrafiltration (UF) are used for sterilization and purification of bio-products. Furthermore, MF, UF and reverse-osmosis (RO) are used for drinking water and wastewater treatment. A common misconception is that membrane filtration is a process solely based on the pore size of the membrane whereas numerous factors can significantly affect the performance. Conventionally, a large number of lab- or full-scale experiments are performed to find the optimum operating conditions for each filtration process. High-throughput (HT) techniques are powerful methods to accelerate the pace of process optimization—they allow for multiple experiments to be run in parallel and require smaller amounts of sample. This thesis focuses on the development of different HT techniques that require a minimal amount of sample for parallel testing and optimization of membrane filtration processes with applications in environmental and biological separations. The introduced techniques can reduce the amount of sample used in each test between 10-50 times and accelerate process development and optimization by running parallel tests.