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.

One major reason of CNS pharmacotherapy's impediment is the existence of "barriers" between blood and CNS, especially the Blood-Brain Barrier (BBB), a neurovascular structure localized at the level of brain microvasculature. Main factors responsible for this barrier function are drug efflux transporters type ABC (ATP-Binding Cassette) and SLC (SoLute Carrier) expressed at BBB level and known to be at the origin of multi-drug resistance phenomenon. Recent researches aim at unraveling the signaling mechanisms regulating these transporters in order to modulate their activity and improve pharmacotherapy in brain diseases. For years, these transporters have been studied in adult organism. But, there is a wide spread belief that the BBB in embryo, fetus, new born and infant is "immature", implying caution in giving drugs to infants. However, current knowledge on the functional status of the BBB in immature organism remains very limited.This study was performed in the aim of understanding: 1) The ontogenesis of ABC and SLC transporters during brain maturation, 2) the functional role of four BBB drug efflux transporters (P-glycoprotein (P-gp), Breast Cancer Resistance Protein (bcrp), Organic Anion Transporter 3 (oat3), and Transporting Peptide 1a4 (oatp1a4) transporters) in children's brain, and 3) the mechanisms that regulate their functional expression under normal and pathological conditions, mostly under inflammatory conditions, because indeed alterations in structural and functional components of the BBB have been reported in a long list of CNS pathologies in adults. Our results showed changing properties of the BBB during ontogenesis, as well as an age-related differential regulation of BBB drug efflux transporters under normal and inflammatory conditions.These findings highlight the importance of considering an age-related response of CNS to drugs and of taking into account the specific properties of juvenile BBB during definition of therapeutic strategies designed to treat childhood brain diseases, and this in the clinical perspective of developing new drugs with enhanced efficacy in children's CNS.

Membrane transport proteins are known to influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. At the onset of this thesis work, only a few structure-activity models, in general describing P-glycoprotein (Pgp/ABCB1) interactions, were developed using small datasets with little structural diversity. In this thesis, drug-transport protein interactions were explored using large, diverse datasets representing the chemical space of orally administered registered drugs. Focus was set on the ATP-binding cassette (ABC) transport proteins expressed in the canalicular membrane of human hepatocytes. The inhibition of the ABC transport proteins multidrug-resistance associated protein 2 (MRP2/ABCC2) and bile salt export pump (BSEP/ABCB11) was experimentally investigated using membrane vesicles from cells overexpressing the investigated proteins and sandwich cultured human hepatocytes (SCHH). Several previously unknown inhibitors were identified for both of the proteins and predictive in silico models were developed. Furthermore, a clear association between BSEP inhibition and clinically reported drug induced liver injuries (DILI) was identified. For the first time, an in silico model that described combined inhibition of Pgp, MRP2 and breast cancer resistance protein (BCRP/ABCG2) was developed using a large, structurally diverse dataset. Lipophilic weak bases were more often found to be general ABC inhibitors in comparison to other drugs. In early drug discovery, in silico models can be used as predictive filters in the drug candidate selection process and membrane vesicles as a first experimental screening tool to investigate protein interactions. In summary, the present work has led to an increased understanding of molecular properties important in ABC inhibition as well as the potential influence of ABC proteins in adverse drug reactions. A number of previously unknown ABC inhibitors were identified and predictive computational models were developed.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) techniques offer the opportunity to understand the physiological processes involved in the distribution of the contrast agent in vivo. This work utilises a liver specific contrast agent (gadoxetate) and demonstrates the potential use of these techniques to study transporter-mediated process in vivo. In vitro experiments investigated gadoxetate’s interaction with uptake and efflux transporters at the cellular level, ideally a prerequisite to understand the contribution of transporter proteins in in vivo pharmacokinetics. MRI was used to measure the accumulation of gadoxetate in fresh rat hepatocytes. Furthermore, LC-MS/MS methodology was optimised in conjunction with two in vitro systems to determine the role of transporters in gadoxetate uptake and efflux. HEK-OATP1B1 transfected cells were used to optimise the LC-MS/MS technique and Caco-2 cell monolayers were used to examine whether gadoxetate is a substrate of the efflux transporters, Pgp and BCRP. Subsequent studies demonstrated the use of DCE-MRI techniques to study transporter-mediated processes. Two pharmacokinetic models were proposed to quantify the uptake and efflux of gadoxetate in vivo. The suitability of the models in describing the liver concentration profiles of gadoxetate was assessed in pre-clinical and clinical reproducibility studies. Further pre-clinical experiments demonstrated the ability of the proposed DCE-MRI techniques to monitor changes in the uptake and efflux rate estimates of gadoxetate into hepatocytes, through co-administration of the transporter inhibitor, rifampicin, at two doses. The work presented demonstrates the potential use of DCE-MRI techniques as a diagnostic probe to assess transporter-mediated processes and drug-drug interactions (DDIs) in vivo.