The nasal route has primarily been used to deliver drugs for the treatment of local diseases such as nasal infections, nasal congestion and allergies. The nasal route can also be used as a non-invasive alternative route to deliver drugs systemically when a rapid onset of action and/or avoidance of hepatic metabolism are desired. Moreover, there is a growing interest in the use of this route for direct transport of drugs from the nose to the brain. Most of the drugs that have been studied for nasal delivery are either small molecules which are lipophilic enough to passively diffuse through the nasal epithelia or macromolecules where bioavailabilities less than 1% are clinically effective and acceptable. This study focused on identifying carrier proteins or transporters in the nasal mucosa that could improve the absorption of specific drug substrates across the nasal respiratory and olfactory epithelia. The presence of drug transporters in the nasal mucosa of humans and commonly used animal models were investigated. DNA microarray results for nasal samples from humans and two commonly used models, mice and rats, were obtained from GenBank and were analyzed in collaboration with the University of Iowa Center for Bioinformatics and Computational Biology. While cow tissues are frequently used in in-vitro nasal permeability analyses, there is limited information available in GenBank for this species. Both DNA microarray analysis and RT-PCR were performed on bovine nasal explants to determine transporter expression. Good agreement between the microarray and RT-PCR results was observed. While human and three animal species commonly used as models in nasal drug delivery research (mouse, rat, and cow) show similar patterns of expression for several transporters, interspecies differences in the level of expression were observed. Therefore, the expression level of transporters remains a factor to consider when translating results obtained using animal models to humans. The nucleoside transporter family was selected for further evaluation of the potential to improve the nasal absorption of substrates. Nucleoside transporters are integral proteins responsible for mediating and facilitating the flux of nucleosides across cellular membranes; they are also known to be responsible for the uptake of nucleoside analog drugs such as anti-cancer and anti-viral agents. RT-PCR and Western blotting were used to verify the presence of two transporter subtypes, ENT1 and CNT3, in the bovine nasal respiratory and olfactory mucosa. The expression level of both transporters in the respiratory mucosa was comparable to that in the olfactory mucosa. Using immunohistochemistry, ENT1 and CNT3 were found to be localized primarily at the apical surface of the nasal epithelial cells. This indicates that the nasal epithelium likely absorbs exogenous nucleosides for intracellular uses such as nucleic acid synthesis and regulating other cellular activities. The contribution of the nucleoside transporters to the permeation of a nucleoside analogue drug, alovudine, across the nasal epithelia was also studied. The transport of alovudine showed a non-linear increase with increasing donor concentration over the range of 50 to 3000 µM which suggests that nucleoside transporters play a role in its uptake. Polarized transport was not observed suggesting that the facilitative nature of ENT1 plays a major role in alovudine transport. S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an ENT1 inhibitor, incompletely decreased alovudine permeability across the nasal mucosa. This demonstrates that at least one transporter, ENT, plays a significant role in the uptake of this nucleoside drug across the nasal mucosa.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and toxikology Student: Zuzana Matiašková Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Role of selected ABC and SLC transporters in transmembrane permeability of maraviroc: effect on transport in placenta Antiretroviral drug maraviroc is an inhibitor of CCR5-trophic HIV virus and belongs to the group of entry inhibitors. Nowadays, maraviroc is administered as part of combination antiretroviral therapy (cART) primarily in adults, children over the age of two and pregnant women to reduce the risk of transmission of HIV to the fetus. The knowledge of interactions of maraviroc with drug transporters in placenta is crucial for optimizing the therapy during pregnancy, both in terms of efficacy and potential adverse effects. Maraviroc is known substrate of ABCB1 transporter, which plays a protective role to the fetus by its efflux activity in the apical membrane of trophoblast. However, the results of recent study employing dually perfused human placenta suggest involvement of other transport mechanisms in the maraviroc transplacental pharmaocokinetics, especially those operating in the opposite direction to ABCB1. The aim of this study was to evaluate in vitro studies whether, besides ABCB1,...

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Current in vitro cellular methods utilised in drug metabolism and pharmacokinetic (DMPK) studies during drug development do not provide the 3D structure and functions of organs found in vivo, such that resulting in vitro-in vivo extrapolation (IVIVE) may not always accurately reflect clinical outcome. This highlights the need for the development of new dynamic in vitro cell models to aid improvement of IVIVE. The aim of this project was to incorporate characterised primary renal cells within a hollow fibre bioreactor for use in DMPK studies investigating renal clearance. Fluorescence based assays were developed to assess the functionality of three drug transporters involved in the renal transport of pharmaceutical compounds: P-gp, BCRP and OCT2. The developed assays were then applied alongside transporter visualisation and genetic expression assays to characterise primary human proximal tubule cells over a series of population doublings. Cells at a population doubling of 5 demonstrated the best transporter activity whilst allowing cells to be expanded in vitro. Polysulfone (PSF) based membranes, which are widely used in dialysis components were developed by blending additives to improve renal cell attachment and culture. The membranes exhibited a characteristic porous internal structure with smooth skin layers on the surface, and were able to be sterilised via autoclaving due to their high thermal stability. PSF blended with polyvinylpyrrolidone (PVP) was the most hydrophilic with cell metabolic activity similar to standard tissue culture plastic. The production of hollow fibres of varying thicknesses and properties from the PSF and PVP blend yielded a marked difference in renal cell attachment and long term viability. Fibres incorporated into glass casings to produce the single hollow fibre bioreactors (HFBs) were able to be sterilised by autoclaving whilst remaining intact. Due to the variation of fibre integrity within the batch, many fibres exhibited tears within the HFBs. This ultimately led to cell depletion within the fibre over the culture period; however, intact fibres demonstrated an increase in cell growth towards the end of the culture period under flow conditions. These results demonstrate the progress made towards a small scale in vitro renal model incorporating characterised primary renal cells to aid the improvement of IVIVE in DMPK research.

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Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Ronette Gehring / Milk composition has a dynamic nature, and the composition varies with stage of lactation, age, breed, nutrition and health status of the udder. The changes in milk composition seem to match the changes in the expression of membrane proteins in secretory mammary epithelial cells that are needed for the movement of molecules from blood to milk and vice versa (Nouws and Ziv, 1982). Thus, an understanding of transporter expression, function and regulation in mammary epithelial cells can provide insight into mammary gland function and regulation. The goal of this project was to elucidate (molecularly and functionally) the role of drug transporters in the barrier function of an epithelial monolayer cultured from an immortalized bovine mammary epithelial cell line (BME-UV). To characterize the regulation (expression and function) of these drug transporters in BME-UV cells after exposure to cytokine TNF-α for selected periods of time. Representative members of drug transporters of the SLC (OCT and OAT) and ABC (P-glycoprotein) superfamilies were chosen for this project. In the first study, the involvement of a carrier-mediated transport system in the passage of organic cation (TEA) and anion (EsS) compounds was elucidated across the BME-UV monolayer. In the second study, molecular and functional expression of bOAT isoforms in BME-UV cells were studied. The final study characterized the effects of cytokine TNF-α on the expression and function of P-glycoprotein, an efflux pump, in BME-UV cells. Cytokine TNF-α exposure induced the expression of ABCB1 mRNA and increased P-glycoprotein production in BME-UV cells, resulting in a greater efflux of digoxin, a known P-glycoprotein substrate, back into the apical fluid. The expression, function, and regulation of these transporters in the mammary gland has important implications for understanding the barrier function of the mammary epithelium and, in more specific, for characterizing the role of these transporters in the accumulation and/or removal of specific substrates from milk and/or plasma. Moreover, this study provides an in vitro cell culture model of mammary epithelium to characterize mammary epithelial cell function during inflammation.

BACKGROUND:Active HIV infection within the central nervous system (CNS) is confined primarily to microglia. The glial cell compartment acts as a viral reservoir behind the blood-brain barrier. It provides an additional roadblock to effective pharmacological treatment via expression of multiple drug efflux transporters, including P-glycoprotein. HIV/AIDS patients frequently suffer bacterial and viral co-infections, leading to deregulation of glial cell function and release of pro-inflammatory mediators including cytokines, chemokines, and nitric oxide.METHODS:To better define the role of inflammation in decreased HIV drug accumulation into CNS targets, accumulation of the antiretroviral saquinavir was examined in purified cultures of rodent microglia exposed to the prototypical inflammatory mediator lipopolysaccharide (LPS).RESULTS:3H]-Saquinavir accumulation by microglia was rapid, and was increased up to two-fold in the presence of the specific P-glycoprotein inhibitor, PSC833. After six or 24 hours of exposure to 10 ng/ml LPS, saquinavir accumulation was decreased by up to 45%. LPS did not directly inhibit saquinavir transport, and did not affect P-glycoprotein protein expression. LPS exposure did not alter RNA and/or protein expression of other transporters including multidrug resistance-associated protein 1 and several solute carrier uptake transporters.CONCLUSIONS:The decrease in saquinavir accumulation in microglia following treatment with LPS is likely multi-factorial, since drug accumulation was attenuated by inhibitors of NF-kappabeta and the MEK1/2 pathway in the microglia cell line HAPI, and in primary microglia cultures from toll-like receptor 4 deficient mice. These data provide new pharmacological insights into why microglia act as a difficult-to-treat viral sanctuary site.

La survenue d’un cancer du sein découvert durant la grossesse est un événement dramatique compliquant entre 1/3000 et 1/10000 grossesses, ce qui en fait le cancer le plus fréquemment rencontré chez la femme enceinte. Sur le plan thérapeutique, certaines molécules anticancéreuses peuvent être utilisées, notamment les taxanes (paclitaxel et docétaxel). Si les études cliniques rétrospectives isolées semblent plutôt rassurantes, les données concernant leur passage transplacentaire sont encore fragmentaires. Quant à leurs effets sur le placenta humain et plus particulièrement sur la fonction de transport placentaire, ils sont pour l’heure inconnus. Nos objectifs étaient de 1) dresser une cartographie de l’expression génique physiologique des différents transporteurs placentaires de médicaments en utilisant un modèle de culture primaire trophoblastique, 2) d’apprécier le passage transplacentaire comparatif des taxanes et leur accumulation placentaire en utilisant le modèle du cotylédon perfusé, 3) d’étudier plus particulièrement les effets du paclitaxel sur le placenta humain et notamment sur l’expression des transporteurs de médicaments, en utilisant en plus des modèles mentionnés, les cotylédons de patientes ayant été traitées par paclitaxel durant leur grossesse. Nos études ont tout d’abord permis de dresser une cartographie originale de l’expression physiologique de plus de 80 transporteurs placentaires de médicaments, et ce comparativement entre le début et la fin de la gestation. De plus, nos expériences ont montré que le passage transplacentaire des taxanes était faible et comparable entre les deux molécules, et que celles-ci semblaient s’accumuler dans les cotylédons placentaires. Enfin, nous avons pu mettre en évidence un effet significatif du paclitaxel sur le placenta humain, notamment sur la modulation de certains transporteurs de médicaments. / The occurrence of breast cancer during pregnancy is a dramatic event reaching roughly 1/3000 to 1/10000 pregnancies, this type of cancer being the most frequent in pregnant women. Regarding therapeutic options, some anticancer agents may be used, especially taxanes (paclitaxel and docetaxel). If most of retrospective data appear to be reassuring, little is known regarding their transplacental transfer. Moreover, to our knowledge, potential effects of taxanes on human placenta, especially on placental transport function are unknown. Our aims were to 1) provide a transcriptional expression cartography of various placental drug transporters throughout pregnancy, using primary trophoblast culture model, 2) assess the comparative transplacental transfer of taxanes and their accumulation in cotyledons, using the perfused placental model, 3) assess potential effects of paclitaxel on human placenta, especially on drug transporter expression, not only using above-described models, but also cotyledons from pregnant-cancer patients treated with paclitaxel during pregnancy. Here, we finally provided an original transcriptional cartography of various drugs transporters in human normal placenta all along pregnancy. Moreover, we found a low and comparable transplacental transfer of paclitaxel and docetaxel that led to a moderate accumulation in cotyledons. Finally, we evidenced a significant effect of paclitaxel on human placenta, especially by modulating drug transporter expression.