Proefschrift Universiteit van Amsterdam. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Purpose: Oral drug development had been hindered by the bioavailability issue despite vast market popularity. Lipid excipients have shown to enhance bioavailability of several reformulated hydrophobic oral drugs, yet the underlying mechanisms of action by lipids are still unclear. One proposed mechanism is that lipid could facilitate drug uptake by altering the activities of apical membrane intestinal efflux transporters. Thus, this study aimed to investigate the effects of specific monoglycerides on the efflux activity and protein expression of multidrug resistance-associated protein 2 (MRP2) in vitro. Methods: A preliminary study was first conducted to determine the effect of Peceol®, a mono- and di-glyceride mixture, on MPR2 efflux activity. Then, the 24- hour non-cytotoxic ranges of specific monoglycerides (1-monopalmitin, 1- monostearin and 1-monoolein) were determined using MTS and LDH assays in Caco-2 cells. Then, the effects of chosen monoglycerides on the functional activity of MRP2 were assessed via rhodamine 123 (Rh123) accumulation and estradiol 17 β-D-glucuronide(E₂17βG) bidirectional transport studies. The dose responses of Rh123 accumulation with each monoglyceride treatment were also determined. Lastly, Western blotting was used to probe the monoglycerides effect on MRP2 protein expression. Results: In the preliminary study, significant increase in Rh123 accumulation and decrease in E₂17βG efflux ratio were observed in Peceol® treated cells. The non-cytotoxic concentration ranges for 1-monopalmitin, 1-monostearin and 1- monoolein were within 1 mM, 1 mM and 500 μM, respectively. Cells treated with 1 mM 1-monoplamitin, 1 mM 1-monostearin, 500 μM 1-monoolein and 50 μM MK571 (a MRP2 inhibitor) resulted in significant increases in Rh123 accumulation and decreases in E₂17βG efflux ratio compared to the control (medium treated only). The three monoglycerides did not show Rh123 accumulation in a dose-responsive manner. MRP2 protein expressions in 1- monopalmitin and 1-monoolein treated cells were decreased by 19% and 35%, respectively; however, there was no change of MRP2 protein expression in 1- monostearin treated cells. Conclusions: These findings suggested that 1-monoolein, 1-monostearin and 1- monopalmitin could attenuate the activity of MRP2 and possibly other efflux transporters in Caco-2 cells. The reduction of efflux activity of MRP2 by 1- monoolein treatment could be partially explained by the non-specific down regulation of MRP2 protein expression.

Purpose: Oral drug development had been hindered by the bioavailability issue despite vast market popularity. Lipid excipients have shown to enhance bioavailability of several reformulated hydrophobic oral drugs, yet the underlying mechanisms of action by lipids are still unclear. One proposed mechanism is that lipid could facilitate drug uptake by altering the activities of apical membrane intestinal efflux transporters. Thus, this study aimed to investigate the effects of specific monoglycerides on the efflux activity and protein expression of multidrug resistance-associated protein 2 (MRP2) in vitro. Methods: A preliminary study was first conducted to determine the effect of Peceol®, a mono- and di-glyceride mixture, on MPR2 efflux activity. Then, the 24- hour non-cytotoxic ranges of specific monoglycerides (1-monopalmitin, 1- monostearin and 1-monoolein) were determined using MTS and LDH assays in Caco-2 cells. Then, the effects of chosen monoglycerides on the functional activity of MRP2 were assessed via rhodamine 123 (Rh123) accumulation and estradiol 17 β-D-glucuronide(E₂17βG) bidirectional transport studies. The dose responses of Rh123 accumulation with each monoglyceride treatment were also determined. Lastly, Western blotting was used to probe the monoglycerides effect on MRP2 protein expression. Results: In the preliminary study, significant increase in Rh123 accumulation and decrease in E₂17βG efflux ratio were observed in Peceol® treated cells. The non-cytotoxic concentration ranges for 1-monopalmitin, 1-monostearin and 1- monoolein were within 1 mM, 1 mM and 500 μM, respectively. Cells treated with 1 mM 1-monoplamitin, 1 mM 1-monostearin, 500 μM 1-monoolein and 50 μM MK571 (a MRP2 inhibitor) resulted in significant increases in Rh123 accumulation and decreases in E₂17βG efflux ratio compared to the control (medium treated only). The three monoglycerides did not show Rh123 accumulation in a dose-responsive manner. MRP2 protein expressions in 1- monopalmitin and 1-monoolein treated cells were decreased by 19% and 35%, respectively; however, there was no change of MRP2 protein expression in 1- monostearin treated cells. Conclusions: These findings suggested that 1-monoolein, 1-monostearin and 1- monopalmitin could attenuate the activity of MRP2 and possibly other efflux transporters in Caco-2 cells. The reduction of efflux activity of MRP2 by 1- monoolein treatment could be partially explained by the non-specific down regulation of MRP2 protein expression.

Purpose: Oral drug development had been hindered by the bioavailability issue despite vast market popularity. Lipid excipients have shown to enhance bioavailability of several reformulated hydrophobic oral drugs, yet the underlying mechanisms of action by lipids are still unclear. One proposed mechanism is that lipid could facilitate drug uptake by altering the activities of apical membrane intestinal efflux transporters. Thus, this study aimed to investigate the effects of specific monoglycerides on the efflux activity and protein expression of multidrug resistance-associated protein 2 (MRP2) in vitro. Methods: A preliminary study was first conducted to determine the effect of Peceol®, a mono- and di-glyceride mixture, on MPR2 efflux activity. Then, the 24- hour non-cytotoxic ranges of specific monoglycerides (1-monopalmitin, 1- monostearin and 1-monoolein) were determined using MTS and LDH assays in Caco-2 cells. Then, the effects of chosen monoglycerides on the functional activity of MRP2 were assessed via rhodamine 123 (Rh123) accumulation and estradiol 17 β-D-glucuronide(E₂17βG) bidirectional transport studies. The dose responses of Rh123 accumulation with each monoglyceride treatment were also determined. Lastly, Western blotting was used to probe the monoglycerides effect on MRP2 protein expression. Results: In the preliminary study, significant increase in Rh123 accumulation and decrease in E₂17βG efflux ratio were observed in Peceol® treated cells. The non-cytotoxic concentration ranges for 1-monopalmitin, 1-monostearin and 1- monoolein were within 1 mM, 1 mM and 500 μM, respectively. Cells treated with 1 mM 1-monoplamitin, 1 mM 1-monostearin, 500 μM 1-monoolein and 50 μM MK571 (a MRP2 inhibitor) resulted in significant increases in Rh123 accumulation and decreases in E₂17βG efflux ratio compared to the control (medium treated only). The three monoglycerides did not show Rh123 accumulation in a dose-responsive manner. MRP2 protein expressions in 1- monopalmitin and 1-monoolein treated cells were decreased by 19% and 35%, respectively; however, there was no change of MRP2 protein expression in 1- monostearin treated cells. Conclusions: These findings suggested that 1-monoolein, 1-monostearin and 1- monopalmitin could attenuate the activity of MRP2 and possibly other efflux transporters in Caco-2 cells. The reduction of efflux activity of MRP2 by 1- monoolein treatment could be partially explained by the non-specific down regulation of MRP2 protein expression. / Pharmaceutical Sciences, Faculty of / Graduate

Le MTX est un agent anticancéreux utilisé à hautes doses pour le traitement des hémopathies malignes et de certaines tumeurs solides. Il présente une importante variabilité pharmacocinétique (PK) traduite par des surexpositions à l'origine de toxicités très sévères, surtout lors d'une administration à haute dose. Les retards d'élimination du MTX surviennent encore de manière inattendue et il n'existe à ce jour aucun biomarqueur qui permette un diagnostic précoce du risque de surexposition. Nos travaux ont focalisé sur les déterminants de l'élimination rénale du MTX, et en particulier le rôle du transporteur MRP2/ABCC2 dans ce processus. Ce travail s'inscrit donc (1) dans la recherche de biomarqueurs métabolomiques urinaires prédictifs de la PK du MTX et (2) dans l'identification de substrats endogènes de MRP2 parmi un panel de 217 acides organiques urinaires analysés par chromatographie gazeuse couplée à la spectrométrie de masse. Nos analyses ont abouti à un profil de 28 anions organiques endogènes, prédictifs de la CL MTX. L'outil était en revanche mal adapté à la prédiction des retards d'élimination. Pour la 2eme partie, nos résultats tendent à montrer que 8 métabolites urinaires sont des bio-marqueurs potentiels de l'activité de MRP2. Leur utilisation en clinique nécessite encore des études confirmatoires. / MTX is an anticancer agent used at high doses for the treatment of malignant haemopathies and some solid tumors. It presents an important pharmacokinetic variability (PK), manifested by overexposures causing very severe toxicities, especially when administered at high doses. Delayed elimination of MTX still occurs unexpectedly and there is currently no biomarker that allows early diagnosis of the risk of overexposure. Our work focused on the determinants of renal elimination of MTX, and particularly on the role of MRP2 / ABCC2 in this process. This work is therefore devoted to (1) the search for metabolomic biomarkers predictive of MTX PK and (2) the identification of endogenous substrates of MRP2, from a panel of 217 urinary organic acids analyzed by gas chromatography-mass spectrometry. Our analyses resulted in a profile of 28 endogenous organic anions, predictive of CL MTX. The tool was, on the other hand, poorly adapted to the prediction of delayed elimination. For the second part, our results tend to show that 8 urinary metabolites are potential biomarkers of MRP2 activity. Their clinical use still requires confirmatory studies.

HINTERGRUND UND ZIELE: Das zentrale Ziel dieser Studie war es der personalisierten Medizin einen Schritt näher zu kommen, bei der für jeden Patienten für die entsprechende Erkrankung das optimale Arzneimittel in der optimalen Dosierung gewählt wird. Dazu ist es notwendig herauszufinden, wie hoch der genetische Anteil auf die Wirkungsweise von Medikamenten ist. Wenn Ergebnisse von klinischen Studien ausreichend belegen, dass der Einfluss von genetischen Faktoren bedeutsam für die Wirkungsweise von Medikamenten ist, kann sich eine genetische Analyse vor Therapiebeginn als sinnvoll erweisen. In dieser Studie erfolgten Untersuchungen zur erblichen Variation der Aktivitäten der Efflux-Transportproteine MDR1 und MRP2. Um den Einfluss von Genen und Umweltfaktoren auf interindividuelle Unterschiede zu erforschen, eignen sich am besten Zwillingsstudien. METHODEN: So haben wir eine Zwillingsstudie mit 20 monozygoten und 9 dizygoten gleichgeschlechtlichen Zwillingen durchgeführt. Dabei wurde Talinolol als in-vivo-Testsubstanz für die Aktivität der Membran-Transportproteine MDR1 und MRP2 analysiert. Es wurden an drei verschiedenen Studientagen, die mindestens eine Woche auseinander liegen mussten, jeweils die gleiche Menge an Talinolol oral verabreicht und anschließend in festgelegten regelmäßigen Abständen die Blutkonzentrationen und weitere pharmakokinetische Parameter bestimmt. Da Talinolol nahezu nicht metabolisiert und unverändert wieder ausgeschieden wird, hängt dessen Bioverfügbarkeit stark von der Funktion und Expression seiner Transporter (MDR1 und MRP 2) ab. Die Erblichkeit wurde mittels drei unterschiedlicher Formeln berechnet. ERGEBNIS: In dieser Studie zeigte sich eine insgesamt große Variabilität der Blutkonzentrationsverläufe sowohl zwischen den Personen (interindividuelle Variabilität) als auch innerhalb einer Person zwischen den unterschiedlichen Studientagen (intraindividuelle Variabilität). Desweiteren war die Variabilität unter monozygoten Zwillingen größer als die unter dizygoten Zwillingen. Diese Konstellationen und die daraus errechnete Erblichkeit sprechen für einen geringen genetischen Einfluss und einen großen Einfluss von Umweltfaktoren auf die Variation in der Pharmakokinetik von Talinolol. Bezüglich der verschiedenen Genvarianten von MDR1 und MRP2 konnten keine signifikanten Unterschiede in den Blutkonzentrationsverläufen gezeigt werden. FAZIT: Es zeigte sich insgesamt ein geringer genetischer Einfluss auf die Variation in der Pharmakokinetik der In-vivo Testsubstanz Talinolol. Desweiteren zeigten die Probanden mit verschiedenen Genvarianten auch keine signifikanten Unterschiede in den Konzentrationsverläufen, sodass in dieser Studie kein relevanter Zusammenhang zwischen dem Vorhandensein bestimmter Genvarianten und der Transporteraktivität von MDR1 und MRP2 gezeigt werden konnte.

Tumorzellen können vielfältige Resistenzmechanismen gegenüber Zytostatika entwickeln. Untersuchungen an drei humanen Tumorzellinien und ihren cisplatinresistenten Varianten zeigten eine Assoziation von erhöhter MRP2-Expression und dem Auftreten von Cisplatinresistenz. Darüberhinaus waren die cisplatinresistenten Zellinien gegenüber Carboplatin kreuzresistent. Um weitere Faktoren im Zusammenhang mit der Cisplatinresistenz zu untersuchen, wurde der Mutationsstatus von p53 und der zelluläre Glutathiongehalt in den Zellinien bestimmt. Der offene Leserahmen von MRP2 aus der cisplatinresistenten Ovarialkarzinomzellinie A2780RCIS wurde für die Transfektion in die cisplatinsensitive Zellinie A2780 genutzt. Die Transfektanten zeigten eine Überexpression von MRP2 und wiesen eine Resistenz gegenüber Cisplatin und Carboplatin auf. Dies konnte in Zellzyklus- und Apoptose-Untersuchungen unter Cisplatinbehandlung gestätigt werden. Durch computergestützte Faltungsanalysen von Abschnitten der MRP2-mRNA wurden zwei potentielle Ribozymschnittstellen ausgewählt. Die konstruierten Anti-MRP2-Hammerhead-Ribozyme RzM1 und RzM2 wurden im zellfreien System auf ihre Schnittaktivität getestet und erwiesen sich als katalytisch aktiv. Es wurden verschiedene kinetische Parameter für RzM1 und RzM2 ermittelt und mit anderen Ribozymen verglichen. Die Ribozyme zeigten eine gute Effektivität bei der Spaltung ihres Zielmoleküls, wobei RzM1 die höhere Effektivität aufwies. Beide Ribozyme wurden auf ihre Wirksamkeit durch Transfektion in die Zellinie A2780RCIS getestet. Die untersuchten Transfektanten zeigten eine geringere Expression von MRP2 auf mRNA- und Proteinebene und wiesen eine verminderte Resistenz gegenüber Cisplatin, Carboplatin, Daunorubicin und Etoposid auf. Die Ribozyme RzM1 und RzM2 waren gleichermaßen für die Expressionsregulierung von MRP2 in Tumorzellen geeignet. In Zellzyklus- und Apoptose-Untersuchungen wurde funktionell bestätigt, daß die A2780RCIS-Anti-MRP2-Ribozym-Transfektanten auf eine Cisplatinbehandlung stärker ansprechen als die cisplatinresistente Ausgangszellinie A2780RCIS. Die Anwendung der Ribozyme RzM1 und RzM2 zur Unterstützung der Chemotherapie von Tumorzellen scheint daher vielversprechend. / Tumour cells can develop a lot of resistance mechanisms against cytostatic drugs. Examinations of three human tumour cell lines and their cisplatinresistant variants showed an association of elevated MRP2 expression and the occurrance of cisplatinresistance. Moreover, the cisplatinresistant cell lines were crossresistant against carboplatin. To examine further factors in context of cisplatinresistance the mutation status of p53 and the cellular glutathione content of the cell lines were determined. The MRP2-open reading frame of the cisplatinresistant ovarian carcinoma cell line A2780RCIS was used for transfection into the cisplatinsensitive cell line A2780. The transfectants showed an overexpression of MRP2 and a resistance against cisplatin and carboplatin. This could be confirmed with analysis of the cell cycle and apoptosis induction after treatment with cisplatin. Using computer aided folding analysis of MRP2 mRNA parts two possible ribozyme cleavage sites were selected. The constructed anti-MRP2 hammerhead ribozymes RzM1 and RzM2 were tested in a cell-free system with respect to their cleavage activities and were found to be catalytic active. Various kinetic parameters of RzM1 and RzM2 were determined and compared with other ribozymes. The ribozymes showed a good effectivity for the substrate cleavage, although RzM1 had the better effectivity. Both ribozymes were tested for their effectiveness after transfection into the cell line A2780RCIS. The transfectants showed a lower MRP2 expression on mRNA and protein level and also a reduced resistance against cisplatin, carboplatin, daunorubicin, and etoposide. The ribozymes RzM1 and RzM2 were both equally suitable for the regulation of MRP2 expression in tumour cells. Analysis of cell cycle and apoptosis induction could confirm functionally the higher sensitivity of the A2780RCIS-anti-MRP2 ribozyme transfectants after treatment with cisplatin in comparison to the cisplatinresistant cell line A2780RCIS. Therefore, the application of the ribozymes RzM1 and RzM2 for the support of cancer chemotherapy seems to be promising.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Streptococcus pneumoniae (S. pneumoniae) is a Gram-positive, encapsulated bacterium capable of causing significant morbidity and mortality throughout the world. A hallmark of S. pneumoniae infection is infiltration of neutrophils (PMNs) that assist in controlling the spread infection but may also contribute to pathology. Paradoxically, studies have shown that limiting PMN infiltration into the lumen of the lung during infection actually betters clinical outcome in experimental S. pneumoniae infection. The final step in PMN luminal trafficking is a Hepoxilin A3 (HXA3)-dependent migration across the pulmonary epithelium. HXA3 is a PMN chemoattractant that forms gradients along the polarized epithelial face, drawing PMNs from the basolateral to the apical surface during proinflammatory responses. HXA3 requires assistance of an integral- membrane protein transporter to escape the cell and form the gradient. The pulmonary HXA3 transporter is currently unidentified. In this work, we identify the pulmonary HXA3 transporter as the ATP-Binding Cassette Transporter (ABC transporter) Multi-drug Resistance Associated Protein 2 (ABCC2, MRP2). We demonstrate that MRP1 and MRP2 are divergent ABC- transporters that control transepithelial PMN migration through efflux of a distinct anti-inflammatory substance and the pro-inflammatory HXA3 in the context of Streptococcus pneumoniae infection. Enrichment of MRP2 on the plasma membrane requires detection of the bacterial virulence factors pneumolysin (PLY) and hydrogen peroxide. PLY and hydrogen peroxide not only coordinate MRP2 apical membrane enrichment but also influence HXA3-dependent PMN transepithelial migration. They influence migration through stimulation of epithelial intracellular calcium increases that are crucial for HXA3 production as well as MRP2 translocation to the plasma membrane. PLY and hydrogen peroxide are not sufficient in their signaling alone, however, and require at least one additional bacterial signal to induce HXA3/MRP2 proinflammatory activities.

Multidrug resistance protein 2 (MRP2) is the second member the C subfamily in the superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) efflux transporters. MRP2 is a critical player for generation of bile acidindependent bile flow and biliary excretion of glutathione, glucuronate and sulfate conjugates of endo- and xenobiotics. Dysfunctional expression of MRP2 is associated with Dubin-Johnson Syndrome. Pathological and physiological states or xenobiotics change the MRP2 expression level. Under some conditions, expression of the human MRP2 and rat Mrp2 proteins are regulated at the translation level. There are several transcription initiation sites in MRP2/Mrp2 gene. The 5’ untranslated regions (5’UTRs) of MRP2/Mrp2 contains multiple translation start codons. The focus of this study, therefore, was investigation of the translational regulatory mechanisms mediated by the upstream open reading frames (uORF) of MRP2/Mrp2. Using in vitro translation assays and transient cotransfection assays in HepG2 cells, we showed that the rat uORF1 starting at position -109 (relative to the ATG of Mrp2) and the human uORF2 starting at position -105 (relative to the ATG of MRP2) are two major cis-acting inhibitors of translation among the rat and human multiple uORFs, respectively. Translational regulation mediated by the uORFs in the rat Mrp2 mRNA is a combined effect of the leaky scanning model and the reinitiation model, and also results from interaction of the multiple uORFs. In addition, by Ribonuclease Protection Assays (RPA), we detected multiple transcription initiation sites of MRP2/Mrp2 gene in tissues. We also found that the relative abundance of the rat Mrp2 mRNA isoforms with different 5’UTRs differed in the rat liver, kidney, jejunum, ileum, placenta, and lung. This is the first study on the translational regulatory mechanisms of the MRP2/Mrp2 gene.