The aims of this thesis were to study the in vivo relevance of membrane transporters for intestinal absorption and the hepatic disposition of drugs in humans and preclinical models. Fexofenadine is a substrate for ABCB1 (P-glycoprotein) and members of the organic anion transporting polypeptide (OATP/SLCO) family. It is marginally metabolised in humans. The influence of known inhibitors of ABCB1 and OATPs on the membrane transport and pharmacokinetics of fexofenadine was investigated in Caco-2 and porcine models and in humans. The permeability of fexofenadine remained low, even when significantly altered by the addition of an inhibitor. Using the Loc-I-Gut® technique in vivo in humans, it was possible to see that the jejunal effective permeability of fexofenadine was unchanged when given with verapamil. However, the systemic exposure and apparent absorption rate of fexofenadine increased. This suggests that the first-pass liver extraction of fexofenadine was reduced by verapamil, probably through the inhibition of sinusoidal OATP-mediated and/or canalicular ABCB1-mediated secretion. The unchanged permeability can be explained by simultaneous inhibition of jejunal apical OATP-uptake and ABCB1-efflux, which would leave fexofenadine to be transported by passive trancellular diffusion. A Loc-I-Gut® perfusion in the porcine model enabling blood sampling in the portal and hepatic veins and bile collection revealed increased jejunal permeability, but no subsequent verapamil-induced elevation in the systemic exposure of fexofenadine. This indicates a species-related difference in the localisation of and/or the substrate specificity of fexofenadine for the transporters involved. The absence of an effect on the first-pass liver extraction in the porcine model might be caused by the observed lower liver exposure of verapamil. Finally, a novel intubation technique enabling dosing of fexofenadine in the jejunum, ileum and the colon showed that fexofenadine was absorbed less along the length the intestine in agreement with the properties of a low permeability drug.

<p>Insects are continually exposed to potentially toxic endogenous compounds and xenobiotics that require rapid elimination from the body. Xenobiotic resistance in insects has evolved predominantly by increasing the activity of detoxification enzymes and/or by increasing toxin excretion via the Malpighian (renal) tubules. The tubules have long been known to transport organic anions at high rates. This thesis examines the mechanisms of excretion and detoxification of the organic anion methotrexate (MTX) by isolated tissues of the fruit fly <em>Drosophila melanogaster</em>. A radioisotope tracer technique and the Ramsay assay were used to measure MTX secretion. Quantitative PCR (qPCR) was used to evaluate the expression of the genes for putative organic anion transporters. My results show that MTX transport across the Malpighian tubule epithelium is active, saturable, Na⁺-independent and inhibited by a wide range of organic anions including MK-571, probenecid and Texas Red. Pharmacological studies and qPCR analyses suggest multiple transporters are involved in the movement of MTX across the Malpighian tubules. Moreover, chronic exposure of larvae to dietary MTX or salicylate dramatically increases the transepithelial transport of MTX by isolated Malpighian tubules, suggesting that excretion of MTX is upregulated by exposure to these organic anions in the diet. In addition, treatments known to increase expression of specific detoxification enzymes, such as the P450 monoxygenases (P450s) and the glutathione-S-transferases (GSTs), also led to an increase in expression levels of multidrug efflux transporter (MET), multidrug resistance like protein 1 (dMRP) as well as to increased secretion of MTX by the tubules. This latter finding suggests a coordinated response to toxin exposure, so that when detoxification pathways are increased, there is a corresponding increase in the capacity for elimination of the products of P450 and GST enzymes. Finally, the last section of this thesis has shown that RNAi knockdown of a single organic anion transporter gene in the principal cells of <em>D. melanogaster</em> Malpighian tubules is associated with reductions in the expression of multiple, functionally-related genes. Importantly, these results indicate that dMRP andMET are not the dominantMTX transporters in the tubules when flies are reared onMTX-enriched diets. However, reductions in the expression of organic anion transporting polypeptide (OATP) are associated with reduced secretion of the organic anionsMTX, fluorescein and Texas Red. Taken together, these results suggest that OATP and at least one additional transporter, as yet unidentified, are required forMTX secretion. In conclusion, the results of my research contribute to our understanding of the mechanisms of organic anion detoxification and excretion in flies exposed to dietary toxins.</p> / Doctor of Science (PhD)

No description available.

Numerous natural products are widely used as first-line/alternative therapeutics and dietary supplements in both western and eastern society. However, the safety and efficacy profiles for herbal products are still limited. Organic anion transporters (OATs; SLC22 family) are expressed in many barrier organs and mediate in vivo body disposition of a broad array of endogenous substances and clinically important drugs. As some dietary flavonoids and phenolic acids were previously demonstrated to interact with OATs, it is necessary to explore the potential interaction of such components found in natural products in order to avoid potential OAT-mediated drug-drug interactions (DDIs). The inhibitory effects of 23 natural products were assessed on the function of human (h) OATs, hOAT1 (SLC22A6), hOAT3 (SLC22A7), and hOAT4 (SLC22A11) and/or the murine (m) orthologs mOat1 and mOat3. For compounds exhibiting marked inhibition at initial screening, dose-response curves (IC50 values) and DDI indices were determined. At the initial screening concentrations, 14, 19, and 2 test compounds exhibited significant inhibition on hOAT1, hOAT3, and hOAT4, respectively. Additionally, all test Danshen (a Chinese herbal medicine) hydrophilic components significantly reduced mOat1- and mOat3-mediated substrate uptake at 1 mM. For selected compounds, the IC50 and Ki values were estimated to be in the micromolar or even nanomolar range. Considering the clinical plasma concentration and unbound fraction in plasma, DDI indices for gallic acid, gentisic acid, lithospermic acid, protocatechuic acid, rosmarinic acid, salvianolic acid B, and tanshinol indicated DDIs may occur in vivo in situations of co-administration of these compounds and clinical therapeutics known to be OAT substrates. Finally, a new, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify gallic acid and gentisic acid in cell lysates in order to measure cellular uptake of these compounds in mOat1- or mOat3-expressing cells. Significant cellular uptake of gallic acid was observed in mOat1-expressing cells, compared with background control cells. The absorptive uptake was completely blocked by probenecid (known OAT inhibitor) at 1 mM. These results indicate that gallic acid is a substrate for mOat1 and suggest that human OAT1 might be involved in the active renal secretion of gallic acid.

Le rein joue un rôle essentiel dans l’élimination des médicaments et de leurs métabolites, de ce fait il assure la défense de l'organisme contre de potentiels xénobiotiques toxiques. Particulièrement, les transporteurs des tubules proximaux rénaux qui ont un rôle dans la sécrétion tubulaire des médicaments. Ainsi, ils sont des déterminants important de la biodisponibilité des xénobiotiques dans l’organisme.Dans cette thèse nous nous sommes intéressés à l’implication des transporteurs rénaux humains hOAT1 et hOAT3 dans des interactions médicamenteuses moyennant des modèles in vitro. Après construction et validation des modèles d’études cellulaires HEK-hOAT1 et HEK-hOAT3, nous avons testé l’effet des inhibiteurs de la pompe à protons sur le transport du méthotrexate par les OATs ainsi que l’effet des antiviraux sur l’influx du tenofovir par ces mêmes transporteurs. Grâce à nos modèles cellulaires nous avons tenté d’expliquer in vitro de probables interactions médicamenteuses décrites en clinique. / The kidney plays an essential role in the elimination of drugs and their metabolites, thus it ensures the defense of the body against potential toxic xenobiotic. Particularly, the secretory transporters in the proximal tubule are major determinants of the disposition of xenobiotic in the body.In this thesis we investigated the involvement of human organic anions transporters hOAT1 and hOAT3 in drug drug interactions through study on in vitro cell models. After construction and validation of cells models studies HEK-hOAT1 and HEK-hOAT3, we tested the effect of proton pump inhibitors on methotrexate transport by OATs and the effect of antivirals on the influx of tenofovir by these two transporters. With our models we tried to explain in vitro probable drug interactions described in the clinic.

Organische Anionen werden aus dem Blut in die proximalen Tubuluszellen durch Organische-Anionen-Transporter 1 und 3 (OAT1 und OAT3) aufgenommen. Die Aufnahme erfolgt im Austausch gegen Dicarboxylate. In dieser Dissertation wurde die Affinität von Dicarboxylaten gegenüber humanen OAT1 und OAT3 untersucht mit dem Ziel mehr Informationen über die Struktur der Transporter zu erhalten. Es sollten Unterschiede zwischen dem OAT1 und OAT3 ermittelt werden, besonders bezüglich deren Substratspezifität. Alle Transporter wurden stabil in HEK293-Zellen exprimiert. Extrazellulär wurden Dicarboxylate als Inhibitoren gegen die 3H-p-Aminohippurat-Aufnahme (OAT1) oder 3H-Östronsulfat-Aufnahme (OAT3) zugefügt. OAT1 zeigt die höchste Affinität gegenüber Glutarat (IC50 3,3 µM), α-Ketoglutarat (IC50 4,7 µM) und Adipat (IC50 6,2 µM), gefolgt von Pimelat (IC50 18,6 µM) und Suberat (IC50 19,3 µM). Die Affinität von OAT1 gegenüber Succinat und Fumarat war gering. Der OAT3 zeigte dieselbe Dicarboxylat-Selektivität mit etwa 13-mal höheren IC50-Werten verglichen mit dem OAT1. Die Daten charakterisieren α-Ketoglutarat als hochaffines Substrat für den OAT1 und den OAT3. Die Ergebnisse deuten auf eine ähnliche Molekülstruktur der Bindungsstellen von OAT1 und OAT3 hin.

No description available.

No description available.

Drug transport across cells and cell membranes in the human body is crucial for the pharmacological effect of drugs. Active transport governed by transport proteins plays an important role in this process. A vast number of transport proteins with a wide tissue distribution have been identified during the last 15 years. Several important examples of their role in drug disposition and drug-drug interactions have been described to date. Investigation of drug-drug interactions at the transport protein level are therefore of increasing interest to the academic, industrial and regulatory research communities. The gene expression of transport proteins involved in drug transport was investigated in the jejunum, liver, kidney and colon to better understand their influence on the ADMET properties of drugs. In addition, the gene and protein expression of transport proteins in cell lines, widely used for predictions of drug transport and metabolism, was examined. The substrate and inhibitor heterogeneity of many transport proteins makes it difficult to foresee whether the transport proteins will cause drug-drug interactions. Therefore, in vitro assays for OCT1 and OATP1B1, among the highest expressed transport proteins in human liver, were developed to allow investigation of the inhibitory patterns of these proteins. These assays were used to investigate two data sets, consisting of 191 and 135 registered drugs and drug-like molecules for the inhibition of OCT1 and OATP1B1, respectively. Numerous new inhibitors of the transport proteins were identified in the data sets and the properties governing inhibition were determined. Further, antidepressant drugs and statins displayed strong inhibition of OCT1 and OATP1B1, respectively. The inhibition data was used to develop predictive in silico models for each of the two transport proteins. The highly polymorphic nature of some transport proteins has been shown to affect drug response and may lead to an increased risk of drug-drug interactions, and therefore, the OCT1 in vitro assay was used to study the effect of common genetic variants of OCT1 on drug inhibition and drug-drug interactions. The results indicated that OCT1 variants with reduced function were more susceptible to inhibition. Further, a drug-drug interaction of potential clinical significance in the genetic OCT1 variant M420del was proposed. In summary, gene expression of transport proteins was investigated in human tissues and cell lines. In vitro assays for two of the highest expressed liver transport proteins were used to identify previously unknown SLC transport protein inhibitors and to develop predictive in silico models, which may detect previously known drug-drug interactions and enable new ones to be identified at the transport protein level. In addition, the effect of genetic variation on inhibition of the OCT1 was investigated.

In vivo studies of hepatobiliary disposition are challenging. The hepatobiliary system is complex, as its physiological localization, complex cellular structure with numerous transporters and enzymes, and the interindividual variability in protein expression and biliary flow will all affect the in vivo disposition of a drug under investigation. The research included in this thesis has focused on the involvement of hepatic transport proteins in the hepatobiliary disposition of rosuvastatin. The impact that several transport inhibitors had on the pharmacokinetics of rosuvastatin was investigated in healthy volunteers and in pigs. The effects were considerable, following inhibition of sinusoidal transport proteins by cyclosporine and rifampicin. These inhibitors significantly reduced the hepatic extraction of rosuvastatin by 50 and 35%, respectively, and the plasma exposure increased by factors of 9.1 and 6.3, respectively. Drug-drug interactions (DDI) resulting in markedly higher plasma exposures are important from a drug safety perspective as increased extrahepatic exposure of statins is associated with an increased risk of severe side-effects, such as myopathy which in rare cases could develop into rhabdomyolysis. The DDI caused by cyclosporine and rifampicin can probably be attributed to inhibition of hepatic uptake transporters. In contrast, inhibition of canalicular transporters by imatinib did not significantly affect the pharmacokinetics of rosuvastatin, which suggests that the intracellular concentration of the inhibitor in the hepatocyte was insufficient to affect the transport of rosuvastatin, or that imatinib is not a sufficiently potent inhibitor in vivo. Furthermore, gemfibrozil administered as a single dose into the jejunum in healthy volunteers and pigs did not affect the plasma or biliary pharmacokinetics of rosuvastatin. The previously reported DDI in humans upon repeated dosing with gemfibrozil might be explained by the accumulation of metabolites able to affect the disposition of rosuvastatin. The investigations presented in this thesis conclude that transport proteins are of considerable importance for the hepatobiliary disposition of rosuvastatin in vivo. The Loc-I-Gut catheter can be applied for the investigation of biliary accumulation and to determine bile specific metabolites, however it has limitations when conducting quantitative measurements. In the porcine model, hepatic bile can be collected for up to six hours and enables the determination of the hepatic extraction in vivo.