The subject of this thesis was to study two intestinal barriers to oral drug bioavailability, drug efflux proteins of the ABC-transporter family, and in particular ABCB1/P-glycoprotein (Pgp), and the drug metabolizing enzyme cytochrome P450 (CYP) 3A4. At the onset of this thesis, similarities between CYP3A4 and Pgp in terms of their tissue distribution and gene regulation, along with overlapping substrate specificities, had generated the hypothesis that CYP3A4 and Pgp may have a complementary function and thus form a coordinated intestinal barrier to drug absorption and gut wall metabolism. In the first part of this thesis, a cell culture model of the intestinal epithelium that expressed both functional Pgp and CYP3A4 was developed. This model was then used to investigate the steroselective drug efflux and metabolism of R/S-verapamil. In summary, the results indicated that the two barriers in the cell culture model were in agreement with those in the human intestine. Both ABC-transporters and CYPs are regulated by drugs that interact with nuclear receptors. However, while the regulation of CYPs is quite well understood, less is known about how repeated drug administration regulates the most abundantly expressed ABC-transporters. Therefore, in the second part of this thesis, the effects of repeated drug administration on the gene regulation of four ABC-transporters and CYP3A4 were studied in intestinal epithelial cell lines in vitro and in the perfused human jejunum in vivo. The in vitro studies revealed that the ABC-transporters are induced by drugs that interact with slightly different sets of nuclear receptors. The in vivo study showed that repeated oral administration of St John’s wort decreased the bioavailability of verapamil, predominantly by induction of intestinal CYP3A4. This part of the thesis provides new information about the regulation of ABC-transporters, shows that the intestinal metabolism is the most significant barrier to oral bioavailability of verapamil and provides evidence for a clinically significant interaction between verapamil and St John’s wort in vivo.

L'intestin assure l'absorption des lipides alimentaires et doit faire face aux variations d'apport en lipides, entre les périodes inter-prandiales et post-prandiales. J'ai donc étudié dans les cellules Caco-2/TC7, la réponse des entérocytes à différents modes d'apports en lipides. Par une approche transcriptomique, j'ai montré que les lipides modulent l'expression de nombreux gènes entérocytaires. Les profils géniques obtenus diffèrent entre l'apport luminal ou sérique en lipides. La comparaison des apports apicaux de micelles inter- ou post-prandiales (MPP) a montré que les MPP modulent spécifiquement l'expression de 46 gènes majoritairement impliqués dans la transduction de signaux, le métabolisme lipidique, et l'architecture cellulaire. Ces résultats, s'ajoutant aux effets spécifiques des MPP obtenus dans l'équipe, suggéraient une détection entérocytaire des lipides alimentaires apportés par ces MPP. J'ai alors montré que les MPP se lient à une protéine dont le poids moléculaire correspond à celui du récepteur SR-BI, induisant sa clusterisation à la membrane apicale et son adressage vers les rafts. Seules les MPP induisent, le trafic de l'apoB, de la membrane apicale vers les compartiments sécrétoires et l'activation de kinases. Ces effets sont abolis quand SR-BI est bloqué par un de ses ligands ou invalidé par ARN interférence. Ces travaux montrent pour la première fois que les entérocytes sont capables de détecter spécifiquement les lipides alimentaires sous forme micellaire. Cette détection implique SR-BI et induit des voies de signalisation aboutissant à la mise en place de paramètres morphologiques et fonctionnels nécessaires au transfert des lipides alimentaires.

<p>The aim of this study was to investigate the effect the plant matrix and the structure of the flavonoid (i.e. whether aglycone or glycoside) may have on the gastrointestinal uptake and metabolism of luteolin derivatives from Artemisia afra traditional plant medicine. Specifically, how these two factors influenced the intestinal uptake and disposition of luteolin derivatives in pure and in Artemisia afra plant extract forms were to be assessed by investigating the uptake and metabolism of the luteolin derivatives in human intestinal epithelial Caco-2 cells and the perfused rat intestinal loop. To realize this aim, the following were determined: (1) identification and characterization of major luteolin derivatives found in Artemisia afra, (2) the effect of the plant matrix on the uptake of luteolin derivatives in Artemisia afra aqueous-extract forms across the Caco-2 cell monolayer, (3) the effect of the plant matrix on the absorption and metabolism of luteolin derivatives in Artemisia afra aqueous-extract forms in the perfused rat small intestine, (4) the effect of gut contents on the uptake and metabolism of luteolin derivatives in intestinal loop and (5) the metabolic profiles of luteolin derivatives obtained for the pure solutions versus plant aqueous extract solutions in Caco-2 cells and the rat intestine.</p>

Previous studies have provided evidence that endothelial cell-based potassium ion selective electrodes possess the ability to quantify substances that have permeability-altering effects on those endothelial cells. The capability of these so-called biosensors to detect elevated concentrations of certain chemical agents found following tumor formation make them useful in the application as an alternative tumor angiogenesis assay. In this study an epithelial cell line, human colon adenocarcinoma epithelial cells (Caco-2), was used to fabricate membranes that were used to test concentrations of these chemical agents, known as cytokines, mimicking the concentrations that have been observed in the serum of healthy individuals as well as the higher concentration found in individuals with cancer. Additionally background information is provided related to the development of whole cell-based biosensors, metabolic pathways related to tumor angiogenesis and the subsequent increase in cytokine concentration, properties of the Caco-2 cell line that make them useful for the application in cell-based biosensors, and the ultimate effect the cytokines have on the permeability of the cells.

Field pea (Pisum sativum L.) seeds have high nutritional value but also contain phytate which can inhibit the absorption and utilization of nutrients. Phytate is the main storage form of phosphorus in the seeds but chelates Fe, Zn and some other micronutrients and is not well digested by monogastrics. Peas with pigmented seed coats contain polyphenols which also have anti-nutritional properties. To increase the nutritional value of field pea seeds, two low phytate lines (1-150-81 and 1-2347-144) containing higher inorganic phosphorus concentration (IN-P) and lower phytate-phosphorus concentration (PA-P) than the normal phytate varieties were developed from CDC Bronco in previous research. The objectives of this research were 1) to determine the effect of genotype and environment on iron bioavailability in a set of five pea varieties differing in phytate concentration and iron concentration using in vitro digestion/Caco-2 cell culture bioassay; 2) to determine the effect of seed coats on iron bioavailability by testing whole seeds compared to dehulled seeds in varieties differing in seed coat pigmentation using in vitro digestion/Caco-2 cell culture bioassay; 3) to determine the inheritance of iron bioavailability in field pea by evaluating recombinant inbred lines differing in phytate concentration using in vitro digestion/Caco-2 cell culture bioassay; 4) to determine the effects of pea with the low phytate trait on body weight and hemoglobin concentration of chickens. Iron concentration (FECON) did not differ significantly between normal and low phytate varieties. Iron bioavailability (FEBIO) of the two low-phytate lines was 1.4 to 1.9 times higher than that of the three normal phytate varieties, and growing environment also had a significant effect on FEBIO. Peas with pigmented seed coats contained 7 times lower FEBIO than peas with non-pigmented seed coat. The removal of the seed coat increased the FEBIO in peas with pigmented seed coat 5 to 6 times. From previous research on PR-15 recombinant inbred lines (RILs) which were developed from a cross between low phytate line 1-2347-144 and a normal phytate variety CDC Meadow, it was found that PA-P was controlled by a single gene. FEBIO, in this study, was also found to follow a bimodal frequency distribution, characteristic of single gene control, and it was highly correlated with PA-P in the PR-15 lines. In vivo studies were used to evaluate iron absorption of chickens fed with low and normal phytate pea diets. The diets containing the low-phytate pea lines had no significant effect on chicken body weight and hemoglobin level, compared with the diets containing normal phytate pea varieties. An unexpected high FECON was discovered in the diets that was traced to the ingredients of limestone and dicalcium-phosphate which likely affected the experimental results.

The main objective of this thesis was to investigate the in vivo relevance of membrane transporters and cytochrome P450 (CYP) 3A4-mediated metabolism in the intestine and liver for the bioavailability of drugs in humans after oral administration. In the first part of the thesis, the main transport mechanisms involved in the intestinal absorption and bioavailability were investigated for fexofenadine, a minimally metabolized drug, which is a substrate for P-glycoprotein (P-gp) and members of organic anion transporting polypeptide (OATP) family. Jejunal perfusion studies revealed that co-perfusion with verapamil increased the bioavailability of fexofenadine by decreasing the first-pass liver extraction as the low intestinal permeability was unchanged by the transport inhibitors studied. The mechanism behind the interaction probably involves inhibition of OATP-mediated sinusoidal uptake and/or P-gp-mediated canalicular secretion of fexofenadine. Results from the Caco-2 model supported that the intestinal absorption of fexofenadine is mainly determined by the low passive permeability of the drug, even though fexofenadine clearly is a P-gp substrate. In the second part of the thesis, the effect of repeated oral administration of the P-gp and CYP3A4 inducer St. John’s wort on the in vivo intestinal permeability and presystemic metabolism of the dual P-gp and CYP3A4 substrate verapamil was investigated in a jejunal perfusion study. St. John’s wort decreased the bioavailability of the enantiomers of verapamil by inducing the CYP3A4-mediated presystemic metabolism, probably mainly in the gut. It was also concluded that induction of efflux transporters, such as P-gp, does not affect the intestinal transport or the gut wall extraction of high permeability substrates like verapamil. Data from Caco-2 cells with induced CYP3A4-activity supported these findings. The plasma levels of the enantiomers of norverapamil also decreased despite an increased formation, which was attributed to induction of CYP3A4 and/or other metabolic routes.

Nutriomics is the study of the whole range of nutritional components (nutriome) in foods. In order to further understand the molecular basis for the positive health benefits of fruits identified from epidemiology, the mass balance through the human digestion and absorption should be studied. The components of the nutriome studied in this research were sugars, carotenoids, phenolics and organic acids, all important for defining dietary – human health relationships and linking to evidence obtained from epidemiological studies. An attempt to approach a realistic human mimetic digestion and absorption model has been carried out in this study using a static in-vitro model of the human digestive system. Two major novelities in this model compared to other in-vitro models are (i) the use of particles of solid fruit products that mimic the products of human chewing and (ii) a cell-based (Caco-2) in-vitro intestinal absorption model. Hence, imitative bioavailability, i.e. releasing nutrients and potential levels of target compounds reaching the portal circulatory system could be assessed. The fruits studied were tomato, mango, papaya; each as fresh, dried and juiced forms. In-vivo chewing suggested 0.5 cm size modes for dried products and 1.5 cm for fresh products. The agglomerates that were obtained from the chewing of dried products disaggregated during in-vitro digestions in the presence of acids (gastric simulation) or sodium bicarbonate at pH 6 (small intestinal simulation). The extent of this disaggregation followed the order: tomato > mango > papaya. Although all fresh samples contained separated cells, their responses to a 5 mm texture analysis probe (mimicking teeth cusps) varied depending on fruit products. All matrices were hardened by drying, becoming more brittle and breaking easier to produce smaller size modes. Variation between individual participants in the size of their chewed particles was lower for fresh products and high for dried products. The in-vitro digestion and absorption model developed had simulated particle sizes of approximately 0.5 cm3 for dried products or 1.5 cm3 (thickness varied with the products) for fresh products in a 9:1 ratio mix with blended samples, and were digested in-vitro using the following steps: 1. ‘Chewing’: pH 6.9; 37 C, 10 min, in a shaking-water bath (55 rpm) with human alpha-amylase (100 U/L). 2. ‘Gastric’ digestion: pH 2; 37 C, 60 min, in a shaking-water (55 rpm) with porcine pepsin (40 µg/L). 3. ‘Intestinal’ digestion: pH 6; 37 C, 60 min, in a shaking-water bath (55 rpm) with porcine pancreatin and bile extract (1.4 µg/L and 8.6 µg/L, respectively). 4. Caco-2 cell monolayer in-vitro passages: aged 22 days post confluent monolayers in a 24 transwell-insert well plate seeded at 105 cells, pH 7.4 with renewal of apical and basolateral solutions every 30 min for bioavailability estimations. In this study, two models of basolateral – apical solution renewals were carried out: both apical and basolateral were renewed (model A) and basolateral only was renewed (model B). To study metabolites produced by Caco-2 cells, the bioassays were carried out for 22 h without renewals of apical and basolateral solutions (model C). An overview of nutriomics analysis of in-vitro digestions of mango, papaya and tomato based on principal component analysis (PCA) suggested: (1) fruit types led to variable nutriome releases: in-vitro digestions affected tomato >mango >papaya; (2) processing varied nutriome releases from fruit products with juicing tended to release more nutriome components, whereas drying and unprocessed (fresh) did not show noticeably different patterns; (3) gastric and simultaneous gastric-intestinal digestions were similar in nutriome releases whereas contributions of intestinal digestion alone were negligible for water soluble nutriome components; and overall (4) during in-vitro digestions there were no interactions among releasing nutriome from the fruit products studied (independent nutriome releasing processes). Phenolic components showed molecular changes during in-vitro digestion and processing, due to, heating effects, pH or enzymic degradations. Caco-2 bioassays using model compounds showed a range of monolayer responses as follows: (1) mannitol, lycopene and catechin were strictly retained in the apical solution; (2) sugars, caffeine and atenolol were translocated in the apical-to-basolateral direction as intact molecules; (3) Beta-carotene partially disappeared from the apical solution without basolateral release. Models A – C consistently confirmed these responses. Low recoveries provided evidence for cellular metabolisms of (particularly) phenolic and carotenoid molecules by the Caco-2 cell monolayers.

Abstract Colorectal cancer is the second most common malignancy in the Western World. The overall 5-year survival is still only 50–60%. Thus, better prognostic markers are needed to improve survival of the disease. Most colorectal cancers develop from pre-existing adenomas including conventional, flat and serrated adenomas. The most important prognostic factors include tumour stage, histologic subtype and poor differentiation. The prognosis of colorectal cancer depends mainly on tumour stage. The growth of colorectal cancer is determined by cell proliferation, differentation and apoptosis. The progression of colorectal cancer is associated with the growth pattern of colorectal cancer and its invasive margin. Cancer cell budding means the presence of cells scattered in the stroma at the invasive margin, and is associated with β-catenin, an adhesion protein involved in the nuclear Wnt/β-catenin pathway. Hormones may be directly involved in the growth of a cancer, for example sex hormones play an important role in the development of most gynaecological cancers. The knowledge about the dependency of cancers on other hormones, such as thyroid hormones, is limited. This thesis focuses on factors affecting growth and prognosis in colorectal cancer. Antibodies for Ki-67, caspase cleavage site for keratin 18, β-catenin and TRβ1 were used to determine their possible associations with colorectal cancer growth patterns and the characteristics of the invasive margin. Apoptosis and proliferation were decreased at the invasive margin, particularly in serrated adenocarcinomas. The invasive margin showed a presence of budding cell clusters in 24.0% of the cases and this predicted a very poor 5-year-survival (15.4%, P &lt; 0.00001), but nuclear β-catenin accumulation did not predict budding. Thyroid hormone receptor TRβ1 was associated with polypoid growth, presence of KRAS mutations and also with a higher WHO histological grade and advanced Dukes' stage, and in in vitro analysis, thyroid hormone T3 had a modulatory effect on colorectal cancer cell protein synthesis and apoptosis. In conclusion, the growth type of colorectal cancer, i.e. conventional polypoid, flat or serrated, has an association with the characteristics of the invasive margin. Budding margin is associated with poor prognosis in colorectal cancer, and could be utilised in diagnostic pathology. Association of TRβ1 expression with polypoid growth pattern and the presence of KRAS mutations suggest that abnormalities in thyroid hormone signalling involving TRβ1 play a role in the development of some types of colorectal adenocarcinomas.

The Caco-2 cell assay is widely used for in vitro permeability measurements. However, a draw back with the assay that this study will focus on improving, is compound adsorption to the plastic material. Lipophilic compounds such as Cyclosporin A and Peptide J, that will be used in this study, tend to bind to the plastic material in the assay. This can result in poor recovery and misleading permeability predictions. Bovine serum albumin (BSA) is an alternative used today to prevent this but is not always successful.    The aim of this study is therefore to improve the Caco-2 permeability assay by adding phospholipid covered silica beads (PLB) to the basolateral chamber. The role of the PLB is to bind the compound of interest and decrease the amount of compound bound to the plastic material and thus better predict the permeability of the compound of interest.   The PLB was produced using phosphatidylcholine and silica beads. Caco-2 cells were seeded and maintained for 21-29 days ahead of the experiment. PLB concentration of 20, 60 and 100 mg/ml were prepared. Samples were analyzed with HPLC-MSMS. The results showed that with increasing PLB concentration we had a significantly decrease in non-specific plastic binding resulting in reliable permeability predictions, concluding that the hypothesis was correct.

Tocopheryl Polyethylene Glycol Succinate 1000 (TPGS 1000) can inhibit P-glycoprotein (P-gp); TPGS 1000 was not originally designed to inhibit an efflux pump. Recent work from our laboratories demonstrated that TPGS activity has a rational PEG chain length dependency. In other recent work, inhibition mechanism was investigated and appears to be specific to the ATPase providing P-gp energy. Based on these observations, we commenced rational surface-active design. The current work summarizes new materials tested in a validated Caco-2 cell monolayer model; rhodamine 123 (10 μM) was used as the P-gp substrate. These results demonstrate that one may logically construct non-ionic surfactants with enhanced propensity to inhibit in vitro efflux. One new surfactant based inhibitor, Tocopheryl Polypropylene Glycol Succinate 1000 (TPPG 1000), approached cyclosporine (CsA) in its in vitro efflux inhibitory potency. Subsequently, TPPG 1000 was tested for its ability to enhance the bioavailability of raloxifene - an established P-gp substrate - in fasted male rats. Animals dosed with raloxifene and TPPG 1000 experienced an increase in raloxifene oral bioavailability versus a control group which received no inhibitor. These preliminary results demonstrate that one may prepare TPGS analogs that possess enhanced inhibitory potency in vitro and in vivo.