The interplay of transporters and enzymes and their transporter-enzyme was examined in Caco-2 cell monolayer and recirculating perfused rat liver preprations via both theoretical and experimental approaches. First, a Caco-2 catenary model that consisted of the apical, cellular, basolateral compartments and encompasses influx, efflux transporters and enzymes was shown to be superior to the single barrier approach for data interpretation on transporter- and enzyme- mediated processes. The kinetics of baicalein, a flavonoid that undergoes glucuronidation and sulfation, were found to be described better by the catenary model for the complex kinetics of substrate inhibition in metabolism. Second, estradiol-17beta-D-glucuronide (E217G), a protypic substrate of Oatp1a1, 1a4, and 1b2 and Mrp2 that underwent futile cycling with its 3-sulfate metabolite (E23S17G) via estrogen sulfotransferase (Sult1e1) and arylsulfatase C, was examined in the perfused rat liver preparation. Solutions of the AUC and clearances were solved to relate the intrinsic clearances of transporters and enzymes to understand how these affected the apparent clearances in the presence of futile cycling. Transporters and enzymes were perturbed experimentally by the intraportal injection of CC531 colon carcinoma cells for tumor induction in Wag/Rij rat livers. The protein expression of Oatp1a1 and Oatp1b2 were reduced to half whereas Sult1e1 was increased by 40% with tumor development versus the sham-operated control. These data were well predicted by the physiologically-based liver model, showing the impact of increased sulfation intrinsic clearance but not the decreased influx clearance. The TR- (Mrp2 mutant) rat model was used to examine how the absence of Mrp2 for biliary secretion of both E217G and E23S17G affected futile cycling. Absence of Mrp2 was found to result in a pseudo steady-state and reduction of the total, excretion, and metabolic clearances in the liver. The work shed new insight on the interplay between enzymes and transporters and how kinetic processes mediated by enzymes or efflux transporters affected futile cycling.

The interplay of transporters and enzymes and their transporter-enzyme was examined in Caco-2 cell monolayer and recirculating perfused rat liver preprations via both theoretical and experimental approaches. First, a Caco-2 catenary model that consisted of the apical, cellular, basolateral compartments and encompasses influx, efflux transporters and enzymes was shown to be superior to the single barrier approach for data interpretation on transporter- and enzyme- mediated processes. The kinetics of baicalein, a flavonoid that undergoes glucuronidation and sulfation, were found to be described better by the catenary model for the complex kinetics of substrate inhibition in metabolism. Second, estradiol-17beta-D-glucuronide (E217G), a protypic substrate of Oatp1a1, 1a4, and 1b2 and Mrp2 that underwent futile cycling with its 3-sulfate metabolite (E23S17G) via estrogen sulfotransferase (Sult1e1) and arylsulfatase C, was examined in the perfused rat liver preparation. Solutions of the AUC and clearances were solved to relate the intrinsic clearances of transporters and enzymes to understand how these affected the apparent clearances in the presence of futile cycling. Transporters and enzymes were perturbed experimentally by the intraportal injection of CC531 colon carcinoma cells for tumor induction in Wag/Rij rat livers. The protein expression of Oatp1a1 and Oatp1b2 were reduced to half whereas Sult1e1 was increased by 40% with tumor development versus the sham-operated control. These data were well predicted by the physiologically-based liver model, showing the impact of increased sulfation intrinsic clearance but not the decreased influx clearance. The TR- (Mrp2 mutant) rat model was used to examine how the absence of Mrp2 for biliary secretion of both E217G and E23S17G affected futile cycling. Absence of Mrp2 was found to result in a pseudo steady-state and reduction of the total, excretion, and metabolic clearances in the liver. The work shed new insight on the interplay between enzymes and transporters and how kinetic processes mediated by enzymes or efflux transporters affected futile cycling.

Ph.D.

Le récepteur ionotropique P2X7 est impliqué dans diverses fonctions physiologiques telles que la prolifération, l’apoptose, la réponse inflammatoire et le trafic membranaire dans plusieurs types cellulaires. Cependant, peu est connu quant aux rôles physiologiques de P2X7 dans les cellules épithéliales intestinales (CEIs). Dernièrement, un collègue a démontré une nouvelle fonction pour P2X7 qui est de réguler l’internalisation du transporteur à glucose GLUT2. Comme l’un des rôles physiologiques majeurs des CEIs est l’absorption du glucose, la régulation de ce transporteur est d’une importance capitale. Par contre, nous ne savons pas ce qui permet de réguler l’expression du récepteur P2X7 dont la disponibilité pourrait être un des facteurs déterminants dans la régulation de l’absorption de glucose par le transporteur GLUT2. Ceci nous a menés à l’hypothèse suivante qui dit que le glucose lui-même pourrait stimuler certaines voies de signalisation menant à l’activation de facteurs de transcription, qui pourraient réguler l’expression du récepteur P2X7. Les objectifs de mes travaux de maîtrise étaient de déterminer si des variations dans la concentration de glucose affectaient l’expression de P2X7 dans les CEIs et d’identifier les facteurs de transcription impliqués dans ce processus. Les résultats obtenus démontrent que l’expression de P2X7 semble être affectée par la concentration de glucose. Cette régulation de l’expression de P2X7 en réponse au glucose semble impliquer les facteurs de transcription C/EBP? et ß. En fait, les essais d’immunoprécipitation de la chromatine (ChIP) effectués confirment que ces facteurs pouvaient lier le promoteur de P2X7 et que la liaison de C/EBPß au promoteur varie en fonction de la concentration de glucose. La capacité de liaison de C/EBPß au promoteur est comparable au niveau d’expression du gène du récepteur P2X7. In vivo, nous avons observé une diminution de l’expression du récepteur P2X7 dans les extraits de jéjunum de souris invalidées pour C/EBPß. Finalement, dans le modèle de souris diabétique NOD, il semble y avoir un dérèglement dans l’expression du récepteur P2X7, ce qui pourrait indiquer que P2X7 a bel et bien un rôle dans la régulation de l’homéostasie du glucose. [symboles non conformes]

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. / Philosophiae Doctor - PhD

The antioxidant capacity of phenolic compounds in red wine is suggested to be responsible for their health-promoting effects. Compared to other wines, little information is available on phenolic compositions and antioxidant capacity of Australian wine. Information related to the fate of these phenolics in the body once consumed is also very limited. The overall aim of this research was to investigate the relevance of red wine consumption as a source of health-giving antioxidants in humans. The phenolic composition of wine was determined using the Folin-Ciocalteu (total phenolic), aluminium chloride (total flavonols), methyl cellulose precipitation (MCP) (total tannins), pH differential (total monomeric anthocyanins), bisulfite bleaching (total polymeric anthocyanin fractions), and liquid chromatographic (LC-MS) (individual phenolics) methods. Antioxidant activities were measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis-93-ethylbenzthiazoline-6-sulfonic acid (ABTS) and oxygen radical absorbance capacity (ORAC) assays. The phenolic and antioxidant data were then used to establish the relationship between these two parameters in wines from different varieties (Shiraz, Cabernet Sauvignon and Merlot) and winemaking stages (crushing, fermentations, oaking and bottling). By using an in vitro digestion model that mimics the upper gastrointestinal tract (GIT) digestion, the stability of the wine phenolics during digestion was examined. Finally, to gain a better understanding of the post-digestion absorption of wine phenolics, their permeability across Caco-2 cell monolayers was evaluated. A total of 8 monomeric anthocyanins and 17 other phenolic compounds were positively identified in the red wines using LC-MS analysis. Most of the phenolic categories showed some positive correlations with the antioxidant activities but none of the individual phenolic compounds showed a strong correlation with the total antioxidant activity of the wine, implying a combined contribution of many wine phenolics to antioxidant effects. The phenolic compositions and antioxidant activities of three of Australia’s most common red wines varieties - Shiraz, Cabernet Sauvignon and Merlot were not different from each other, possibly due to the variability within each grape cultivar. During the winemaking process, the total phenolic content and the associated antioxidant activity of the wine increased during the fermentation process, as more phenolics are being extracted from grape skin, seeds and stems into the wine. During oak and bottle ageing, the total phenolic contents were stabilised. Most of the wine phenolics were more stable under acidic conditions (pH 2 and 5.5) than neutral or alkaline conditions (pH 7.4 and 9). This may partly explain the stability of the wine phenolics subjected to the acidic (pH 2) gastric digestion and their loss following simulated pancreatic digestion (pH 7.4). In addition, sample pre-treatment procedures prior to LC-MS analysis may have removed some antioxidants in the form of degradation products and/or new polymeric compounds following the in vitro gastric and pancreatic digestion processes. The missing products appeared to be detected by both the Folin-Ciocalteu method and ORAC assay, which measured the phenolic compounds and their antioxidant activity, after the pancreatic digestion. This suggests that the instability of phenolic compounds at pH 7.4, results in the transformation of most of the oral phenolic antioxidants into more stable forms in the GIT, which in turn contribute positively to the overall antioxidant activities of the ingested wine. All of the original wine phenolics had very low permeabilities across Caco-2 cell monolayers, except for syringic acid, p-coumaric acid and an unknown phenolic acid. Limited surface area for absorption (0.33 cm2) and the limited peak detection sensitivity in the LC method may have contributed towards the difficulty in detecting and identifying compounds with low permeability. In addition, extensive metabolism of absorbed phenolics by the Caco-2 cells may occur based on the appearance of several new peaks. However, due to their low concentrations and lack of reference, the identities of the new products and metabolites remain unknown. The present in vitro study suggests that upon ingestion, most of the original phenolic compounds in red wine are lost either through degradation to new compounds and/or complexation with other compounds. However, these products seem to possess some antioxidant activity and may be the key compounds responsible for the health-promoting effects of red wine. The limitation of the present study in detecting and fully identifying these breakdown products and metabolites should be addressed in future studies.

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No. of bitstreams: 1 boni_fi_me_arafcf.pdf: 2349970 bytes, checksum: 94103feab0447379967a227a0fcd3977 (MD5) Previous issue date: 2017-09-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A nanotecnologia farmacêutica é uma relevante ferramenta tecnológica para o desenvolvimento de novos sistemas para a veiculação de fármacos, pois devido às suas dimensões reduzidas são capazes de circular por capilares que irrigam os tecidos, escapar da fagocitose por células do sistema imunológico e permear passivamente células e epitélios. Além disso, esses sistemas possibilitam a encapsulação de fármacos de baixa estabilidade, protegendo-os contra degradação prematura e/ou permitindo a modulação de suas propriedades físico-químicas, bem como a interação biológica na biointerface. O metotrexato (MTX) é um dos fármacos mais utilizados no tratamento de tumores sólidos e doenças inflamatórias intestinais, no entanto, por ser altamente citotóxico e não seletivo promove diversos efeitos colaterais. Sua eficácia terapêutica é comprometida devido à resistência adquirida pelas células tumorais e por sua baixa permeabilidade intestinal, principalmente por meio do mecanismo de efluxo da forma livre. Nesse trabalho, nanopartículas poliméricas (NPs) orais compostas por quitosana (QS), ácido hialurônico (AH) e ftalato de hidroxipropilmetilcelulose (HP), polímeros que possuem propriedades como solubilidade pH dependente, mucoadesividade e de funcionalização, foram desenvolvidas como plataforma tecnológica para a vetorização do MTX para o cólon, visando o tratamento local de patologias intestinais. As análises de peso molecular e coeficiente viral demostraram a adequação dos polímeros e solventes para a obtenção das nanoestruturas pelo método de complexação polieletrolítica. A avaliação da influência do pH no potencial zeta dos polímeros permitiu a seleção do valor ótimo (pH 5,5) para obtenção das nanoestruturas. O diâmetro médio das NPs sem fármaco, contendo ou não HP variou de 451,73-574,95 nm e a incorporação do MTX reduziu significativamente esses valores para a faixa de 216,83-345,03 nm (p<0,05). A adição do HP, bem como do MTX promoveu a redução do potencial zeta das NPs (p<0,05). O índice de polidispersão das NPs contendo ou não MTX variou de 0,21-0,39 a 0,26-0,33, respectivamente, indicando a homogeneidade de tamanho. A forma esférica das partículas foi também foi demonstrada. Os espectros de absorção na região IV indicaram a formação dos complexos polieletrolíticos e as análises de DRX a formação de estruturas cristalinas. A eficiência de incorporação do MTX variou de 20,06% a 36,18%, sendo as NHP0,5-1 e NHP0,2-5 as que apresentaram as maiores porcentagens (32,21% e 36,18%, respectivamente). Embora o MTX tenha promovido a redução dos valores de mucoadesão (p<0,05), a elevada mucoadesividade (71,22% a 91,53%) de todas as NPs foi evidenciada. De acordo com as isotermas de adsorção, os dados de interação com a mucina apresentaram correlação com o modelo de Freundlich, que indica a adsorção reversível em multicamadas. Em pH 6,8, a interação da mucina com as NPs promoveu a redução do potencial zeta, o que pode estar relacionado a maior capacidade de adsorção nesse valor de pH. A incorporação do MTX nas NPs permitiu a redução da liberação em meio ácido em até 40%, em relação ao fármaco livre, enquanto a adição de HP à matriz das NPs reduziu a liberação em até 10%, neste pH. O mecanismo de liberação do fármaco foi avaliado por diferentes modelos matemáticos. A viabilidade nas linhagens Caco-2 e HT29-MTX para os polímeros e NPs (0,01 - 1 μg.mL-1) foi demonstrada. Para as NPs contendo MTX, nas concentrações de 10 μg.mL-1 e 100 μg.mL- 1, a viabilidade na linhagem Caco-2 após 24 h de incubação foi de aproximadamente 50%. Nos estudos de permeabilidade in vitro, as NPs sem adição de HP permearam aproximadamente 3 vezes mais as monocamadas de Caco-2 e de co-cultura tripla de células, em comparação às amostras contendo HP e ao fármaco livre. A maior associação celular para o fármaco incorporado às NPs, em comparação ao MTX livre, demonstrou a capacidade do sistema em modular a interação biológica. / The pharmaceutical nanotechnology is a relevant technological tool for the development of new drug delivery systems, because of their small size they are able to circulate through capillaries that irrigate tissues, escape from phagocytosis by the immune system cells and passively permeate cells and epithelia. In addition, these systems enable the encapsulation of low stability drugs, protecting them against premature degradation and/or allowing the modulation of their physico-chemical properties, as well as the biological interaction in the biointerface. Methotrexate (MTX) is one of the most used drug in the treatment of solid tumors and inflammatory bowel diseases, however, because MTX highly cytotoxic and nonselectivity promotes several side effects. Its therapeutic efficacy is compromised due to the resistance acquired by tumor cells and it low intestinal permeability, mainly through the efflux mechanism of the free form. In this work, oral nanoparticles composed of chitosan (CS), hyaluronic acid (HA) and hydroxypropylmethylcellulose (HP) phthalate, polymers that have properties such as pH-dependent solubility, mucoadhesiveness and functionalization have been developed as a technological platform for the target of MTX to the colon, aiming the treatment of local pathologies. Molecular weight and second viral coefficient analyzes demonstrated the suitability of polymers and solvents to obtain nanostructures by the polyelectrolyte complexation method. The evaluation of the pH influence on the zeta potential of the polymers allowed to the selection of the optimum value (pH 5.5) to obtain the nanostructures. The mean diameter of the MTX free nanoparticles, containing or not HP ranged from 451.73-574.95 nm and the incorporation of MTX significantly reduced these values for the range of 216.83-345.03 nm (p <0.05). The addition of HP as well as MTX promoted reduction of zeta potential of nanoparticles (p <0.05). The polydispersity index of the nanoparticles containing or not MTX ranged from 0.21-0.39 to 0.26-0.33, respectively, indicating homogeneity of size. The spherical shape of the particles has been demonstrated. The absorption spectra in the IV region indicated the formation of the polyelectrolyte complexes and the DRX analyzes, the formation of crystalline structures. The efficiency of drug incorporation ranged from 20.06% to 36.18%, with the NHP0.5-1 and NHP0.2-5 samples having the highest percentages (32.21% and 36.18%, respectively). Although MTX promoted the reduction of mucoadhesion values (p<0.05), the high mucoadhesiveness (71.22% to 91.53%) of all nanoparticles was evidenced. According to the adsorption isotherms, the interaction data with the mucin presented a correlation with the Freundlich model, which indicates the reversible adsorption in multilayers. At pH 6.8, the interaction of mucin with the nanoparticles promoted the reduction of the zeta potential, which may be related to the higher adsorption capacity at this pH value. The incorporation of MTX in the nanoparticles allowed to the reduction of MTX release in acidic media by up to 40% in relation to the free drug and the addition of HP to the nanoparticle matrix reduced the release by up to 10% at pH 1.2. The mechanism of drug release was evaluated by different mathematical models. The viability of Caco-2 and HT29-MTX lines for polymers and NPs (0.01-1 μg.mL-1) was demonstrated. For nanoparticles containing MTX, at concentrations of 10 μg.mL-1 and 100 μg.mL-1 , the viability of Caco-2 after 24 h incubation was approximately 50%. In the in vitro permeability studies, samples without HP permeated approximately 3- fold more monolayers of Caco-2 and triple co-culture compared to samples containing HP and the free drug. The higher cellular association for the drug incorporated into the nanoparticles, compared to free MTX, demonstrated the ability of the system to modulate the biological interaction / FAPESP: 2015/21176-5 / FAPESP BEPE: 2016/20360-0

Summary Natural phytochemicals are currently used in the treatment of many diseases. Cancers are just ones of them and they are ranked among the most common and the most serious. Phytochemicals in the form of cytostatics are used in chemotherapeutic treatment of cancer. In future there could be included among cytostatics also some alkaloids from the family of Amaryllidaceae, whose testing for a selective cytostatic effect on tumor cell lines of colorectal carcinoma Caco-2 and HT-29 and on normal cell lines of human intestinal epithelial FHs 74 Int is a subject of this thesis. There were tested 17 alkaloids isolated from plants of Chlidanthusfragrans, Zephyranthes robusta and Nerine bowdenii. Particularly alkaloids from plant Zephyranthes robusta namely haemanthamine with this values: IC50 = 0.99 plus/minus 0.14 microM for tumor cells, Caco-2, 0.59 plus/minus 0.01 microM for tumor cells HT-29 and 19.47 plus/minus 8.86 microM for normal cells FHs 74 Int, Lycorine with values IC50 = 0.99 plus/minus 0.08 microM for tumor cells Caco-2, 1.2 plus/minus 0.01 microM for tumor cells HT-29 and 22.68 plus/minus 0.09 microM for normal cells FHs 74 Int and Haemanthidin with values IC50 = 3.29 plus/minus 0.91 microM to tumor cells Caco-2, 1.72 plus/minus 0.11 microM to tumor cells HT-29, and 11.63 plus/minus 0.86 microM for normal cells FHs 74 Int proved a significant anti-proliferative activity. From these results there is evident the selectivity against colorectal cancer cell lines. For this reason, those tested alkaloids are suitable for further testing and for study of their biological activity against tumor cells in the terms of in vitro and in vivo.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Alice Mlčochová Supervisor: PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: In vitro transport of abacavir across the monolayer of Caco-2 cells; interaction with etravirine and rilpivirine. Abacavir belongs among nucleoside reverse transciptase inhibitors (NRTIs) representing a basic component of combined antiretroviral therapy used in treatment of HIV-positive patients [1]. Etravirine and rilpivirine are newer non-nucleoside reverse transcriptase inhibitors (NNRTIs) combined in cART together with NRTI. ATP-dependent transporters, so called ABC transporters, are able to affect pharmacokinetic properties of drugs, thus they are important site of drug-drug interactions affecting absorption, distribution and excretion level. P-glycoprotein (Pgp, ABCB1) and BCRP (ABCG2) belong among the most clinically important ABC transporters able to cause drug-drug interactions. The aim of this thesis was to introduce and optimize the method for evaluation of drug absorption using monolayers of Caco-2human intestine cell lines, whose integrity was verified by evaluating TEER (transepithelial electrical resistance). This model was also used for abacavir transport studies. Significant...

Doctor of Philosophy / Department of Human Nutrition / Brian Lindshield / Fortified blended foods (FBFs), in particular, corn-soybean blend (CSB), are food aid commodities widely used in infant and young children supplementary feeding programs. A United States Agency for International Development (USAID) Food Aid Quality Review report recommended developing novel FBFs using local alternative commodities such as sorghum and improving the nutritional quality of FBFs using extrusion processing. Extruded sorghum-cowpea, sorghum-soy and corn-soy FBFs were developed and compared with the non-extruded FBFs corn-soy blend 13 (CSB13) and corn-soy blend plus (CSB+) using the in-vitro digestion/Caco-2 cell model. Dry FBFs’ iron and vitamin A content ranged from 8.0 to 31.8 mg/100g and 0.54 to 1.67 mg/100g, respectively. Following in-vitro digestion, bioavailable iron and vitamin A levels were determined by measuring Caco-2 cell ferritin and vitamin A levels in response to 12-hour and 4-hour treatments, respectively, with aqueous fractions collected from digested FBFs. Most extruded FBFs’ aqueous fraction iron levels were 2- to 7-fold higher (p<0.05) than CSB13 and CSB+. However, Caco-2 cell ferritin and vitamin A levels were not significantly different among FBFs. These results suggest that consumption of newly developed extruded sorghum-cowpea, sorghum-soy and corn-soy FBFs will result in bioavailable iron and vitamin A levels comparable to traditional non-extruded CSB13 and CSB+. Thus, extruded sorghum-cowpea FBF may be a suitable alternative to corn-soybean based FBFs. Saw palmetto supplements are one of the most commonly consumed products by men with prostate cancer and/or benign prostatic hyperplasia (BPH). Some studies have found significant improvements in BPH with saw palmetto supplementation, whereas others found no benefits. The variation in the efficacy in these trials may be a result of differences in the putative active components, fatty acids and phytosterols, of the saw palmetto supplements. We quantified fatty acids and phytosterols in 20 commercially available liquid, powder, dried berry, and tincture saw palmetto supplements. Liquid saw palmetto supplements contained significantly higher (p<0.05) concentrations of total fatty acids (908.5 mg/g), individual fatty acids, total phytosterols (2.04 mg/g), and individual phytosterols, than the other supplement categories. Our findings suggest that liquid saw palmetto supplements may be the best choice for individuals who want to take a saw palmetto supplement.