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.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate Mgr. Jan Dušek Supervisor Prof. PharmDr. Petr Pávek, Ph.D. Title of Doctoral Thesis Development of novel Constitutive androstane receptor (CAR) ligands Constitutive androstane receptor is nowadays known as the established nuclear receptor that regulates the expression of several key cytochrome P450 enzymes, predominantly CYP3A4 and CYP2B6. Recently it has been shown that CAR has also essential role in the regulation of endogenous metabolism of glucose, lipids, cholesterole or bile acids. Simultaneously, this receptor is considered to have proliferative effect on human hepatocytes and protective effects against toxic and dietary damage of liver parenchyme. Given the possible therapeutic utilization of CAR, its therapeutic options are being intensively studied. Unfortunately, currently known ligands of human or mouse CAR are either poorly selective or indirect. The aim of my doctoral thesis was to find new ligands of mouse and human CAR, that would enable more detailed study of the receptor.

Changes in the human gastrointestinal microbiome are associated with several diseases. To infer causality, experiments in representative models are essential, but widely used animal models exhibit limitations. Here we present a modular, microfluidics-based model (HuMiX, human-microbial crosstalk), which allows co-culture of human and microbial cells under conditions representative of the gastrointestinal human-microbe interface. We demonstrate the ability of HuMiX to recapitulate in vivo transcriptional, metabolic and immunological responses in human intestinal epithelial cells following their co-culture with the commensal Lactobacillus rhamnosus GG (LGG) grown under anaerobic conditions. In addition, we show that the co-culture of human epithelial cells with the obligate anaerobe Bacteroides caccae and LGG results in a transcriptional response, which is distinct from that of a co-culture solely comprising LGG. HuMiX facilitates investigations of host-microbe molecular interactions and provides insights into a range of fundamental research questions linking the gastrointestinal microbiome to human health and disease.

La laccase de Myceliophtora thermophila oxyde certains acides hydroxycinnamiques avec formation éphémère d’intermédiaires de différentes couleurs avant que ceux-ci n’évoluent ensuite spontanément en polymères brun noir. Quand cette oxydation est effectuée en milieu biphasique (tampon phosphate pH 7,5 / acétate d’éthyle), une partie de ces intermédiaires colorés peut être récupérée dans la phase organique et ainsi soustraite à la polycondensation ultérieure. Dans le cas de l’acide férulique, on peut ainsi isoler une fraction colorée en jaune orangé surtout composée de dimères résultant de la condensation des semiquinones initialement formées. La synthèse de ces colorants peut être favorisée et le rendement amélioré en contrôlant la réaction par un ajout minimum régulé d’oxygène. Ces colorants conservent le pouvoir antioxydant de l’acide férulique parent mais à concentration élevée (100 à 200 mg/ml), ils présentent une cytotoxicité plus élevée vis-à-vis de cellules humaines normales (HUVEC) et cancéreuses (Caco-2) ce qui limite éventuellement leur intérêt comme colorants naturels. Les semi quinones de l’acide férulique ne forment pas de dimères mixtes, quand on effectue l’oxydation laccasique de cet acide en présence d’autres acides phénoliques car la laccase de M. thermophila effectue une oxydation séquencée en fonction de sa plus ou moins grande affinité pour les différents substrats mis en jeu. En présence d’un polyoside aminé insoluble comme le chitosane, les intermédiaires d’oxydation laccasique de différents composés phénoliques (acides férulique, sinapique, syringique et catéchine) réagissent avec les groupements NH2 pour former des liaisons de covalence et conduire ainsi à des chitosanes colorés doués de nouvelles propriétés dues au greffage d’entités phénoliques. Ces chitosanes fonctionnalisés conservent les propriétés filmogènes du chitosane natif mais en plus, forment des solutions plus visqueuses et sont devenus solubles en milieux acide et basique. Ils permettent la croissance de cellules HUVEC. Ils ont surtout acquis des propriétés antioxydantes et forment des films imperméables à l’oxygène ce qui laisse entrevoir de multiples applications intéressantes / The laccase of Myceliophtora thermophila oxidizes some hydroxycinnamic acids with ephemeral formation of intermediates of different colors before these evolve spontaneously in dark brown polymers. When this oxidation is performed in biphasique medium (phosphate buffer pH 7.5 and ethyl acetate), a part of these colored intermediates can be recovered in the organic phase and it can be subtracted from an ulterior polycondensation. In the case of ferulic acid, we could isolate an orange yellow fraction, which is especially composed of dimers resulting from the condensation of the semiquinones initially formed. The synthesis of these colorants can be enhanced and their yield can be improved by controlling the reaction through a regulated minimum addition of oxygen. They keep the antioxidant power of related ferulic acid, but in high concentration (from 100 to 200 mg/ml), their cytotoxicity toward the human normal cells (HUVEC) and cancerous one (Caco-2) is important and consequently, their interest is limited as natural colorants. The semiquinone of ferulic acid doesn't form any mixed dimers, when the laccase-catalysed ferulic acid oxidation is performed in the presence of other phenolic acids. This can be explained by the fact that laccase from M. thermophila performes a multioxidation in the function of its high or little affinity for the involved substrates. In presence of an insoluble amino polyoside as chitosan, the intermediates of laccase-catalysed oxidation of different phenolic compounds (ferulic, sinapic, syringic acids and catechin) react with its NH2 groups forming covalent liaisons. Actually, this type of link leads to colored chitosans endowed of news properties due to the grafting of phenolic entities. These functionalized chitosans keep the filmogens properties of the native one. Moreover, also it can form more viscous solutions and become soluble in acidic and basic medium. It can permit the growth of HUVEC cells. They especially acquired some antioxidant properties and formed impermeable films to the oxygen which highlights multiple interesting applications

The present study aimed at characterizing pineapple, apple and melon by-products and evaluating their potential as an ingredient for the partial substitution of wheat flour in cookie formulations. The addition of fruit by-products contributed to rise the cookies fiber content. Melon by-product was prominent for its mineral content, whereas apple by-product presented more relevant results for phenolic compounds and antioxidant capacity (DPPH and ABTS). Such behaviors reflected in the cookie formulations. Eight phenolic compounds (vanillic acid, gallic acid, sinapic acid, salicylic acid, p-coumaric acid, catechin, epicatechin and rutin) were identified and quantified by HPLC in both the by-products and cookies. Regarding the antinutritional factors and mineral bioavailability, it was observed that the addition of fruit by-products in cookies reduced the contents of phytate and oxalate and promoted changes in calcium, iron and zinc bioavailability. Facing the technological aspects, it was observed that the use of by-products interfered in the color of the cookies, making them slightly darker and also weakened the gluten formed, promoting variations in diameter and expansion factor. On the other hand, it was observed that increasing concentrations of by-products led to higher values of hardness, making the cookies firmer and crunchier. In the sensory point of view, it was verified that the cookie with 15% of pineapple by-product demonstrated the highest acceptance, followed by cookies with 15% of apple by-product, control and 15% of melon by-product. By quantitative descriptive analysis (QDA), it was found that the addition of fruit by-products altered the sensory profile of the cookies. From the results obtained, it can be concluded that the fruit by-products are potential ingredients for cookie formulation, attributing to them specific physicochemical, technological, nutritional and sensory properties which can vary according to the by-products employed. / O presente estudo visou caracterizar subprodutos de abacaxi, maçã e melão e avaliar seu potencial como ingrediente para substituição parcial de farinha de trigo em formulações de biscoitos. A adição de subprodutos de frutas contribuiu para elevar o teor de fibras dos biscoitos. O subproduto de melão se destacou por seu conteúdo mineral, enquanto que o subproduto de maçã apresentou resultados mais relevantes para compostos fenólicos e capacidade antioxidante (DPPH e ABTS). Tais comportamentos se refletiram nas formulações de biscoitos. Oito compostos fenólicos (ácido vanílico, ácido gálico, ácido sinápico, ácido salicílico, ácido p-cumárico, catequina, epicatequina e rutina) foram identificados e quantificados por HPLC tanto nos subprodutos como nos biscoitos. Com relação aos fatores antinutricionais e biodisponibilidade mineral, observou-se que a adição de subprodutos de frutas em biscoitos diminuiu os teores de fitato e oxalato e promoveu mudanças na biodisponibilidade de cálcio, ferro e zinco. Frente aos aspectos tecnológicos, observou-se que o uso de subprodutos interferiu na cor dos biscoitos, tornando-os levemente mais escuros e também enfraqueceu a rede de glúten formada, promovendo variações do diâmetro e fator de expansão. Por outro lado, observou-se que quanto maior a concentração de subprodutos, maiores foram os valores de dureza (textura), sendo as amostras consideradas mais firmes e crocantes. Do ponto de vista sensorial, verificou-se que o biscoito com 15% de subproduto de abacaxi foi o que demonstrou maior aceitação, seguido pelos cookies com 15% de subproduto de maçã, controle e 15% de subproduto de melão. Por meio de análise descritiva quantitativa (ADQ), observou-se que a adição de subprodutos de frutas alterou o perfil sensorial dos biscoitos. A partir dos resultados obtidos, concluiu-se que os subprodutos de frutas se apresentam como potenciais ingredientes para formulações de cookies, atribuindo-lhes propriedades físico-químicas, tecnológicas, nutricionais e sensoriais específicas as quais podem variar de acordo com o subproduto empregado.

The present study aimed at characterizing pineapple, apple and melon by-products and evaluating their potential as an ingredient for the partial substitution of wheat flour in cookie formulations. The addition of fruit by-products contributed to rise the cookies fiber content. Melon by-product was prominent for its mineral content, whereas apple by-product presented more relevant results for phenolic compounds and antioxidant capacity (DPPH and ABTS). Such behaviors reflected in the cookie formulations. Eight phenolic compounds (vanillic acid, gallic acid, sinapic acid, salicylic acid, p-coumaric acid, catechin, epicatechin and rutin) were identified and quantified by HPLC in both the by-products and cookies. Regarding the antinutritional factors and mineral bioavailability, it was observed that the addition of fruit by-products in cookies reduced the contents of phytate and oxalate and promoted changes in calcium, iron and zinc bioavailability. Facing the technological aspects, it was observed that the use of by-products interfered in the color of the cookies, making them slightly darker and also weakened the gluten formed, promoting variations in diameter and expansion factor. On the other hand, it was observed that increasing concentrations of by-products led to higher values of hardness, making the cookies firmer and crunchier. In the sensory point of view, it was verified that the cookie with 15% of pineapple by-product demonstrated the highest acceptance, followed by cookies with 15% of apple by-product, control and 15% of melon by-product. By quantitative descriptive analysis (QDA), it was found that the addition of fruit by-products altered the sensory profile of the cookies. From the results obtained, it can be concluded that the fruit by-products are potential ingredients for cookie formulation, attributing to them specific physicochemical, technological, nutritional and sensory properties which can vary according to the by-products employed. / O presente estudo visou caracterizar subprodutos de abacaxi, maçã e melão e avaliar seu potencial como ingrediente para substituição parcial de farinha de trigo em formulações de biscoitos. A adição de subprodutos de frutas contribuiu para elevar o teor de fibras dos biscoitos. O subproduto de melão se destacou por seu conteúdo mineral, enquanto que o subproduto de maçã apresentou resultados mais relevantes para compostos fenólicos e capacidade antioxidante (DPPH e ABTS). Tais comportamentos se refletiram nas formulações de biscoitos. Oito compostos fenólicos (ácido vanílico, ácido gálico, ácido sinápico, ácido salicílico, ácido p-cumárico, catequina, epicatequina e rutina) foram identificados e quantificados por HPLC tanto nos subprodutos como nos biscoitos. Com relação aos fatores antinutricionais e biodisponibilidade mineral, observou-se que a adição de subprodutos de frutas em biscoitos diminuiu os teores de fitato e oxalato e promoveu mudanças na biodisponibilidade de cálcio, ferro e zinco. Frente aos aspectos tecnológicos, observou-se que o uso de subprodutos interferiu na cor dos biscoitos, tornando-os levemente mais escuros e também enfraqueceu a rede de glúten formada, promovendo variações do diâmetro e fator de expansão. Por outro lado, observou-se que quanto maior a concentração de subprodutos, maiores foram os valores de dureza (textura), sendo as amostras consideradas mais firmes e crocantes. Do ponto de vista sensorial, verificou-se que o biscoito com 15% de subproduto de abacaxi foi o que demonstrou maior aceitação, seguido pelos cookies com 15% de subproduto de maçã, controle e 15% de subproduto de melão. Por meio de análise descritiva quantitativa (ADQ), observou-se que a adição de subprodutos de frutas alterou o perfil sensorial dos biscoitos. A partir dos resultados obtidos, concluiu-se que os subprodutos de frutas se apresentam como potenciais ingredientes para formulações de cookies, atribuindo-lhes propriedades físico-químicas, tecnológicas, nutricionais e sensoriais específicas as quais podem variar de acordo com o subproduto empregado.

Prior work using a contractility bioassay determined that the serotonin (5-HT) receptor subtype 5-HT2A is present in bovine lateral saphenous veins and plays a role in ergot alkaloid-induced vascular contraction in steers grazing endophyte-infected (Epichloë coenophiala) tall fescue (Lolium arundinaceum). A study was conducted to determine what 5-HT receptors are involved in vasoconstriction of bovine gut vasculature. The findings of this study indicate that 5-HT2A is present and may play a role in ergot alkaloid induced vasoconstriction. A second study was conducted to determine if ergot alkaloids were transported in the small intestine. The active transporter, peptide transporter 1 (PepT1), was evaluated for its role in the transport of various concentrations of ergot alkaloids across Caco-2 cell monolayers. Results indicate that CEPH, ERT, EXT, and LSA do move across Caco-2 cell monolayers, but appear to utilize PepT1 at larger concentrations. Overall, the demonstrated presence of 5-HT2A receptors in the bovine gut vasculature established a potential for vascular interference by ergot alkaloids entering the bloodstream through transepithelial absorption.

Ces travaux de thèse ont porté sur l'utilisation d'un nouveau vecteur particulaire lipidique (LNCs) destiné à la voie orale. Ce système devrait permettre d'encapsuler une molécule anticancéreuse et améliorer sa biodisponibilité. Notre premier axe de recherche a été d'étudier les propriétés in vitro des LNCs encapsulant du paclitaxel en vue d'une administration par voie orale. La stabilité in vitro dans les milieux gastro-intestinaux simulés a été démontrée. De plus, les études de transport à travers l'épithélium intestinal (modèle cellulaire Caco-2) ont montré que les LNCs permettaient d'augmenter le transport du paclitaxel grâce à un mécanisme d'endocytose actif clathrine- et cavéole-dépendant situé dans le microenvironnement de la P-gp. Nous avons aussi mis en évidence les interactions entre l'endocytose des nanocapsules et l'activité de la P-gp. Notre second axe de recherche a porté sur l'application des LNCs pour encapsuler un nouvel anticancéreux : le Sn38 qui est le métabolite actif de l'irinotécan. Une formulation modifiée de nanocapsules lipidiques a été obtenue en utilisant un co-tensioactif et une huile hydrophile en plus des excipients standards. Les propriétés physico-chimiques et la stabilité, l'absorption epithéliale et la toxicité in vitro de cette formulation ont ensuite été vérifiées. Enfin, une activité antitumorale de courte durée après administration orale chez la souris a été observée. Ce travail fait donc la preuve de concept de l'absorption d'anticancéreux après administration par voie orale à l'aide de vecteurs colloïdaux. Néanmoins, certaines optimisations restent encore à réaliser pour prévoir une application clinique.

<p>Drug transport across the intestinal epithelium is roughly predicted from permeability values obtained from Caco-2 cell monolayers. This thesis examines the important role of <i>pH</i> and extracellular additives for increasing the reliability and predictivity of the <i>in vitro</i> screening system, Caco-2.</p><p>It was shown that the passive transport of ionizable compounds may be biased by a false efflux or uptake component, when applying a physiological <i>pH</i>-gradient across the membrane. <i>pH</i> also affected the amount of compound available at the transporter-binding site. Therefore, <i>pH</i> dependence should be considered in studies of such compounds and of drug-drug interactions involving efflux transporters. It was also shown that proton-dependent apical uptake or basolateral efflux should be studied both with and without a <i>pH</i> gradient over the whole monolayers. </p><p>The two extracellular additives, bovine serum albumin (BSA) and the solubilizing agent, Cremophor^® EL, also influenced Caco-2 permeabilities. BSA applied to the receiver side increases, and to the donor side decreases drug permeation according to the drug’s protein binding capacity. Thus, the absorptive transport for both passive and active compounds is favoured, giving a physiologically sound improvement of the Caco-2 cell model. Inclusion of BSA increased both the predictivity and quality of permeability studies, particularly of highly lipophilic, BCS class II compounds. Passive and active transport processes could also be distinguished after accounting for unbound concentrations. The overall effect of Cremophor^® EL on the permeability to a drug was compound-specific and probably dependent on micellar incorporation. Cremophor^® EL can therefore not be recommended. </p><p>Neither <i>pH</i> nor BSA affect the functionality of transporters such as P-glycoprotein. However, efflux ratios of ionizable or protein bound drugs are altered in the presence of a <i>pH</i>-gradient or BSA, indicating that an experimental system without protein or <i>pH</i> gradient can over- or underestimate active and passive efflux in drug transport.</p>

<p>Over the past decade, the development of macromolecular drugs based on peptides, proteins and nucleic acids has increased the interest in microparticulate drug delivery, i.e., the delivery of drug systems in the nanometer and micrometer ranges. However, little is known so far about the effect that microparticulate systems have on various tissues after administration. Additionally, the knowledge of mechanisms responsible for the uptake and transport of microparticles across the human intestine is incomplete and requires further investigation to improve both the safety profiles and the efficiency of these drug delivery systems.</p><p>This thesis is comprised of two parts. The first one investigates gene expression responses obtained from DNA arrays in local and distal tissues after microparticulate drug delivery. The second part focuses on the mechanisms responsible for the transport of microparticles across epithelial cells lining the intestine.</p><p>The results presented in the first part demonstrated that gene expression analysis offers a detailed picture of the tissue responses after intramuscular or pulmonary administration of microparticulate drug delivery systems compared to the traditional techniques used for such evaluations. In addition, DNA arrays provided a useful and sensitive tool for the initial characterization and evaluation of both local and distal tissue responses, making it possible to distinguish between gene expression patterns related to each studied delivery system.</p><p>The results presented in the second part demonstrated that the surface properties of the microparticle were important for the extent of transport across an <i>in vitro</i> model of the follicle-associated epithelium (FAE), comprised of intestinal epithelial cells specialized in particle transport (M cells). Another important finding was that the enteropathogen bacterium, <i>Yersinia pseudotuberculosis</i>, induced microparticle transport across the normal intestinal epithelium, represented by Caco-2 cells and excised human ileal tissue. This transport was most probably mediated by an increased capacity for macropinocytosis in the epithelial cells.</p>