Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2015-03-27T14:27:58Z No. of bitstreams: 2 Dissertação - Laís Cristina da Silva - 2014.pdf: 4002977 bytes, checksum: c54a0aa96c3be46a95b8df8e9a407c1d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-03-27T15:49:49Z (GMT) No. of bitstreams: 2 Dissertação - Laís Cristina da Silva - 2014.pdf: 4002977 bytes, checksum: c54a0aa96c3be46a95b8df8e9a407c1d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-27T15:49:49Z (GMT). No. of bitstreams: 2 Dissertação - Laís Cristina da Silva - 2014.pdf: 4002977 bytes, checksum: c54a0aa96c3be46a95b8df8e9a407c1d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-09-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The main predictive models of absorption of potential new drugs in preclinical stage are focused on the gastrointestinal mucosa, given the predominance of this pathway in drug administration. Often, the fraction absorbed (Fa) can be predicted in ex vivo models (p.e. Ussing chambers), in vitro (p.e. Caco-2 cells monolayers), intestinal perfusion studies in situ and in vivo absorption. In the present study, from an adaptation of Snapwell ™ inserts, a new ex vivo model to evaluate the permeability of substances passively absorbed is proposed. High permeable drugs (metoprolol, caffeine and theophylline) and low permeable drugs (atenolol, ranitidine and cimetidine) were maintained in an incubator at 37 ° C under constant stirring (60 rpm) and carbogenic atmosphere (5% CO2). The viability of the jejunal membrane (52 Ω.cm2 ± 8.0) was observed remaining above 20 Ω.cm2 for 120 min incubation, under all conditions evaluated, including the addition of co-solvents (1% DMSO and 1% EtOH). Values of apparent permeability coefficients obtained (Papp) were characteristic of ex vivo permeation studies (3.8 to 12.6 x10-6 cm / s). Strong correlation was observed between the data obtained here versus data intestinal perfusion in vivo (r = 0.89), as well as the fraction absorbed in humans (r = 0.85), reported in the literature. Additionally, the model features high sensitivity and accuracy compared to other commonly used models in classification permeability of substances. In line, we can infer that the MTSSNAPWELL model demonstrates, yet, potential application in studies of screening for selection of low molecular weight, such as potential phytochemicals, as well as their synthetic analogues evaluated with low amount of sample (ca 10 mg). / Os principais modelos preditivos da absorção de potenciais novos fármacos na etapa pré-clinica são focados na mucosa gastrointestinal, haja vista a predominância desta via na administração medicamentosa. Frequentemente, a fração absorvida (Fa) pode ser predita em modelos ex vivo em câmaras de Ussing, in vitro em monocamadas de células Caco-2, perfusão intestinal in situ e estudos de absorção in vivo. No presente estudo, a partir de uma adaptação do aparato Snapwell™, um novo modelo ex vivo de avaliação da permeabilidade para substâncias absorvidas por difusão passiva é proposto. Substâncias de alta (metoprolol, cafeína e teofilina) e baixa (atenolol, ranitidina e cimetidina) permeabilidade, foram mantidos em incubadora à 37OC, sob agitação constante (60 rpm) e atmosfera carbogênica (5% CO2). A viabilidade da membrana jejunal (52 ± 8,0 Ω.cm2) foi observada mantendose acima de 20 Ω.cm2 por até 120 min de incubação, sob todas condições avaliadas incluindo a adição de co-solventes (DMSO 1% e EtOH 1%). Os valores de coeficientes de permeabilidade aparente obtidos (Papp) mostraram-se característicos de estudos ex vivo de permeação (3,8 – 12,6 x10-6 cm/s). Forte correlação foi observada entre os dados aqui obtidos versus dados de perfusão intestinal in vivo (r = 0,89), assim como da fração absorvida em humanos (r = 0,85), relatados na literatura. Adicionalmente, o modelo apresenta elevada sensibilidade e precisão frente aos demais modelos comumente utilizados na classificação da permeabilidade de substâncias. Em consonância, pode-se inferir que o modelo MTSSNAPWELL demonstra, até o momento, potencial aplicação em estudos de screening para seleção de moléculas de baixo peso molecular, tais como potenciais fitofármacos, assim como seus análogos sintéticos avaliados com baixa quantidade de amostra (c.a. 10 mg).

Blood-brain barrier (BBB) is a unique membranous barrier, which segregates brain from the circulating blood. It works as a physical and metabolic barrier between the central nervous system (CNS) and periphery. In mammals, endothelial cells were shown to be of BBB and are characterized by the tight junctions along with efflux system which are responsible for the restriction of movement of molecules within the cells. Efflux system consists of multidrug resistance proteins such as P-glycoprotein (P-gp). P-gp removes substances out back from the brain to the blood before they reach to the brain. So the barrier is impermeable to many compounds such as amino acids, ions, small peptides and proteins, making it the most challenging factor for the development of new drugs for targeting CNS. Curcumin is a bioactive compound that has a number of health promoting benefits such as anti-inflammatory, anticancer, anti-oxidant agent; as well as a role in neurodegenerative diseases, but low oral bioavailability is the major limiting factor. Low water solubility and rapid metabolism are the two important factors responsible for poor bioavailability of curcumin. Galaxolide is a musk compound and previously known for the bioaccumulation of toxic components in the aquatic animals by interference with the activity of multidrug/multixenobiotic resistance efflux transporters (MDR/MXR). The bioavailability of curcumin can be enhanced when administered with galaxolide. This study was carried out to investigate the effect of galaxolide on the permeation of curcumin through the epithelial cell monolayers. MDCKII-MDR1 cell monolayer is used an in vitro blood-brain barrier model while Caco-2 monolayer is used as an in vitro intestinal model, which also expresses the P-glycoprotein. The curcumin and galaxolide were separately solubilised in the DMSO and used in combination to perform permeation study, to determine the effect of galaxolide on curcumin permeation through epithelial cell monolayers. The galaxolide shows an efflux protein inhibition activity and this activity was used to enhance permeation of curcumin through the Caco-2 monolayer. In summary, galaxolide is a novel permeation enhancer molecule, which can be used for the improvement of drug delivery of other bioactive compounds in future. / Department of Social Welfare, Govt. of Maharashtra (India)

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The field of organ-on-chip is a relatively new area of research and builds upon the principle of engineering microfluidic systems to mimic the body’s internal environment as precisely as possible. Eventually these models could hopefully simulate whole organ-systems and enable the examination of the cell’s or organ’s reaction to foreign substances like new pharmaceuticals in a better way than current models. Previously this has been done with in vitro models such as petri dishes that only offer static culturing conditions. These are not very realistic environments compared to the human body where the cells are exposed to both variations in pressure and flows among other things. The purpose of this bachelor’s thesis project has been to evaluate and improve the design of an organ-on-chip system developed by the EMBLA-group at Ångströmslaboratoriet, Uppsala university. This has been done by evaluating the manufacturing process to find areas of improvements of the current chip design, as well as conducting a literature study to understand key components of similar organ-on-chip systems and see if it is possible to implement relevant parts to the organ-on-chip of this project. One of these important parts is a so-called bubble trap. A bubble trap is a construction that enables the capturing and elimination of bubbles in the system since the bubbles can harm the chips components, kill the cells, and compromise measurements.  A first prototype of the bubble trap was developed in Polydimethylsioxane (PDMS) and integrated on the EMBLA-group’s chip design. The principle behind the bubble trap was to use the natural buoyancy of the bubbles to trap them. This was done by introducing an upwards going slope before the inlets to the chip. In this manner the bubbles would float up to the top of the slope and accumulate at the roof as the liquid moved on into the chip without bubbles. To make the bubbles leave the chip a low-pressure chamber was added on top of the bubble trap to help the process of the bubble’s diffusion through the roof and out of the chip. The development of an improved chip design turned out to be a time-consuming endeavor and the time left for evaluation the functionality of the chip became too short. One test was performed which showed that the bubbles did accumulate at the top of the slope as expected, but it rapidly became full and thus started to let bubbles through to the microfluidic chip. The bubbles did not diffuse as efficiently as required and the removal of the bubbles became inefficient. To understand and correct the problem areas of this bubble trap design further tests and experiments will have to be conducted. / Organ-på-chip (Organ-on-chip eller OoC) är ett relativt nytt forskningsområde som bygger på att mikrofluidiksystem utvecklas till att efterlikna människokroppen i så stor utsträckning som möjligt. Detta då det är attraktivt att kunna undersöka cellers/organs beteende vid tillförsel av vissa substanser, till exempel nya läkemedel. I tidigare in vitro modeller har det endast observerats och utförts tester på celler odlade i statiska förhållanden vilket inte är likt den omgivning cellerna har i människokroppen där de tex utsätts för olika vätskeflöden och tryckförändringar.    Syftet med detta examensarbete har varit att utvärdera och förbättra designen på ett OoC system utvecklat av EMBLA-gruppen på Ångströmlaboratoriet vid Uppsala universitet. Detta har gjorts genom att studera den nuvarande tillverkningsprocessen för att hitta relevanta förbättringsområden samt att genom en litteraturstudie undersöka viktiga delar som bör ingå i dessa typer av system. En av dessa delar är en bubbelfälla (bubble trap eller BT) vilket innebär att det i chippet bör finnas ett sätt att eliminera/fånga upp bubblor. Detta eftersom bubblorna kan orsaka stor skada på både chipet, cellerna och mätningarna som skall utföras. En första prototyp av en BT design i Polydimetylsiloxan (PDMS) utvecklades och integrerades på EMBLA-gruppens OoC design. Principen bakom BT-designen var att utnyttja bubblornas flytkraft vilket gjordes genom att introducera en uppåtgående backe innan ingångskanalen. Bubblorna kan därmed flyta upp till toppen av lutningen och vätskan kan fortsätta in i mikrochipset utan bubblor. För att bubblorna ska ta sig ut ur chippet integrerades en tryckkammare ovanpå BT-designen för att få bubblorna att diffundera ut genom taket i den uppåtgående kammaren och ut ur chippet. Utvecklingen av den förbättrade chip-designen visade sig var tidskrävande och tiden för att utvärdera designens funktionalitet blev för kort. Ett test gjordes på den nya chip-designen vilket visade att den utvecklade BT som väntat fångade upp bubblor men att den snabbt blev full i och med att bubblorna inte diffunderade ut genom taket i den takt som behövdes. Vidare undersökningar och experiment behövs för att evaluera vad som orsakade detta och rätta till eventuella felkällor i design och experimentuppställning.