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Browsing by Subject "transporter"

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  • Tikkanen, Alli (2019)
    Organic Anion Transporting Polypeptide 2B1 (OATP2B1) is an influx transporter expressed widely throughout the body in tissues such as intestine, liver, brain, placenta and skeletal muscle. Since many clinically used drugs are transported by OATP2B1, changes in the function of the transporter due to genetic polymorphism could lead to altered pharmacokinetics or -dynamics of OATP2B1 substrate drugs. The aim of this Master’s thesis was to create and optimize a cellular uptake assay to study the function of OATP2B1. Furthermore, the aim was to study the effects of six naturally occurring nonsynonymous single nucleotide variants on OATP2B1 transport function in vitro. With site-directed mutagenesis, single nucleotide changes were introduced into the gene coding for OATP2B1. OATP2B1 variants were expressed in human derived HEK293 cell line using baculovirus expression system. A cellular uptake assay with estrone-3-sulfate and a fluorescent probe 4’, 5’-dibromofluorescein (DBF) as substrates was set up and optimized. With the assay, OATP2B1-mediated uptake of variants was compared to the transport activity of OATP2B1 wild type. Amino acid changes Ser486Phe and Cys520Ser impaired OATP2B1 transport function severely. In addition, variant Thr318Ile transported DBF and estrone-3-sulfate less efficiently compared to OATP2B1 wild type, but Arg312Gln, Thr392Ile and Ser532Arg transport function was not affected. A method to study OATP2B1 function was created successfully. According to the results, single amino acid changes in OATP2B1 can impair OATP2B1 function. The results and method can be utilized to understand findings from pharmacogenetic studies in vivo, and to predict consequences of especially rare variants, which can be difficult to detect in small sample populations in clinical studies. However, further studies on the expression level and cellular localization of OATP2B1 variants are needed to fully characterize the impact of the variants studied.
  • Kaugonen, Olga (2017)
    Investigating the role of cell membrane proteins has increased over the last decade, as drugdrug interactions and genetic polymorphisms have been found to cause changes in drug pharmacokinetics and dynamics. In this study the characteristics of the OATP1B1 transporter were reviewed and new in vitro research method to study protein functions was developed. Human Embryonic Kidney cells (HEK) is a human derived mammalian cell-line that is widely used in the study of OATP1B1 transporter. The Sf9 cell line is isolated from Spodoptera frugiperda insect and is one of the standard in vitro tools in a genetic engineering study. In the experimental part of this thesis the goal was to express OATP1B1 transporter in Sf9 and HEK293 cell lines. The wild-type SLCO1B1-gene encoding the OATP1B1 was virulent with baculovirus into the cells by the Bac-to-Bac® Baculovirus Expression System. For expression in the Sf9 cells, the aim of the study was to clone the SLCO1B1-gene into the pFastBac vector. The cloning was not successful in this study although attempts were made for several approaches. The expression of OATP1B1 transporter in HEK293 cells was successful. HEK293 cells expressing OATP1B1 transporter are well suited for the study of the SLCO1B1-gene. The in vitro method developed in this study remains in the research team as a tool to investigate the polymorphisms of the SLCO1B1-gene, the inhibition of the transporter and possible drug interactions.
  • Munsterhjelm, Nina (2012)
    The liver is the major site of drug metabolism and excretion. Within the liver endogenous and exogenous compounds are eliminated through many metabolizing enzymes. Drug removal is not only dependent on metabolic enzymes, but also on transporters. Before cellular metabolism can occur, a drug must first enter the hepatocyte. Very lipophilic drugs enter the cell membrane through passive diffusion, but polar or ionized organic compounds can enter the cell membrane only by transporters. Transporters in the basolateral membrane of the hepatocyte facilitate drug entry and access to drug metabolizing enzymes. Transporters in the canalicular domain (apical) of the hepatocyte faclitate removal of drugs or metabolites from the cell interior. Recent studies have shown that transporters can mediate drug-drug interactions, and transporter genes are subject to genetic polymorphism which may affect pharmacokinetic parameters of a drug, such as absorption, distribution, and excretion. This Master's thesis consists of two parts, a literature review and an experimental section. In the literature review two transporters, OATP1B1 and MRP2, are discussed in detail. OATP1B1 is expressed on the basolateral and MRP2 on the apical membrane of the hepatocyte. These transporters are responsible for the vectorial transcellular hepatobiliary transport of various organic anions in humans. The experimental section aims at modelling vectorial hepatobiliary transport of three compounds in a double-transfected (OATP1B1/MRP2) MDCKII cell line. All three compounds studied, rosuvastatin, estrone sulphate, and estradiol glucuronide, are substrates of both transporters. Wild type (WT) MDCKII cells were used as a control. Tight junctions form a barrier between cells. This barrier regulates the paracellular passage of, for example, water, ions, large molecules, and drugs. In the experimental section the tight junctions were reversibely opened to distinguish between trans- and paracelluar routs of transport of the three compounds studied. Permeation of rosuvastatin and estradiol glucuronide in the basolateral to apical direction was faster in the double-transfected cell line compared to the MDCKII-WT cell line. Permeation of estrone sulphate, however, behaved unexpectedly in the double-transfected cell line. The permeation of this compound was almost equal in the apical to basolateral and basolateral to apical direction. The reason for this unexpected finding remains unclear. By opening the tight junctions the permeation of all compounds in both cell lines was increased, indicating that the compounds studied preferred the paracellular route and the importance of transporters were reduced. The double-transfected MDCKII cell line is a useful in vitro model of hepatic vectorial transport of organic anions in humans.
  • Koskenkorva, Tiina (2012)
    Elucidation of transporter- and/or metabolic enzyme-mediated drug interactions is important part of early drug development. However the knowledge about clinical consequences of transporter-mediated drug-drug interactions is still limited and more investigation is needed to improve our understanding. MDR1 transporter, widely distributed on the pharmacokinetic barriers in the body (e.g. intestine) and has been shown no limit the bioavailability of drugs. Substrates of MDR1 are exposed to limited intestinal drug absorption and intestinal drug-drug interactions due to inhibition of the transporter. In predicting the clinical significance of an interaction, the principal obstacle has been the limited ability to appropriately scale the preclinical data into in vivo situation. In vitro-in vivo correlations on the extent of MDR1's influence on absorption and standardized predicting methods for drug-drug interactions using the inhibitory constants (IC50 and Ki) would greatly increase the value of in vitro studies. Current in vitro and in silico methods for prediction of the influence of MDR1 on intestinal absorption and related drug-drug interactions are discussed in the literature review. In addition, the latest regulatory draft guidances (FDA, EMA) are reviewed. Aliskiren has been shown to be a sensitive MDR1 substrate in vivo and high affinity substrate for the transporter in vitro. The objective of the experimental work was to study the MDR1-mediated transport of aliskiren and the related drug-drug interactions in vitro and in silico. Vesicular transport assay was used to obtain kinetic parameters for aliskiren (Km and Vmax) and inhibitor potencies (IC50) for ketoconazole, verapamil, itraconazole and its metabolite hydroxyitraconazole. Ki was further calculated for itraconazole and hydroxyitraconazole. Aliskiren showed high affinity to MDR1 transporter with a Km value 5 µM, consistent to what was reported previously in different assay systems. The interactions between aliskiren and the inhibitors in vitro correlated to the observed interactions in vivo in humans. In addition, hydroxyitraconazole was shown to be a potent inhibitor of MDR1-mediated transport of aliskiren in vitro. This suggests that hydroxyitraconazole may contribute to the pronounced interaction observed between aliskiren and itraconazole in a clinical interaction study. A compartmental absorption and transit (CAT) model with added enterocyte compartments and MDR1 efflux was used to describe the influence of MDR1 on intestinal absorption of aliskiren in humans. The integration of kinetic parameters (Km) from in vitro studies requires further optimization on how to describe the intracellular drug concentrations in the model. Aliskiren is however suitable MDR1 probe substrate to be used in in vitro and in vivo trials in humans and therefore gives a good basis for developing vitro-in vivo predictive models.
  • Tepponen, Tuomas (2017)
    Multidrug resistance protein 1 (MDR1, p-glycoprotein) belongs to the ATP-binding cassette transporter family and it's encoded by ABCB1/MDR1 gene. It is a protein which transports many different kinds of compounds out of cells, for example from endocytes to the lumen with the use of energy from ATP. MDR1 is there for a restrictive factor for several orally administered drugs. It`s important to have knowledge about MDR1-inhibitors, in order to avoid harmful drug-drug and food-drug interactions that might affect medical treatment. The purpose of this master's thesis was to optimize an in vitro MDR1-vesicle uptake method and use it to screen inhibitors from compound libraries. To optimize the method, the effect of cholesterol loading on ATP-dependent transport of test substrate N-methylquinidine (NMQ) was evaluated, transport kinetics of the vesicles and kinetics of known inhibitors were also tested. With the optimized method, screening was done with a library of 25 food additives and a library of 42 synthetic compounds. The chemical structures of the synthetic compounds were analyzed manually in order to find factors that could explain their ability to inhibit MDR1. Only one inhibitor was found among food additives: curcumin. Other additives didn't increase or decrease the ATP-dependent transport of NMQ. Several inhibitors were found from the library of synthetic compounds, also a couple of compounds were found to increase the active transport of NMQ. Results indicate, that the additives used in this study have low risk to cause MDR1 mediated interactions, if curcumin is excluded. The inhibitory effect of curcumin should be investigated in in vivo-situation, because vesicle-based in vitro-results have tendency to overestimate results. Screening results of the synthetic compounds gives more confirmation to the usefulness of the screening method. The MDR1-inhibition screening method described in this Master`s thesis is valid, and it can be used to screen different compound libraries for MDR1-inhibitors. In the future it could be used to screen different kinds of compounds, which might end up inside humans and cause interactions with drugs.
  • Suvanto, Satu (2014)
    P-glycoprotein is an ATP-dependent efflux protein expressed in many tissues which participate in absorption, distribution and elimination of drug molecules. It can mediate clinically significant drug-drug interactions. Characteristics of P-gp have been studied widely and crystal structure of mouse P-gp has been successfully determined. P-gp binds its substrates either directly from cell membrane or from cytosol and it has at least three separate binding sites. P-gp has wide selection of substrates from many therapeutical groups. According to the latest computational models, a typical P-gp substrate can be defined with the help of molecule structural factors rather than physicochemical properties. However function of P-gp is very complex which is why drug-drug interactions should be studied for each drug pair separately. In addition expression of P-gp is regulated by nuclear receptors PXR and CAR thus P-gp induction is separate, which also complicates P-gp mediated interactions. P-gp substrates celiprolol, talinolol, aliskiren and fexofenadine have in vivo interactions with P-gp inhibitors or inducers. The objective the experimental work was to study suitability of two in vitro methods, MDCKII-cell permeability assay and MDR1-vesicle transport assay, for predicting in vivo effect of drug-drug interaction. ATP-dependent transport of substrates was determined in membrane vesicles extracted from human P-gp expressing Sf9 cells. Cell assay was used to determine efflux ratio (ER) for all the substrates alone and efflux ratio with P-gp inhibitor itraconazole for the substrates which have reported in vivo interaction with itraconazole. All compounds showed ATP dependent transport in MDR1-vesicles and celiprolol, talinolol and fexofenadine showed ER over 1 in MDCKII-MDR1 cells thus according to vesicle assay and ER-value they are P-gp substrates. However ER of talinolol and fexofenadine was not affected by inhibitor itraconazole, thus the drugs did not fulfil the inhibition criteria of FDA for P-gp substrates. The performing of interaction test was possible failed due lack of pre-incubation of the cells with the inhibitor. Talinolol had the highest ER in thus according to cell experiments talinolol has P-gp dependent transport. Aliskiren ER was not obtained because of the low recovery of the drug but it had clear ATP-dependent transport in the vesicle assay as was expected according to in vivo results. According to in vitro results and in vivo studies celiprolol is a poor P-gp substrate whereas fexofenadine showed P-gp mediated transport both in vitro and in vivo. The results suggest that significance of drug interaction is difficult to predict with the vesicle and the cell assay but they can be used to recognize P-gp substrates.