Browsing by Subject "transportteri"

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  • Kaugonen, Olga (Helsingfors universitet, 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.
  • Tepponen, Tuomas (Helsingfors universitet, 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.
  • Jaakkonen, Liina (Helsingin yliopisto, 2022)
    OATP1B1 is an influx transporter that is predominantly expressed in the liver, and it mediates the uptake of many clinically important endogenous compounds and drugs from portal vein blood into hepatocytes. OATP1B1-mediated uptake affects the rate of hepatic elimination of substrate drugs, directly affecting their plasma concentrations. Some naturally occurring single nucleotide variants (SNVs) in the SLCO1B1 gene encoding OATP1B1 can alter the transport function of the transporter resulting in alterations in pharmacokinetics, efficiency and toxicity of substrate drugs. The aim of this master´s thesis was to examine the effect of four naturally occurring SNVs of the SLCO1B1 gene on transport activity, expression, and localization of the OATP1B1 transporter in vitro. SNVs 170G>A (R57Q), 388A>G (N130D), 452A>G (N151S) and 758G>A (R253Q) were created using site-directed mutagenesis in the SLCO1B1 gene presenting in the pENTR221 plasmid. Recombinante baculoviruses were produced in Sf9 cells using the Bac-to-Bac® Baculovirus Expression System and used to transduce HEK293 cells for the overexpression of OATP1B1 wild type and variant proteins. An uptake assay was used to study the transport activity of the OATP1B1 variants in HEK293 cells. Western blotting was used to study the expression of OATP1B1 proteins in membrane vesicles. Immunofluorescence staining was used to determine the localization of OATP1B1 wild type and variants in HEK293 cells. Transport activity of the OATP1B1 variants R57Q and R253Q was significantly decreased compared to wild type. In contrast, transport activity of the N130D ja N151S variants was not significantly altered. The reasons for the changes in transport activity could not be reliably estimated due to the failure to measure the expression levels of OATP1B1 proteins by Western blotting. However, immunofluorescence microscopy revealed that the localization and expression of the all the studied OATP1B1 in baculovirus transduced HEK293 cells were comparable to the wild type. The results of this master´s thesis indicate that SNVs 170G>A and 758G>A can impair the transport activity and substrate uptake functions of OATP1B1 in vitro. Additional in vitro studies of transport activity, expression and localization of the variants R57Q and R253Q will be required to confirm these results. In the future, the R57Q and R253Q variants should be also studied for their possible clinical significance in pharmacokinetics and pharmacodynamics of substrate drugs, as SNVs 170G>A and 758G>A may increase the exposure and the risk for adverse effects of OATP1B1 substrate drugs.