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Browsing by Subject "OATP1B1"

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  • Hämäläinen, Kreetta (2021)
    Personalized medicine tailors therapies for the patient based on predicted risk factors. Some tools used for making predictions on the safety and efficacy of drugs are genetics and metabolomics. This thesis focuses on identifying biomarkers for the activity level of the drug transporter organic anion transporting polypep-tide 1B1 (OATP1B1) from data acquired from untargeted metabolite profiling. OATP1B1 transports various drugs, such as statins, from portal blood into the hepatocytes. OATP1B1 is a genetically polymorphic influx transporter, which is expressed in human hepatocytes. Statins are low-density lipoprotein cholesterol-lowering drugs, and decreased or poor OATP1B1 function has been shown to be associated with statin-induced myopathy. Based on genetic variability, individuals can be classified to those with normal, decreased or poor OATP1B1 function. These activity classes were employed to identify metabolomic biomarkers for OATP1B1. To find the most efficient way to predict the activity level and find the biomarkers that associate with the activity level, 5 different machine learning models were tested with a dataset that consisted of 356 fasting blood samples with 9152 metabolite features. The models included both a Random Forest regressor and a classifier, Gradient Boosted Decision Tree regressor and classifier, and a Deep Neural Network regressor. Hindrances specific for this type of data was the collinearity between the features and the large amount of features compared to the number of samples, which lead to issues in determining the important features of the neural network model. To adjust to this, the data was clustered according to their Spearman’s rank-order correlation ranks. Feature importances were calculated using two methods. In the case of neural network, the feature importances were calculated with permutation feature importance using mean squared error, and random forest and gradient boosted decision trees used gini impurity. The performance of each model was measured, and all classifiers had a poor ability to predict decreasead and poor function classes. All regressors performed very similarly to each other. Gradient boosted decision tree regressor performed the best by a slight margin, but random forest regressor and neural network regressor performed nearly as well. The best features from all three models were cross-referenced with the features found from y-aware PCA analysis. The y-aware PCA analysis indicated that 14 best features cover 95% of the explained variance, so 14 features were picked from each model and cross-referenced with each other. Cross-referencing highest scoring features reported by the best models found multiple features that showed up as important in many models.Taken together, machine learning methods provide powerful tools to identify potential biomarkers from untargeted metabolomics data.
  • Leppänen, Riikka (2017)
    The effect of genes on drug response is studied in the field of pharmacogenetics. Genetic polymorphism occurs in several genes that code drug metabolizing enzymes or drug transporters. A protein coded by a variant gene may be dysfunctional, which can affect the efficiency and safety of the substrate drug individually. The common polymorphisms of the gene ABCG2 coding the efflux transporter BCRP and the gene SLCO1B1 coding the influx transporter OATP1B1 are associated with the interindividual variation in the effectiveness and tolerability of the cholesterol-lowering statins. In this study, the effects of the polymorphisms ABCG2 c.421C>A and SLCO1B1 c.521T>C on rosuvastatin concentration in plasma and the liver were studied with two different pharmacokinetic models. The developed liver model illustrating the enterohepatic circulation of drugs was compared to a commercial Simcyp model. According to the simulations with both models, the effect of the polymorphisms of OATP1B1 and BCRP on the plasma concentration of rosuvastatin is additive. The plasma concentration increases up to fourfold if the same individual has homozygous polymorphic forms of both the OATP1B1 and the BCRP. Based on the modellings, the change of the rosuvastatin concentration in the liver owing to polymorphism does not follow the same pattern as in plasma. In consequence of the polymorphism of the BCRP, the rosuvastatin concentration rises two to three times larger in the liver, which is the site of action of the statins. The polymorphism of the OATP1B1 instead causes the liver concentration to decrease little compared to the wild type. In conclusion, the efflux transporter BCRP seems to have a greater significance on regulating the concentration of rosuvastatin in the liver than the influx transporter OATP1B1. Computer modelling is worth exploiting as a supportive method of other study methods in the pharmacogenetic research, for example when the relative significance of separate transporter proteins is evaluated.
  • 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.
  • Jaakkonen, Liina (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.