Browsing by Subject "kuljetusaktiivisuus"

The ABCG2-protein is an ATP-dependent half transporter. It is found on apical membranes in intestine, liver, kidney, blood-brain barrier and placenta where it regulates absorption, distribution and elimination of many drugs, but also natural compounds and endogenous metabolites. Natural variation found on the ABCG2-gene can alter protein expression and transport activity. The altered function has been linked to pharmacokinetic changes and developing of diseases like gout. Studying natural ABCG2-variants and their effect gathers knowledge not only on their effect on pharmacokinetics but also on the ABCG2- transporters’ mechanism of function. The aim of this study was to combine an activating (I456V or H457R) and an inactivating (Q141K, F431L or T542A) non-synonymous single nucleotide variant in the same gene to study their combined effect on the ABCG2-transporter expression and active transport. Mutations were incorporated into the ABCG2- gene by site directed mutagenesis and the protein was expressed on HEK293-cells. The transport activity for Lucifer-Yellow and estrone sulfate was measured using HEK293-ABCG2-vesicles produced from cell membranes. The protein expression was measured with Western blot and mass spectrometry proteomics. Based on this study, different mutations together can alter each other’s effects, but the combined result is not always equal to the sum of variations. T542A-mutation did not show significant increase on the protein expression on any of the T542A-combinations, even though it has had such an effect in earlier studies. I456V, earlier expressed like wild type ABCG2, seemed to increase protein expression in all combinations. Q141K, F431L and T542A -mutations had lowering not expression dependent effect on the transport activity. F431L-mutation being so dominant that either of the two activating mutations could not restore the active transport in combinations. As seen before, H457R-variant seemed to cause a significant substrate specific activating effect on transport activity also in this study when combined with other mutations. However, H457R had a strong lowering effect on the protein expression and two of the combinations did not produce enough protein for active transport. As seen in this study, the ABCG2-doublemutations can cause altered ABCG2-function and lead to pharmacokinetic changes. These types of in vitro studies are important in studying these less common genetic variants which in lack of study subjects can be hard to study on clinical trials.

P-glycoprotein is an efflux transporter of the ABC family. It is expressed mainly in tissues that have a role in limiting the absorption and distribution of xenobiotics in the body or their elimination. P-glycoprotein is known to have an important role for example in the blood-brain barrier and in protecting the fetus from xenobiotics in the mother’s blood stream. Genetic polymorphisms in transporter proteins can cause individual differences in the pharmacokinetics of drug substances, which can lead to differences in drug efficacy or side effects. In the ABCB1 gene, which codes for p-glycoprotein, several polymorphisms have been discovered. The frequencies of these polymorphisms vary in different ethnic populations. Previous studies have shown that the effects of these polymorphisms are often substrate-dependent. Since there are several confounding factors usually present in clinical association studies, in vitro studies are needed to clarify the effects of individual polymorphisms. Polymorphisms can be studied in vitro by making intentional mutations to the gene sequence and expressing the variant gene in a suitable cell line. In this study four variant p-glycoprotein genes (c.781A>G, c.1199G>T, c.2005C>T and c.3421T>A) were created by site-directed mutagenesis, and expressed in HEK293 cells using a baculovirus recombinant protein expression method. The effects of the polymorphisms were studied by determining the expression level and the transport acitivity of the variant proteins compared to the wild-type. Western blot was used to determine the expression level and a calcein accumulation assay in HEK293 cells was used to compare the transport activities. Also a membrane vesicle transport assay with n-methyl quinidine was set up and optimized, but the variants were not yet studied with this method during this study. In this study no statistically significant differences were found in the transport activities of any of the four variants compared to the wild-type p-glycoprotein. Also the differences in protein expression level between wild-type and variant proteins were small. However, because of the previously reported substrate dependency of polymorphism effects, it would be beneficial to study the variants with at least one other substrate and one other assay method, and thus the membrane vesicle transport assay would be useful to further compare the transport activities of variant proteins to the wild-type p-glycoprotein.

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.