Browsing by Subject "BCRP"
Now showing items 1-4 of 4
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(Helsingfors universitet, 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.
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(Helsingin yliopisto, 2021)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.
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(2017)To study the function and expression of nine naturally occurring single-nucleotide polymorphisms (G406R, F431L, S441N, P480L, F489L, M515R, L525R, A528T and T542A) that are predicted to reside in the transmembrane regions of the ABC transporter ABCG2. The transport activity of the variants was tested in inside-out membrane vesicles from Sf9 insect and human derived HEK293 cells overexpressing ABCG2. Lucifer Yellow and estrone sulfate were used as probe substrates of activity. The expression levels and cellular localization of the variants was compared to the wild-type ABCG2 by western blotting and immunofluorescence microscopy. All studied variants of ABCG2 displayed markedly decreased transport in both Sf9-ABCG2 and HEK293-ABCG2 vesicles. Impaired transport could be explained for some variants by altered expression levels and cellular localization. Moreover, the destructive effect on transport activity of variants G406R, P480L, M515R and T542A is, to our knowledge, reported for the first time. These results indicate that the transmembrane region of ABCG2 is sensitive to amino acid substitution and that patients harboring these ABCG2 variant forms could suffer from unexpected pharmacokinetic events of ABCG2 substrate drugs or have an increased risk for diseases such as gout where ABCG2 is implicated.