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Browsing by Subject "MRP2"

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  • Järvinen, Erkka (2016)
    UDP-glucuronosyltransferases (UGTs) catalyse glucuronidation reactions between glucuronic acid and drug molecules, which contain nucleophilic groups, mostly hydroxyls, amines or carboxylic acids. Glucuronidation is the most important reaction in the conjugative drug metabolism. Because these conjugates are not usually able to cross cell membranes passively, they need active efflux transport. Efflux transporters mostly belong to superfamily of ATP-binding cassette transporters (ABC). Subfamily C of ABC transporters (ABCC) are known to be involved in efflux transport of glucuronides. Especially MRP2 (ABCC2) and MRP3 (ABCC3) play key roles in the elimination of glucuronide conjugates of drugs. MRP2 is localized in the apical membranes of hepatocytes and enterocytes, whereas MRP3 is localized in the basolateral membranes of the respective cells. On the other hand, UGT1A1 and UGT2B7 are highly expressed in liver and small intestine and are the most important UGTs in drug metabolism. It is known, that UGTs and efflux transporters work together forming interplay to eliminate drugs. Therefore, studying both of them in the same in vitro system is in important focus of drug metabolism studies. The Madin Darby canine kidney cell line (MDCK) is one of the standard in vitro tools in drug metabolism studies. In this study, MDCK was chosen for a cell line to co-express UGTs (UGT1A1 or UGT2B7) and efflux transporters (MRP2 and MRP3 simultaneously. Therefore, cloning of the UGT2B7 cDNA and the ABCC3 cDNA encoding MRP3 was aimed in this study. On the other hand, the UGT1A1 cDNA was already cloned in-house and MRP2 expressing MDCK cells were established earlier. Cloning of the UGT2B7 cDNA was not successful in this study despite of several different strategies such as PCR-amplification of the cDNA fragment using kidney or liver sscDNA as template. Cloning of the ABCC3 cDNA encoding MRP3 was achieved and a mammalian expression vector containing this cDNA was constructed. In addition, the mammalian expression vector containing the UGT1A1 cDNA was used to establish MDCK-UGT1A1 cells and this cell line was characterized regarding the expression of UGT1A1 mRNA and UGT1A1 protein amount. Furthermore, establishment of MDCK-UGT1A1-MRP2 cell line was attempted in this study without success. The mammalian expression vector containing the ABCC3 cDNA encoding MRP3 could be used for future experiments to achieve novel cell lines such as MDCK-UGT1A1-MRP3 and MDCK-UGT1A1-MRP2-MRP3 for drug metabolism studies. In addition, the novel cell line MDCK-UGT1A1 could be used for drug metabolism studies in further experiments, but also as a cell line for further establishment of above cell lines. On the other hand, the cloning of the UGT2B7 cDNA needs optimization and several different strategies should be used to achieve the mammalian expression vector containing this cDNA.
  • Holvikari, Kira (2015)
    MRP2 is an efflux-transporter from the group of ABC-transporters located in the apical side of cell membranes mainly in the liver, intestine, kidneys and lungs. This transporter is associated with multidrug resistance, a phenomenon where the absorption of a drug to the cell is prevented by the transporter as it transports the compound out of the cell. To overcome this phenomenon, inhibitors and substrates for MRP2 are constantly studied. Several flavonoids have been presented of being inhibitors and the research of these compounds continues. Pharmaceutical excipients are also another major group of compounds that possess inhibitory effects towards MRP2. Excipients, as well as flavonoids, are an increasingly studied section of drug interactions and today it may be evaluated that excipients are not thought as inert compounds as has been presented for several years. For now the research of MRP2 interactions focuses mainly on in vitro studies. In the experimental part of this thesis the effects of natural compounds and pharmaceutical excipients are studied towards MRP2 with the vesicular transport assay (VT-assay) with MRP2- Spodoptera frugiperda 9 (Sf9)-membrane vesicles using 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF) as probe. A total of 157 compounds are screened using this in vitro method and hits are further experimented studying IC50 and Ki values. Potential compounds are also tested with two types of particle size measurements (Dynamic light scattering and nephelometer) to evaluate inhibition caused by microaggregates. Some compounds are also studied with liquid chromatograph-mass spectrometry (LC-MS) to determine possible substrates for MRP2. 19 (12%) hits were found from the library of 157 compounds. These hits included 6 stimulators (CDCF transport increased ≥ 150%) and 13 inhibitors (CDCF transport decreased ≤ 50%). IC50 determination was conducted for 12 inhibitors with best-fit values of: Ellagic acid 10.4 µM, gossypin 17.4 µM, morin dihydrate 19.4 µM, myricetin 27.1 µM, nordihydroguaiaretic acid (NDGA) 36.2 µM, octyl gallate 20.3 µM, silybin 52.3 µM, pluronic ®F98 6.9 µM, lutrol F127 ~ 8.2 µM and tannic acid 1.99 µM. Ki determination was conducted for 3 compounds where best-fit values were myricetin 42.9 ± 47.4 µM, gossypin 19.4 ± 12.5 µM and tannic acid 0.0538 ± 0.0398 µM. Ki determination allowed determination of inhibition type: competitive inhibition for tannic acid and gossypin, noncompetitive inhibition for myricetin. Particle sizes studied with dynamic light scattering (DLS) and a nephelometer did not show any significant aggregate formation and inhibition by that mechanism can be ruled out granted that the measurement method should be optimised. Stimulators baicalein, baicalin, digitoxigenin and inhibitors myricetin, gossypin and tannic acid were studied finally with the VT-assay with LC-MS as detector in search of substrates for MRP2. With significant changes in ‚àíATP and +ATP at 50 µM was gossypin. To conclude, gossypin possesses competitive inhibition towards MRP2 and exhibits sings of being a substrate for the transporter as well. Further studies need to be performed to confirm these findings.
  • 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.