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Browsing by Subject "MRP2 substraatti"

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  • 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.