ABSTRACT
<p><b>OBJECTIVE</b>To establish a precolumn chiral derivatization method for determination of fexofenadine enantiomers, a chiral substrate of OATP1B1, in cellular model.</p><p><b>METHODS</b>R-(+)-phenylethyl isocyanate was selected as chiral derivatization reagent, which was reacted with fexofenadine to form carbamate derivatives. Enantiomers were identified by LC/MS and separated by RP-HPLC.</p><p><b>RESULTS</b>Under the experimental conditions, the fexofenadine enantiomers were separated completely. The standard curve was linear over the concentration range of 25-100 ng/ml (R(2)=0.9992, 0.9989). Accuracy was 101.1% and 98.3%, intra-precision was 2.4% and 3.1%, inter-precision was 3.1% and 4.0% for D1 and D2, respectively.</p><p><b>CONCLUSION</b>The method established is sensitive and accurate for determination of fexofenadine enantiomers in cells.</p>
Subject(s)
Chromatography, High Pressure Liquid , Methods , Stereoisomerism , TerfenadineABSTRACT
To establish a pig kidney cell line LLC-PK1/BCRP in which human breast cancer resistance protein was highly expressed, the expression vector pcDNA3.1(+)-BCRP which contained BCRP gene was constructed and transfected into LLC-PKI cells via liposomes. After selecting with G418, population doubling time, flow cytometry and Western blotting analysis were used to evaluate the cell line. MTT assays were employed to determine the drug resistance index of mitoxantrone and doxorubicin. Invert fluorescent microscope was used to observe the efflux of fluorescence dye Hoechst 33342 by BCRP, furthermore, the BCRP's inhibitor GF120918 was applied to reverse the efflux of Hoechst 33342. The experiment results showed that the expression of BCRP protein increased in LLC-PK1/BCRP cell. The population doubling time of LLC-PK1/BCRP cell was a little longer than that of the parental cell LLC-PK1. The resistance indexes to mitoxantrone and doxorubicin were 51.95 and 6.09 times, respectively, higher than LLC-PK1 cell. The efflux of Hoechst 33342 was significantly enhanced and could be reversed by GF120918. So a LLC-PK1/BCRP cell line was established, which highly expressed BCRP protein successfully. This cell line could be a valuable model to further investigate the biological profile of BCRP and select the substrate and inhibitor of BCRP.
Subject(s)
Animals , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters , Genetics , Metabolism , Acridines , Pharmacology , Benzimidazoles , Metabolism , Cell Cycle , Cell Proliferation , Doxorubicin , Pharmacology , Drug Resistance, Multiple , Genetic Vectors , LLC-PK1 Cells , Cell Biology , Metabolism , Mitoxantrone , Pharmacology , Neoplasm Proteins , Genetics , Metabolism , Plasmids , Swine , Tetrahydroisoquinolines , Pharmacology , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To establish a chiral separation method for determination of fluvastatin enantiomer with in vitro cellular model.</p><p><b>METHODS</b>The determination was performed on Chiralpak AD column (4.6 mm × 250 mm); and the phase consisted of hexane-isopropanol-trifluoroacetic acid (90:10:0.1) at a flow rate of 0.5 ml/min with UV detection of 239 nm.</p><p><b>RESULT</b>The standard curve was linear over the concentration range of 20 μmol/L-300 μmol/L (r² = 0.9993, r² = 0.9997). The recovery for this assay was (99.4 ± 0.8)%, precision for inter-assay and intra-assay was <10 %.</p><p><b>CONCLUSION</b>The normal-phase HPLC chiral separation method was accurate and suitable for study on the stereoselectivity of fluvastatin with in vitro cellular model.</p>