HepG2 cell line as a model for studying of the statins’ hepatic uptake

Abstract

Statins (β-hydroxy-β-methyl-glutaryl-CoA reductase inhibitors) are the main class of drugs for the treatment of dyslipidemia. To improve the safety of therapy with their use, a test system is needed to assess their penetration into hepatocytes using OATP1B1/OATP1B3 transporter proteins. The aim of the study was to develop a method for assessing the penetration of statins into hepatocytes on HepG2 cells (human hepatocellular carcinoma). Materials and methods. Cells were cultured in 6- and 24-well plates. The presence of OATP1B1 in HepG2 cells was assessed using the Western blot method. Penetration of statins into cells was analyzed using atorvastatin as an example. It was added to the cell monolayer at concentrations of 1 and 10 μM and incubated for 30 minutes. The cells were then removed from the wells, lysed in various ways, and the amount of atorvastatin was determined by high performance liquid chromatography with tandem mass selective detection (HPLC-MS/MS). Results. Western blot showed the presence of OATP1B1 in HepG2 cells, the main protein that transports statins into hepatocytes. The best way to lyse the cells was a three-cycle freeze-thaw cycle at -80 °C. The analytical range of the method for the quantitative determination of atorvastatin in the lysate of HepG2 cells by HPLC-MS/MS was 0.5-200 nmol/l, which made it possible to perform transport experiments with the addition of atorvastatin at a concentration of 1 μM and incubation for up to 30 min. The use of the OATP1B1/OATP1B3 inhibitor rifampicin (100 μM) reduced the penetration of atorvastatin into HepG2 cells, which confirms the adequacy of the proposed method. Conclusions. A technique for assessing the penetration of statins into hepatocytes on HepG2 cells has been developed.