Absolute Quantification Of Drug Transporters In Human Hepatocytes And Its Application Based Liquid Chromatography-Tandem Mass Spectrometry

PurposeThis research is to explore the correlation between transporter absolute expression quantity and its biological functions, which will offer new method for new drug development and clinical medication safety. MethodDrug transporter OATP1B1, OATP1B3, OATP2B1 and NTCP were selected as the research object. Firstly, the pretreatment processes of drug transporter membrane protein was optimized, including extraction of membrane protein, the selection of solubilation solvent and the ratio of trypsin to protein. The liquid chromatography-tandem mass spectrometry(LC-MS/MS) method was developed and validated for simultaneous determination of OATP1B1, OATP1B3, OATP2B1 and NTCP expression level in 23 donors of Chinese human cryopreserved hepatocytes, with synthesizing surrogate peptides of OATP1B1, OATP1B3, OATP2B1 and NTCP as calibrated standard, and corresponding stable isotope labeled surrogate peptides as internal standard and optimizing the chromatograph and mass spectrometry conditions.With the model of sandwich cultured human hepatocytes, hepatic uptake activities of the substrates including deuterium sodium taurocholate and rosuvastatin were determined in different batches of human hepatocytes. With HEK293 cells stably transfected human OATP1B1, OATP1B3, OATP2B1 and NTCP gene, the values of Km and Vmax of deuterium sodium taurocholate and rosuvastatin were determinedand applied in correlation analysis between transporter protein expression and uptake activity..In the uptake inhibition assay, the inhibition effect of Danhong injection on transporter OATPs and NTCP was explored by the inhibition of Danhong injection on uptake of rosuvastain in transfected cells and human hepatocytes. And drug-drug interaction between Dandong injection and rosuvastain was analysed based on this research results. ResultThis study optimized the pretreatment processes of drug transporter membrane protein, and the results revealed that membrane protein extraction method membrane extraction kit had good reproducibility; and sodium laurate, used as a solubilizer of transporter membrane protein, had better dissolving efficency than sodium deoxycholate a and did not inhibit the ionization of surrogate peptides; the optimal ratio of trypsin to protein was 1:5. The LC-MS/MS method was established and validated for simultaneous quantification of OATP1B1, OATP1B3, OATP2B1 and NTCP. Both the intra-day and inter-day precision and accuracy of analytes were all within the acceptable criterion of macromolecules bioanalysis. The standard curves were demonstrated linear in the range of 0.2-50 nM for OATP1B1,OATP1B3 and OATP2B1 and 0.4-100 nM for NTCP, respectively( r>0.995). The average protein expression level of OATP1B1, OATP1B3, OATP2B1 and NTCP in 23 donors of Chinese human cryopreserved hepatocytes was 4.74, 1.39, 3.02 and 3.69 fmol/μg, respectively. The results showed that transporter protein expression had individual differences.In the uptake studies, combined with transporter quantification, NTCP played a major contribution in the hepatic uptake of rosuvastain and deuterium sodium taurocholate with 68% and 89% of overall active uptake, respectively. Besides, the hepatic uptake clearance mediated by transporters was determined with sandwich cultured human hepatocytes in vivo. As the comparison of the values of predicting clearance and determined hepatic active clearance, linear regression correlation coefficient was 0.74 and 0.66 respectively for deuterium sodium taurocholate and rosuvastain. And the results with spearman analysis showed positive correlation with rs were 0.1319 and 0.3604, respectively.In the uptake inhibition study, Danhong injection showed different inhibition effect on the uptake of rosuvastatin with different stable, the IC50 values on OATP1B1,OATP1B3,OATP2B1 and NTCP were 0.38mL/100 m L, 0.14 mL/100 m L, 0.72 m L/100 m L and >3.2 m L/100 mL, respectively. The results showed Danhong injection had the strongest inhibitory effect on OATP1B3, and the weakest inhibition on NTCP. And Danhong injection also inhibited on hepatic uptake of rosuvastain in human hepatocyte model, and the IC50 value was 0.85 m L / 100 mL. Hence, there will be a potential drug-drug interaction risk when Danhong injection is used in clinical medication combined with rosuvastain and other substrates of OATP.