Statistical analysis. Data are expressed as means with SDs or SEMs, as indicated in the text. In the BSEP inhibition screen, compounds that significantly selleck chemical Bicalutamide decreased BSEP transport were identified by 1-way ANOVA with Bonferroni��s post hoc test, as implemented in Prism 5 (GraphPad Software, Inc, La Jolla, CA). Differences in the proportions of DILI-inducing agents in each ADR class were determined using normality tests. The Marascuillo procedure was used to determine statistical differences of mild and severe DILI distribution in the different ADR sections. Analysis of SCHH accumulation data were conducted using 2-way ANOVA with Bonferroni��s post hoc test. RESULTS TA Transport in Inverted Membrane Vesicles To investigate the impact of drugs on BSEP-mediated TA transport, we used inverted membrane vesicles from cells overexpressing human BSEP.

This system allows direct measurement of drug interactions with BSEP, thereby eliminating certain confounding factors that affect whole-cell systems (such as drug metabolism and the need for sufficient membrane permeability to reach transporter binding sites). The ATP-dependent transport of TA into inverted membrane vesicles was linear for up to 15min (Fig. 2A). The passive TA transport was approximately 5% of the active uptake under the conditions applied. The kinetics of the TA transport was determined from the data in Figure 2B using nonlinear regression. This resulted in a K m of 17.8��5.0��M and a V max of 286.2��28.2 pmol/mg protein/min, in good agreement with previously published data (Kis et al., 2009; Yabuuchi et al.

, 2008). Two inhibitors (cyclosporine A and indocyanine green) were used as controls throughout the experiments and gave low interday (n = 45) variability of 6% and 7%, respectively. Indocyanine green and cyclosporine A inhibited TA uptake with IC50 values of 3.7��1.3 and 4.6��1.2��M, respectively, as determined by fitting a sigmoidal dose-response relationship to the inhibition data (Figs. 2C and and2D2D). FIG. 2. Kinetics of taurocholate (TA) transport in bile salt export pump (BSEP)�Coverexpressing inverted Sf9 membrane vesicles. A, Linearity of TA transport. The TA transport kinetics was linear up to 15min (squares). At 5-min incubation (used in the BSEP … Inhibition of BSEP Transport in Inverted Membrane Vesicles Of the 250 compounds screened for inhibition of TA transport, 86 were identified to significantly (p < .

05) inhibit BSEP at 50��M (Fig. 3). Thirty-seven of these have, to our knowledge, not been previously reported (Table 2). No transport stimulation was detectable at the statistical significance level used (p < .05). Among Brefeldin_A the 86 inhibitors, 53 reduced BSEP-mediated TA transport by more than 50% compared with the controls (without inhibitor) and were therefore defined as strong inhibitors (as described in the Materials and Methods section).