When projected onto this PCA, the BSEP data set (n = 250) was shown … The DILI classifications were product info conducted on approved drugs using the U.S. FDA��s drug labels; nondrug compounds were therefore not included in our analysis. Of the compounds investigated for BSEP inhibition, 180 were identified as the active component in FDA-approved drugs. Two withdrawn drugs (troglitazone and benzbromarone) for which FDA classification data could be retrieved from the literature were also included in the DILI analysis (Chen et al., 2011). A subset of 15 model compounds was selected for further investigation in SCHH. These compounds were chosen to cover the possible combinations of different degrees of BSEP inhibition (inhibitors and noninhibitors) and DILI potential (severe and mild/no DILI).

BSEP inhibitors that increase the risk of severe DILI were represented by cyclosporine A, ritonavir, rosiglitazone, and troglitazone. BSEP inhibitors with no or mild reported DILI were exemplified by mifepristone, isradipine, budesonide, and glyburide. Representative BSEP noninhibitors for severe DILI were valproic acid, flutamide, and zidovudine, whereas BSEP noninhibitors with no or only mild reported DILI were represented by omeprazole, cimetidine, haloperidol, and chlorpromazine. BSEP-dependent TA transport assay. Taurocholate transport was determined, in a 96-well plate format, in inverted membrane vesicles from Sf9 cells overexpressing human BSEP. Statistical experimental design, as implemented in Modde version 7.

0 (Umetrics, Ume?, Sweden), was used to optimize experimental parameters with regard to (1) amount of membrane vesicles per well (10�C50 ��g/well), (2) TA concentration (1�C10��M), and (3) incubation time (1�C10min), at 5 levels per evaluated parameter. On the basis of the experimental design optimization (data not shown), 10 ��g vesicles were used in each well and were incubated with 2��M TA for 5min. All experiments were performed using a rapid filtration technique modified from Pedersen et al. (2008). Briefly, transport buffer (10mM Tris-HCl [pH 7.4], 250mM sucrose, 10mM MgCl2, and 10mM phosphocreatine) was used to dilute dimethyl sulfoxide (DMSO) stock solutions to final substrate concentrations of 2��M (0.8��M/0.1 ��Ci 3H-TA). Final DMSO concentrations were consistently <0.3%, which in our laboratory has been shown to have negligible effects on the TA transport.

BSEP membrane vesicles were quickly thawed from ?85��C to 37��C and diluted in transport buffer to a final concentration of 0.2 ��g/��l. Vesicle solution (50 GSK-3 ��l) including substrate (2��M TA) was preincubated at 37��C for 10min, after which transport was initiated by the addition of 4mM adenosine triphosphate (ATP) and 90U/ml creatine kinase. All plates included control samples in which 4mM ATP was replaced by 4mM Adenosine monophosphate (AMP) to determine passive uptake of TA in the vesicles.