TY - THES ID - 134902583 TI - Development of a membrane vesicle-based assay for in vitro identification of bile salt export pump (BSEP) inhibitors AU - Jacobs, Elisa AU - Annaert, Pieter AU - KU Leuven. Faculteit Farmaceutische Wetenschappen. Opleiding Master in de geneesmiddelenontwikkeling (nieuw programma vanaf 2017-2018) (Leuven) PY - 2020 PB - Leuven KU Leuven. Faculteit Farmaceutische Wetenschappen DB - UniCat UR - https://www.unicat.be/uniCat?func=search&query=sysid:134902583 AB - Introduction: Drug-induced liver injury (DILI) covers a spectrum of rare but potentially severe adverse effects. DILI diagnosis is an exclusion process of other possible causes, with the occasional misdiagnosis. DILI accounts for high attrition rates in drug development and drug withdrawal from the market. The development of an early (preclinical) DILI predictive model is therefore of high importance. Cholestatic DILI is a result of (drug-induced) cholestasis, which is an alteration in bile acid homeostasis and bile flow. The mechanisms involved in both drug-induced cholestasis and liver injury are complex and not fully cleared up yet. However, several pathways have shown a correlation with cholestatic liver injury, one being the malfunctioning of the bile salt export pump (BSEP). BSEP is the main hepatic efflux transporter for conjugated bile acids. Two genetic diseases (PFIC-2 and BRIC-2) are caused by mutations in the gene coding for BSEP and clinical presentation includes cholestasis. The correlation between BSEP malfunction and cholestasis has led to the hypothesis that the inhibition of BSEP by drugs is a mechanism involved in cholestatic DILI. The development of an in vitro BSEP inhibition assay is therefore seen as a promising tool for the preclinical prediction of the DILI potential of drugs. Materials and methods: Inside-out BSEP membrane vesicles were used as in vitro model system to determine BSEP-mediated substrate uptake and the influence of inhibitors. Fluorescent substrates used were tauro-nor-THCA-24-DBD and cholyl-L-lysyl-fluorescein (CLF), while glycocholic acid (GCA) was used as the endogenous substrate. Bioanalysis was performed by fluorescence spectrometry for fluorescent substrates and LC-MS/MS for GCA. Net uptake was calculated by subtracting uptake in BSEP membrane vesicles by uptake in MOCK membrane vesicles. Results: Inhibition experiments with tauro-nor-THCA-24-DBD showed high interexperimental variability. Adsorption of the substrate to the glass-fiber filter plate was seen to highly influence substrate uptake measurements. This problem could not be overcome by the use of filter plates lacking a glass-fiber layer, as uptake rates were even lower. Additionally, the dynamic range was not sufficient. CLF was used as alternative probe substrate in an exploratory experiment and highest uptake was seen at 15 min incubation time and 20 µM substrate concentration. However, the dynamic range was not increased compared to tauro-nor-THCA-24-DBD. GCA showed increasing uptake with prolonged incubation times and in function of increasing substrate concentration. The dynamic range was comparable to tauro-nor-THCA-DBD and CLF, but could still be increased by assay optimization. Conclusion: Tauro-nor-THCA-24-DBD nor CLF were found suitable substrates for membrane vesicle-based assessment of BSEP activity and evaluation of BSEP inhibitors. Dynamic ranges were too narrow, primarily due to non-specific binding to the in vitro assay setup. However, the endogenous bile acid GCA was found to be remarkably suitable for the purpose of this BSEP assay. Despite the disadvantage that LC-MS/MS-based bioanalysis is required, this assay is currently being optimized to increase its sensitivity and robustness. ER -