Effect of a Common Genetic Variant (p.V444A) in the Bile Salt Export Pump on the Inhibition of Bile Acid Transport by Cholestatic Medications

The bile salt export pump (BSEP) is the primary canalicular transporter responsible for the secretion of bile acids from hepatocytes into bile canaliculi, and inhibition of this transporter has been associated with drug-induced liver injury (DILI). A common variant (rs2287622; p.V444A) in the gene encoding BSEP has been associated with an increased risk of cholestatic DILI. Although p.444V BSEP (reference) and p.444A BSEP (variant) do not differ in their transport kinetics of taurocholic acid (TCA), transport of the more abundant glycocholic acid (GCA) has not been investigated. Importantly, differences in the susceptibility of p.444V and p.444A BSEP to inhibition by drugs causing cholestatic DILI have not been investigated. To address these issues, the transport kinetics of GCA were evaluated by incubating membrane vesicles expressing either p.444V or p.444A BSEP with GCA over a range of concentrations (1, 10, 25, 50, and 100 μM). The abilities of commonly used cholestatic medications to inhibit the transport of TCA and GCA by the reference and variant proteins were compared. Resulting data indicated that GCA transport kinetics for reference and variant BSEP followed Michaelis-Menten kinetics and were not statistically different [ Vmax values of 1132 ± 246 and 959 ± 256 pmol min-1 (mg of protein)-1, respectively, and Km values of 32.7 ± 18.2 and 45.7 ± 25.5 μM, respectively]. There were no statistically significant differences between the reference and variant BSEP in the inhibition of TCA or GCA transport by the cholestatic drugs tested. In conclusion, differential inhibition of TCA or GCA transport cannot account for an association between the variant BSEP and the risk for cholestatic DILI due to the drugs tested

FUNCTIONAL IMPACT OF GENETIC VARIANTS, MEDICATIONS, AND DISEASE ON HEPATIC BILE ACID TRANSPORTERS

Hepatic transporters mediate the movement of substrates including medications and endogenous compounds (e.g., bile acids) across membranes. Transporters are important determinants of pharmacokinetics, and altered transporter function can lead to clinically significant changes in drug disposition and safety. The objective of this dissertation was to characterize changes in hepatic transporter function due to patient specific factors (e.g., genetic variants, concomitant medications, and disease). The association of cholestatic drug-induced liver injury and a common variant (p.V444A) in the bile salt export pump (BSEP) was evaluated to identify underlying mechanisms. The kinetics of glycocholate transport mediated by reference and variant BSEP, and transport inhibition by cholestatic medications, were tested. No differences were found, raising important questions about the mechanistic explanation for the increased risk of cholestasis reported with this BSEP variant. A framework to improve the efficiency of drug-transporter inhibition screenings was developed and applied to identify novel inhibitors of multidrug resistance-associated protein (MRP) 3. A computational model was used to conduct a virtual screen to predict likely inhibitors, these predictions were tested in vitro, and three strong MRP3 inhibitors were identified. This approach can be applied to conduct efficient inhibitor screenings for additional transporters. The impact of nonalcoholic steatohepatitis (NASH), an increasingly prevalent liver disease characterized by steatosis and inflammation, on transporter function was evaluated. Patients with NASH exhibited reduced hepatic uptake clearance mediated by organic anion transporting polypeptide (OATP) 1B1 and OATP1B3, and decreased MRP2-mediated biliary clearance of 99mTechnetium-mebrofenin, an imaging probe. The disposition of medications that are substrates of these transporters could be altered in NASH. Additionally, patients with NASH exhibited increased total serum bile acid concentrations and altered bile acid profiles. Genetic variants in transporters and demographics were important sources of variability that need to be considered when examining specific bile acids as potential biomarkers. Methods and translational approaches utilized in these experiments can be used to improve our understanding of transporter function. This dissertation research underscores the importance of assessing transporter function in the context of patient-specific factors, which will help improve our ability to predict drug disposition and safety in the clinic.Doctor of Philosoph