Regulation of hepatic uptake transporters by tyrosine kinases

ABSTRACT Hepatic drug uptake transporters are major regulators of systemic clearance and patient outcome, however, characterization of post-translational modifications that mediate the function of these transporters have remained greatly under studied. Therefore, the long-term goal associated with this proposal is to define the extent and significance of post-translational-mediated regulation of pharmacologically important drug-transporters, such as OATP1B1. The overall objective of this proposal is to delineate the consequences of tyrosine kinase inhibitor- (TKI) mediated inhibition of OATP1B1. The central hypothesis is that specific tyrosine kinase(s) are essential regulators of OATPB1, and that interference with this process will decrease hepatic accumulation and increase plasma concentrations of OATP1B1 substrates, resulting in pharmacokinetic (PK) and pharmacodynamic (PD) drug-drug interactions (DDIs). The rationale for this project is that elucidation of the regulatory mechanisms that affect OATP1B1 function will contribute to refinement of therapeutic strategies that have unfortunately to date yielded a variety of unexpected DDI-mediated life-threatening toxicities, which includes statin-induced rhabdomyolysis. The central hypothesis will be tested by pursuing two specific aims: 1) Identify and elucidate the role of regulatory kinases involved in OATP1B1 function; and 2) Investigate the in vivo effects of regulatory kinases on OATP1B1 activity. Under the first aim, LC/MS/MS-based proteomics will be used to measure the ability of kinase inhibitors to modulate OATP1B1 phosphorylation in vitro and ex vivo, while genetic manipulation and comprehensive kinase inhibition or binding assays will be utilized to identify the regulatory kinase of OATP1B1 function. Under the second aim, human transgenic OATP1B1, as well as transporter- and kinase- deficient in vivo mouse models will be used to assess PK/PD changes of OATP1B1 substrates in the presence or absence of TKIs. This will provide mechanistic insight into the role of tyrosine kinases in regulating OATP1B1- mediated hepatic handling of substrates. The collective findings of Aim 2 will also be utilized to develop physiologically-based PK/PD models to predict outcome of TKI-OATP1B1 substrate interactions. The research proposed in this application is innovative because it represents a substantive departure from the status quo by exploring tyrosine kinase activity as a regulator of OATP1B1 function and as a mediator of systemic levels of both endogenous biomarkers and xenobiotics. The proposed research is significant because it is expected to provide important new insights into regulatory mechanisms essential to the function of OATP1B1, the molecular kinase pathways modulating this transporter, and their role in the hepatic handling of the many relevant endogenous and xenobiotic substrates. Ultimately, such knowledge is expected to contribute to the foundation of preventing life-threatening DDIs in patients.