The Polyspecific Organic Cation Transporters: Role in Cancer Drug Targeting, Xenobiotic Disposition, and Drug-drug Interactions

Abstract

The polyspecific organic cation transporters (pOCTs) include the organic cation transporters (OCT1-3), the multidrug and toxin extrusion proteins (MATE1/2K) and the plasma membrane monoamine transporter (PMAT). These transporters have been implicated in the absorption, distribution and excretion of many drugs and xenobiotics, and are considered major determinants in the pharmacokinetics, pharmacodynamics, and toxicity of its substrates. Additionally, the OCT2 and MATE1/2K renal transporters are considered important sites for transporter-mediated drug-drug interactions (DDIs), capable of impacting systemic and intrarenal concentrations of xenobiotics. There are still many gaps in our knowledge of pOCTs, including their role in cancer-drug targeting, their contribution to the disposition and toxicity of environmental xenobiotics, and how to improve the in vitro to in vivo prediction of renal transporter-mediated DDIs. The overall goal of this dissertation research is to fill in some of these gaps by: (1) investigating the expression of pOCTs in neuroblastoma and their potential role in tumor disposition of the theranostic agent meta-iodobenzylguanidine (mIBG), (2) characterizing the interactions and uptake kinetics of pOCTs with benzalkonium chlorides (BACs) and their role in tissue accumulation of these environmental compounds, and (3) exploring the use of a double-transfected system to improve prediction of OCT2/MATE1-mediated renal DDIs.Neuroblastoma is a childhood cancer with poor survival rates in high-risk patients. 131-iodine labeled mIBG (131I-mIBG) kills tumor cells by exposure to radiation and has emerged as a promising therapy for high-risk neuroblastoma. mIBG enters neuroblastoma cells via the norepinephrine transporter (NET), however expression of NET alone cannot predict clinical response to mIBG. Previously, our lab identified that mIBG is an excellent substrate of OCTs and MATEs, and thus the expression of pOCTs in neuroblastoma could impact tumor disposition and response to 131I-mIBG. As part of this dissertation research, I investigated the expression of pOCTs and other monoamine transporters in neuroblastoma cell lines, in local tumor samples, and using neuroblastoma genomic data available at the NCI TARGET database. Our results revealed that PMAT is a previously unrecognized transporter highly expressed in neuroblastoma and that its expression level is positively associated with overall survival of high-risk patients without MYCN oncogene amplification. Additionally, we showed that PMAT efficiently transports mIBG, is mainly localized in mitochondria of neuroblastoma cells and mediates mitochondrial uptake of mIBG. Together, these results support a role of PMAT in intracellular disposition of mIBG in neuroblastoma, potentially impacting tumor exposure and response to 131I-mIBG therapy. The pOCTs have the potential to impact not only human exposure to drugs, but also our exposure to environmental xenobiotics. BACs are quaternary ammonium compounds used as disinfectants and as preservatives in several consumer products. However, multiple studies report that BACs are cytotoxic and may be involved in biochemical interactions, and thus their safety has been questioned by the FDA. Humans are chronically exposed to BACs, and these compounds have been found to broadly distribute and accumulate in tissues with known expression of pOCTs such as the kidneys. To characterize the interaction of BAC of varying alkyl chain length (C8, C10, C12 and C14) with the human OCT1-3 and MATE1/2K, we conducted in vitro uptake and inhibition assays in HEK293 cells transfected with the transporters. We showed that all investigated BACs are inhibitors OCTs and MATEs, and that C8 and C10 are substrates of these transporters. We further demonstrated that BAC C8 and C10 are transported across a OCT2/MATE1 double-transfected MDCK monolayer, and that intracellular accumulation of these compounds is much higher in OCT2/MATE1-expressing cells in comparison to vector-transfected cells, suggesting a role of these transporters in the intrarenal accumulation of short chain BACs. We propose that OCTs and MATEs mediate tissue distribution and accumulation of short chain BACs and thus may represent important determinants of organ susceptibility to BAC toxicity in humans as a result of chronic environmental exposure. The research presented in this dissertation highlights that many xenobiotics can interact with pOCTs as substrates and/or inhibitors, raising the concern for transporter-mediated DDIs. Accurately predicting clinical DDI potential based on in vitro data is challenging and this is especially evident for MATE inhibitors, for which the use of plasma unbound maximal inhibitor concentration (Imax,u) and IC50 values determined in single transporter-transfected cells often lead to false or overprediction of DDI potential. With the goal of improving DDI risk assessment, I explored the use of OCT2/MATE1 double-transfected MDCK cells in a Transwell system as a new in vitro tool to assess the inhibitory potential of compounds. Our results revealed that some potent in vitro inhibitors of MATE1 (hydroxychloroquine, brigatinib and famotidine) failed to inhibit the transepithelial flux of metformin in the double-transfected system. In contrast, the classical OCT2/MATE1 inhibitors pyrimethamine and cimetidine dose-dependently inhibited metformin transepithelial flux. We hypothesize that the different behaviors of these MATE1 inhibitors in the double-transfected system vs single-transfected model could be explained by their different abilities to gain intracellular access and reach MATE1 site of inhibition. Additionally, we propose a new parameter reflecting inhibitory potential on overall OCT2/MATE1-mediated secretion (IC50,flux), and concluded that the IC50,flux performs better than individual transporter IC50 values when predicting in vivo DDIs using a static model. Together, our findings suggest that the use of OCT2/MATE1 double-transfected cells in DDI risk assessment is promising and has the potential to reduce the burden of unnecessary clinical DDI investigations by identifying in vitro MATE1 inhibitors unlikely to result in DDIs in vivo. In summary, this dissertation research has contributed significantly to our knowledge of the clinical significance of pOCTs, exploring their role in mIBG disposition in neuroblastoma, their contribution to the tissue-disposition and toxicity of BACs, and providing new tools to improve in vitro to in vivo prediction of OCT2/MATE1-mediated renal DDIs.