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Background
Transporters in the hepatocyte basolateral membrane are responsible for carrier-mediated processes involved in the uptake of xenobiotics from the systemic circulation. Hepatic clearance can be thought to begin with movement of xenobiotics from the blood into hepatocytes by either passive diffusion or active transport through the cell membrane. In hepatocytes, active basolateral uptake has been shown to be mediated by various pathways including organic anion transporters (e.g. OATP1B1, OATP1B3, OATPB2B1, OATP1A2, OAT2, OAT5), organic cation transporters (e.g. OCT1), and the Na-taurocholate cotransporting protein NTCP. Multiple transporters are also known to mediate the excretion of compounds from hepatocytes back into the blood via basolateral membrane efflux transporters or excretion into the bile via outwardly directed efflux transporters including the ATP-binding cassette (ABC) protein family. Collectively these and other transport and metabolism processes work in concert to carry out essential hepatic detoxification drug/xenobiotic clearance functions.
Drug-drug interactions involving hepatic membrane transporters are thought to arise via direct (competitive) or allosteric inhibition of hepatic transporters or via perturbations in transporter cytosolic membrane localization or gene expression levels. The resulting changes in transport activity can alter the drug pharmacokinetics that can result in elevated levels of a co-administered compound due to impaired clearance pathways. Therefore, evaluation of the substrate and inhibitor potential of a drug candidate for the uptake hepatic transporters in vitro can be useful in understanding xenobiotic clearance pathways and predicting the potential for drug-drug interactions (e.g. the hepatitis C drugs alphainterferon and S-acyl-2-thioethyl esters or the HMGCoA inhibitors (statins)).
In addition to drug-drug interactions, hepatic transporters also play an important role in detoxification, and have been linked with toxicities including cholestasis and hyperbilirubinemia. Drug-induced hepatotoxicity is a major liability in drug development and there is growing evidence that inhibition of biliary transporters is a contributing mechanism (1).
Primary hepatocytes in suspension, attached to tissue culture matrices, or in sandwich-culture have all been shown to be useful models to assess hepatic transport potential. The contribution of transporter-mediated uptake to hepatic clearance (CLH) was recognized when CLH was consistently under-predicted for many series of chemotypes using just metabolic stability for the calculations. Factoring in transporter-mediated hepatic uptake, along with metabolic clearance using hepatocytes in suspension, improved these predictions (2).
Important notes
Critical materials and reagents
Equipment
* Important note: Repeat this assay using cold (4˚C) buffer, dose solution, cold wash buffer and incubation temperatures in order to account for non-specific uptake. Plates should be kept on ice for the entire cold procedure.
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