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The Role of BSEP/MRP Transporter Interactions in Cholestatic Drug-induced Liver Injury

With the advent of more complex drug therapies and combinations, drug-induced liver injury (DILI) is becoming more problematic. DILI is the leading cause of acute liver failure and describes different clinical manifestations of liver toxicity following drug exposure, for example the development of cholestatic DILI due to impaired hepatic biliary function. Bile, formed within the liver, is essential for the elimination of environmental toxins, carcinogens, drugs and their metabolites, allowing for healthy liver function. ATP-binding cassette (ABC) transporters, most commonly categorised as exporters, utilise ATP binding and hydrolysis to translocate the above mentioned substances across membranes. In particular, ABCB11 (BSEP) and ABCC2 (MRP2) play a crucial role in the biliary secretion of bile acids across the canalicular membrane, through the bile ducts and into the gastrointestinal tract. In addition, an adaptive response against bile acid-mediated hepatotoxicity is established by upregulation of ABCC3 (MRP3) and ABCC4 (MRP4) in situations where BSEP and/or MRP2 are dysfunction or inhibited. It is recognised that inhibition of any of these bile acid transporters can result in increased hepatocellular concentrations of bile acids which could lead to necrotic and apoptotic cell death. It is therefore important that new potential drug compounds are investigated for their potential to influence the function of BSEP, MRP2, MRP3 and MRP4. The propensity of drugs to inhibit these key transporters and the associated DILI risk can be predicted using in vitro methods.

ABC transporters function to efflux drug substrates from the cell and the typical conventional in vitro methodology used to study such transporters is bidirectional transport across polarised cell monolayers. Using such methodology, to enable interaction with the apical efflux transporter, the substrate must first gain entry to the cell by crossing the cell membrane. However, this can present a challenge if the compound has poor passive membrane permeability, rendering it unable to interact with the transporter in vitro. To overcome this barrier, Cyprotex have validated inside-out membrane vesicles overexpressing the BSEP, MRP2, MRP3 or MRP4 transporters for investigating inhibitory potential of compounds. The inside-out configuration allows compounds to have direct (unimpeded) access to the transporter from the incubation buffer. The vesicles are incubated with a radiolabelled probe substrate and increasing concentrations of test compound in the presence of ATP. Transporter-mediated uptake of the probe substrate is monitored by liquid scintillation counting (LSC) and a decrease in uptake is used to calculate an IC50 value (test compound concentration which results in 50 % inhibition) which can then be contextualised together with compound exposure to establish the interaction (and therefore DILI) risk. In addition, substrate identification assays using vesicle test systems can be conducted to provide a more comprehensive picture of the disposition of more polar compounds and/or conjugated metabolites. These compounds may have limited passive permeability making it difficult to evaluate them as substrates of efflux transporters in the conventional polarised cell monolayer based systems.

Overall the addition of these assays to our repertoire of regulatory in vitro test systems increases the understanding of a compound’s disposition pathways and interactions specifically within hepatocytes, so that prediction of DILI is more inclusive. Such knowledge should reduce the chances of later stage failures of compounds that reach clinical trials.

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