Drug-Induced Liver Injury – DILI – is one of the most common and potentially severe drug side-effects, which may lead to acute liver failure or develop into chronic liver disease in affected patients or trial volunteers [1-2]. Among the possible underlying mechanisms of DILI, cholestatic liver injury accounts for the majority of such outcomes, where the excessive accumulation of bile acids and salts (BA and BS, respectively) inside the hepatocytes leads to cell toxicity and liver damage [3-4].
A total of 63 drugs were withdrawn from the market between 1964 and 2018 due to toxic liver effects, demonstrating the serious impact of DILI [5-8], and while the related safety and regulatory measures have become increasingly strict and more detailed over the years, 11 of these withdrawals still occurred after 1998, with the latest being Flupirtine which was withdrawn in 2018 [9-11]. As the impact of DILI may have severe, even life-threatening consequences for patients and trial participants, identification of risk factors associated with DILI early in development is an important safety consideration. While post-market discovery of potential DILI-inducing effects of a drug is arguably the most severe scenario, hepatotoxicity is also a common cause for clinical stage project terminations [12-16]. Any such termination imposes a significant cost for drug developers and their investors in both time and monetary value.
Early detection of potential hepatotoxic properties of drug candidates in development is therefore beneficial, as it can significantly reduce the risk of later-stage clinical failure due to DILI. While hepatotoxicity-assessment assays are usually part of early safety testing using liver-derived cell lines such as HepG2 or HepaRG cells, the in vitro cellular systems commonly applied in these experiments are not suitable for assessing toxic bile accumulation and cholestatic cytotoxicity [17-18]. In contrast, isolated primary hepatocytes grown in sandwich cultures form bile canaliculi and produce significant quantities of bile salts and can therefore be used for assessing cholestatic effects [17, 19]. One drawback of this in vitro model however is that it is relatively complex and costly, and thus not typically used in early discovery. Furthermore, while preclinical in vivo toxicity studies can be carried out to investigate signs of liver toxicity, these may fail to accurately predict the risk of cholestatic DILI due to species differences. This can be particularly problematic when using rodents are used, as BA composition and associated metabolic pathways in rodents are quite different from those found in humans [20-24].
A review of drug withdrawals due to DILI over the past decades has identified transporter involvement in 78% of cholestasis cases . Cholestatic DILI most often develops due to the interaction of a compound with one or more hepatic BA/BS transporters, such as BSEP (Bile Salt Export Pump), MRPs (Multidrug Resistance-associated Proteins) or NTCP (Sodium/Taurocholate Co-transporting Polypeptide) [5, 25-27]. Inhibition of these transporters can be tested in in vitro systems, such as the vesicular transport (VT) assay for efflux pumps or a cellular uptake assay for uptake carriers. Unlike the cellular methods mentioned above, these assays are not only significantly more cost-efficient, but also lend themselves better for evaluating multiple test articles (TAs) in discovery type screening setups to enable quick turnaround of data.
Bile acids (BA) and bile salts (BS) undergo the process of enterohepatic recirculation, in which they are excreted from the systemic circulation via the liver into the bile and then released in the small intestine to aid in digestion of fats from the diet, followed by reabsorption across the gut enterocytes back into the systemic circulation from where they are taken up again into hepatocytes [28-29]. Uptake of bile acids/salts from the blood into the hepatocytes and their excretion into the bile canaliculi are mediated by transporters. Under normal conditions, BA/BS efflux is mediated mainly by BSEP and MRP2: BSEP is responsible for the secretion of monovalent BS, which constitute the majority of human bile secretion [30-32], while efflux of glucuronide or sulfate conjugates of monovalent BA/BS is mediated by MRP2 [Figure 1a] [33-34].
Reuptake of BA/BS into the hepatocytes through the sinusoidal membrane is mediated by uptake transporters. Conjugated BS, representing the main fraction of circulating BA/BS, are taken up by NTCP in a sodium-dependent manner, while the rest is reabsorbed by sodium-independent transport via different mechanisms [35-37].
Inhibition of BA/BS transporters expressed in the liver may lead to DILI due to different mechanisms and events: Inhibition of BSEP by xenobiotics disrupts the normal excretion of bile acids into the bile canaliculi resulting in increased intracellular accumulation of bile salts  [Figure 1b]. Inhibitors of BSEP that also inhibit NTCP may mitigate intracellular BA/BS levels by blocking uptake , but if NTCP function is not impacted the intracellular concentration of BA/BS may further increase due to their continued active uptake into the hepatocytes [Figure 1d]. This is turn may lead to the development of cholestatic liver damage.
The efflux transporters MRP3 and MRP4 are expressed at relatively low levels and localized to the apical side of the hepatocytes. Under normal physiological conditions they do not significantly contribute to the efflux of BA into the systemic circulation. However, upon the increase of intracellular BA/BS levels, their expression is induced, leading to increased efflux of bile acids from the hepatocytes [26, 38-39] to the blood and easing cholestatic conditions [Figure 1c]. A similar role has been proposed for MRP2, which – unlike MRP3 and MRP4 – resides in the basolateral (canalicular) membrane of hepatocytes and which both mediates biliary BA/BS secretion and also excretes high amounts of reduced glutathione (GSH) which is a driving force for overall bile secretion [35, 40-41]. Inhibition of MRP2 can therefore contribute to cytotoxic effects of BA/BS accumulation in hepatocytes via two separate mechanisms.
Figure 1. Schematic representation of transporters involved in bile salt and bile acid (BS/BA) transport and DILI a) main transporters mediating efflux enterohepatic recirculation of BS/BA, b) cholestatic intracellular bile salt accumulation in hepatocytes upon BSEP inhibition, MRP3 and MRP4 upregulation, c) increased intracellular BS/BA levels upon MRP3, MRP4 and MRP2 inhibition, and d) reduced cholestatic hepatotoxicity upon co-inhibition of NTCP.
In summary, any drug or drug candidate that inhibits not only BSEP, but also one or more of the MRP2, MRP3, or MRP4 transporters while not inhibiting NTCP may exhibit an increased liability for cholestatic liver damage and DILI .
As expected, the role and relative importance of each of the hepatic BA/BS transporters mentioned (BSEP, MRP2, MRP3, MRP4 and NTCP) is reflected in the impact of their inhibition on DILI risk. BSEP is the primary culprit for possible transporter-mediated DILI, with MRP3 and MRP4 following closely on the list of transporters most likely to be involved in drug-induced cholestasis  [Figure 2].
Figure 2. Relative frequencies (expressed as percentages) of transporter involved in drug-induced cholestatic liver injury. Relative frequencies are calculated by dividing the times that a molecular mechanism is involved in drug-induced cholestatic liver injury by the sum of all reported mechanisms for drugs withdrawn due to cholestatic DILI between 1964 and 2016. Adapted from Deferm et al., 2019 .
As described above, potential inhibition of different transporters involved in BA/BS transport are a concern for drug developers worldwide as clinical stage terminations and market withdrawals due to DILI remain a tangible risk. Obtaining data to address the possible risk of these events at an early stage of discovery or development can therefore be a useful and cost-effective approach to select safe lead molecules during candidate selection, identify and deprioritize (or discontinue) molecules with increased DILI risk, or - if possible - minimize DILI risk via selection of appropriate dosage and treatment intervals for a safe application.
To assist drug developers in characterizing the potential transporter-mediated DILI risk of their chemical library, SOLVO has developed the DILIScreen™ service package to investigate potential transporter interactions at an early stage of drug discovery. The DILIScreen™ package has been optimized for discovery projects with emphasis on:
Furthermore, as with all SOLVO services, our expert team will be at your disposal for any follow-up discussion regarding your results.
* Data delivery within up to 3 business days from test article arrival is possible for vesicular transport assays (BSEP, MRP2, MRP3, MRP4) upon pre-agreed conditions and reservation of lab capacity, depending on the number of test articles – for more information on timelines contact your SOLVO representative.
SOLVO’s DILIScreen™ assay package includes the key transporters involved in DILI: BSEP, MRP2, MRP3 and MRP4 efflux pumps and the NTCP uptake carrier. When addressing DILI risk, a minimal setup to assess BSEP inhibition is advised, however, including MRPs from the start may be considered to obtain more information at an early stage.
|DILIScreenTM||Transporter(s)||Assay conditions||System used|
|Efflux||BSEP, MRP2, MRP3, MRP4||1cc “spot-check”
|Vesicular transport (VT) assay|
|Uptake||NTCP||Cellular uptake (Upt) assay|
Assay setups are available for testing TAs for transporter inhibition at a single concentration, referred to as a “spot-check”, or for IC50 determination using multiple TA concentrations. While a spot-check can already provide important indication of potential DILI risk, IC50 values – where applicable – can yield a significantly more robust liver safety dataset. For early assessment of transporter-mediated DILI, a tiered approach has been proposed , in which potential inhibition of BSEP is tested first, followed by the MRPs. While BSEP is the transporter most commonly involved in DILI, multiple groups have reported that assessing drug interaction with MRPs in addition can improve predictivity [26, 42, 46]. In the event that significant inhibition is observed for one or more efflux transporters, a follow-up investigation of NTCP inhibition is recommended in order to provide further insight into whether this mechanism could contribute to an overall reduced risk of DILI.
Did you know…?
Bile acid transporters, such as BSEP, MRP2, MRP3, MRP4 or NTCP are not commonly involved in mediating drug-drug interactions (DDI), therefore they are not required to be studied for regulatory submission at most agencies, including the FDA . The 2013 EMA guidance highlights BSEP as a potential DILI mediator , however BSEP inhibition data is not always included in regulatory submissions for DDI purposes. Nonetheless, the International Transporter Consortium (ITC) has been advocating for the inclusion of BSEP-inhibition assessment studies at an early phase in drug development and issued a white paper on the topic in 2018 . While in this publication, other bile acid transporters are not selected for screening, advances in the development of in silico models for DILI prediction based on in vitro-in vivo extrapolation (IVIVE) of transporter interaction data (among others) highlighted the importance of looking beyond BSEP for accurate risk assessment [26, 42, 46].
DILIScreenTM has been optimized for the early discovery pipeline: thanks to our semi-automated assay design, we can provide a medium-high throughput screening service where large numbers of TAs can be tested cost efficiently with short turnaround time. For assessing inhibition of BSEP, MRP2, MRP3 and MRP4 (efflux transporters), a Vesicular Transport assay system is utilized, whereby the effect of the TAs on the uptake of a radiolabeled probe substrate into inside-out membrane vesicles generated from transporter-overexpressing cells is measured . NTCP inhibition is assessed in a cellular uptake assay whereby the inhibitory effect on the intracellular accumulation of a probe substrate is measured . Assay setups have been optimized for this semi-automated workflow (outlined for VT assays in Figure 3a) using carefully selected conditions and controls that ensure outstanding data quality and reproducibility (demonstrated in Figure 3b using a series of BSEP inhibitors).
We have generated data on BSEP inhibition using a reference data set of over 60 known inhibitors to determine IC50 values using the automated DILIScreenTM platform. As shown in Figure 3b, variability among data points from 3 technical replicates using DILIScreenTM remained within 1.4-fold, demonstrating that this quick and cost-effective setup provides reliable and high-quality data.
Figure 3. a) Outline of the DILIScreenTM semi-automated assay workflow for Vesicular Transport assays. Most steps are automated, with addition of the scintillation cocktail carried out manually. b) BSEP inhibition IC50 values obtained in the semi-automated DILIScreenTM VT assay setup normalized to the average IC50 value for each respective TA obtained from at least 3 separate experiments. A total number of 61 individual known BSEP inhibitors were tested as represented on the X-axis, orange dots show IC50 values generated from each individual experiment. Variability among data points from at least 3 technical replicates remains within 1.4-fold.
Beyond DILIScreen™, SOLVO also offers an assay package for thoroughly investigating one or more compounds using assay setups specially designed for in silico prediction of DILI in the DILIsym platform from Simulations Plus. Aside from cholestatic effects due to BA/BS transport inhibition, additional mechanisms may be responsible for DILI, including disruption of bilirubin- or phospholipid trafficking, that are often mediated by transporters, such as MDR3, ASBT, OATP1B1, OAPT1B3 or OSTα/β, all for which assays are available at SOLVO. Complementary services are also available including HepatoPac® and B-clear® services which are conducted in advanced cellular models using primary hepatocytes and therefore most suitable for detailed investigations using lead compounds. We invite you to browse our Transporters A-Z collection or request your own personal copy of our Transporter Book to learn more.
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