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Research ArticleSpecial Section on Transporters in Toxicity and Disease

Drug-Induced Perturbations of the Bile Acid Pool, Cholestasis, and Hepatotoxicity: Mechanistic Considerations beyond the Direct Inhibition of the Bile Salt Export Pump

A. David Rodrigues, Yurong Lai, Mary Ellen Cvijic, Lisa L. Elkin, Tatyana Zvyaga and Matthew G. Soars
Drug Metabolism and Disposition April 2014, 42 (4) 566-574; DOI: https://doi.org/10.1124/dmd.113.054205

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Abstract

The bile salt export pump (BSEP) is located on the canalicular plasma membrane of hepatocytes and plays an important role in the biliary clearance of bile acids (BAs). Therefore, any drug or new chemical entity that inhibits BSEP has the potential to cause cholestasis and possibly liver injury. In reality, however, one must consider the complexity of the BA pool, BA enterohepatic recirculation (EHR), extrahepatic (renal) BA clearance, and the interplay of multiple participant transporters and enzymes (e.g., sulfotransferase 2A1, multidrug resistance–associated protein 2, 3, and 4). Moreover, BAs undergo extensive enzyme-catalyzed amidation and are subjected to metabolism by enterobacteria during EHR. Expression of the various enzymes and transporters described above is governed by nuclear hormone receptors (NHRs) that mount an adaptive response when intracellular levels of BAs are increased. The intracellular trafficking of transporters, and their ability to mediate the vectorial transport of BAs, is governed by specific kinases also. Finally, bile flow, micelle formation, canalicular membrane integrity, and BA clearance can be influenced by the inhibition of multidrug resistant protein 3- or ATPase-aminophospholipid transporter–mediated phospholipid flux. Consequently, when screening compounds in a discovery setting or conducting mechanistic studies to address clinical findings, one has to consider the direct (inhibitory) effect of the parent drug and metabolites on multiple BA transporters, as well as inhibition of BA sulfation and amidation and NHR function. Vectorial BA transport, in addition to BA EHR and homoeostasis, could also be impacted by drug-dependent modulation of kinases and enterobacteria.

Introduction

Interest in the liver canalicular bile salt export pump (BSEP) has grown in recent years because it plays an important role in the biliary clearance of numerous bile acids (BAs). Specifically, focus has shifted to BSEP inhibition as a cause of drug-induced cholestasis (DIC) and possibly drug-induced liver injury (DILI) (Stepan et al., 2011; Kubitz et al., 2012; Bjornsson and Jonasson, 2013; Yang et al., 2013). Interest in BSEP (ABC11) has been fueled also by reports that subjects carrying certain loss-of-function/expression ABC11 alleles are predisposed to DILI (Ulzurrun et al., 2013). As a result, a considerable amount of effort has been made to set up high-throughput in vitro inhibition screens in an attempt to mitigate drug interactions involving BSEP inhibition and DIC (Morgan et al., 2010; Dawson et al., 2012; Warner et al., 2012; Pedersen et al., 2013). Some groups have gone on to attempt to relate BSEP inhibitory potency in vitro to cholestasis and DILI. Unfortunately, such exercises have rendered mixed results even after consideration of drug exposure (Morgan et al., 2010; Dawson et al., 2012).

There is no doubt that BSEP plays an important role in the biliary clearance of BAs. However, is it the major player in DIC and DILI? When one considers the complexity of the BA pool (Fig. 1), the differential properties of BAs (hydrophobicity, toxicity, etc.), and the complex and dynamic interplay of multiple participant transporters and enzymes (Fig. 2A), is it overly simplistic to think that a drug (at a threshold exposure) needs only to directly inhibit BSEP to bring about cholestasis and DILI? Is it possible that the direct inhibition of BSEP is simply the “trigger” event that leads to elevations in BA levels and perturbation of the BA pool, followed by an adaptive response and shunting of BAs to alternative (salvage) pathways? Such a concept is well established and is consistent with the observed increases in serum BAs (∼50-fold) in cholestatic versus noncholestatic subjects (Xie et al., 2001; Stahl et al., 2008; Humbert et al., 2012). From the standpoint of the pharmaceutical industry, however, what has not received as much attention is the concept that a drug (or new chemical entity) can trigger BA pool perturbations by alternative mechanisms, or combinations of mechanisms, in addition to (or instead of) the direct inhibition of BSEP. Furthermore, a drug could not only act as a “trigger” but also interfere with the adaptive response mounted by the different organs participating in the enterohepatic recycling (EHR) of BAs. Could the latter be the important step that leads to DILI? It is worth noting that increases in circulating BAs are not always associated with DILI, and concerns arise only when additional “liver signals” are evident (Ozer et al., 2008). Careful review and interpretation of clinical data are warranted (see Supplemental Fig. 1), because hepatoxicity could lead to hepatocellular damage and reduced bile flow. In such a scenario, cholestasis would be a secondary phenomenon. On the other hand, the direct impact of a drug on BA homeostasis, by whatever mechanism(s), could lead to an increase in the intracellular concentrations of toxic BAs. In this second scenario, cholestasis would be reflective of the primary event leading to hepatoxicity. Given such complexity, and the known species differences in enzyme and transporter expression, activity, and inhibition, as well as the composition of the BA pool, it is not surprising that animal models often fail to predict DIC and DILI in human subjects. Despite the known species differences, however, rodent data (e.g., Bsep knockout mouse) do support the concept of a stunted adaptive response leading to severe intrahepatic cholestasis (Wang et al., 2009).

Fig. 1.
Fig. 1.

Structures of representative BAs.

Fig. 2.
Fig. 2.

Liver BA transporters and their regulation. Hepatic transporters involved in the vectorial transport of BAs (A) and their regulation by NHRs (B).

In short, when it comes to drug-induced perturbations of the BA pool, DIC, and DILI, it is likely that one has to consider additional factors beyond the direct inhibition of hepatic BSEP. In reality, the pharmacokinetic-absorption-distribution-metabolism-excretion-toxicity (PK-ADME-TOX) profiles of individual BAs are complex and must be viewed in light of their EHR (Ballatori et al., 2009; Gonzalez, 2012), renal clearance, vectorial flux by multiple transporters, transport out of cells back into blood, 3-O-sulfation, glucuronidation, amidation, and the nuclear hormone receptor (NHR)-mediated “adaptive” responses of the liver, kidney, and intestine (Fig. 2B; liver shown). A given drug can inhibit (or modulate) any one or more of these important processes and alter the composition of the BA pool (“balance of BAs in the pool”) and increase intracellular concentrations of toxic BAs.

Major Considerations

Complex and Dynamic BA Pool.

Primary BAs [cholic acid (CA) and chenodeoxycholic acid (CDCA)] (Fig. 1) are synthesized from cholesterol in the liver via two multistep biosynthetic pathways (initiated by cholesterol 7- and 27-hydroxylation) involving various cytochromes P450 (Gonzalez, 2012). Once formed, they can undergo extensive enzyme-catalyzed taurine and glycine conjugation to form “amidated” BAs [taurocholic acid (T-CA), glycocholic acid (G-CA), glycochenodeoxycholic acid (G-CDCA), and taurochenodeoxycholic acid (T-CDCA)]. In turn, the mixture can be actively transported out of the liver and into the bile (Table 1). Under normal conditions, such transport renders a very concentrated BA pool in the gallbladder (∼100 mM total BAs) compared with liver tissue (∼20 µM), small intestine lumen (2–10 mM), serum (∼2 µM), and urine (∼1 µM) (Northfield and McColl, 1973; Takikawa et al., 1985; Garcia-Canaveras et al., 2012; Humbert et al., 2012). Via the bile ducts, BAs travel to the gallbladder and are released into the upper small intestine (duodenum). Along the small intestine, BAs can be absorbed by passive diffusion and, on reaching the ileum, are subjected to active uptake via apical sodium-dependent bile acid transporter (ASBT) and basolateral organic solute transporter (OST). While in the gut, CA [to deoxycholic acid (DCA)] and CDCA [to lithocholic acid (LCA) and ursodeoxycholic acid (UDCA)] are metabolized by bacteria to form secondary BAs (Fig. 1) (Trauner and Boyer, 2003; Ridlon et al., 2006; Ballatori et al., 2009; Gonzalez, 2012). Any taurine- or glycine-conjugated BAs in the intestine are also subjected to deamidation by enterobacteria, especially in the mid to lower ileum and large intestine (Northfield and McColl, 1973; Ridlon et al., 2006). As shown in Table 1, the BA pool in the cecum (vs. liver tissue and gallbladder bile) is dominated by nonamidated LCA (17.5%), DCA (29.5%), CDCA (20.1%), CA (14.8%), and UDCA (3.5%).

View this table:
TABLE 1

Summary quantitation of different bile acids (BAs) in human serum, urine, liver tissue, cecum, bile, and feces

During the EHR process, ∼90% of the BA pool in the gut is absorbed. A small fraction of the BA pool (∼10%) is lost in feces and replaced by de novo synthesis in the liver (Trauner and Boyer, 2003; Ridlon et al., 2006; Ballatori et al., 2009; Gonzalez, 2012). Of the fraction absorbed, most of the hepatic portal BA pool (∼90%) is extracted by the liver, and the remainder circulates and is cleared via the kidneys (Van Berge Henegouwen et al., 1976; Ballatori et al., 2009; Humbert et al., 2012). Once in the liver, re-extracted BAs enter the BA pool therein and undergo amino acid conjugation and vectorial transport to bile; it has been reported that a given BA will undergo ∼20 cycles of EHR before elimination (Gonzalez, 2012). Cholestasis can, therefore, be caused by “prehepatic” (e.g., inhibition of liver uptake), “intrahepatic” (e.g., inhibition of biliary efflux), or “posthepatic” (e.g., bile duct injury) events. Moreover, the kidneys serve as backup clearance organs when hepatic function is impaired, so factors impacting renal function could also perturb BA homeostasis.

In addition to conjugation with glycine and taurine, a number of BAs undergo sulfotransferase 2A1 (SULT2A1)-mediated 3-O-sulfation and UDP-glucuronosyltransferase-catalyzed glucuronidation. Consequently, it is not surprising that gallbladder bile, liver tissue, serum, and urine samples render complex, and distinctive, BA signature profiles (Takikawa et al., 1985; Rossi et al., 1987; Hamilton et al., 2007; Garcia-Canaveras et al., 2012; Humbert et al., 2012; Trottier et al., 2013). Such “signatures” are reflective of the PK-ADME properties of individual BAs (Table 1), imparted by their unique transporter-enzyme-NHR profile [e.g., BSEP-sodium-taurocholate cotransporting polypeptide (NTCP)-organic anion transporting peptide (OATP)-multidrug resistance-associated protein 2 (MRP-2)- farnesoid X receptor (FXR)-SULT2A1] (Heuman et al., 1989; Meier et al., 1997; Parks et al., 1999; Staudinger et al., 2001; Hayashi et al., 2005; Huang et al., 2010). In agreement, for individual BAs, one can find reports describing a wide range of uptake rates for human BSEP vesicles (∼30-fold), sulfation rates for human SULT2A1 (>2000-fold), human NTCP- (∼20-fold) and OATP- (∼20-fold) mediated cell uptake rates, and human FXR- (7-fold) and pregnane X receptor (PXR; ∼7-fold)-mediated reporter [chloramphenicol acetyltransferase (CAT) activity] induction in cells (Table 2).

View this table:
TABLE 2

Summary of the in vitro properties of different bile acids reported in the literature

Based on the available literature, it appears that the multidrug resistance-associated protein 4 (MRP4)-mediated uptake rate is less varied among different BAs (∼5-fold) (Table 2; Rius et al., 2006). Although additional data are needed, it implies that MRP4 may have a broader BA substrate selectivity; MRP4 serves as “salvage transporter” and is upregulated in the liver during cholestasis (Gradhand et al., 2008). Unfortunately, even less is known regarding the selectivity of multidrug resistance-associated protein 3 (MRP3), which also likely plays a role in the efflux of certain BAs (Fig. 2A). Only one report, by Zeng et al. (2000), describes G-CA as a human MPR3 substrate. Under the same assay conditions; however, no uptake of T-CA into MRP3 vesicles was detected. Does this mean that MRP3 is more selective (vs. MRP4) as a basolateral BA efflux transporter?

One of the most toxic and hydrophobic BAs (i.e., LCA) is a poor human BSEP substrate in vitro but is a relatively good SULT2A1 substrate (Table 2) (Hayashi et al., 2005; Huang et al., 2010) and undergoes extensive conjugation with glycine and taurine in human subjects (Cowen et al., 1975). It is the amidated/3-O-sulfated form of LCA that serves as a BSEP substrate (Hayashi et al., 2005). So is the inhibition of BSEP irrelevant in the case of LCA itself? The same could be said for other hydrophobic BAs such as G-LCA, T-LCA, and DCA, although no BSEP data are available (Table 2). By comparison, T-DCA (taurodeoxycholic acid), G-CDCA, T-CDCA, G-CA, and T-CA are better BSEP substrates, relatively less hydrophobic, poorer SULT2A1 substrates (Table 2) and together are major components of the BA pool in gallbladder bile (5.4%, 26%, 13%, 26%, 11% of total BA, respectively) (Table 1). Potent inhibition of BSEP in the liver would likely impact the biliary clearance of these five BAs.

In toto, when attempting to understand DIC mechanistically, it is evident that the PK-ADME, transporter, conjugation (amino acid and sulfation), and EHR properties of each individual BA must be considered. This becomes even more critical in light of each BA’s physicochemical and cytotoxicity profile.

Transporter-Enzyme Interplay.

As shown in Fig. 2A, hepatic canalicular BSEP functions coordinately with sinusoidal NTCP to enable vectorial transport of circulating BAs into the bile. However, this “NTCP-BSEP axis” should be viewed as only one possible mechanism by which BAs are transported. BAs such as LCA, glycolithocholic acid (G-LCA), and taurolithocholic acid (T-CLA) can also undergo SULT2A1-catalyzed sulfation to form a 3-O-sulfate conjugate that can serve as a substrate of the “OATP-MRP2 axis.” Therefore, SULT2A1 can be regarded as an important junction point between the two BA transport axes (Alnouti, 2009; Huang et al., 2010). Beyond NTCP, BSEP, OATP, and MRP2, liver OST and additional multidrug resistance-associated proteins (MRP3 and MRP4) also play an important role in BA transport (Fig. 2A). Such transporters mediate the transport of BAs into the blood (from the liver) and enable “hopping” between individual hepatocytes, and more efficient extraction, as the blood flows along the sinusoids. Such a concept was introduced by van de Steeg et al. (2012).

As described already herein, SULT2A1 can play an important role in the clearance and disposition of certain BAs. The enzyme is expressed in liver (∼1500 ng/mg cytosol protein), small intestine (∼400 ng/mg cytosol protein), and kidneys (∼5 ng/mg cytosol protein); but, as expected, the rate of BA sulfation is highest in the liver and not detectable in kidneys (Loof and Wengle, 1979; Riches et al., 2009). SULT2A1 could, therefore, determine the composition of the BA pool in both the liver and intestine. In the absence of renal BA sulfation, it is noteworthy that BA 3-O-sulfates represent ∼75% of the BA pool in urine (sulfated forms of G-UDCA, G-LCA, T-LCA, T-UDCA, DCA, UDCA, and CDCA represent 23.6%, 21.8%, 12.7%, 6.3%, 5.8%, 2.9%, and 2.3% of the BA pool in urine, respectively). By comparison, sulfated BAs represent only a small fraction (<5%) of the BA pool in the bile (Table 1). Under normal conditions, it is likely that 3-O-sulfate forms of BAs are substrates of MRP3 and MRP4, circulate and are taken up by renal transporters. Unfortunately, the transporters involved in the renal clearance of sulfated and nonsulfated BAs have not been reported. To date, there is information for only two nonsulfated BAs (CA and T-CA). Both are detectable in urine (Table 1) and have been shown to be substrates of organic anion transporter 3 (hOAT3) in vitro (Chen et al., 2008; Brandoni et al., 2012). Interestingly, it has been reported that OAT3 expression is elevated in kidneys of cholestatic subjects (Chen et al., 2008; Brandoni et al., 2012). It is accepted that OAT3 will likely function coordinately with other renal transporters, possibly apical organic anion transporter 4 and MRP2, to mediate the vectorial transport of BAs in kidneys.

From the standpoint of BA clearance and disposition, therefore, one has to consider the inhibition of multiple transporters in liver and kidney. For example, the combined inhibition of liver BSEP, OST, MRP2, MRP3, or MRP4 could trigger intrahepatic cholestasis, inhibition of liver OATP or NTCP could lead to prehepatic cholestasis, and the inhibition of renal BA transporters could give rise to extrahepatic cholestasis. Interestingly, during the preparation of the present manuscript, Morgan et al. (2013) reported that integration of exposure data and knowledge of drug effect to not only BSEP but also one or more of the MRPs is a useful tool for informing the potential for DILI resulting from altered bile acid transport. Therefore, investigators are moving beyond BSEP data alone and starting to integrate inhibition data for additional BA transporters like MRP2, MRP3, and MRP4. In terms of BA sulfation, LCA, G-LCA, and T-LCA are relatively hydrophobic and serve as good SULT2A1 substrates (Table 2). Consequently, any drug or metabolite that inhibits SULT2A1 in the liver (or small intestine) could alter the composition of the BA pool and impact the levels of hydrophobic BAs during EHR. Although a theoretical consideration, the inhibition of SULT2A1 by known cholestatic compounds warrants investigation.

It is important to note that, as described as follows, NHRs mount an adaptive response when intracellular BA levels rise. Such a response leads to increases in transporter (e.g., OST and MRPs) and SULT2A1 expression. So any combination of BSEP, OST, MRP, and SULT2A1 inhibition could not only trigger DIC but also stunt any NHR-mediated adaptive response and exacerbate cholestatic liver injury.

Role of NHRs.

As discussed previously, the liver is able to respond to increases in BA levels and BA pool perturbations. This response is possible via the coordinated interplay of at least three NHRs: FXR, PXR, and constitutive androstane receptor (CAR) (Fig. 2B). Numerous BAs have been shown to be agonists of these receptors (Guo et al., 2003; Stahl et al., 2008; Li and Chiang, 2013). For example, CDCA, DCA, and UDCA are relatively good FXR agonists, whereas LCA behaves as a good FXR and PXR agonist (Parks et al., 1999; Staudinger et al., 2001). FXR agonism increases BSEP, MRP2, and OST expression and represses NTCP and OATP expression in hepatocytes. CAR or PXR agonism increases OATP, SULT2A1, MRP2, MRP3, and MRP4 expression (Xie et al., 2001; Stahl et al., 2008; Halilbasic et al., 2013). The net effect of this NHR interplay, and its modulation by members of the BA pool, is increased efflux of BAs out of the hepatocyte, as well as shunting of BAs toward MRPs, especially after sulfation to 3-O-sulfates that are excreted in the urine (Van Berge Henegouwen et al., 1976; Keppler, 2011; Humbert et al., 2012). This effect is reflected in the serum BA profile of cholestatic versus healthy subjects: elevations in primary BAs (amidated with or without sulfation) accompanied by decreases in secondary-hydrophobic/nonamidated/nonsulfated BAs. The NHRs “compete” with each other and can elicit differential effects on transporter expression. Such a phenomenon is exemplified by the opposing effect of CAR (upregulation) and FXR (downregulation) on MRP4 expression (Renga et al., 2011).

If a given drug disrupts NHR function (e.g., by interfering with transcription factor or coactivator function), then the adaptive ability of the liver will be muted and the toxic effects of BAs can be exacerbated. Ketoconazole is one example of a drug that has been shown to inhibit the activation of PXR (Huang et al., 2007). Similarly, the sulfate conjugates of certain hormones (e.g., progesterone) are known to inhibit FXR (Abu-Hayyeh et al., 2013). So it is possible that one must consider BSEP inhibition in concert with NHR inhibition. As in the case of SULT2A1, inhibition of NHRs (e.g., PXR and FXR) by known cholestatic drugs warrants further study.

Inhibition of BA Conjugation.

All BAs undergo extensive conjugation with glycine and taurine. In fact, the amidated forms of DCA, CDCA, and CA dominate the BA pool (>90%) in liver and bile (Table 1). Such conjugation is catalyzed (stepwise) by two enzymes that are highly expressed in the liver: bile acid-CoA ligase or bile acid-CoA synthetase (BACS) and bile acid-CoA: amino acid (glycine/taurine) N-acetyltransferase (BAAT) (Falany et al., 1994; Solaas et al., 2000; O’Byrne et al., 2003). Therefore, inhibition of one or both enzymes can perturb the BA pool and the balance of toxic versus less toxic BAs. For example, cyclosporine (which is cholestatic) has been shown to inhibit BA conjugation in vitro (Vessey and Kelley, 1995), and lack of BA conjugation (related to BAAT genotype) has been associated with cholestasis (Hadžićet al., 2012). Hydrophobic BAs like LCA and DCA are extensively conjugated with glycine and taurine, so one can imagine the consequences of inhibiting BACS and/or BAAT.

Additional Considerations (Involving the Direct Inhibition of BA Transporters)

To date, as in the case of BSEP, investigators have largely focused on transporter (direct) inhibition screening in vitro (Morgan et al., 2010; Dawson et al., 2012; Warner et al., 2012; Pedersen et al., 2013). Typically, this requires a fully automated or semiautomated high-throughput (multiwell plate-based) assay using a well-characterized transporter substrate. However, based on current literature, it is apparent that one must evaluate metabolites as transporter inhibitors also. Moreover, the possibility of “trans” inhibition, especially in the case of canalicular transporters like BSEP, has to be considered.

Metabolites as Transporter Inhibitors.

Although current screening efforts focus on parent molecules, there is a growing appreciation that some metabolites may inhibit transporters. For example, progesterone metabolites have been shown to inhibit NTCP (Abu-Hayyeh et al., 2010), whereas the sulfate conjugate of troglitazone is a more potent inhibitor (10-fold) of rat bsep than is the parent troglitazone (Funk et al., 2001). Given the high levels of some metabolites in the liver and bile, it is likely that many could behave as transporter cosubstrates (inhibitors) and impact BA clearance also. Therefore, careful metabolic profiling of human bile would be warranted in such cases, and the major metabolites would be screened as transporter inhibitors. Because human ADME data are not available in early discovery, there is some risk when advancing parent molecules that have been shown to be weak inhibitors of human BA transporters in vitro.

Cis- Versus Trans-Inhibition.

Classically, drugs are thought to inhibit BSEP activity by impacting ATP-dependent transport. This is known as “cis-inhibition” and involves binding to the (intracellular) ATP-binding site or substrate binding site. Typically, this renders competitive inhibition. “trans-inhibition,” on the other hand, involves the interplay of BSEP with at least one additional canalicular transporter. For example, certain glucuronides (and other metabolites) are transported into the bile via a second transporter (e.g., MRP2), are present at high concentrations, and inhibit BSEP once in the bile canaliculus (Pauli-Magnus and Meier, 2006). In such a scenario, a drug (or metabolite) would inhibit BSEP only after its biliary secretion by a second transporter.

Additional Considerations (Involving Indirect Effects Leading to DIC)

Although direct inhibition of BA transporters and BA-metabolizing enzymes by drugs is important, one must consider the possibility that DIC, and possibly DILI, may be caused by indirect effects. In this instance, a given parent drug (or a metabolite) does not have to bind to, or interact directly with, a BA transporter. Three possibilities are described below.

Modulation of Kinases.

Numerous publications have described the regulation of transporter (intracellular) trafficking by various kinases (Roma et al., 2008; Boaglio et al., 2010; Crocenzi et al., 2012). “Translocation” of OATP and NTCP from the basolateral membrane of hepatocytes to endosomes is stimulated by activation of Ca2+-dependent protein kinase C (PKC). Likewise, the trafficking of MRP2 and BSEP away from the canalicular membrane is also mediated by PKC. Trafficking from endosomes can be disrupted also by activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B pathway. Conversely, trafficking of transporters to the basolateral and canalicular membranes is mediated by protein kinase A (PKA). Because the activity of these various kinases is linked to the levels of oxidative stress in hepatocytes, any pro-oxidant drug (or metabolite) can cause cholestasis by elevating Ca2+ levels and decreasing the concentration of reduced glutathione; transporters such as BSEP and MRP2 are known to be internalized under conditions of oxidative stress (Perez et al., 2006; Sekine et al., 2011). Therefore, a given drug or metabolite could decrease the vectorial transport of BAs by modulating the balance of PKA, PKC, PI3K activity without direct inhibition of transporters such as BSEP and MRP2. The internalization of key BA transporters would negate any effort by the liver to mount a NHR-mediated adaptive response.

Impact on Phospholipid Flux.

At least two transporters function coordinately to maintain membrane integrity and mediate phospholipid flux across the canalicular plasma membrane of hepatocytes (Groen et al., 2011). Loss-of-function phenotype in either case has been associated with intrahepatic cholestasis; therefore, inhibition of either transporter could give rise to DIC.

The first is MDR3 (ABCB4), also known as a phosphatidylcholine translocase or “floppase,” which is the locus of progressive familial intrahepatic cholestasis type 3 (Harris et al., 2005; Groen et al., 2011; Dzagania et al., 2012). It plays a major role in the secretion of phospholipids out of the liver and into bile. Such phospholipids are important components of BA-containing biliary micelles. So it is likely that MDR3 and BSEP function together to form mixed (BA-phospholipid) micelles (Groen et al., 2011). Therefore, inhibition of MDR3 could bring about cholestasis and impact the biliary clearance of BAs. Compared with BSEP, however, inhibition of MDR3 has received relatively little attention to date, and only one report has described the inhibition of MDR3 by a drug (itraconazole) (Yoshikado et al., 2011).

The second transporter, ATP8B1 (ATPase-aminophospholipid transporter), is associated with progressive familial intrahepatic cholestasis type 1 (Harris et al., 2005; Groen et al., 2011). Unlike MDR3, canalicular ATP8B1 acts as a “flippase” and mediates the translocation of phosphatidylserine from the outer to the inner leaflet of the canalicular membrane. The continued inward flux of phosphatidylserine is thought to be essential for the maintenance of membrane integrity in the presence of high concentrations of detergent BAs (Groen et al., 2011). To date, there are no reports of any drug inhibiting ATP8B1.

Impact on Gut Bacteria.

As described earlier, gut bacteria are involved in the metabolism of BAs (amino acid and sulfate deconjugation, as well as 7α/β-dehydroxylation) during EHR (Robben et al., 1989; Ridlon et al., 2006). This is reflected in the BA profiles of stool and caecal contents (LCA, DCA, and CDCA represent >70% of the BA pool, low levels of amidated and sulfated BAs detected; Table 1). Moreover, there is evidence that the expression of host BA transporters (e.g., MRP2 and ASBT) in the intestine can be modulated by enterobacteria (Mercado-Lubo and McCormack, 2010; Miyata et al., 2011). Therefore, any orally dosed drug that perturbs gut bacteria could impact BA homeostasis, alter the composition of the BA pool (primary vs. secondary BAs, amidated vs. nonamidated BAs, sulfated versus nonsulfated BAs) by altering BA metabolism or transport, and give rise to cholestasis.

Conclusions (Going Beyond “BSEP Bias”)

The growing awareness of the complex and dynamic interplay between transporters, enzymes, and NHRs in multiple organs, and its likely impact on BA EHR and disposition, will compel industry researchers and regulators to garner a more “systems view” of DIC. As described herein, DIC could be triggered not only by direct inhibition of BSEP but by any combination of direct and indirect mechanisms involving enterobacteria, BA-metabolizing enzymes, kinases, and other participating BA transporters (Fig. 3, liver shown). From the standpoint of DILI, the impact of drug or new chemical entity on any subsequent NHR-mediated (adaptive) responses could be very important and should also be considered. Therefore, it is likely that efforts will be made to expand inhibition screening in vitro beyond BSEP and include assays for additional transporters, NHRs, kinases, and phase II (conjugation) enzymes.

Fig. 3.
Fig. 3.

Summary of the potential mechanism(s) by which a drug or metabolite can impact the hepatobiliary disposition of BAs.

Ultimately, progress will be enabled by the development and wide use of validated and more sophisticated in vitro models, the availability of complete in vitro data sets for greater numbers of individual BAs (Table 2), and improved modeling and simulation tools that support in vitro-in vivo extrapolations and provide mechanistic insight. Integrated strategies will also require the development of robust analytical methods that facilitate the more routine profiling of individual BAs and the generation of BA “signatures” in human subjects. Ideally, individual subject BA profiles would be interpreted in light of genotype data (see Supplemental Fig. 2).

As evidenced by the recent reports of Thompson et al. (2012), Chang et al. (2013), and Morgan et al. (2013), researchers are already migrating to assay panel-based (integrative) strategies to enable high-throughput compound screening, support the building of structure-activity relationships, and facilitate risk assessment in a discovery setting. Therefore, the widespread and rapid transition from “BSEP inhibition” screens to “cholestasis assay panels” and “assay batteries” is envisioned. Concomitantly, there will also be a push for “physiologically relevant” primary cell-based screens, coupled to multiplexed readouts (e.g., human hepatocyte cocultures, sandwich cultures, and three-dimensional cultures), and more sophisticated data integration strategies (Godoy et al., 2013; Pedersen et al., 2013; Vinken et al., 2013).

Despite the obvious challenges, it is hoped that systematic approaches that enable investigation of the potential causes of DIC and DILI (beyond direct BSEP inhibition) will support the discovery and development of new chemical entities that are less cholestatic and hepatotoxic.

Authorship Contributions

Participated in research design: Rodrigues, Soars, Lai.

Wrote or contributed to the writing of the manuscript: Rodrigues, Soars, Lai, Zvyaga, Elkin, Cvijic.

Footnotes

Abbreviations

ASBT
apical sodium-dependent bile acid transporter
ATP8B1
ATPase-aminophospholipid transporter
BA
bile acid
BAAT, bile acid-CoA
amino acid (glycine/taurine) N-acetyltransferase
BACS
bile acid-CoA ligase (bile acid-CoA synthetase)
BSEP
bile salt export pump
CA
cholic acid
CAR
constitutive androstane receptor
CAT
chloramphenicol acetyltransferase
CDCA
chenodeoxycholic acid
DCA
deoxycholic acid
DIC
drug-induced cholestasis
DILI
drug-induced liver injury
EHR
enterohepatic recirculation
FXR
farnesoid X receptor
G-CA
glycocholic acid
G-CDCA
glycochenodeoxycholic acid
G-DCA
glycodeoxycholic acid
G-LCA
glycolithocholic acid
G-UDCA
glycoursodeoxycholic acid
LCA
lithocholic acid
MDR3
multidrug resistant protein 3
MRP
multidrug resistance-associated protein
NHR
nuclear hormone receptors
NTCP
sodium-taurocholate cotransporting polypeptide
OAT
organic anion transporter
OATP
organic anion transporting peptide
OST
organic solute transporter
PI3K
phosphoinositide 3-kinase
PKA
protein kinase A
PK-ADME-TOX
pharmacokinetic-absorption-distribution-metabolism-excretion-toxicity
PKC
protein kinase C
PXR
pregnane X receptor
SULT2A1
sulfotransferase 2A1
T-CA
taurocholic acid
T-CDCA
taurochenodeoxycholic acid
T-DCA
taurodeoxycholic acid
T-LCA
taurolithocholic acid
T-UDCA
tauroursodeoxycholic acid
UDCA
ursodeoxycholic acid

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Research ArticleSpecial Section on Transporters in Toxicity and Disease

Mechanisms Leading to Drug-Induced Cholestasis

A. David Rodrigues, Yurong Lai, Mary Ellen Cvijic, Lisa L. Elkin, Tatyana Zvyaga and Matthew G. Soars
Drug Metabolism and Disposition April 1, 2014, 42 (4) 566-574; DOI: https://doi.org/10.1124/dmd.113.054205
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