Pharmaceutical Candidate Optimization, Bristol-Myers Squibb ......2013/10/10 · Finally, bile...

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Commentary

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

Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Princeton, New Jersey

(A.D.R., Y.L.); Pharmaceutical Candidate Optimization, Bristol-Myers Squibb,

Wallingford, Connecticut (M.S.); Leads Discovery and Optimization, Bristol-Myers

Squibb, Princeton, New Jersey (M.E.C.); and Leads Discovery and Optimization, Bristol-

Myers Squibb, Wallingford, Connecticut (L.E., T.Z.)

DMD Fast Forward. Published on October 10, 2013 as doi:10.1124/dmd.113.054205

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on October 10, 2013 as DOI: 10.1124/dmd.113.054205

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Running title: Mechanisms leading to drug-induced cholestasis

Correspondence Address:

A. David Rodrigues, PhD

Pharmaceutical Candidate Optimization

Bristol-Myers Squibb, Mail Stop F12-04

P.O. Box 4000, Princeton, New Jersey 08543

U.S.A

TEL: (609) 252-7813

FAX: (609) 252-6802

[email protected]

Number of text pages: 38

Number of tables: 2

Number of figures: 3

Number of references: 73

Number of words in Abstract: 249

Number of words in Introduction: 733

Total number of words: 8,744


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Abbreviations: Bile acid, BA; SULT2A1, sulfotransferase 2A1; FXR, farnesoid X

receptor; PXR, pregnane X receptor; BSEP, bile salt export pump; MRP, multidrug

resistance-associated protein; OATP, organic anion transporting peptide; NTCP, sodium-

taurocholate co-transporting polypeptide; ASBT, apical sodium-dependent bile acid

transporter; OST, organic solute transporter; PI3K, phosphoinositide 3-kinase; PKA,

protein kinase A; PKC, protein kinase C; NHR, nuclear hormone receptors; EHR,

enterohepatic recirculation; DILI, drug-induced liver injury; DIC, drug-induced

cholestasis; BACS, bile acid-CoA ligase (bile acid-CoA synthetase); BAAT, bile acid-

CoA: amino acid (glycine/taurine) N-acetyltransferase; Akt, protein kinase B; OAT,

organic anion transporter; CAR, constitutive androstane receptor; MDR3, multidrug

resistant protein 3; PK-ADME-TOX, pharmacokinetic-absorption-distribution-

metabolism-excretion-toxicity; LCA, lithocholic acid; G-LCA, glycolithocholic acid; T-

LCA, taurolithocholic acid; G-UDCA, glycoursodeoxycholic acid; T-UDCA,

tauroursodeoxycholic acid; UDCA, ursodeoxycholic acid; DCA, deoxycholic acid; G-

DCA, glycodeoxycholic acid; T-DCA, taurodeoxycholic acid; CDCA, chenodeoxycholic

acid; G-CDCA, glycochenodeoxycholic acid; T-CDCA, taurochenodeoxycholic acid;

CA, cholic acid; G-CA, glycocholic acid; T-CA, taurocholic acid; CAT, chloramphenicol

acetyltransferase; ATP8B1, ATPase-aminophospholipid transporter.


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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 has to 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. Importantly, the 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

MDR3 (multidrug resistant protein 3)- or ATP8B1 (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 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.


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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) (Yang et al., 2013;

Kubitz et al., 2012; Bjornsson and Jonasson, 2013; Stepan et al., 2011). 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 too simplistic to think that a drug (at a threshold exposure)

only needs 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


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versus non-cholestatic 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 only arise when additional “liver signals”

are evident (Ozer et al., 2008). Careful review and interpretation of clinical data is

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/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

intra-hepatic cholestasis (Wang et al., 2009).

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 PK-ADME-TOX (pharmacokinetic-absorption-distribution-


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metabolism-excretion-toxicity) profiles of individual BAs are complex and have to 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, the 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 multi-step 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) when compared to liver tissue (~20 µM),

small intestine lumen (2-10 mM), serum (~2 µM) and urine (~1 µM) (Humbert et al.,

2012; Garcia-Canaveras et al., 2012; Takikawa et al., 1984; Northfield and McColl,

1973). 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


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diffusion and, upon reaching the ileum, are subjected to active uptake via apical sodium-

dependent bile acid transporter [ABST] 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) (Ridlon et al., 2006; Ballatori et al., 2009; Gonzalez, 2012; Trauner and Boyer,

2003). Any taurine- or glycine-conjugated BAs in the intestine are also subjected to de-

amidation 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 caecum (versus liver tissue and gallbladder bile) is dominated by non-amidated LCA

(17.5%), DCA (29.5%), CDCA (20.1%), CA (14.8%) and UDCA (3.5%).

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 (Ridlon et al., 2006; Ballatori et al., 2009; Gonzalez, 2012; Trauner and Boyer,

2003). Of the fraction absorbed, the majority of the hepatic portal BA pool (~90%) is

extracted by the liver and the remainder circulates and is cleared via the kidneys

(Ballatori et al., 2009; Van Berge Henegouwen et al., 1976; 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 prior to elimination (Gonzalez, 2012). Cholestasis can,

therefore, be caused by “pre-hepatic” (e.g., inhibition of liver uptake), “intra-hepatic”

(e.g., inhibition of biliary efflux) or “post-hepatic” (e.g., bile duct injury) events.

Moreover, the kidneys serve as back-up clearance organs, when hepatic function is

impaired, so factors impacting renal function could also perturb BA homeostasis.


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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 (Humbert et al., 2012; Garcia-Canaveras et al., 2012; Hamilton et

al., 2007; Rossi et al., 1987; Takikawa et al., 1984; 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-NTCP [sodium-

taurocholate co-transporting polypeptide]-OATP [organic anion transporting peptide]-

MRP2 [multidrug resistance-associated protein 2]-FXR [farnesoid X receptor]-

SULT2A1) (Heuman et al., 1989; Huang et al., 2010; Hayashi et al., 2005; Meier et al.,

1997; Parks et al., 1999; Staudinger et al., 2001). 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).

Based on the available literature, it appears that multidrug resistance-associated

protein 4 (MRP4)-mediated uptake rate is less varied amongst 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

up-regulated 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


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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?

Importantly, one of the most toxic and hydrophobic BAs (LCA) is a poor human

BSEP substrate in vitro, but is a relatively good SULT2A1 substrate (Table 2; Huang et

al., 2010; Hayashi et al., 2005) 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 5 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 have to 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,


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this “NTCP-BSEP axis” should only be viewed as one possible mechanism by which

BAs are transported. BAs such as LCA, G-LCA (glycolithocholic acid) and T-LCA

(taurolithocholic acid) 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 has been introduced by van de Steeg et al (2012).

As described above, 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 (Riches et al., 2009; Loof and Wengle, 1979). 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


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and non-sulfated BAs have not been reported. To date, there is only information for two

non-sulfated 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 (Brandoni et al.,

2012; Chen et al., 2008). Interestingly, it has been reported that OAT3 expression is

elevated in kidneys of cholestatic subjects (Brandoni et al., 2012; Chen et al., 2008). 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 and/or MRP4 could trigger intra-

hepatic cholestasis, inhibition of liver OATP and/or NTCP could lead to pre-hepatic

cholestasis, and the inhibition of renal BA transporters could give rise to extra-hepatic

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 due to 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.

While a theoretical consideration, the inhibition of SULT2A1 by known cholestatic

compounds warrants investigation.


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Importantly, as described below, 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.

The Role of NHRs

As discussed previously, the liver is able to respond to increases in BA levels and

BA pool perturbations. This is possible via the coordinated interplay of at least 3 NHRs;

FXR, PXR and CAR (constitutive androstane receptor) (Fig. 2B). Numerous BAs have

been shown to be agonists of these receptors (Li and Chiang, 2013; Stahl et al., 2008;

Guo et al., 2003). 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/PXR agonism increases

OATP, SULT2A1, MRP2, MRP3 and MRP4 expression (Halilbasic et al., 2013; Xie et

al., 2001; Stahl et al., 2008). 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 towards MRPs, especially after sulfation to 3-O-sulfates that are

excreted in the urine (Keppler, 2011; Van Berge Henegouwen et al., 1976; Humbert et

al., 2012). This 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/non-sulfated-BAs. Importantly, the

NHRs “compete” with each other and can elicit differential effects on transporter


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expression. Such a phenomenon is exemplified by the opposing effect of CAR (up-

regulation) and FXR (down-regulation) on MRP4 expression (Renga et al., 2011).

If a given drug disrupts NHR function (e.g., by interfering with transcription

factor, and/or co-activator function, etc), 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 has to 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 (step-wise) 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’Bryne et 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 (Hadzic et al., 2012). Importantly, 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.


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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- or semi-automated high-

throughput (multi-well plate-based) assay employing a well-characterized transporter

substrate. However, based on current literature, it is apparent that one has to 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

While 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

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 co-substrates (inhibitors)

and impact BA clearance also. Therefore, careful metabolic profiling of human bile

would be warranted in such cases and the major metabolites 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


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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 has to 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 (Crocenzi et al., 2012; Roma et al., 2008; Boaglio et al.,

2010). “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


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by activation of the PI3K-Akt (phosphoinositide 3-kinase/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). Importantly, loss of function phenotype in either case has been

associated with intra-hepatic cholestasis. Therefore, inhibition of either transporter could

give rise to DIC.

The first is MDR3 (multidrug resistant protein 3; ABCB4), also known as a

“phosphatidylcholine translocase” or “floppase”, which is the locus of progressive

familial intra-hepatic cholestasis type 3 (“PFIC-3”) (Dzagania et al., 2012; Harris et al.,

2005; Groen et al., 2011). 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


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(BA-phospholipid) micelles (Groen et al., 2011). Therefore, inhibition of MDR3 could

bring about cholestasis and impact the biliary clearance of BAs. Compared to BSEP,

however, inhibition of MDR3 has received relatively little attention to date and there is

only one report describing the inhibition of MDR3 by a drug (itraconazole) (Yoshikado et

al., 2011).

The second transporter, ATP8B1 (ATPase-aminophospholipid transporter), is

associated with progressive familial intra-hepatic cholestasis type 1 (“PFIC-1”) (Groen et

al., 2011; Harris et al., 2005). 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 (Ridlon et

al., 2006; Robben et al., 1989). 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 ABST) in the intestine can be modulated by

enterobacteria (Mercado-Lubo et al., 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 non-amidated BAs; sulfated vs.


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non-sulfated BAs), by altering BA metabolism and/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 mportant and should be considered also. 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.

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).


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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 co-cultures, sandwich cultures, and 3-

dimensional cultures), and more sophisticated data integration strategies (Godoy et al.,

2013; Vinken et al., 2013; Pedersen 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.


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Authorship Contributions

Participated in research design: Rodrigues, Soars, Lai

Conducted experiments: Not applicable

Contributed to new reagents or analytic tools: Not applicable

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

Elkin, Cvijic


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Figure Legends:

Figure 1. Structures of representative BAs

Figure 2. Liver BA transporters and their regulation

Hepatic transporters involved in the vectorial transport of BAs (A) and their regulation by

NHRs (B). OST, MRP3/4, OATP1B1, NTCP, BSEP, and MRP2 represent organic solute

transporter, multidrug resistance-associated protein 3 and 4, organic anion transporting

peptide 1B1, sodium-taurocholate co-transporting polypeptide, bile salt export pump, and

multidrug resistance-associated protein 2, respectively. SULT2A1, FXR, PXR, and CAR

represent sulfotransferase 2A1, farnesoid X receptor, pregnane X receptor and

constitutive androstane receptor, respectively.

Figure 3. Summary of the potential mechanism(s) by which a drug or metabolite

can impact the hepatobiliary disposition of BAs

ATP8B1, PKC, PI3K/Akt, MDR3, BACS and BAAT represent ATPase-

aminophospholipid transporter, protein kinase C, phosphoinositide 3-kinase, protein

kinase B, multidrug resistant protein 3, bile acid-CoA ligase (bile acid-CoA synthetase)

and bile acid-CoA: amino acid (glycine/taurine) N-acetyltransferase, respectively. See

Figure 2 for additional abbreviations.


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Table 1 Summary quantitation of different BAs in human serum, urine, liver tissue, caecum, bile and feces

BAe

Mean % Total BA (n = number subjects)

Serum (n = 39)a

Urine (n = 39)a

Liver Tissue (n = 8)b

Gallbladder Bile (n = 14)c

Caecal Contents (n = 15)d

Stool (n = 19)a

Non-Sulfatede

LCA ≤ 0.5 ≤ 0.5 ≤ 0.5

≤ 0.5 17.5 32.1

G-LCA ≤ 0.5 ≤ 0.5 1.5

≤ 0.5 ≤ 0.5 ≤ 0.5

T-LCA ≤ 0.5 ≤ 0.5 0.9

≤ 0.5 ≤ 0.5 ≤ 0.5

DCA 11.2 2.2 ≤ 0.5

≤ 0.5 29.5 60.6

G-DCA 11.8 ≤ 0.5 17.5

10.3 ≤ 0.5 0.61

T-DCA 2.2 ≤ 0.5 6.9

5.4 ≤ 0.5 ≤ 0.5

CDCA 8.2g ≤ 0.5 ≤ 0.5

≤ 0.5 20.1 1.73

G-CDCA 32.6 ≤ 0.5 33.1

26 1.3 0.70

T-CDCA 3.9 ≤ 0.5 17.5

13 1.3 ≤ 0.5

CA 7.8g 14.6 ≤ 0.5

≤ 0.5 14.8 1.41

G-CA 5.2 5.6 16.9

26 ≤ 0.5 ≤ 0.5

T-CA 1.9 1.9 6.9

11 ≤ 0.5 ≤ 0.5

UDCA 2.7 ≤ 0.5 ≤ 0.5

≤ 0.5 3.5 0.85

G-UDCA 4.6 ≤ 0.5 1.6

1.4 ≤ 0.5 ≤ 0.5

T-UDCA ≤ 0.5 ≤ 0.5 ≤ 0.5

0.7 ≤ 0.5 ≤ 0.5

Sulfated

LCA-3S ≤ 0.5 ≤ 0.5 -f - 1.4 ≤ 0.5

CA-3S ≤ 0.5 ≤ 0.5 - - ≤ 0.5 ≤ 0.5

CDCA-3S ≤ 0.5 2.3 - - 3.9 ≤ 0.5

UDCA-3S ≤ 0.5 2.9 - - ≤ 0.5 ≤ 0.5

DCA-3S ≤ 0.5 5.8 - - 1.2 ≤ 0.5

T-UDCA-3S ≤ 0.5 6.3 - - ≤ 0.5 ≤ 0.5

T-LCA-3S 2.5 12.7 -

≤ 0.5 ≤ 0.5 ≤ 0.5

G-LCA-3S 3.5 21.8 -

≤ 0.5 ≤ 0.5 ≤ 0.5

G-UDCA-3S 1.8 23.6 - - 0.6 ≤ 0.5

Total 100.0 99.8 102.7

93.8 96.1 97.9


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Table 1.

LCA, lithocholic acid; G-LCA, glycolithocholic acid; T-LCA, taurolithocholic acid; G-

UDCA, glycoursodeoxycholic acid; T-UDCA, tauroursodeoxycholic acid; UDCA,

ursodeoxycholic acid; DCA, deoxycholic acid; G-DCA, glycodeoxycholic acid; T-DCA,

taurodeoxycholic acid; CDCA, chenodeoxycholic acid; G-CDCA,

glycochenodeoxycholic acid; T-CDCA, taurochenodeoxycholic acid; CA, cholic acid; G-

CA, glycocholic acid; T-CA, taurocholic acid; T-UDCA-3S, tauroursodeoxycholic acid

3-O-sulfate; G-UDCA-3S, glycoursodeoxycholic acid 3-O-sulfate; UDCA-3S,

ursodeoxycholic acid 3-O-sulfate; T-LCA-3S, taurolithocholic acid 3-O-sulfate; CA-3S,

cholic acid 3-O-sulfate; LCA-3S, lithocholic acid 3-O-sulfate; G-LCA-3S,

glycolithocholic acid 3-O-sulfate; LCA-3S, lithocholic acid 3-O-sulfate; DCA-3S,

deoxycholic acid 3-O-sulfate; CDCA-3S, chenodeoxycholic acid 3-O-sulfate.

aData reported by Humbert et al (2012) as nM, but calculated and presented as mean % of

total BA. Low levels of amidated and sulfated BAs, as well as the high levels of LCA and

DCA (93% of total BA), in the stool is the result of metabolism by enterobacteria.

Approximately 75% and 8% of the BA pool in the urine and serum represents 3-O-

sulfated BAs, respectively. It should be noted that Takikawa et al. (1984) have reported

the presence of both BA glucuronide and sulfate conjugates in human serum and urine.

bData reported as fmol/mg tissue (Garcia-Canaveras et al., 2012), but calculated and

presented as mean % of total BA in the table above. Note high levels of amino acid

conjugated BAs in liver tissue (>99% of total BA).

cRossi et al., 1987 (gallbladder bile samples of healthy subjects). The authors do not

report the levels of amidated or non-amidated CDCA, DCA and CA 3-O-sulfate or 3-O-


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glucuronide conjugates. However, Takikawa et al. (1984) have reported that the levels of

sulfated or glucuronidated BAs in human bile are low (<1% total BA).

dHamilton et al., 2007 (authors reported % total BA, but only mean data are shown). It is

assumed that the low level of amidated and sulfated BAs, as well as the high levels of

LCA, DCA and CDCA (~70% of total BA), in caecum is the result of metabolism by

enterobacteria.

eNon-sulfated BAs are ranked in terms of decreasing hydrophobicity index (see Table 2).

fNot reported

gTrottier et al (2013) report that 38% of serum CDCA is in the form of 3-O-glucuronide

(amidated versus non-amidated not specified). The same authors report that about 14%

of serum CA is in the form of an acyl glucuronide (non-amidated).


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Table 2 Summary of the in vitro properties of different BAs reported in the literature

BA SULT2A1 Vmax/Km (µL/min per mg)b

BSEP Uptake (pmol/mg per 30sec)c

MRP4 Uptake Vmax/Km (µL/min per mg)d

NTCP (OATP) Cell Uptake (fmol/min)e

FXR (PXR) (CAT Activity Fold-Increase)f

Hydrophobicity Indexg

LCA 23.3 <45 -a - 35 (15) -

G-LCA 22.5 - - - - +1.05 T-LCA 16.7 - - - - +1.0 DCA 0.63 - - - 40 (3) +0.72

G-DCA 0.17 - 17 - - +0.65 T-DCA 0.14 600 - - - +0.59 CDCA 0.12 <45 - - 120 (3) +0.59

G-CDCA 0.16 1000 16 - - +0.51 T-CDCA 0.01 1400 23 29 (0.9) - +0.46

CA <0.01 45 5 2 (0.3) 5 (2) +0.13 G-CA <0.01 100 7 14 (0.1) - +0.07 T-CA <0.01 400 20 30 (0.1) - 0 UDCA 0.91 <45 - - 10 (-) -0.31

G-UDCA 1.02 300 10 - - -0.43 T-UDCA 0.96 400 17 40 (1.8) - -0.47

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

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Table 2:

LCA, lithocholic acid; G-LCA, glycolithocholic acid; T-LCA, taurolithocholic acid; G-

UDCA, glycoursodeoxycholic acid; T-UDCA, tauroursodeoxycholic acid; UDCA,

ursodeoxycholic acid;DCA, deoxycholic acid; G-DCA, glycodeoxycholic acid; T-DCA,

taurodeoxycholic acid; CDCA, chenodeoxycholic acid; G-CDCA,

glycochenodeoxycholic acid; T-CDCA, taurochenodeoxycholic acid; CA, cholic acid; G-

CA, glycocholic acid; T-CA, taurocholic acid.

aNo data reported.

bHuang et al., 2010 (authors reported kinetic parameters for the sulfation of each BA after

incubation with recombinant human sulfotransferase 2A1, SULT2A1).

cHayashi et al., 2005 (authors reported BA uptake rate by human bile salt export pump

[BSEP] vesicles and vector control vesicles; data represent difference between the two).

dRius et al., 2005 (authors reported kinetic parameters for BA uptake by human multidrug

resistance-associated protein 4 [MRP4] vesicles).

eMeier et al., 1997 (authors reported BA uptake rate into oocytes containing human

sodium-taurocholate co-transporting polypeptide [NTCP] or human organic anion

transporting peptide [OATP]).

fParks et al., 1999; Staudinger et al., 2001 (human farnesoid X receptor (FXR)- and

human pregnane X receptor [PXR]-mediated increase in chloramphenicol

acetyltransferase [CAT] activity in CV-1 cells).

gHeuman et al., 1989.


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Bile Acid R1 (α) R2 (α) R3 (β) R4 (α)

Ursodeoxycholic acid (UDCA) H OH H H

Cholic acid (CA) H OH OH OH

Chenodeoxycholic acid (CDCA) H OH OH H

Deoxycholic acid (DCA) H H H OH

Lithocholic acid (LCA) H H H H

Free Bile Acids R5 = OH

Glyco-Conjugated Bile Acids (G) R5 = NHCH2COOH

Tauro-Conjugated Bile Acids (T) R5 = NHCH2CH2SO3H

Non-Sulfated Bile Acids R6 = OH

Sulfated Bile Acids (S) R6 = OSO3H

Figure 1


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Figure 2

A

B


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Direct InhibitionIndirect Effects

Inhibition of BA biliary clearance?

Inhibition of BAamidation and/orsulfation?

Inhibition of BA hepatic uptake and efflux?

Impact on NHR-mediated adaptiveresponse to BApool perturbations

Figure 3

Impact on BA poolcomposition

Inhibition of BA biliary clearance?

Inhibition of NHRs?

Inhibition of BA hepatic uptake and efflux?

Impact on intracellularBA levels

Impact on intracellularBA levels

Impact on serum vs. liver BA levels

Impact on serum vs. liver BA levels


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