INTRODUCTION

Bile salts are synthesized in the liver and are secreted from hepatocytes into bile via bile salt export pump (BSEP; ABCB11). After temporary storage in the gallbladder, bile is released into the small intestine upon food intake. The apical sodium-dependent bile salt transporter (ASBT or IBAT; SLC10A2) transports bile salts back into the ileocyte, after which organic solute transporter alpha/beta (OSTα/β; SLC51A/B) on the basolateral membrane releases bile salts into the splanchnic venous system. Na+-taurocholate cotransporting polypeptide (NTCP; SLC10A1) concludes the enterohepatic circulation by transporting bile salts back into the hepatocyte [1].

Bile salts aid in the emulsification of fat and nutrients in the small intestine, but are also known as potent receptor activators of among others Farnesoid X receptor (FXR; NR1H4) and Takeda G-protein-coupled receptor 5 (TGR5; GPBAR1). FXR is highly expressed in the liver and small intestine, where its activation limits bile salt synthesis by repressing cytochrome P450 7A1 (CYP7A1) and regulates bile salt transporter expression. TGR5 is expressed on the primary cilium of cholangiocytes where it has a sensory function and alters cholangiocyte fluid and ion secretion accordingly. Besides this, TGR5 is expressed in other cell types, including immune cells underlining that bile salts not only act in the enterohepatic circulation but have systemic effects.

There is a wide range of hepatobiliary diseases underlying cholestasis (diminished or entirely blocked bile flow from the liver to small intestine) ranging from identified congenital defects in Alagille syndrome and primary familial intrahepatic cholestasis (PFIC), including multiple subtypes, to immune-mediated diseases where genetic makeup and environmental factors also play a role such as primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and IgG4-related cholangitis (IRC), and finally idiopathic / multifactorial diseases with a largely unclear pathogenesis such as intrahepatic cholestasis of pregnancy (ICP). Yet a common denominator in all these cholestatic liver diseases is an apparent role for bile salts causing damage to the hepatobiliary system and therefore modulation of bile salt abundance and composition is an attractive therapeutic strategy. In this review we outline the current clinical advances and developments for treating cholestatic liver diseases through modulation of bile salt transport and signalling. We also discuss novel preclinical findings targeting these processes and most recent pathophysiological insights. 

Box 1:

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CLINICAL RESEARCH Primary biliary cholangitis

The majority of primary biliary cholangitis (PBC) patient responds well to ursodeoxycholic acid (UDCA), yet for those who are inadequate responders second-line therapy of the approved FXR agonist obeticholic acid (OCA) [2,3▪▪] or off-label therapeutics such as fibrates may be added on [4]. Although cholestasis is improving, PBC patients frequently suffer from pruritus and a relatively common adverse effect of FXR agonists is a dose-dependent exacerbation of the pruritus leading to treatment discontinuation in up 10% of patients [5,6]. The FITCH trial showed that bezafibrate caused a clear reduction in pruritus compared to placebo in UDCA treated PBC patients [7]. Intestinal ASBT inhibitors are used to lower hepatic bile salt load by blocking intestinal bile salt re-uptake, and are currently also clinically under investigation for treatment of PBC. Even though intestinal ASBT inhibitors are still considered experimental in pruritus management, Linerixibat gave a significant dose-dependent reduction in itch, according to the mean worst daily itch score in PBC patients [8]. Consistent with the mechanisms of action, i.e. the high colonic bile salt load, diarrhoea is a frequent and dose-dependent side effect.

Sclerosing cholangiopathies (immunoglobulin G4-related cholangitis and primary sclerosing cholangitis)

Sclerosing cholangiopathies include different aetiologies, with a final common pathway of fibrosis/stricturing of the bile ducts often with cholestasis, yet require different treatment strategies. IRC patients generally respond well to immunosuppressive therapy, whilst in PSC there is principle no place for immunosuppression unless there are biochemical and histological features of an autoimmune hepatitis component [9]. Clinical trials investigating drugs modulating bile salt transport or signalling in IRC are lacking, although anticholestatic and anti-inflammatory effects of UDCA in IRC patients have been observed.

Therapeutic drugs able to slow down disease progression in PSC are still an unmet need in patient care. Normal dose 15–20/mg/kg/day UDCA (weak recommendation in PSC; 2022 EASL CPG on sclerosing cholangitis) has shown improvement of liver biochemistry, yet a benefit on survival was still lacking [9]. More recently, a large retrospective study on long-term outcomes from a Japanese PSC patient registry has been published [10▪]. 325 PSC patients were included, of which 278 were receiving UDCA, and with a cox regression model UDCA treatment was associated with liver-transplant free survival. In a phase 2 clinical trial with over 160 PSC patients enrolled, the side chain shortened derivate 24-norUDCA (norUDCA) effectively lowered alkaline phosphatase (ALP) compared to placebo [11]. Similar results were found for the secondary endpoints, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma-glutamyl transferase (gGT). The use of norUDCA in PSC is currently being further investigated in a phase 3 trial (ClinicalTrials.gov number: NCT03872921). Although PSC patients are at increased risk for gallbladder carcinoma, recent work has shown that an enlarged gallbladder (frequently occurring in PSC) may protect against bile salt overload [12]. Patients with enlarged gallbladders had lower ALP, lower tauro-/glyco- conjugate ratio's and higher UDCA/total bile salt ratio's [12]. Cholecystectomized patients on the other hand had worse bile duct strictures and dilatations.

Intestinal ASBT inhibition improved pruritus and lowered serum bile salt levels in PSC patients, while liver biochemistry was not changed after 14 weeks of treatment [13▪]. In line with previous studies on ASBT inhibitors, around half of the treatment associated adverse events were diarrhoea [13▪]. FXR agonists are also widely studied in the context of PSC. Cilofexor, reduced liver biochemistry and serum bile salt levels after 96 weeks of treatment [14]. Remarkably, a phase 3 clinical trial investigating the safety and efficacy of cilofexor in noncirrhotic PSC was terminated early due to the low probability of achieving its primary endpoint of reduced risk on fibrosis progression [15]. Aldafermin (previously called NGM282 or M70), a nontumorigenic fibroblast growth factor 19 (FGF19) analogue, mimics FXR-FGF19 signalling to the liver and thereby represses bile salt synthesis. Aldafermin lowers serum bile salt levels, specifically hydrophobic bile salts, and circulating Pro-C3, which is associated with fibrogenesis [16].

Intrahepatic cholestasis of pregnancy

Women with intrahepatic cholestasis of pregnancy (ICP) typically present with pruritus in the third trimester and have elevated serum bile salt concentrations and transaminases. A recent study demonstrated that the gut microbiota not only differed between ICP patient and healthy pregnant women, but that colonization with the gut microbiota of ICP patients in mice resulted in cholestasis [17▪]. A particular difference was the abundance of Bacteroides fragilis (B. fragilis), which was able to inhibit FXR signalling, thereby causing increased bile salt synthesis.

UDCA is safe to use during pregnancy and recommended to treat pruritus in ICP patients, although the magnitude of itch reduction may be limited [18,19]. In ICP patients with serum bile salts ≥40 μmol/l, UDCA treatment should also be offered to reduce the risk of preterm birth or stillbirth. Elevated hydrophobic cholates could be responsible for foetal arrhythmia and cardiac dysfunction, while UDCA treatment in ICP partially ameliorated these findings [20]. A large randomized controlled clinical trial (PITCHES) investigating whether UDCA reduces adverse perinatal outcomes in ICP did not find a clear-cut effect, however the majority of included patients had serum bile salts <40 μmol/l at the start [21]. A subsequent meta-analysis went on to show that UDCA treatment did protect against adverse perinatal outcomes when serum bile salts were ≥40 μmol/l [22].

Congenital cholestatic syndromes (Alagille syndrome and primary familial intrahepatic cholestasis)

Intestinal ASBT inhibition in paediatric Alagille syndrome patients leads to both improved pruritus and quality of life scores [23]. Remarkably, ALT increased and total bilirubin did not change. In the ICONIC phase 2 trial, a significant relationship between reduced itch and the intestinal ASBT inhibitor Maralixibat was found, leading to improved quality of life [24]. Similarly, intestinal ASBT inhibition in PFIC patients lowered circulating plasma bile salt levels and improved pruritus [25]. Interestingly, a phase 2 study (INDIGO) using Maralixibat in PFIC patients, found that positive results with ASBT inhibition were dependent on PFIC subtype [26]. Patients with PFIC1 (due to ATP8B1 mutations) for example had less improvements in serum bile salt levels, compared to the other subtypes. Lastly, in a phase 3 clinical trial with paediatric PFIC1 and PFIC2 patients, ASBT inhibition improved pruritus and serum bile salt levels, the latter especially in the PFIC2 subgroup [27▪▪]. Furthermore, 24 weeks of treatment led to reduced ALT and AST, although this was not significantly different from placebo treated patients. Unfortunately, diarrhoea was a more frequently occurring adverse event in the treatment group compared to placebo treated patients.

PRE-CLINICAL RESEARCH Farnesoid X receptor activation

Preclinical studies are still investigating the effects and underlying mechanisms of OCA, other FXR agonists and combinations thereof. In mice, OCA tended to ameliorate liver injury in obstructive cholestasis via fibroblast growth factor 15 (FGF15) signalling to the hepatocyte [28▪]. Interestingly, OCA treatment did not improve the cholestatic phenotype of cytochrome P450 2C70 (Cyp2c70) deficient mice with a humanized more hydrophobic bile salt pool [29]. The nonsteroidal FXR agonist cilofexor also improved cholestatic liver injury in multidrug resistance protein 2 (Mdr2) knock-out mice, marked by amongst others improved ALP and ALT, and reduced hepatic fibrosis [30▪]. To date, novel FXR agonists are still being discovered. Even though results are very preliminary, Licraside for example was found to bind FXR and relieve alpha-naphthylisothiocyanate (ANIT)-induced cholestasis in mice, which was measured by a reduction in total bile salts but also reduced ALP, ALT and AST [31].

Apical sodium-dependent bile salt transporter inhibition

Interrupting the enterohepatic circulation by inhibiting intestinal ASBT, blocks bile salt re-uptake in the small intestine resulting in increased faecal bile salt excretion and a lack of bile salt return to the liver. In Cyp2c70 knock-out mice, pharmacological inhibition of ASBT improved liver histological markers and reduced inflammatory marker gene expression [32]. In line with this, strategies to knock down ASBT in Mdr2 knock-out mice lowered plasma ALT, AST and ALP, liver fibrosis and inflammation [33]. Surprisingly in this model, total bile salt content in the liver and small intestine were not affected and faecal bile salt excretion was reduced.

Other than the small intestine and cholangiocytes, ASBT is also expressed in the proximal renal tubule of the kidneys, where it prevents bile salts from being excreted via the urine. Systemic ASBT inactivation, to simultaneously target intestinal and renal ASBT, improves liver histology in bile duct ligated mice, and dramatically increased urinary bile salt excretion [34]. A systemic ASBT inhibitor called A3907 improved ALP, ALT and AST in Mdr2 knock-out mice while it also reduced serum bile salts [35]. Even though urinary bile salt excretion was not changed in Mdr2 knock-out mice treated with A3907, its potential was shown in bile duct ligated mice, where A3907 increases urinary bile salt excretion by up to 90-fold compared to vehicle treated controls [35] while completely preventing cholemic nephropathy [36▪▪]. Another strategy is to combine (intestinal) ASBT inhibition with repression of bile salt synthesis, in order to lower the hepatic bile salt load. This was first shown in female Cyp2c70 deficient mice, where a combination of intestinal ASBT inhibitor with FGF15 treatment reversed the cholestatic phenotype while ASBT inhibitor monotherapy was not as effective [37]. Recently, combination therapies were tested in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-cholestasis mouse model. In particular combinations of the nonsteroidal FXR agonist cilofexor or Aldafermin (FGF19 analogue) with intestinal ASBT inhibitor are effective in improving liver histology, ALT and AST and reducing inflammatory and fibrotic markers [28▪].

Na+-taurocholate cotransporting polypeptide inhibition

Besides being the main hepatic bile salt uptake transporter, NTCP is also an entry receptor for hepatitis B and delta virus (HBV/HDV). Myrcludex B (Bulevirtide) is a synthetic peptide approved for treatment of chronic HDV infections by the EMA whilst it also inhibits bile salt transport [38]. It was shown in various cholestatic mouse models that pharmacological NTCP inhibition protects against severe liver injury [39]. Myrcludex B however requires daily subcutaneous injection in order to reach maximum efficiency. Therefore, multiple orally available NTCP inhibitors are currently under development. The inhibitor A2342 blocks HBV infection and bile salt uptake in HEK293 cells expressing NTCP [40]. In addition, another orally available NTCP inhibitor completely blocked HBV infection in a humanized liver mouse model [41]. Efficacy of these novel compounds in cholestasis models is still under investigation.

Novel dual NTCP/ASBT inhibitors with varying potencies were developed and well tolerated in organic anion transporting polypeptide 1a/1b (Oatp1a/1b) deficient mice [42], a model preventing Na+-independent uptake of organic endo- and exogenous compounds such as bile salts, bilirubin and numerous drugs [43]. Both dual inhibitors with NTCP or ASBT specificity increased serum and urinary bile salt levels, while faecal bile salt concentrations were only increased by the inhibitor with ASBT specificity [42].

Biliary secretagogues and defence machinery

UDCA, a hydrophilic bile salt, acts through enhancing bicarbonate-rich bile flow [44,45], thereby promoting choleresis and limiting cholangiocyte damage. UDCA treatment reverses cholangiopathy and biliary fibrosis in female Cyp2c70 deficient mice [46], while it also prevents development of cholestasis in neonatal Cyp2c70 deficient mice [47]. norUDCA, which is able to undergo cholehepatic shunting independent of bile salt transporters such as ASBT, increased bile flow, biliary bicarbonate concentration and bicarbonate flow in mice [48▪]. Besides anticholestatic effects, norUDCA also attenuates a CD8+ T cell-driven hepatic immune response, which is also relevant in treating immune-mediated cholestatic liver diseases [49].

Previously, bicarbonate secretion appeared hampered in PBC patients and improved after 8 months of UDCA treatment, yet no positive changes were observed after stimulation with secretin [50]. Recently, a reduction of secretin and the secretin receptor was found in PBC livers and in livers of the TGF-β receptor II late stage PBC mouse model [51]. Secretin treatment reduced inflammation, ductular reaction and hepatic bile salt levels. Although interesting findings, evidence that secretin holds promise for a novel therapeutic strategy is lacking.

TGR5 promotes epithelial cell secretion and cell barrier function integrity. For the first time it was shown that biliary TGR5 expression was markedly reduced in cholangiocytes of PSC livers and Abcb4 knock-out mice livers [52▪▪]. The inflammatory liver phenotype of Abcb4 knock-out mice was rescued by TGR5 overexpression and intriguingly norUDCA treatment of Abcb4 knock-out mice restored the reduced TGR5 expression in cholangiocytes.

In IRC, autoantibodies have been described directed against annexin A11, laminin 511-E8, galectin-3 and prohibitin 1 [53–56]. Annexin A11 trafficks the calcium-activated chloride channel TMEM16A to the apical membrane of cholangiocytes [57▪]. TMEM16A maintains an adequate chloride gradient to allow for bicarbonate excretion, thereby protecting cholangiocytes from bile salt-induced damage. The other autoantigen, laminin 511-E8, has been shown to promote differentiation of iPSCs towards cholangiocytes through upregulation of secretory machinery such as TGR5 and the secretin receptor [58]. Furthermore, laminin 511 strengthens barrier function in endothelial cells and prevents extravasation of leukocytes [59]. We have recently demonstrated that a subset of IRC patients has autoantibodies against laminin 511-E8 and that laminin 511 protects cholangiocytes against hydrophobic bile salts and T lymphocyte-induced barrier dysfunction [60,61▪]. For galectin-3 and prohibitin 1 protective roles in cholangiocytes have not been observed.

The gut microbiome and immune cells

Besides the primary to secondary conversion of bile salts by the microbiome, bile salts shape the composition of the microbiome [62]. Novel amino acid conjugated bile salts, phenylalanocholic acid (Phe-chol), tyrosocholic acid (Tyr-chol), and leucocholic acid mediated by the microbiome have been identified and were partially elevated in inflammatory bowel disease and cystic fibrosis [63]. Oral gavage of mice with Phe-chol and Tyr-chol resulted in increased expression of FXR effector genes (Fgf15, Shp) and suppression of bile salt synthesis genes (Cyp8b1, Cyp7a1) [63]. Similarly, Mdr2 knock-out mice treated with broad spectrum antibiotics showed loss of FXR signalling, increased hepatic bile salt concentrations and phenotypically worse cholangitis features [64]. Alpha diversity in IRC and PSC faecal samples was reduced compared to healthy controls and despite common alteration, each had distinct host-microbe associations [65]. Surprisingly, in a mouse model for PSC-inflammatory bowel disease (IBD) intestinal inflammation improved the cholestatic liver injury and downstream liver fibrosis, likely due to nuclear factor kappa B (NF-κB)-mediated suppressed bile salt metabolism [66].

In PSC an interleukin 17 (IL17)/T helper 17 (Th17) cell response is observed and may be elicited by microbial stimulation [67]. Among other T cell subsets, circulating Th17 cells can also be found in IRC [68]. Notably, derivates of litocholic acid (LCA) were identified to regulate T cell populations in mice [69]. Administration of LCA derivates in mice induced a reduction in Th17 and an increase in T regulatory cell (Treg) differentiation demonstrating that bile salt metabolites can skew the immune response. Mechanistically, human gut bacteria converted LCA to 3-oxoLCA, which suppressed Th17 cell differentiation through inhibiting the Th17 promoting transcription factor RAR-related orphan receptor gamma (RORγt) [70▪▪]. The levels of 3-oxoLCA were reduced in IBD patients indicating the potential relation between an altered gut microbiome, changes in bile salt pool composition and the downstream effects on dominant T cell subsets and potentially disease phenotype.

CONCLUSION

Despite different diseases aetiologies, cholestatic liver diseases have the common burden of accumulating bile salts causing toxicity. Therefore, pharmacological targeting of proteins involved in bile salt synthesis, transport and signalling are an attractive treatment strategy (Fig. 1).

FIGURE 1:

Current therapeutic strategies modulating bile salt homeostasis in biliary diseases and novel pathogenic insights. Bile salt transport is targeted by intestinal or systemic ASBT inhibition and has been combined with NTCP inhibition. Bile salt synthesis is repressed by activating the FXR–FGF15/19 axis and has been combined with ASBT inhibition. The bile salt receptor TGR5 appears reduced in PSC, and the IRC autoantigens annexin A11 and laminin 511 contribute to cholangiocyte protection. The gut microbiome and bile salt pool have a bidirectional interaction with downstream effects on dominant T-cell populations and the immune response. Created with BioRender.com. ASBT, apical sodium-dependent bile salt transporter; FGF, fibroblast growth factor; FXR, Farnesoid X receptor; NTCP, Na+-taurocholate cotransporting polypeptide; TGR5, Takeda G-protein-coupled receptor 5; UDCA, ursodeoxycholic acid.

Besides OCA, other nonsteroidal FXR agonists are being investigated in earlier trial phases for PBC. The reporting on pruritus as a common side effect remains. Although retrospective, normal dose UDCA improved liver transplant free survival in PSC. Furthermore, UDCA partially ameliorates the prenatal complications likely caused by cholates in ICP. For congenital cholestatic syndromes ASBT inhibitors are currently being investigated and show promising outcomes, yet the frequently reported side effect bile acid diarrhoea (BAD) remains.

Regarding drug development, combination therapies are being investigated including ASBT inhibition alongside FXR agonism or NTCP inhibition. Given the promising clinical and preclinical data, and the ability to undergo cholehepatic shunting, norUDCA could be interesting to combine with ASBT inhibition. A role for bile salt receptor TGR5 dysfunction has emerged in the pathogenesis of PSC, whilst in IRC recent advances have been made in understanding the roles of autoantigens annexin A11 and laminin 511-E8 in cholangiocytes. Bile salts can shape the microbiome composition, which is altered in sclerosing cholangiopathies, and can skew dominant T cell subsets. The interplay between gut microbiome, bile salt pool composition, immune response and disease phenotype will hopefully be further clarified in the near future.

Acknowledgements

None.

Financial support and sponsorship

This work was supported by Vici grant 09150182010007 from the Netherlands Organization for Scientific Research to SFJvdG. This work was further supported by a Gastrostart grant of the Netherlands Society of Gastroenterology (NVGE) / the Netherlands Association for the Study of the Liver (NASL) and an Amsterdam UMC/AMC PhD Scholarship to D.C.T.

Conflicts of interest

S.F.J.vdG. declares consulting activities for ProQR Therapeutics. D.C.T. and R.F.K. have no conflicts of interest to declare.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

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