Bile Salt Export Pump Research Articles

Structure based classification for bile salt export pump (BSEP) inhibitors using comparative structural modeling of human BSEP

The bile salt export pump (BSEP) actively transports conjugated monovalent bile acids from the hepatocytes into the bile. This facilitates the formation of micelles and promotes digestion and absorption of dietary fat. Inhibition of BSEP leads to decreased bile flow and accumulation of cytotoxic bile salts in the liver. A number of compounds have been identified to interact with BSEP, which results in drug-induced cholestasis or liver injury. Therefore, in silico approaches for flagging compounds as potential BSEP inhibitors would be of high value in the early stage of the drug discovery pipeline. Up to now, due to the lack of a high-resolution X-ray structure of BSEP, in silico based identification of BSEP inhibitors focused on ligand-based approaches. In this study, we provide a homology model for BSEP, developed using the corrected mouse P-glycoprotein structure (PDB ID: 4M1M). Subsequently, the model was used for docking-based classification of a set of 1212 compounds (405 BSEP inhibitors, 807 non-inhibitors). Using the scoring function ChemScore, a prediction accuracy of 81% on the training set and 73% on two external test sets could be obtained. In addition, the applicability domain of the models was assessed based on Euclidean distance. Further, analysis of the protein–ligand interaction fingerprints revealed certain functional group-amino acid residue interactions that could play a key role for ligand binding. Though ligand-based models, due to their high speed and accuracy, remain the method of choice for classification of BSEP inhibitors, structure-assisted docking models demonstrate reasonably good prediction accuracies while additionally providing information about putative protein–ligand interactions.

Troglitazone Inhibits Bile Acid Amidation: A Possible Risk Factor for Liver Injury

Troglitazone and pioglitazone were developed as thiazolidinedione-type antidiabetes drugs, but only troglitazone was withdrawn from the markets due to severe liver injury. As both troglitazone and its sulfate metabolite are strong inhibitors of the bile salt export pump (BSEP), troglitazone-induced bile acid (BA) retention is thought to be one of the underlying mechanisms of liver injury. However, pioglitazone is also a strong BSEP inhibitor, indicating other mechanisms may also be involved in troglitazone-induced BA retention. Although retention of hydrophobic BAs (eg, chenodeoxycholic acid [CDCA]: a nonamidated BA) is known to cause hepatocyte injury, little is known about the hepatic conversion of nonamidated, hydrophobic BA species into less toxic hydrophilic BAs (eg, glycochenodeoxycholic acid: amidated BA) as a mechanism of drug-induced liver injury. In this study, we, therefore, investigated the effects of troglitazone and pioglitazone on BA amidation and the role of amidated BAs in troglitazone-associated BA-mediated hepatotoxicity. We also evaluated the intracellular BA composition of human hepatocytes treated with nonamidated BA species (CDCA or deoxycholic acid [DCA]) in the presence of troglitazone or pioglitazone. Amidation of CDCA and DCA was significantly inhibited by troglitazone (IC50: 5 and 3 µmol/l, respectively), but not pioglitazone. Moreover, treatment with troglitazone led to the retention of CDCA and DCA and decrease of glycine-amidation in hepatocytes. From these results, we suggest that troglitazone-induced liver injury might be caused by the accumulation of nonamidated BAs in hepatocytes due to inhibition of BA amidation.

Transporter-Mediated Disposition, Clinical Pharmacokinetics and Cholestatic Potential of Glyburide and Its Primary Active Metabolites.

Glyburide is widely used for the treatment of type 2 diabetes. We studied the mechanisms involved in the disposition of glyburide and its pharmacologically active hydroxy metabolites M1 and M2b and evaluated their clinical pharmacokinetics and the potential role in glyburide-induced cholestasis employing physiologically based pharmacokinetic (PBPK) modeling. Transport studies of parent and metabolites in human hepatocytes and transfected cell systems imply hepatic uptake mediated by organic anion-transporting polypeptides. Metabolites are also subjected to basolateral and biliary efflux by P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated proteins, and are substrates to renal organic anion transporter 3. A PBPK model in combination with a Bayesian approach was developed considering the identified disposition mechanisms. The model reasonably described plasma concentration time profiles and urinary recoveries of glyburide and the metabolites, implying the role of multiple transport processes in their pharmacokinetics. Predicted free liver concentrations of the parent (∼30-fold) and metabolites (∼4-fold) were higher than their free plasma concentrations. Finally, all three compounds showed bile salt export pump inhibition in vitro; however, significant in vivo inhibition was not apparent for any compound on the basis of a predicted unbound liver exposure-response effect model using measured in vitro IC50 values. In conclusion, this study demonstrates the important role of multiple drug transporters in the disposition of glyburide and its active metabolites, suggesting that variability in the function of these processes may lead to pharmacokinetic variability in the parent and the metabolites, potentially translating to pharmacodynamic variability.

Phytosterols Have Limited Direct Effect on Hepatocyte Transporter Expression but Synergize with Endotoxin to Enhance Kupffer Cell Inflammatory Response

Phytosterols, non‐nutritive components of soybean oil‐based lipid emulsions (SO), have been implicated in the development of parenteral nutrition associated liver disease (PNALD). Phytosterols may drive the pathogenesis of PNALD through a two‐hit mechanism: suppression of hepatic bile clearance through downregulation of the bile salt export pump (BSEP) and increased hepatic inflammation. Inflammation can further suppress BSEP and potentially the ATP‐binding cassette transporters ABCG5/8, the hepatic phytosterol clearance transporter, creating a feed‐forward loop leading to further hepatic injury. Two nuclear hormone receptors, Farnesoid X Receptor (FXR) and Liver X Receptor (LXR) regulate bile acid metabolism and have been shown to suppress the inflammatory response. The aim of our study was to first, determine if the hepatic transport dysfunction and inflammatory response is hepatocyte independent or requires activation of the resident macrophages, Kupffer cells; second, determine if ligand‐mediated activation of FXR and LXR can restore inflammation‐induced suppression of transporter function. Preterm piglets were administered parenteral nutrition for 10 d containing SO lipid emulsion. On day 10, piglets were given an 8‐h infusion of saline or LPS (10 μg*kg−1*h−1). In a separate experiment, primary hepatocytes and Kupffer cells were isolated from neonatal piglets. Cells were treated with media supplemented with 1% SO with or without the addition of a phytosterol mixture containing β‐sitosterol, stigmasterol, and campesterol. After incubation for 24 h, cells were additionally treated with FXR ligand Obeticholic acid (OCA), or LXR ligand GW3694 in the presence or absence of 50 ng/mL LPS or 10 ng/mL IL‐1β for 24 h. In piglets, administration of LPS lead to a significant suppression of BSEP and ABCG5/8 expression compared to saline infused piglets. In cell culture, administration of IL‐1β, but not LPS or phytosterols, led to suppression of both BSEP and ABCG5/8. In hepatocytes co‐treated with IL‐1β and OCA or GW3694, only BSEP expression was rescued. In Kupffer cells, phytosterols alone failed to activate expression of cytokines, however, when treated with both LPS and phytosterols increased cytokine expression significantly more than with LPS alone. From this study, we conclude inflammation can suppress the transporters for bile acid and phytosterol clearance. Our in vitro data shows that Kupffer cells are susceptible to enhanced activation of cytokine expression by phytosterols when cells are primed with LPS. These results suggest that phytosterols may contribute to the progression of liver disease through inflammation.Support or Funding InformationAmerican Society of Parenteral and Enteral Nutrition Rhoads Research Foundation, National Institute of Health Grant DK‐094616

Effects of troglitazone on bile acid disposition in Bsep KO rats

The inhibition of bile salt export pump (BSEP) by troglitazone (TGZ) and its sulfate metabolite (TS) is one of the mechanisms for TGZ‐induced hepatotoxicity. Here, we evaluated the bile acid profiles in wild type (WT) and Bsep knock‐out (KO) rats following repeated oral dose of TGZ (200 mg/kg/day, 7 days). With the remove of 8 base‐pair of nuclear acids, the mRNA levels of Shp, Mrp2, Mrp3 and Sult2A1 were significantly upregulated in KO rats. Following 7 day TGZ treatment, no signs of liver toxicity were observed in WT and KO rats, but Cyp7A1 was significantly increased in both groups. The plasma exposures of TGZ were not different between WT and KO rats, and are similar on Day‐1 and Day‐7. The exposures of TS were 7.6 and 9.3 fold lower in KO rats than in WT on Day‐1 and Day‐7, respectively. However, the formation of TS in liver S9 incubation was similar between WT and KO, indicating that the decreased TS plasma exposures in KO rats could be from increased biliary excretion, rather than altered metabolism. In general, dosing vehicle had minimal effects on bile acid profiles in WT and KO rats, but plasma exposure of individual bile acids demonstrated variable changes in KO rats when compared with WT rats. Many bile acids were increased with the first dose of TGZ in WT, but the increases were partially restored on last dose. In KO rats, alterations of most bile acids were only observed after 7 doses of TGZ. Therefore, rats seem able to regulate bile acid homeostasis through bile acid synthesis and transporter regulation in the presence of Bsep impairment caused by genetic modification or drug inhibition.

Genetic and Proteomic characterization of Bile Salt Export Pump (BSEP) in Snake Liver

Snake gallbladder, a traditional Chinese medicine, has been believed in various Asian countries to improve visual acuity and alleviate rheumatism. Bile acids, a major component of the gallbladder, are toxic to the liver and kidney in humans and animals due to its detergent effects, while also exhibiting therapeutic effects due to an increase in the gallbladder contractions of muscle strips in patients with cholesterol gallstones. Secretion of bile acids in human and mammals depends on the bile salt export pump (BSEP), a liver-specific adenosine triphosphate (ATP)-binding cassette transporter encoded by ABCB11. However, the presence of BSEP in snakes has not been thoroughly explored. Here we confirm the existence of BSEP and its coding DNA sequence in snakes on both the proteomic and genetic level. This work provides information on the snake ABCB11 sequence and helps further potential genetic manipulation to affect bile salt metabolism. Our study provides the foundation for research on bile acid production from snakes by using modern genetic and proteomic methodologies.

POTENTIAL FOR DRUG INTERACTION OF THE NOVEL COMT INHIBITOR OPICAPONE

ObjectivesThe effect of opicapone, a third generation COMT inhibitor, on metabolic enzymes and drug transporters was evaluated in vitro to anticipate potential drug‐drug interactions.Methodsin vitro studies using human liver microsomes and cell‐lines overexpressing a panel of selected drug‐transporters.ResultsWhen tested against P450 cytochromes (CYP), opicapone was found to be reversible inhibitor of CYP2C8 (IC50 = 3.6 μg/ml and IC50 = 6.7 μg/ml, respectively) and CYP2C9 (IC50 = 9.6 μg/ml, and IC50 = 20.7 μg/ml, respectively). No metabolism dependent inhibition by opicapone was observed for all tested CYPs. Opicapone was a substrate for the efflux transporters breast cancer resistance protein (BCRP) and P‐glycoprotein (P‐gp) and for the organic anion transporting polypeptide (OATP1B3). Opicapone showed moderate inhibition of bile salt export pump (BSEP), and organic anionic transporters (OAT1 and OAT3), but inhibited OATP1B1 and OATP1B3 at lower concentrations (IC50 values of 0.45 and 1.4 μg/ml, respectively).ConclusionsWhen the 99.9% binding of opicapone to human serum proteins is considered, the 1.55 μg/mL Cmax in humans receiving 50 mg/day of opicapone equates to 0.002 μg/mL of unbound material. This is 225‐fold lower than the lowest IC50 against transporters (OATP1B1, OATP1B3) and metabolic enzymes (CYP2C8 and CYP2C9) measured in the present study. As such, the observed in vitro effect upon these biological targets is highly unlikely to have any clinical impact.

In Vitro Drug-Induced Liver Injury Prediction: Criteria Optimization of Efflux Transporter IC50 and Physicochemical Properties.

Drug-induced liver injury (DILI) is a severe drug adverse response, which cannot always be reliably predicted in preclinical or clinical studies. Lack of observation of DILI during preclinical and clinical drug development has led to DILI being a leading cause of drug withdrawal from the market. As DILI is potentially fatal, pharmaceutical companies have been developing in vitro tools to screen for potential liver injury. Screens for physicochemical properties, mitochondrial function, and transport protein inhibition have all been employed to varying degrees of success. In vitro inhibition of the bile salt export pump (BSEP) has become a major risk factor for in vivo DILI predictions, yet discrepancies exist in which methods to use and the extent to which BSEP inhibition predicts clinical DILI. The presented work focuses on optimizing DILI predictions by comparing BSEP inhibition via the membrane vesicle assay and the hepatocyte-based BSEPcyte assay, as well as dual and triple liabilities. BSEP transport inhibition of taurcholic acids and glycocholic acids were similar for up to 29 drugs tested, in both the vesicle and hepatocyte-based assays. Positive and negative DILI predictions were optimized at a 50-µM cutoff value for 50 drugs using both NIH Livertox and PharmaPendium databases. Additionally, dual inhibition of BSEP and other efflux transporters (multidrug resistance-associated protein [MRP]2, MRP3, or MRP4) provided no observable predictive benefit compared with BSEP inhibition alone. Eighty-five percent of drugs with high molecular weight (>600 Da), high cLogP (>3), or a daily dose >100 mg and BSEP inhibition were associated with DILI. Triple liability of BSEP inhibition, high molecular weight, and high cLogP attained a 100% positive prediction rate.

Defects in myosin VB are associated with a spectrum of previously undiagnosed low γ-glutamyltransferase cholestasis.

Hereditary cholestasis in childhood and infancy with normal serum gamma‐glutamyltransferase (GGT) activity is linked to several genes. Many patients, however, remain genetically undiagnosed. Defects in myosin VB (MYO5B; encoded by MYO5B) cause microvillus inclusion disease (MVID; MIM251850) with recurrent watery diarrhea. Cholestasis, reported as an atypical presentation in MVID, has been considered a side effect of parenteral alimentation. Here, however, we report on 10 patients who experienced cholestasis associated with biallelic, or suspected biallelic, mutations in MYO5B and who had neither recurrent diarrhea nor received parenteral alimentation. Seven of them are from two study cohorts, together comprising 31 undiagnosed low‐GGT cholestasis patients; 3 are sporadic. Cholestasis in 2 patients was progressive, in 3 recurrent, in 2 transient, and in 3 uncategorized because of insufficient follow‐up. Liver biopsy specimens revealed giant‐cell change of hepatocytes and intralobular cholestasis with abnormal distribution of bile salt export pump (BSEP) at canaliculi, as well as coarse granular dislocation of MYO5B. Mass spectrometry of plasma demonstrated increased total bile acids, primary bile acids, and conjugated bile acids, with decreased free bile acids, similar to changes in BSEP‐deficient patients. Literature review revealed that patients with biallelic mutations predicted to eliminate MYO5B expression were more frequent in typical MVID than in isolated‐cholestasis patients (11 of 38 vs. 0 of 13). Conclusion: MYO5B deficiency may underlie 20% of previously undiagnosed low‐GGT cholestasis. MYO5B deficiency appears to impair targeting of BSEP to the canalicular membrane with hampered bile acid excretion, resulting in a spectrum of cholestasis without diarrhea. (Hepatology 2017;65:1655‐1669).

Analysis of surgical interruption of the enterohepatic circulation as a treatment for pediatric cholestasis.

This was a multicenter analysis of nontransplant surgical approaches to intrahepatic cholestasis. Approaches vary, are well tolerated, and generally, although not uniformly, result in improvement of pruritus and cholestasis. (Hepatology 2017;65:1645-1654).

A preliminary investigation on single nucleotide polymorphism rs2287622 of bile salt export pump gene in patients with chronic hepatitis C virus infection in Hunan, China

BackgroundEuropean researchers have underscored associations between single nucleotide polymorphism (SNP) rs2287622 of the hepatobiliary bile salt export pump (BSEP) gene and the risk of hepatitis C virus (HCV) infection. The distributions of SNP rs2287622 are racially specific. This study was aimed to preliminarily investigate the distribution of BSEP gene SNP rs2287622 in the Han patients with chronic HCV-infection (CHC) in Hunan, China.MethodsBSEP gene SNP rs2287622 of 165 CHC patients, 99 patients with chronic hepatitis B virus infection (CHB) and 99 healthy individuals were analyzed by polymerase chain reaction-restriction fragment length polymorphism analysis and nucleotide sequencing.ResultsThe overall frequencies of the C allele of BESP gene SNP rs2287622 in the CHC patients, CHB patients and healthy individuals were 74.2, 72.7 and 74.2%, respectively (P > 0.05). The overall odds ratios (ORs) aiming at predicting CHC risk by comparing the ratios of the frequency distribution of alleles or genotypes in the CHC group with those in the non-CHC group had no statistical significance (P > 0.05). However, the CHC ORs of CC vs TT, TC vs TT and CC + CT vs TT among the individuals aged over 40 years were 2.680, 3.122 and 2.824 respectively (P < 0.05), and the higher risk did not relate to gender, HCV genotypes and presence of HCV-related liver cirrhosis.ConclusionsAmong the Han individuals aged over 40 years in Hunan, China, genotype CC or CT of BSEP gene SNP rs2287622 may correlate with higher risk of CHC in comparison with genotype TT. Further study with a larger cohort is essential.

Assessing the Risk of Drug-Induced Cholestasis Using Unbound Intrahepatic Concentrations.

Inhibition of the bile salt export pump (BSEP) has been recognized as a key factor in the development of drug-induced cholestasis (DIC). The risk of DIC in humans has been previously assessed using in vitro BSEP inhibition data (IC50) and unbound systemic drug exposure under assumption of the "free drug hypothesis." This concept, however, is unlikely valid, as unbound intrahepatic drug concentrations are affected by active transport and metabolism. To investigate this hypothesis, we experimentally determined the in vitro liver-to-blood partition coefficients (Kpuu) for 18 drug compounds using the hepatic extended clearance model (ECM). In vitro-in vivo translatability of Kpuu values was verified for a subset of compounds in rat. Consequently, unbound intrahepatic concentrations were calculated from clinical exposure (systemic and hepatic inlet) and measured Kpuu data. Using these values, corresponding safety margins against BSEP IC50 values were determined and compared with the clinical incidence of DIC. Depending on the ECM class of a drug, in vitro Kpuu values deviated up to 14-fold from unity, and unbound intrahepatic concentrations were affected accordingly. The use of in vitro Kpuu-based safety margins allowed separation of clinical cholestasis frequency into three classes (no cholestasis, cholestasis in ≤2%, and cholestasis in >2% of subjects) for 17 out of 18 compounds. This assessment was significantly superior compared with using unbound extracellular concentrations as a surrogate for intrahepatic concentrations. Furthermore, the assessment of Kpuu according to ECM provides useful guidance for the quantitative evaluation of genetic and physiologic risk factors for the development of cholestasis.

Prognostic roles of tetrahydroxy bile acids in infantile intrahepatic cholestasis

Tetrahydroxy bile acids (THBAs) are hydrophilic and are present at minimal or undetectable levels in healthy human adults, but are present at high levels in bile salt export pump (abcb11)-knockout mice. The roles of THBAs in human cholestatic diseases are unclear. We aimed to investigate the presence of THBAs in patients with infantile intrahepatic cholestasis and its correlation with outcome. Urinary bile acids (BAs) were analyzed by GC-MS. Data were compared between good (n = 21) (disease-free before 1 year old) and poor prognosis groups (n = 19). Good prognosis patients had a higher urinary THBA proportion than poor prognosis patients [25.89% (3.45-76.73%) vs. 1.93% (0.05-48.90%)]. A urinary THBA proportion >7.23% predicted good prognosis with high sensitivity (95.24%), specificity (84.21%), and area under the curve (0.91) (P < 0.0001). A THBA proportion 7.23% was an independent factor for decreased transplant-free survival (hazard ratio = 7.16, confidence interval: 1.24-41.31, P = 0.028). Patients with a confirmed ABCB11 or tight junction protein 2 gene mutation (n = 7) had a minimally detectable THBA proportion (0.23-2.99% of total BAs). Three patients with an ATP8B1 mutation had an elevated THBA proportion (7.51-37.26%). In conclusion, in addition to disease entity as a major determinant of outcome, a high THBA level was associated with good outcome in the infantile intrahepatic cholestasis patients.

Tissue Biomarkers in Hepatocellular Tumors: Which, When, and How.

Few tissue markers are currently available to pathologists in the study of hepatocellular tumors. These markers should be used carefully taking into consideration not only morphology but also, and sometimes even more important, the clinical setting where the lesion to be diagnosed had developed. Glypican-3, heat shock protein 70, and glutamine synthetase (GS) are markers currently used, as a single panel, to discriminate the nature of a <2 cm hepatocellular lesion lacking radiological features of hepatocellular carcinoma (HCC) detected in a cirrhotic patient under surveillance. Their use, which can be improved by clathrin heavy chain, is mostly requested on liver biopsy. Hepatocyte paraffin 1, arginase-1, polyclonal carcinoembryonic antigen, CD10, and bile salt export pump are tissue markers used to confirm, at histology, the diagnosis of HCC made by imaging before enrollment for phase III studies on novel anti-HCC drugs. In this setting, pathologists are usually requested a conclusive diagnosis on a liver biopsy of a poorly differentiated, necrotic, enriched in stem-phenotype, carcinoma. Liver fatty acid-binding protein, serum amyloid A, C-reactive protein, prostaglandin D2 synthetase, GS, and β-catenin can be used either on biopsy or surgical specimen to classify hepatocellular adenoma into hepatocyte nuclear factor (HNF-1α) inactivated (steatotic), inflammatory, with dysregulation of sonic hedgehog and prostaglandin pathways, β-catenin mutated, and unclassified.

Identification of urine tauro-β-muricholic acid as a promising biomarker in Polygoni Multiflori Radix-induced hepatotoxicity by targeted metabolomics of bile acids

Polygoni Multiflori Radix (PMR) has been widely used as a tonic for centuries. However, hepatotoxicity cases linked to PMR have been frequently reported and appropriate biomarkers for clinical diagnosis are currently lacking. Here, an approach using UPLC-QqQ/MS-based targeted metabolomics of bile acids (BAs) complemented with biochemistry and histopathology was applied to characterize the development and recovery processes of PMR-induced hepatotoxicity in rats and to identify biomarkers. The expression of bile salt export pump (Bsep) and sodium taurocholate cotransporting polypeptide (Ntcp) were evaluated to investigate the underlying mechanism. Steatosis and inflammatory cell infiltration were observed in PMR-treated rats, which were accompanied by the elevation of serum biochemistry. The metabolic profiles of BAs were analyzed by Principal Component Analysis, hyodeoxycholic acid (HDCA) in serum and tauro-β-muricholic acid (TβMCA) in urine were identified as potential biomarkers for PMR-induced hepatotoxicity. The elevated expression of Bsep and decreased expression of Ntcp in the liver of PMRtreated rats indicated that hepatotoxicity was related to the disorders of BAs metabolism. Our study demonstrated that BAs may be used for clinical diagnosis of PMR-induced hepatotoxicity. Urine TβMCA was identified as a promising biomarker to facilitate the clinical monitoring of PMR-induced hepatotoxicity and may serve as potential therapeutic target.

ABCB4 missense mutations D243A, K435T, G535D, I490T, R545C, and S978P significantly impair the lipid floppase and likely predispose to secondary pathologies in the human population

Bile salts are natural detergents required to solubilise dietary fat and lipid soluble vitamins. They are synthesised in hepatocytes and secreted into the luminal space of the biliary tree by the bile salt export pump (BSEP), an ATP-binding cassette (ABC) transporter in the canalicular membrane. BSEP deficiency causes cytotoxic accumulation of bile salts in the hepatocyte that results in mild-to-severe forms of cholestasis. The resulting inflammation can also progress to hepatocellular cancer via a novel mechanism involving upregulation of proliferative signalling pathways. A second ABC transporter of the canalicular membrane is also critical for bile formation. ABCB4 flops phosphatidylcholine into the outer leaflet of the membrane to be extracted by bile salts in the canalicular space. These mixed micelles reduce the detergent action of the bile salts and protect the biliary tree from their cytotoxic activity. ABCB4 deficiency also causes cholestasis, and might be expected to cause cholangitis and predispose to liver cancer. Non-synonymous SNPs in ABCB4 have now been described in patients with liver cancer or with inflammatory liver diseases that are known to predispose to cancer, but data showing that the SNPs are sufficiently deleterious to be an etiological factor are lacking. Here, we report the first characterisation at the protein level of six ABCB4 variants (D243A, K435T, G535D, I490T, R545C, and S978P) previously found in patients with inflammatory liver diseases or liver cancer. All significantly impair the transporter with a range of phenotypes exhibited, including low abundance, intracellular retention, and reduced floppase activity, suggesting that ABCB4 deficiency is the root cause of the pathology in these cases.

Paeoniflorin ameliorates cholestasis via regulating hepatic transporters and suppressing inflammation in ANIT-fed rats.

Paeoniflorin has shown the obvious effect on cholestasis according to our previous research. However, its mechanism has not been absolutely explored yet. This study aims at evaluating the potential effect of paeoniflorin on alpha-naphthylisothiocyanate (ANIT) -induced cholestasis by inhibiting nuclear factor kappa-B (NF-κB) and simultaneously regulating hepatocyte transporters. Cholestasis was induced by administration of ANIT. The effect of paeoniflorin on serum indices such as total bilirubin (TBIL), direct bilirubin (DBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyltranspeptidase (γ-GT), total bile acid (TBA) and histopathology of liver were determined. Liver protein levels of NF-κB, interleukin 1β (IL-1β) and the hepatocyte transporters such as Na+/taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2) and cholesterol 7α-hydroxylase (Cyp7a1) were investigated by western blotting. The results demonstrated that paeoniflorin could decrease serum ALT, AST, ALP, γ-GT, TBIL, DBIL and TBA in ANIT-treated rats. Histological examination revealed that rats treated with paeoniflorin represented fewer neutrophils infiltration, edema and necrosis in liver tissue compared with ANIT rats. Moreover, paeoniflorin significantly reduced the over expressions of NF-κB and IL-1β induced by ANIT in liver tissue. In addition, the relative protein expressions of NTCP, BSEP, MRP2 but not Cyp7a1 were also restored by paeoniflorin. The potential mechanism of paeoniflorin in alleviating ANIT-induced cholestasis seems to be related to reduce the over expressions of NF-κB and hepatocyte transporters such as NTCP, BSEP as well as MRP2.

Fasiglifam (TAK-875): Mechanistic Investigation and Retrospective Identification of Hazards for Drug Induced Liver Injury.

TAK-875, a GPR40 agonist, was withdrawn from Phase III clinical trials due to drug-induced liver injury (DILI). Mechanistic studies were conducted to identify potential DILI hazards (covalent binding burden (CVB), hepatic transporter inhibition, mitochondrial toxicity, and liver toxicity in rats) associated with TAK-875. Treatment of hepatocytes with radiolabeled TAK-875 resulted in a CVB of 2.0 mg/day, which is above the threshold of 1 mg/day considered to be a risk for DILI. Covalent binding to hepatocytes was due to formation of a reactive acyl glucuronide (AG) and, possibly, an acyl-CoA thioester intermediate. Formation of TAK-875AG in hepatocytes and/or in vivo was in the order of non-rodents > human (in vitro only) > rat. These data suggest that non-rodents, and presumably humans, form TAK-875AG more efficiently than rats, and that AG-mediated toxicities in rats may only occur at high doses. TAK-875 (1000 mg/kg/day) formed significant amounts of AG metabolite (≤32.7 μM) in rat liver that was associated with increases in ALT (×4), bilirubin (×9), and bile acids (×3.4), and microscopic findings of hepatocellular hypertrophy and single cell necrosis. TAK-875 and TAK-875AG had similar potencies (within 3-fold) for human multi-drug resistant associated protein 2/4 (MRP2/4) and bile salt export pump, but TAK-875AG was exceptionally potent against MRP3 (0.21 μM). Inhibition of MRPs may contribute to liver accumulation of TAK-875AG. TAK-875 also inhibited mitochondrial respiration in HepG2 cells, and mitochondrial Complex 1 and 2 activities in isolated rat mitochondria. In summary, formation of TAK-875AG, and possibly TAK-875CoA in hepatocytes, coupled with inhibition of hepatic transporters and mitochondrial respiration may be key contributors to TAK-875-mediated DILI.

Nimesulide and 4'-Hydroxynimesulide as Bile Acid Transporters Inhibitors Are Contributory Factors for Drug-Induced Cholestasis.

Nimesulide (NIM) is a classic nonsteroidal anti-inflammatory drug. However, some patients treated with NIM experienced cholestatic liver injury. For this reason, we investigated the potential mechanism underlying NIM-induced cholestasis by using in vivo and in vitro models. Oral administration of 100 mg/kg/day NIM to Wistar rats for 5 days increased the levels of plasma total bile acids, alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase by 1.49-, 1.31-, 1.60-, and 1.29-fold, respectively. In sandwich-cultured rat hepatocytes, NIM and 4'-hydroxynimesulide (M1) reduced the biliary excretion index of d8-taurocholic acid (d8-TCA) and 5 (and 6)-carboxy-2',7'-dichlorofluorescein in a concentration-dependent manner, indicating the inhibition of the efflux transporters bile salt export pump and multidrug resistance-associated protein 2, respectively. In suspended rat hepatocytes, NIM and M1 inhibited the uptake transporters of d8-TCA for Na+-taurocholate cotransporting polypeptide at IC50 values of 21.3 and 25.0 μM, respectively, and for organic anion-transporting proteins at IC50 values of 45.6 and 39.4 μM, respectively. By contrast, nitro-reduced NIM and the further acetylated metabolite did not inhibit or only marginally inhibited these transporters at the maximum soluble concentrations. Inhibitory effects of NIM and M1 on human bile acid transporters were also confirmed using sandwich-cultured human hepatocytes. These data suggest that the inhibition of bile acid transporters by NIM and M1 is one of the biologic mechanisms of NIM-induced cholestasis.

Generation of a bile salt export pump deficiency model using patient-specific induced pluripotent stem cell-derived hepatocyte-like cells

Bile salt export pump (BSEP) plays an important role in hepatic secretion of bile acids and its deficiency results in severe cholestasis and liver failure. Mutation of the ABCB11 gene encoding BSEP induces BSEP deficiency and progressive familial intrahepatic cholestasis type 2 (PFIC2). Because liver transplantation remains standard treatment for PFIC2, the development of a novel therapeutic option is desired. However, a well reproducible model, which is essential for the new drug development for PFIC2, has not been established. Therefore, we attempted to establish a PFIC2 model by using iPSC technology. Human iPSCs were generated from patients with BSEP-deficiency (BD-iPSC), and were differentiated into hepatocyte-like cells (HLCs). In the BD-iPSC derived HLCs (BD-HLCs), BSEP was not expressed on the cell surface and the biliary excretion capacity was significantly impaired. We also identified a novel mutation in the 5′-untranslated region of the ABCB11 gene that led to aberrant RNA splicing in BD-HLCs. Furthermore, to evaluate the drug efficacy, BD-HLCs were treated with 4-phenylbutyrate (4PBA). The membrane BSEP expression level and the biliary excretion capacity in BD-HLCs were rescued by 4PBA treatment. In summary, we succeeded in establishing a PFIC2 model, which may be useful for its pathophysiological analysis and drug development.