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Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive fibro-stenotic strictures and destruction of the biliary tree. Currently, there is no effective treatment which can delay its progression or ameliorate the transplant-free survival. Moreover, a major chontroversy in PSC is whether to use UDCA. More recently, novel pharmacological agents emerged aiming at: i) modulation of bile composition; ii) immunomodulation; iii) targeting the gut microbiome; iv) targeting fibrosis. Successful PSC therapy, however, will be most likely a personalized combination of different drugs plus endoscopic treatment. This review aims at offering an overview on the experimental pharmacological strategies currently exploited for PSC treatment., (Copyright © 2021. Published by Elsevier Ltd.)

Objective: To investigate the pathogens' distribution and antimicrobial resistance in the bile of acute biliary tract infection patients. Methods: The data of bile bacterial culture and drug sensitivity test of 223 acute biliary tract infection patients who underwent gallbladder puncture or endoscopic retrograde cholangiopancreatography drainage from July 2009 to July 2019 were analyzed retrospectively at Department of General Surgery,Xinhua Hospital,Affiliated to Shanghai Jiao Tong University School of Medicine.There were 141 males and 82 females with age of 67.3 years(range:28 to 93 years).Three to five milliliter of bile was extracted from each patient and sent to the laboratory for bacterial culture,identification and drug sensitivity test.The patients were divided into two groups according to the visiting time: the former group ( n =124) was admitted from July 2009 to July 2014,and the latter group( n =99) was admitted from August 2014 to July 2019.The distribution of pathogenic bacteria and the changing trend of drug resistance rate of common bacteria in the two groups were compared.The results of drug sensitivity test were analyzed by WHONET software provided by WHO bacterial surveillance network.The drug resistance rates in different time periods were compared by χ 2 test. Results: In this study,there were 147 cases of acute cholangitis and 76 cases of acute cholecystitis.A total of 376 strains of pathogenic bacteria were cultured.Among them,98 strains(26.1%) were gram-positive bacteria,269 strains(71.5%) were gram-negative bacteria and 9 strains(2.4%) were fungi.The top three gram-positive bacteria were Enterococcus faecium (49.0%,48/98), Enterococcus faecalis (20.4%,20/98),and Enterococcus luteus (7.1%,7/98).The top 5 gram-negative bacteria were Escherichia coli (33.5%,90/269), Klebsiella pneumoniae (13.8%,37/269), Pseudomonas aeruginosa (13.0%,35/269), Acinetobacter baumannii (12.6%,34/269),and Enterobacter cloacae (4.8%,13/269).From 2009 to 2019,there was no significant change in the proportion of gram-positive bacteria (former group vs. latter group: 25.3% vs. 28.2%) and gram-negative bacteria(former group vs. latter group: 74.7% vs. 71.8%) in the bile of patients with acute biliary tract infection.Gram-positive bacteria were mainly Enterococci (85.7%,84/98) and gram-negative bacteria were Escherichia coli (33.5%,90/269). Acinetobacter baumannii accounted for 7.8%(11/142) of gram-negative bacteria in the former group and 18.1%(23/127) in the latter group,an increase of 10.3% over previous five years. Pseudomonas aeruginosa had a downward trend,16.9% in the former group(24/142) and 8.7% in the latter group (11/127),the proportion decreased by 8.2%,and the other changes were not significant.The drug resistance rates of common gram-positive bacteria were relatively stable,and the drug resistance rates of Enterococcus faecium to many antibiotics were higher than those of Enterococcus faecalis .The resistance rates of gram-negative bacteria to most antibiotics showed an upward trend,among which Klebsiella pneumoniae showed an upward trend to most of antibiotics(former group: 0/15－4/13, latter group: 55.0%－70.0%; χ 2 =3.996－16.942, P =0.000－0.046).The drug resistance rates of Acinetobacter baumannii was generally higher,but there were no significant changes in the drug resistance rates of Acinetobacter baumannii between the two groups.The drug resistance rates of Pseudomonas aeruginosa to most antibiotics increased,and the overall drug resistance rates of Escherichia coli were stable and showed a slight upward trend. Conclusions: The main pathogens in bile of patients with acute biliary tract infection are gram-negative bacteria.The constituent ratio of various gram-negative bacteria had no significant change from 2009 to 2019,but the drug resistance rates shows an upward trend.Among the gram-negative bacteria, Escherichia coli is the most important pathogen,and the proportion has no significant change.The proportion of Acinetobacter baumannii in the former group was significantly higher than that in the former group.And the proportion of Pseudomonas aeruginosa has a decreased trend.

Pancreatic cancer (PC) is one of the leading causes of mortality rate globally and is usually associated with obstructive jaundice (OJ). Up to date, there is no clear consensus on whether biliary decompression should be performed prior to surgery and how high levels of serum bile affects the outcome of PC. Therefore, our study aims were to characterise the effect of bile acids (BAs) on carcinogenic processes using pancreatic ductal adenocarcinoma (PDAC) cell lines and to investigate the underlying mechanisms. Liquid chromatography-mass spectrometry was used to determine the serum concentrations of BAs. The effects of BAs on tumour progression were investigated using different assays. Mucin expressions were studied in normal and PDAC cell lines and in human samples at gene and protein levels and results were validated with gene silencing. The levels of BAs were significantly higher in the PDAC + OJ group compared to the healthy control. Treating PDAC cells with different BAs or with human serum obtained from PDAC + OJ patients enhanced the rate of proliferation, migration, adhesion, colony forming, and the expression of MUC4. In PDAC + OJ patients, MUC4 expression was higher and the 4-year survival rate was lower compare to PDAC patients. Silencing of MUC4 decreased BAs-induced carcinogenic processes in PDAC cells. Our results show that BAs promote carcinogenic process in PDAC cells, in which the increased expression of MUC4 plays an important role. Based on these results, we assume that in PC patients, where the disease is associated with OJ, the early treatment of biliary obstruction improves life expectancy.

Background and Aims: Estrogen is an important risk factor for cholesterol gallstone disease because women are twice as likely as men to form gallstones. The classical estrogen receptor α (ERα), but not ERβ, in the liver plays a critical role in the formation of estrogen-induced gallstones in female mice. The molecular mechanisms underlying the lithogenic effect of estrogen on gallstone formation have become more complicated with the identification of G protein-coupled receptor 30 (GPR30), an estrogen receptor., Approach and Results: We investigated the biliary and gallstone phenotypes in ovariectomized female GPR30 -/- , ERα -/- , and wild-type mice injected intramuscularly with the potent GPR30-selective agonist G-1 at 0 or 1 μg/day and fed a lithogenic diet for 8 weeks. The activation of GPR30 by G-1 enhanced cholelithogenesis by suppressing expression of cholesterol 7α-hydroxylase, the rate-limiting enzyme for the classical pathway of bile salt synthesis. These metabolic abnormalities led to an increase in biliary cholesterol concentrations in company with hepatic hyposecretion of biliary bile salts, thereby inducing cholesterol-supersaturated gallbladder bile and accelerating cholesterol crystallization. G-1 also impairs gallbladder emptying, leading to sluggish gallbladder motility and promoting the development of biliary sludge in the early stage of gallstone formation. The prevalence rates of gallstones were 80% in wild-type and ERα -/- mice treated with G-1 compared to 10% in wild-type mice receiving no G-1. However, no gallstones were formed in GPR30 -/- mice treated with G-1., Conclusions: GPR30 produces additional lithogenic actions, working independently of ERα, to increase susceptible to gallstone formation in female mice; both GPR30 and ERα are potential therapeutic targets for cholesterol gallstone disease, particularly in women and patients exposed to high levels of estrogen., (© 2020 by the American Association for the Study of Liver Diseases.)

Background: In primary bile acid diarrhoea, feedback by farnesoid X receptor (FXR) and fibroblast growth hormone 19 (FGF19) on hepatic bile acid production is impaired., Aims: To evaluate the safety, mechanisms and efficacy of negative feedback by FXR activation with tropifexor, a non-bile acid FXR agonist, in patients with primary bile acid diarrhoea., Methods: In this double-blind, multicentre, randomised, cross-over study, patients received tropifexor 60 µg or placebo once daily for 14 days in each of two treatment periods. Primary objectives included tropifexor safety and tolerability, and on stool frequency and form. Other assessments included pharmacokinetic and pharmacodynamic measures, biochemical markers and gastrointestinal transit., Results: Twenty patients (tropifexor 60 µg/placebo [N = 10]; placebo/tropifexor 60 µg [N = 10]) were enrolled. Adverse event rates were lower with tropifexor vs placebo (52.9% vs 73.7%). No patient had pruritus during tropifexor intake. There were no significant differences in stool frequency, stool form or loperamide use between treatments. Tropifexor increased FGF19 and decreased 7α-hydroxy-4-cholesten-3-one (C4) levels for up to 8 h. Plasma tropifexor concentrations peaked at 5 hours post-dose on days 1 and 12. At day 12, tropifexor caused reduction in peak total bile acid concentration (33%, P = 0.032) and exposure (36%, P = 0.005). Moreover, tropifexor showed a significant increase in ascending colon half-emptying time (P = 0.036)., Conclusions: Tropifexor 60 µg once daily had acceptable safety and tolerability. Changes in FGF19 and C4 showed effective target engagement; however, higher doses may be required to observe stool frequency changes. Slowing of ascending colon emptying suggests therapeutic potential of tropifexor in patients with primary bile acid diarrhoea. ClinicalTrials.gov number: NCT02713243., (© 2020 John Wiley & Sons Ltd.)

Ethnopharmacological Relevance: Semen Arecae (SA) is one of the most commonly used Traditional Chinese Medicine. Charred Semen Arecae (CSA) is the processed product of SA. Alkaloids are considered as pharmacological mechanisms of SA and CSA on gastrointestinal motility. Recent studies have shown alkaloids decreased quickly after procession. However, the promoting on gastrointestinal motility were not decreased. Is gastrointestinal motility related to alkaloids of CSA? This study explored the effects of SA, CSA, Semen Arecae-Removal (SA-R), and Charred Semen Arecae-Removal (CSA-R) on gastrointestinal motility, Gastric Inhibitory Polypeptide (GIP), Glucagon Like Peptide-1 (GLP-1), gastric juice and bile in rats., Material and Methods: Rats were randomly divided into six groups, including the Control group, SA group, CSA group, SA-R group, CSA-R group, and Positive drug group (Mosapride). Alkaloids of samples were knocked out by using the "target constituent removal" strategy. Gastric residue and intestinal propulsion rate were evaluated in rats. Serum levels of GIP and GLP-1 were measured by Enzyme-Linked Immunosorbent Assay (ELISA). Gastric juice and bile were examined, respectively., Results: CSA-R and SA-R have been investigated by the Preparative Thin-layer Chromatography (PTLC) method. Intestinal propulsion and gastric residue assessments confirmed the effectiveness of CSA and CSA-R. CSA-R was higher than SA-R in the GLP-1, pepsin activity, the secretion of bile, Bilirubin (BIL), and Cholesterol (CHO). The statistical comparison demonstrated that there is no difference between the CSA group and CSA-R group., Conclusions: After processing, the promoting gastrointestinal motility might be not related to alkaloids. Maillard reaction could be produced to promote the secretion of GLP-1, bile, and CHO for gastrointestinal motility. Our findings provide a pharmacological reference for the clinical application of SA and CSA in the treatment of digestive diseases., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder, characterised by chronic diarrhoea, xanthomas, cataracts, and neurological deterioration. CTX is caused by CYP27A1 deficiency, which leads to abnormal cholesterol and bile acid metabolism. Urinary bile acid profiling (increased m/z 627: glucuronide-5β-cholestane-pentol) serves as diagnostic screening for CTX. However, this led to a false positive CTX diagnosis in two patients, who had received total intravenous anaesthesia (TIVA) with propofol., Methods: To determine the influence of propofol on bile acid profiling, 10 urinary samples and 2 blood samples were collected after TIVA with propofol Fresenius 7 to 10 mg/kg/h from 12 subjects undergoing scoliosis correction. Urinary bile acids were analysed using flow injection negative electrospray mass spectrometry. Propofol binding to recombinant CYP27A1, the effects of propofol on recombinant CYP27A1 activity, and CYP27A1 expression in liver organoids were investigated using spectral binding, enzyme activity assays, and qPCR, respectively. Accurate masses were determined with high-resolution mass spectrometry., Results: Abnormal urinary profiles were identified in all subjects after TIVA, with a trend correlating propofol dose per kilogramme and m/z 627 peak intensity. Propofol only induced a weak CYP27A1 response in the spectral binding assay, minimally affected CYP27A1 activity and did not affect CYP27A1 expression. The accurate mass of m/z 627 induced by propofol differed >10 PPM from m/z 627 observed in CTX., Conclusions: TIVA with propofol invariably led to a urinary profile misleadingly suggestive of CTX, but not through CYP27A1 inhibition. To avoid further misdiagnoses, propofol administration should be considered when interpreting urinary bile acid profiles., (© 2020 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Purpose: The bile salt export pump (BSEP), a key player in hepatic bile acid clearance, has been the center of research on drug-induced cholestasis. However, such studies focus primarily on the direct inhibition of BSEP, often overlooking the potential impact of transcriptional repression. This work aims to explore the disruption of bile acid efflux caused by drug-induced BSEP repression., Methods: BSEP activity was analyzed in human primary hepatocytes (HPH) using a traditional biliary-clearance experiment and a modified efflux assay, which includes a 72-h pretreatment prior to efflux measurement. Relative mRNA and protein expressions were examined by RT-PCR and Western blotting, respectively., Results: Metformin concentration-dependently repressed BSEP expression in HPH. Although metformin did not directly inhibit BSEP activity, longer metformin exposure reduced BSEP transport function in HPH by down-regulating BSEP expression. BSEP repression by metformin was found to be AMP-activated protein kinase-independent. Additional screening of 10 reported cholestatic non-BSEP inhibitors revealed that the anti-cancer drug tamoxifen also markedly repressed BSEP expression and reduced BSEP activity in HPH., Conclusions: Repression of BSEP alone is sufficient to disrupt hepatic bile acid efflux. Metformin and tamoxifen appear to be prototypes of a class of BSEP repressors that may cause drug-induced cholestasis through gene repression instead of direct BSEP inhibition.

The G-protein bile acid receptor 1 (GPBAR1) has emerged in the last decade as prominent target for the treatment of metabolic and inflammatory diseases including type 2 diabetes, obesity, and non-alcoholic steatohepatitis. To date numerous bile acid derivatives have been identified as GPBAR1 agonists, however their clinical application is hampered by the lack of selectivity toward the other bile acid receptors. Therefore, non-steroidal GPBAR1 ligands able to selectively activate the receptor are urgently needed. With this aim, we here designed, synthesized and biologically evaluated ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl) urea derivatives as novel potent GPBAR1 agonists. Particularly, compounds 9 and 10 induce the mRNA expression of the GPBAR1 target gene pro-glucagon and show high selectivity over the other bile acid receptors FXR, LXRα, LXRβ and PXR, and the related receptors PPARα and PPARγ. Computational studies elucidated the binding mode of 10 to GPBAR1, providing important structural insights for the design of non-steroidal GPBAR1 agonists. The pharmacokinetic properties of 9 and 10 suggest that the ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl)ureydil scaffold might be exploited to achieve effective drug candidates to treat GPBAR1 related disorders.

Our recent metagenomics analysis has uncovered remarkable modifying effects of green tea polyphenols (GTP) on gut-microbiota community structure and energy conversion related gene orthologs in rats. How these genomic changes could further influence host health is still unclear. In this work, the alterations of gut-microbiota dependent metabolites were studied in the GTP-treated rats. Six groups of female SD rats (n=12/group) were administered drinking water containing 0%, 0.5%, and 1.5% GTP (wt/vol). Their gut contents were collected at 3 and 6 months and were analyzed via high performance liquid chromatography (HPLC) and gas chromatography (GC)-mass spectrometry (MS). GC-MS based metabolomics analysis captured 2668 feature, and 57 metabolites were imputatively from top 200 differential features identified via NIST fragmentation database. A group of key metabolites were quantitated using standard calibration methods. Compared with control, the elevated components in the GTP-treated groups include niacin (8.61-fold), 3-phenyllactic acid (2.20-fold), galactose (3.13-fold), mannose (2.05-fold), pentadecanoic acid (2.15-fold), lactic acid (2.70-fold), and proline (2.15-fold); the reduced components include cholesterol (0.29-fold), cholic acid (0.62-fold), deoxycholic acid (0.41-fold), trehalose (0.14-fold), glucose (0.46-fold), fructose (0.12-fold), and alanine (0.61-fold). These results were in line with the genomic alterations of gut-microbiome previously discovered by metagenomics analysis. The alterations of these metabolites suggested the reduction of calorific carbohydrates, elevation of vitamin production, decreases of bile constituents, and modified metabolic pattern of amino acids in the GTP-treated animals. Changes in gut-microbiota associated metabolism may be a major contributor to the anti-obesity function of GTP., (Copyright © 2018 Elsevier Inc. All rights reserved.)

1. 14  C-Labelled E/Z isomers of a synthetic pyrethroid metofluthrin ((E/Z)-(1 R,3 R)-2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylate, abbreviated as RTE/RTZ, respectively) were used for rat metabolism studies. 14  C-RTE or RTZ labelled at the carbonyl-carbon [acid- 14 C] or the methoxymethylbenzyl-α-carbon [alcohol- 14  C] was administered orally to rats at 1 and 20 mg/kg. 2. Dosed compounds were mostly absorbed, metabolised, and rapidly excreted. Dose-related increase in blood AUC suggested no saturation of absorption at the high dose. Blood 14  C was maximal at 3-8 h and decreased with a half-life of 52-163 h. Radioactivity in tissues, blood and plasma decreased basically at the same rate and the sum fell below 0.2% of the dose at 168 h. 3. Although the major metabolic pathways of the isomers, that is, ester cleavage, O-demethylation and ω-oxidation, were similar, there was a notable difference. The RTZ double bond commonly undergoes epoxidation while RTE double bond mainly undergoes glutathione conjugation, which causes faster elimination from plasma and greater excretion into faeces on RTE. Faster urinary excretion and elimination from blood were observed for the alcohol moiety than the acid moiety. 4. In conclusion, this study described the overall metabolic profiles of metofluthrin and identified the differences in metabolic breakdown between the isomers. No marked sex-/dose-related differences were observed.

OBJECTIVE To investigate effects of prednisolone administration on gallbladder emptying rate and gallbladder bile composition in dogs. ANIMALS 6 healthy Beagles. PROCEDURES Prednisolone was administered (2 mg/kg, SC, once daily for 2 weeks) to each dog and tapered over 2 weeks. Gallbladder emptying rate and bile composition were evaluated before and after administration of prednisolone for 2 weeks as well as 1 week after cessation of prednisolone administration. RESULTS Gallbladder emptying rate decreased significantly after prednisolone administration (median, 27%; range, 0% to 38%), compared with rate before administration (median, 59%; range, 29% to 68%), but then increased 1 week after cessation of administration (median, 45%; range, 23% to 48%). Gallbladder bile mucin concentration decreased significantly after prednisolone administration (median, 8.8 mg/dL; range, 6.2 to 11.3 mg/dL), compared with concentration before administration (median, 13.1 mg/dL; range, 10.7 to 21.7 mg/dL), but then increased 1 week after cessation of administration (median, 14.3 mg/dL; range, 9.6 to 26.7 mg/dL). Gallbladder taurochenodeoxycholic acid concentration decreased significantly after prednisolone administration (8.1 mmol/L; range, 6.8 to 15.2 mmol/L), compared with concentration before administration (median, 27.2 mmol/L; range, 22.0 to 31.9 mmol/L), but then increased 1 week after cessation of administration (median, 26.4 mmol/L; range, 15.1 to 31.5 mmol/L). CONCLUSIONS AND CLINICAL RELEVANCE A lower gallbladder emptying rate caused by prednisolone administration may be involved in the pathogenesis of gallbladder disease in dogs. Further studies are required to determine the clinical importance of lower gallbladder bile mucin concentrations caused by glucocorticoid administration in the pathogenesis of gallbladder disease in dogs.

The metabolic fate of the human hepatotoxin fenclozic acid ([2-(4-chlorophenyl)-1,3-thiazol-4-yl]acetic acid) (Myalex) was studied in normal and bile-cannulated chimeric mice with a humanized liver, following oral administration of 10 mg/kg. This in vivo animal model was investigated to assess its utility to study "human" metabolism of fenclozic acid, and in particular to explore the formation of electrophilic reactive metabolites (RMs), potentially unique to humans. Metabolism was extensive, particularly involving the carboxylic acid-containing side chain. Metabolism resulted in the formation of a large number of metabolites and involved biotransformation via both oxidative and conjugative routes. The oxidative metabolites detected included a variety of hydroxylations as well as cysteinyl-, N-acetylcysteinyl-, and cysteinylglycine metabolites. The latter resulted from the formation of glutathione adducts/conjugates providing evidence for the production of RMs. The production of other classes of RMs included acyl-glucuronides, and the biosynthesis of acyl carnitine, taurine, glutamine, and glycine conjugates via potentially reactive acyl-CoA intermediates was also demonstrated. A number of unique "human" metabolites, e.g., those providing evidence for side-chain extension, were detected in the plasma and excreta of the chimeric liver-humanized mice that were not previously characterised in, e.g., the excreta of rat and C57BL/6 mice. The different pattern of metabolism seen in these chimeric mice with a humanized liver compared to the conventional rodents may offer clues to the factors that contributed to the drug-induced liver injury seen in humans.

This current study aimed to elucidate the effects and possible underlying mechanisms of long-term supplementation with dietary luteolin (LU)-enriched artichoke leaf (AR) in high-fat diet (HFD)-induced obesity and its complications (e.g., dyslipidemia, insulin resistance, and non-alcoholic fatty liver disease) in C57BL/6N mice. The mice were fed a normal diet, an HFD, or an HFD plus AR or LU for 16 weeks. In the HFD-fed mice, AR decreased the adiposity and dyslipidemia by decreasing lipogenesis while increasing fatty acid oxidation, which contributed to better hepatic steatosis. LU also prevented adiposity and hepatic steatosis by suppressing lipogenesis while increasing biliary sterol excretion. Moreover, AR and LU prevented insulin sensitivity by decreasing the level of plasma gastric inhibitory polypeptide and activity of hepatic glucogenic enzymes, which may be linked to the lowering of inflammation as evidenced by the reduced plasma interleukin (IL)-6, IL-1β, and plasminogen activator inhibitor-1 levels. Although the anti-metabolic syndrome effects of AR and LU were similar, the anti-adiposity and anti-dyslipidemic effects of AR were more pronounced. These results in mice with diet-induced obesity suggest that long-term supplementation with AR can prevent adiposity and related metabolic disorders such as dyslipidemia, hepatic steatosis, insulin resistance, and inflammation.

The bile salt export pump (BSEP) is expressed at the canalicular domain of hepatocytes, where it mediates the elimination of monovalent bile salts into the bile. Inhibition of BSEP is considered a susceptibility factor for drug-induced liver injury that often goes undetected during nonclinical testing. Although in vitro assays exist for screening BSEP inhibition, a reliable and specific method for confirming Bsep inhibition in vivo would be a valuable follow up to a BSEP screening strategy, helping to put a translatable context around in vitro inhibition data, incorporating processes such as metabolism, protein binding, and other exposure properties that are lacking in most in vitro BSEP models. Here, we describe studies in which methods of quantitative intravital microscopy were used to identify dose-dependent effects of two known BSEP/Bsep inhibitors, 2-[4-[4-(butylcarbamoyl)-2-[(2,4-dichlorophenyl)sulfonylamino]phenoxy]-3-methoxyphenyl]acetic acid (AMG-009) and bosentan, on hepatocellular transport of the fluorescent bile salts cholylglycyl amidofluorescein and cholyl-lysyl-fluorescein in rats. Results of these studies demonstrate that the intravital microscopy approach is capable of detecting Bsep inhibition at drug doses well below those found to increase serum bile acid levels, and also indicate that basolateral efflux transporters play a significant role in preventing cytosolic accumulation of bile acids under conditions of Bsep inhibition in rats. Studies of this kind can both improve our understanding of exposures needed to inhibit Bsep in vivo and provide unique insights into drug effects in ways that can improve our ability interpret animal studies for the prediction of human drug hepatotoxicity., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Introduction: Endotoxins, in the form of lipopolysaccharides (LPS), are potent inducers of biliary injury. However the mechanism by which injury develops remains unclear. We hypothesized that hepatic macrophages are pivotal in the development of endotoxin-induced biliary injury and that no injury would occur in their absence., Material and Methods: Clodronate liposomes were used to deplete macrophages from the liver. Forty-eight rats were equally divided across six study groups: sham operation (sham), liposome treatment and sham operation (liposomes+sham), 1mg/kg LPS i.p. (LPS), liposome treatment and LPS administration (liposomes+LPS), hepatic ischaemia-reperfusion injury with LPS administration (IRI+LPS) and liposome treatment followed by IRI+LPS (liposomes+IRI+LPS). Following 6h of reperfusion, blood, bile, and liver tissue was collected for further analysis. Small bile duct injury was assessed, serum liver tests were performed and bile composition was evaluated. The permeability of the blood-biliary barrier (BBB) was assessed using intravenously administered horseradish peroxidase (HRP)., Results: The presence of hepatic macrophages was reduced by 90% in LPS and IRI+LPS groups pre-treated with clodronate liposomes (P<0.001). Severe small bile duct injury was not affected by macrophage depletion, and persisted in the liposomes+IRI+LPS group (50% of animals) and liposomes+LPS group (75% of animals). Likewise, BBB impairment persisted following macrophage depletion. LPS-induced elevation of the chemokine Mcp-1 in bile was not affected by macrophage depletion., Conclusions: Depletion of hepatic macrophages did not prevent development of biliary injury following LPS or LPS-enhanced IRI. Cholangiocyte activation rather than macrophage activation may underlie this injury. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The indocyanine green (ICG) clearance, presented as plasma disappearance rate is, presently, a reliable method to estimate the hepatic "function". However, this technique is not instantaneously available and thus cannot been used intra-operatively (during liver surgery). Near-infrared spectroscopy enables to assess hepatic ICG concentration over time in the liver tissue. This article proposes to extract more information from the liver intensity dynamics by interpreting it through a dedicated pharmacokinetics model. In order to account for the different exchanges between the liver tissues, the proposed model includes three compartments for the liver model (sinusoids, hepatocytes and bile canaliculi). The model output dependency to parameters is studied with sensitivity analysis and solving an inverse problem on synthetic data. The estimation of model parameters is then performed with in-vivo measurements in rabbits (El-Desoky et al. 1999). Parameters for different liver states are estimated, and their link with liver function is investigated. A non-linear (Michaelis-Menten type) excretion rate from the hepatocytes to the bile canaliculi was necessary to reproduce the measurements for different liver conditions. In case of bile duct ligation, the model suggests that this rate is reduced, and that the ICG is stored in the hepatocytes. Moreover, the level of ICG remains high in the blood following the ligation of the bile duct. The percentage of retention of indocyanine green in blood, which is a common test for hepatic function estimation, is also investigated with the model. The impact of bile duct ligation and reduced liver inflow on the percentage of ICG retention in blood is studied. The estimation of the pharmacokinetics model parameters may lead to an evaluation of different liver functions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Sandwich-cultured rat hepatocytes (SCRH) have become an invaluable in vitro model to study hepatic drug disposition. SCRH are maintained between two layers of extracellular matrix. In this configuration, culture periods of 4days are typically applicable. The aim of the present study was to modify conventional SCRH by applying an additional collagen overlay to prolong the hepatic phenotype in SCRH and thus to extend the applicability of the model., Methods: The cultures receiving an extra top layer ('SCRH-plus' cultures) were compared with the conventional SCRH by testing the morphology, cell functionality, metabolic capacity and Mrp2-activity., Results: In the SCRH-plus cultures, cell functionality, evaluated by measuring urea production, was increased from day 5 onwards, compared to conventional cultures. Furthermore, these cells retained the appearance of typical hepatocytes, in contrast with conventional sandwich cultures which showed rapid dedifferentiation. SCRH-plus exhibited significantly improved metabolic clearance mediated by cytochrome P450 3A compared to conventional SCRH whereas UDP-glucuronosyltransferase-mediated metabolism was unaffected. Both conventional SCRH and SCRH-plus showed extensive biliary networks at day 4 of culture. However, from day 4 onwards, a decline in biliary excretion index (BEI) was observed in the conventional SCRH, while BEI values in SCRH-plus cultures did not decrease until day 7., Discussion: The application of an extra top layer of collagen on the SCRH prolongs their useful life-span to 7days. Therefore, SCRH-plus cultures will broaden the applications of SCRH in terms of long-term toxicity evaluation and when determining metabolism of low turnover compounds., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Rhein (RH), 4,5-dihydroxyanthrauinone-2-carboxylic acid, is found in rhubarb (Dahuang), a traditional herbal medicine. RH has reportedly demonstrated multiple pharmacologic properties. Previous studies have also shown that RH induced hepatotoxicity, but the mechanisms of the adverse effect remain unknown. The major objective of the present study was to study the metabolic pathways of RH in order to identify potential reactive metabolites. One mono-hydroxylation metabolite (M1) was detected in urine and bile of rats given RH. M1 was also observed in rat and human liver microsomal incubations after exposure to RH. A total of three (GSH) conjugates (M2, M3 and M5) were detected in bile of rats treated with RH. We concluded that M2-M3 were directly derived from parent compound RH through spontaneous reaction with GSH. M5 was derived from M1 by reaction with GSH, which required cytoslic GSTs. M5 was further metabolized to the corresponding NAC conjugate (mercapturic acid) and was excreted in urine. P450 2C9 was mainly involved in the oxidation of RH., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Water-soluble analogue of quercetin, corvitin is used in patients with myocardial infarction as blocker of 5-lipoxygenase. However, its effects on secretion, lipid content and physico-chemical properties of bile have not been understood yet. We investigated the effect of corvitin, applied in different doses, on the level of bile flow, the content of bile free and esterified cholesterol, phospholipids, triacylglycerols, and free fatty acids. In order to determine stability of the bile colloidal system, we examined the relationship between different lipid components. The rats were injected intraportally with a bolus of corvitin. At doses of 2.5, 5, and 10 mg/kg, the latter increased bile flow and concentration of total cholates, as well as free fatty acids. Corvitin (5 mg/kg) elevated phospholipids and cholesterol content, but at a dose of 10 mg/kg it increased the concentration of bile cholesterol esters and triacylglycerols. Corvitin applied at doses of 2.5 and 10 mg/kg increased total cholates/cholesterol ratio, but at a dose of 10 mg/kg, the drug reduced cholesterol / esterified cholesterol ratio. The results suggest that corvitin exerts choleretic effect and improves stability of bile colloidal system.

The distribution, metabolism, excretion and hepatic effects of the human hepatotoxin fenclozic acid were investigated following single oral doses of 10 mg/kg to normal and bile duct-cannulated male C57BL/6J mice. Whole body autoradiography showed distribution into all tissues except the brain, with radioactivity still detectable in blood, kidney and liver at 72 h post-dose. Mice dosed with [ 14 C]-fenclozic acid showed acute centrilobular hepatocellular necrosis, but no other regions of the liver were affected. The majority of the [ 14 C]-fenclozic acid-related material recovered was found in the urine/aqueous cage wash, (49%) whilst a smaller portion (13%) was eliminated via the faeces. Metabolic profiles for urine, bile and faecal extracts, obtained using liquid chromatography and a combination of mass spectrometric and radioactivity detection, revealed extensive metabolism of fenclozic acid in mice that involved biotransformations via both oxidation and conjugation. These profiling studies also revealed the presence of glutathione-derived metabolites providing evidence for the production of reactive species by mice administered fenclozic acid. Covalent binding to proteins from liver, kidney and plasma was also demonstrated, although this binding was relatively low (less than 50 pmol eq./mg protein).

Background: Biliary lithiasis, or sludge, and nephrolithiasis have been reported as a possible complication of ceftriaxone therapy. However, no study related to cefotaxime-induced biliary pseudolithiasis or nephrolithiasis was observed in the literature. Therefore, we investigated the comparative formation of biliary pseudolithiasis and nephrolithiasis after cefotaxime and ceftriaxone therapies., Methods: The patients treated with ceftriaxone or cefotaxime were enrolled during the study period. Ultrasound imaging of the biliary and urinary tract was performed in all patients before and after the treatment. The patients with a positive sonographic finding at the end of treatment were followed up with monthly ultrasonography for 3 months., Results: The present study showed that abnormal biliary sonographic findings were demonstrated in 18 children (20.9%) treated with ceftriaxone, 13 (15.1%) had biliary lithiasis, 5 (5.8%) had biliary sludge and 1 (1.2%) had nephrolithiasis. Abnormal biliary sonographic findings were demonstrated in only four (5.9%) children treated with cefotaxime who had biliary sludge and only one (1.5%) had nephrolithiasis. It was observed that older age was at significantly higher risk of developing biliary sludge or stone formation. Receiver operating characteristic analysis was performed to determine the residual risk and analysis found that 4.5 years was the cut-off value for age., Conclusions: The present study is unique in the literature for reporting for the first time gall bladder sludge and nephrolithiasis associated with cefotaxime use. Therefore, patients treated with cefotaxime should be monitored for serious complications like patients treated with ceftriaxone. Nevertheless, if third-generation cephalosporin is used, cefotaxime is recommended to be used rather than ceftriaxone.

Disruption of the vectorial bile acid transport in the liver is a key feature of cholestatic drugs, although many causal and mechanistic aspects are still unknown. The aim of the present study was to explore if cholestatic drugs can repress or induce the expression of hepatic transporters. To this end, sandwich-cultured rat hepatocytes were treated with cholestatic and non-cholestatic (steatotic, non-hepatotoxic, etc.) drugs and the mRNA expression of 10 uptake and efflux biliary transporters was measured. Results evidenced that all cholestatic drugs cause extensive alterations in the mRNA expression of most biliary transporters. Surprisingly, nearly all steatotic drugs also affected the expression of these genes. The most frequent alterations triggered by both types of drugs were the repression of OATP1A1, NTCP and BSEP, and the induction of MRP2/3/4, MDR2 and ABCG5/8. The majority of these alterations were also observed in vivo, in the livers of treated rats. The common signature of cholestatic and steatotic drugs was the repression of OATP1A1. Indeed, ROC curve analysis indicated that OATP1A1 mRNA is a very sensitive marker to identify drugs with cholestatic or steatotic potential, with a maximal sensitivity and specificity of 0.917 and 0.941, respectively. We conclude that alteration of expression of hepatobiliary transporters is a hallmark of both cholestatic and steatotic drugs, lending support to a connection between these two mechanisms of hepatotoxicity. Moreover, OATP1A1 mRNA is proposed as a very simple and useful screening biomarker for the prediction of new cholestatic or steatotic drugs in early drug development., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) represent the major clinical entities of chronic cholestatic liver diseases. Both disorders are characterized by portal inflammation and slowly progress to obliterative fibrosis and eventually liver cirrhosis. Although immune-pathogenic mechanisms have been implicated in the pathogenesis of PBC and PSC, neither disorder is considered to be a classical autoimmune disease, as PSC and PBC patients do not respond to immune-suppressants. Furthermore, the decreased bile flow resulting from the immune-mediated tissue assault and the subsequent accumulation of toxic bile products in PBC and PSC not only perpetuates biliary epithelial damage, but also alters the composition of the intestinal and biliary microbiota and its mutual interactions with the host. Consistent with the close association of PSC and inflammatory bowel disease (IBD), the polyclonal hyper IgM response in PBC and (auto-)antibodies which cross-react to microbial antigens in both diseases, an expansion of individual microbes leads to shifts in the composition of the intestinal or biliary microbiota and a subsequent altered integrity of epithelial layers, promoting microbial translocation. These changes have been implicated in the pathogenesis of both devastating disorders. Thus, we will discuss here these recent findings in the context of novel and alternative therapeutic options., Competing Interests: The author declares no conflict of interest.

Increasing human activities in the Arctic raise the risk of petroleum pollution, thus posing an elevated risk for Arctic organisms to be chronically exposed to petroleum compounds. The endocrine disrupting properties of some of these compounds (i.e. polycyclic aromatic hydrocarbons [PAHs]) present in crude oil may have negative effects on the long and energy intensive reproductive development of polar cod (Boreogadus saida), an Arctic keystone species. In the present study, selected reproductive parameters were examined in feral polar cod exposed to crude oil via a natural diet (0.11, 0.57 and 1.14μg crude oil/g fish/day [corresponding to low, medium and high treatments, respectively]) for 31 weeks prior to spawning. Fish maturing in the current reproductive period made up 92% of the experimental population while 5% were immature and 3% were identified as resting fish. Phase I metabolism of PAHs, indicated by ethoxyresorufin-O-deethylase (EROD) activity, showed a dose-dependent increase in high and medium crude oil treatments at week 6 and 22, respectively. Decreasing EROD activity and increasing PAH bile metabolite concentrations over the experimental period may be explained by reproductive maturity stage. Significant alterations in sperm motility were observed in crude oil exposed males compared to the controls. The investigated somatic indices (gonad and hepatic), germ cell development and plasma steroid levels (estradiol-17β [females], testosterone [males and females] and 11-ketotestosterone [males]) were not significantly altered by chronic dietary exposure to crude oil. The environmentally realistic doses polar cod were chronically exposed to in this study were likely not high enough to induce adverse effects in this ecologically important fish species. This study elucidated many baseline aspects of polar cod reproductive physiology and emphasized the influence of maturation state on biomarkers of PAH biotransformation (EROD and PAH bile metabolites)., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase (NOS) and aromatic amino acid hydroxylases. BH4 and 7,8-dihydrobiopterin (BH2) are metabolically interchangeable at the expense of NADPH. Exogenously administered BH4 can be metabolized by the body, similar to vitamins. At present, synthetic BH4 is used as an orphan drug for patients with inherited diseases requiring BH4 supplementation. BH4 supplementation has also drawn attention as a means of treating certain cardiovascular symptoms, however, its application in human patients remains limited. Here, we tracked biopterin (BP) distribution in blood, bile, urine, liver, kidney and brain after BH4 administration (5 mg/kg rat, i.v.) with or without prior treatment with probenecid, a potent inhibitor of uptake transporters particularly including organic anion transporter families such as OTA1 and OAT3. The rapid excretion of BP in urine was driven by elevated blood concentrations and its elimination reached about 90% within 120 min. In the very early period, BP was taken up by the liver and kidney and gradually released back to the blood. BH4 administration caused a considerable decrease in the BH4% in blood BP as an inevitable compensatory process. Probenecid treatment slowed down the decrease in blood BP and simultaneously inhibited its initial rapid excretion in the kidney. At the same time, the BH4% was further lowered, suggesting that the probenecid-sensitive BP uptake played a crucial role in BH2 scavenging in vivo. This suggested that the overproduced BH2 was taken up by organs by means of the probenecid-sensitive process, and was then scavenged by counter-conversion to BH4 via the BH4 salvage pathway. Taken together, BH4 administration was effective at raising BP levels in organs over the course of hours but with extremely low efficiency. Since a high BH2 relative to BH4 causes NOS dysfunction, the lowering of the BH4% must be avoided in practice, otherwise the desired effect of the supplementation in ameliorating NOS dysfunction would be spoiled., Competing Interests: The authors have declared that no competing interests exist.

Port Phillip Bay, Australia, is a large semi-closed bay with over four million people living in its catchment basin. The Bay receives waters from the Yarra River which drains the city of Melbourne, as well as receiving the discharges of sewage treatment plants and petrochemical and agricultural chemicals. A 1999 study demonstrated that fish inhabiting Port Phillip Bay showed signs of effects related to pollutant exposure despite pollution management practices having been implemented for over a decade. To assess the current health status of the fish inhabiting the Bay, a follow up survey was conducted in 2015. A suite of biomarkers of exposure and effects were measured to determine the health status of Port Phillip Bay sand flathead (Platycephalus bassensis), namely ethoxyresorufin-O-deethylase (EROD) activity, polycyclic aromatic hydrocarbons (PAH) biliary metabolites, carboxylesterase activity (CbE) and DNA damage (8-oxo-dG). The reduction in EROD activity in the present study suggests a decline in the presence of EROD activity-inducing chemicals within the Bay since the 1990s. Fish collected in the most industrialised/urbanised sites did not display higher PAH metabolite levels than those in less developed areas of the Bay. Ratios of PAH biliary metabolite types were used to indicate PAH contaminant origin. Ratios indicated fish collected at Corio Bay and Hobsons Bay were subjected to increased low molecular weight hydrocarbons of petrogenic origin, likely attributed to the close proximity of these sites to oil refineries, compared to PAH biliary metabolites in fish from Geelong Arm and Mordialloc. Quantification of DNA damage indicated a localised effect of exposure to pollutants, with a 10-fold higher DNA damage level in fish sampled from the industrial site of Corio Bay relative to the less developed site of Sorrento. Overall, integration of biomarkers by multivariate analysis indicated that the health of fish collected in industrialised areas was compromised, with biologically significant biomarkers of effects (LSI, CF and DNA damage) discriminating between individuals collected in industrialised areas from observations made in fish collected in less developed areas of the Bay., Competing Interests: The authors have declared that no competing interests exist.

A method using liquid chromatography-electrospray ionization ion trap mass spectrometry was established for the identification of metabolites in feces, urine and bile in rats after oral administration of 3',4'-dimethoxy flavonol-3-β-d-glucopyranoside (abbreviated DF3G). Seven metabolites in rat feces, urine and bile were firstly identified on the basis of their MS fragmentation behaviors. Three metabolites were identified in the feces, 6 in the urine and 2 in the bile, which suggested that demethylation, deglycosylation and deglycosylation followed by glucuronide conjugation were the major metabolic pathways for DF3G in vivo. Hydrolyzation might be the first step in the absorption and metabolism of DF3G. The possible metabolic pathway was proposed for the first time. The established method was simple, reliable and sensitive, revealing that it could be used to rapidly screen and identify the structures of metabolites of DF3G to better understand its metabolism in vivo.

Background: Chronic application of third generation progestagens as contraceptives or hormone replacement therapy (HRT) could influence the serum lipid profile, and consequently the bile and gallstone composition. The aim of this study was to determine components of serum, bile and gallstones in women of reproductive age or postmenopausal women using hormonal third generation for at least two years., Methods: We enrolled 101 Caucasian women with cholelithiasis. The study included 45 women of reproductive age and 56 postmenopausal women who were divided into subgroups receiving or not exogenous female hormones. In patients we determined serum levels of 17β-estradiol, triglycerides, HDL and LDL cholesterol as well as composition of gallstones and bile., Results: The postmenopausal women showed a significant reduction in the concentration of bile acids in serum while the application of HRT caused an increase in their contents. Serum total and LDL cholesterol in postmenopausal women was higher than in women without hormonal contraception and postmenopausal patients with HRT. Moreover, women taking the exogenous hormones showed a reduced content of calcium ions in both serum, bile and gallstones., Conclusions: Our observations confirm that the chronic use of oral contraceptives and hormone replacement therapy cause an increase in bile lithogenity.

Background/aims: Bile is the main cause of poor bowel preparation for capsule endoscopy (CE). We aimed to determine whether cholestyramine and hydrotalcite can eliminate bile in the bowel., Patients and Methods: Patients undergoing CE were randomized into two groups. Group A patients (n = 75) received 250 mL 20% mannitol and 1 L 0.9% saline orally at 20:00 hours on the day before and at 05:00 hours on the day of CE and 20 mL simethicone 30 min before CE. Group B patients (n = 73) were treated identically, except for taking oral cholestyramine and hydrotalcite, starting 3 days before CE. Greenish luminal contents were assessed by four tissue color bar segments using Color Area Statistics software. Bowel cleanliness was evaluated by visualized area percentage assessment of cleansing (AAC) score., Result: Bowel cleanliness (82.7% [62/75] vs 46.6% [34/73]; χ2 = 14.596, P = 0.000). and detected greenish luminal contents (20.0% [15/75] vs 8.2% [6/73]; χ2 = 4.217, P = 0.040) were significantly greater in Group A than in Group B. Greenish luminal contents in the two groups differed significantly in the captured small-bowel (t = -13.74, P = 0.000) segments and proximal small-bowel (t = -0.7365, P = 0.000) segments, but not for the distal small-bowel (t = -0.552, P = 0.581) segments., Conclusions: Cholestyramine and hydrotalcite were ineffective in eliminating bile and improving small-bowel preparation.

Renal dysfunction in cholestatic liver disease is multifactorial. Acute kidney injury may develop secondary to renal vasoconstriction in the setting of peripheral vasodilation and relative hypovolemia, tubular obstruction by bile casts, and direct tubular toxicity from bile. Anabolic steroids are frequently used by athletes to boost endurance and increase muscle mass. These agents are a recently recognized cause of hepatotoxicity and jaundice and may lead to acute kidney injury. To increase awareness about this growing problem and to characterize the pathology of acute kidney injury in this setting, we report on a young male who developed acute kidney injury in the setting of severe cholestatic jaundice related to ingestion of anabolic steroids used for bodybuilding. Kidney biopsy showed bile casts within distal tubular lumina, filamentous bile inclusions within tubular cells, and signs of acute tubular injury. This report supports the recently re-emerged concept of bile nephropathy cholemic nephrosis.

Aim: To determine the incidence of biliary sludge (BS) and to evaluate the clinical efficacy of Chophytol in patients with nonalcoholic steatohepatitis (NASH) and BS in the presence of metabolic syndrome (MS)., Subjects and Methods: Clinical laboratory examination was made in 72 patients with NASH in the presence of MS. BS was identified in 52 (72.2%) examinees who were divided in two (study and control) groups. The efficacy of Chophytol of plant origin, prepared from the purified extract of fresh artichoke (Cynara scolymus) leaf juice, which was used at a dose of 3 tablets thrice daily for a month, was evaluated., Results: The use of Chophytol was established to promote BS elimination in 87% of the patients. This ceased pain syndrome in 87% and dyspeptic manifestations in 65.2% and normalized the levels of lipids and the biochemical indicators of liver process activity. No adverse drug reactions were seen., Conclusion: Chophytol used in patients with HASH and BS may soon be possible to make general condition better, to alleviate the symptoms of diseases of the liver and biliary system, and to appreciably improve the biochemical indicators of blood and bile.

Efficacy of the ozonotherapy application as an important component of complex treatment in purulent cholangitis (PCH) was studied. In choledocholithiasis (without infectioning of bile) ozonotherapy may be prescribed as additional component at complex treatment. In PCH ozonotherapy application have promoted the improvement of laboratory indices in 6.6 times, comparing with such, occurring after basic therapy.

This study was to compare pharmacokinetics and bile transformation of R-enantiomer bambuterol with its racemate. Pharmacokinetics of R-enantiomer was investigated after single-dose intravenous and three doses of oral administration to rats and beagle dogs. To compare the pharmacokinetics with racemic bambuterol, the same oral doses of racemic bambuterol were also administrated; the blood and bile samples were collected by cannulation. A validated LC-MS/MS method was used to assess the level of bambuterol in plasma and bile. After single intravenous administration, no significant differences were observed between the two drugs in pharmacokinetic data. After oral dosing of R-bambuterol, the AUCs of R-enantiomer presented linear correlation. After same oral dosing of R-enantiomer and its racemate, all the pharmacokinetic parameters were equivalent. However, the clearance and apparent distribution had different results due to species and administration route difference. The bile transformation of these two compounds was similar and implicated that liver transformation accounted for the major metabolism of them. The bioavailability of R-enantiomer and racemate were comparative and relatively high in beagle dogs. Thus, R-enantiomer had a comparative pharmacokinetic profile and bile transformation with racemic bambuterol in rats and beagle dogs. These findings provided references for further clinical study.

Nomegestrol acetate (NOMAC), a synthetic progestogen derived from 19-norprogesterone, is an orally active drug with a strong affinity for the progesterone receptor. NOMAC inhibits ovulation and is devoid of undesirable androgenic and estrogenic activities. The aim of this study was to evaluate the pharmacokinetics, tissue distribution, and excretion of NOMAC in female rats. Sprague-Dawley female rats were orally administered a single dose of NOMAC (10, 20 or 40 mg/kg) and drug plasma concentrations at different times were determined by RP-HPLC. Tissue distribution at 1, 2, and 4 h and excretion of NOMAC into bile, urine, and feces after dosing were investigated. The results showed that NOMAC was rapidly absorbed after oral administration, with [Formula: see text] of 1-2 h. The plasma concentration-time curves were fitted in a two-compartment model. The exposure to NOMAC ([Formula: see text] and [Formula: see text]) increased dose proportionally from 10 to 40 mg/kg. The average CL and [Formula: see text] were 5.58 L/(h·kg) and 10.8 h, respectively. The highest concentrations of NOMAC in ovary, liver, kidney, lung, heart, brain, spleen, muscle, and uterus were observed at 2 h, whereas the highest concentrations in stomach, pituitary, and hypothalamus appeared at 1 h. The total cumulative excretion of NOMAC in feces (0-72 h), urine (0-72 h), and bile (0-48 h) was ~1.06, 0.03, and 0.08 % of the oral administered dose, respectively. This study indicated that NOMAC had a widespread distribution in tissues, including ovary, pituitary, and hypothalamus, which are main target tissues where NOMAC inhibits ovulation. NOMAC was excreted via both feces and urine with few unchanged NOMAC excreted. Enterohepatic circulation was found in the drug elimination; however, it did not significantly affect [Formula: see text].

The formation of cholesterol gallstones involves very complex imbalances, such as alterations in the secretion of biliary lipids (which involves the ABCG5, ABCG8, ABCB4 and ABCB11 transporters), biochemical and immunological reactions in the gallbladder that produce biliary sludge (mucins), physicochemical changes in the structure of cholesterol (crystallization), alterations in gallbladder motility, changes in the intestinal absorption of cholesterol (ABCG5/8 transporters and Niemann-Pick C1L1 protein) and alterations in small intestine motility. Some of these proteins have been studied at the clinical and experimental levels, but more research is required. In this review, we discuss the results of studies on some molecules involved in the pathophysiology of gallstones that may be future therapeutic targets to prevent the development of this disease, and possible sites for treatment based mainly on the absorption of intestinal cholesterol (Niemann-Pick C1L1 and ABCG5/8 proteins)., (Copyright © 2015. Published by Elsevier B.V.)

Background & Objectives: Dietary fibre-rich tender cluster beans (Cyamopsis tetragonoloba; CB) are known to exert beneficial cholesterol lowering influence. We examined the influence of a combination of dietary tender CB and garlic (Allium sativum) in reducing the cholesterol gallstone formation in mice., Methods: Cholesterol gallstones were induced in Swiss mice by feeding a high-cholesterol diet (HCD) for 10 wk. Dietary interventions were made with 10 per cent CB and 1 per cent garlic included individually or together along with HCD. A total of 100 mice were divided into five groups of 20 mice each., Results: Dietary CB, garlic and CB+garlic reduced the formation of cholesterol gallstones by 44, 25 and 56 per cent, respectively, lowered cholesterol by 23-48, 16-24, and 24-58 in bile, serum, and liver, respectively. Cholesterol saturation index in bile and cholesterol: phospholipid ratio in circulation and hepatic tissue were significantly lowered by these dietary interventions, with highest beneficial effect from CB+garlic. Activities of hepatic cholesterol metabolizing enzymes were modulated by CB, garlic and CB+garlic. Elevation in lipid peroxides caused by HCD was also countered by these dietary interventions, the combination producing the highest effect., Interpretation & Conclusions: The results showed that the prevention of experimentally induced formation of cholesterol gallstones by dietary CB and garlic was due to decreased biliary cholesterol secretion and increased cholesterol saturation index. In addition of anti-lithogenic effect, dietary CB and garlic in combination had a beneficial antioxidant effect.

Background: A disruption of cholesterol homeostasis characterized by the physical-chemical imbalance of cholesterol solubility in bile often results in formation of cholesterol gallstones. Our earlier studies revealed that curcumin (1000 mg/kg) could prevent formation of gallstones. It has been proved that curcumin is poorly absorbed while piperine is a bioavailability-enhancer. Nevertheless, whether curcumin combined with piperine could enhance the effect of curcumin in preventing gallstones is still awaited., Method: C57BL6 mice were fed on a lithogenic diet concomitant with curcumin at 500 or 1000 mg/kg and/or piperine at 20 mg/kg for 4 weeks. The ratio of gallbladder stone formation was recorded and samples of blood, bile, gallbladder, liver and small intestine were also collected. The volume of gallbladder and weight of liver were calculated, and blood and bile samples were analyzed through biochemical methods. Intestinal NPC1L1 and SREBP2 mRNA and protein expression were detected by real-time PCR and Western blot., Result: Combining with piperine can significantly enhance the effect of curcumin, thus preventing the development of gallbladder stones, lowering the saturation of blood lipids and cholesterol in bile, as well as decreasing the expression of NPC1L1 and SREBP2 in both mRNA and protein levels., Conclusion: Curcumin can prevent the formation of cholesterol gallstones induced by high fat diet in mice and SREBP2 and NPC1L1 may participate in this process. Piperine can increase curcumin's bioavailability, thereby enhancing the effect of curcumin.

Ethnopharmacological Relevance: Whole herb of Lysimachia christinae has long been used as a remedy for cholelithiasis extensively in China. This study was aimed to validate the effect of L. christinae on eliminating preestablished cholesterol gallstone (CGS) in model animals., Materials and Methods: Acute toxicity of aqueous extract of L. christinae (LCAE) was estimated in male C57BL/6 mice, so was the efficiency of LCAE on preformed CGS induced by lithogenic diet. The effects of LCAE were also examined on bile secretion rate, the lipid profiles of bile and serum, body weight, main visceral organ indexes, and histomorphology of main visceral organs., Results: Single dose of LCAE did not lead to death and changes on body weight gain, main visceral organ indexes, histomorphology of main visceral organs, and blood hemogram and biochemical indexes, even at dose of 50g material/kg body weight. Preestablished CGSs were almost entirely eliminated after administration of LCAE for 2wk at high dose or for 4wk at low dose. LCAE promoted bile secretion and lowered cholesterol levels in either cystic bile or hepatic bile. LCAE also decreased serum cholesterol content, especially LDL-C content, tremendously, reduced the levels of serum HDL-C, phospholipid, and triglycerine a little, and lowered body weight and liver index significantly. After medication of LCAE for 8wk , neither visceral indexes nor histomorphology of heart, kidney, and spleen were influenced, but fatty degeneration of liver induced by high fat and high cholesterol diet was reverted., Conclusion: L. christinae can be considered as non-toxic. It showed prominent efficiency of eliminating preexisted CGS in mice and indicated a hypolipidaemic effect., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Biliary cholesterol may contribute to the formation of cholesterol gallstones, and regulation of these levels could be a useful therapeutic strategy for gallstones disease. Piperine (PA) is a potential cholesterol lowering agent. In this study, we assessed the effect and mechanism of PA in preventing cholesterol gallstones formation induced by feeding lithogenic diet containing high cholesterol levels to mice. C57BL/6 inbred mice were fed lithogenic or chow diets for 10 weeks, with or without PA (15, 30 and 60 mg/kg) or ursodeoxycholic acid (UDCA, 60 mg/kg) administration. Cholesterol, phospholipids and crystals in bile, the lipid in serum, pathological changes and proteins expression in liver were analyzed. The results showed that PA could decrease the cholesterol potency and crystals in bile, reduce total cholesterol (TC), triglycerides (TG) and increase high-density lipoprotein/low-density lipoprotein (HDL/LDL) levels in serum. Furthermore, PA treatment reduced liver lipid peroxidation and protected hepatobiliary cells from liver injury by decreasing malondialdehyde (MDA) and increasing superoxide dismutase (SOD). In addition, PA inhibited the expression of ATP-binding cassette transporters G5/8 (ABCG5/8) and liver X receptor (LXR) in liver, and reduced cholesterol transport from the hepatocytes to the gallbladder. It may be the mechanism of PA in preventing cholesterol gallstones formation. PA as a potential drug for prevention cholesterol gallstones merits further investigation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Danshu capsule (DSC) is a medicinal compound in traditional Chinese medicine (TCM). It is commonly used for the treatment of acute & chronic cholecystitis as well as choleithiasis. To study its choleretic effect, healthy rats were randomly divided into DSC high (DSCH, 900mg/kg), medium (DSCM, 450mg/kg), and low (DSCL, 225mg/kg) group, Xiaoyan Lidan tablet (XYLDT, 750mg/kg), and saline group. The bile was collected for 1h after 20-minute stabilization as the base level, and at 1h, 2h, 3h, and 4h after drug administration, respectively. Bile volume, total cholesterol, and total bile acid were measured at each time point. The results revealed that DSC significantly stimulated bile secretion, decreased total cholesterol level and increased total bile acid level. Therefore, it had choleretic effects. To identify the active components contributing to its choleretic effects, five major constituents which are menthol (39.33mg/kg), menthone (18.02mg/kg), isomenthone (8.18mg/kg), pluegone (3.31mg/kg), and limonene (4.39mg/kg) were tested on our rat model. The results showed that menthol and limonene could promote bile secretion when compared to DSC treatment (p > 0.05); Menthol, menthol and limonene could significantly decrease total cholesterol level (p<0.05 or p<0.01) as well as increase total bile acid level (p<0.05 or p<0.01); Isomenthone, as a isomer of menthone, existed slightly choleretic effects; Pluegone had no obvious role in bile acid efflux. These findings indicated that the choleretic effects of DSC may be attributed mainly to its three major constituents: menthol, menthone and limonene., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Bile acids, synthesized by hepatocytes from cholesterol, are specific and quantitatively important organic components of bile, where they are the main driving force of the osmotic process that generates bile flow toward the canaliculus. The bile acid pool comprises a variety of species of amphipathic acidic steroids. They are not mere detergent molecules that play a key role in fat digestion and the intestinal absorption of hydrophobic compounds present in the intestinal lumen after meals, including liposoluble vitamins. They are now known to be involved in the regulation of multiple functions in liver cells, mainly hepatocytes and cholangiocytes, and also in extrahepatic tissues. The identification of nuclear receptors, such as farnesoid X receptor (FXR or NR1H4), and plasma membrane receptors, such as the G protein-coupled bile acid receptor (TGR5, GPBAR1 or MBAR), which are able to trigger specific and complex responses upon activation (with dissimilar sensitivities) by different bile acid molecular species and synthetic agonists, has opened a new and promising field of research whose implications extend to physiology, pathology and pharmacology. In addition, pharmacological development has taken advantage of advances in the understanding of the chemistry and biology of bile acids and the biological systems that interact with them, which in addition to the receptors include several families of transporters and export pumps, to generate novel bile acid derivatives aimed at treating different liver diseases, such as cholestasis, biliary diseases, metabolic disorders and cancer. This review is an update of the role of bile acids in health and disease.

In acute experiments on laboratory male rats we have shown that serotonin (10 mkg/kg, intraportal) increased the oxygen consumption of by liver on 28.8% (P < 0.001) and reduced oxygen tension levels on 19.3% (P < 0.001). The action of serotonin on tissue respiration in liver realized through 5-HT(2) receptors because previous blockade by ketanserin (3 mg/kg) led to remove the effects of exogenous serotonin and inhibition of the action of endogenous autacoid. Serotonin reduced the amount of secreted bile on 13.5% (P < 0.05), and increases the concentration of conjugated bile acids and decreases the content of free cholate, indicating enhanced conjugation with taurine and glycine in the liver cells. However, serotonin didn't stimulate synthesis of primary bile acids. Introduction of serotonin in the conditions of 5-HT2 receptors blockade by ketanserin also led to speed decrease of bile secretion, but in this case stimulating effect of autacoid on bile acid conjugation with taurine and glycine wasn't manifested and content of free cholate wasn't reduced.

Objective: To study the concentrations and pharmacokinetics of 6 different kinds of antibiotics in rabbit bile, and evaluate their microbicidal potential., Methods: Thirty-six health rabbits were randomly divided into 6 groups, and each group was 6 rabbits. After anaesthesia, the common bile duct of rabbit was isolated and cumulated with a silicone tube. The rabbits were administered intravenously with the equal-effect dose of antibiotics. Bile (1.5 ml) was collected at different time points after administration, and the concentration of antibiotics of bile was assayed by high performance liquid chromatography. The bile drug concentration-time data were processed by software to figure out the pharmacokinetic parameters such as maximum concentration (C(max)), peak time (T(max)), half-life time (T(1/2)), clearance (CL) and apparent volume of distribution (VD). The bile antibiotics concentration contrasted to the minimum inhibitory concentration (MIC), and attained the bactericidal index (C(max)/MIC) and the time when the drug concentration exceeded the MIC (T(>MIC))., Results: The C(max) and T1/2 of each antibiotic were as the followings: piperacillin (7 950 ± 3 023) mg/L and (1.97 ± 1.23) h, ceftriaxone (1 104 ± 248) mg/L and (3.14 ± 0.57) h, cefoperazone (5 215 ± 2 225) mg/L and (0.89 ± 0.13) h, meropenem (31.97 ± 12.44) mg/L and (0.36 ± 0.11) h, levofloxacin (66.3 ± 36.9) mg/L and (3.32 ± 2.57) h, metronidazole (28.2 ± 10.2) mg/L and (0.81 ± 0.33) h, respectively. Piperacillin/tazobactam and cefoperazone/sulbactam had the largest bactericidal index and the longest T(>MIC), and their bactericidal indexes were (62.1 ± 23.6) - (993.8 ± 377.9) and (164.8 ± 69.0) - (659.3 ± 275.9), their T(>MIC) were (6.00 ± 2.53) - (8.00 ± 0.00) h and (6.33 ± 1.97) - (8.00 ± 0.00) h. The bactericidal index and T(>MIC) of levofloxacin were the smallest, which were (2.1 ± 1.2) - (8.3 ± 4.6) and (0.54 ± 0.25) - (2.67 ± 1.03) h . Ceftriaxone and meropenem were as the medium, and their bactericidal indexes and T(>MIC) were (4.3 ± 1.0) - (69.2 ± 15.5) , (1.42 ± 0.65) - (8.00 ± 0.00) h and (2.0 ± 0.8) - (1 031.3 ± 401.4) , (0.29 ± 0.10) - (1.83 ± 0.26) h. The bactericidal index of metronidazole to anaerobic ranged from 7.4 to 294.9, and the T(>MIC) ranged from 1.88 to 5.00 h., Conclusions: The bile concentrations of six antibiotics all exceed their effective bactericidal concentrations. The concentration-time curves of piperacillin, cefoperazone, meropenem and metronidazole conformed to one-compartment model, and ceftriaxone and levofloxacin are conformed to two-compartment model. Piperacillin/tazobactam and cefoperazone/sulbactam have the largest bactericidal index and the longest T(>MIC), so they can be chosen as the first choice for the therapy of hepatobiliary infection.For the anaerobic, the microbicidal potential of metronidazole is high.

The study aimed to characterize the pharmacokinetics and pharmacodynamics of pazufloxacin (PZFX) in bile and to identify optimal dosing regimens. Pazufloxacin 500 mg was administered via a 0·5-hour intravenous infusion to 10 patients with endoscopic nasal bile drainage before or after biliary pancreatic surgery. Both blood and bile samples were collected pre-dose and at the end of infusion (0·5 hours) and for up to 5 hours thereafter. Concentrations of PZFX were determined using high-performance liquid chromatography. Noncompartmental and compartmental pharmacokinetic parameters were estimated, and Monte Carlo simulation was conducted to evaluate the pharmacodynamic exposure of PZFX in bile. The bile/plasma ratios were 3·58±1·15 in the area under the drug concentration-time curve (AUC) and 2·13±0·74 in the maximum drug concentration (Cmax). The delay in the time to Cmax, from plasma to bile, was 0·75±0·18 hours The probability of attaining pharmacodynamic targets (both AUC/MIC = 100 and Cmax/MIC = 8) in bile against a minimum inhibitory concentration (MIC) of 2 mg/l was >90% when PZFX was administered by a 0·5-hour infusion with 500 mg every 8 hours or 1000 mg every 12 hours These regimens provided an adequate antibacterial effect against the most common pathogens of biliary tract infections, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae with their MICs<2 mg/l.

Reverse cholesterol transport (RCT) is pivotal in the return of excess cholesterol from peripheral tissues to the liver for excretion in bile and eventually feces. RCT from macrophages is a critical anti-atherogenicity mechanism of HDL. As the cholesterol absorption inhibitor ezetimibe promoted RCT in mice, which lack cholesterol ester transfer protein (CETP), we investigated its effects in hamsters, which have CETP. A high-cholesterol diet (HC) increased cholesterol levels throughout lipoprotein fractions and ezetimibe markedly reduced VLDL/LDL cholesterol levels under both normal chow (NC) and HC. However, ezetimibe did not affect and reduced HDL-cholesterol levels under NC and HC, respectively. Intraperitoneal injection of (3)H-cholesterol pre-labeled macrophages in an in vivo RCT assay increased tracer accumulation in the liver but reduced it in bile under HC, and these changes were completely cancelled by ezetimibe. Under both NC and HC, ezetimibe reduced tracer levels in the liver but increased them in feces, indicating promotion of RCT in vivo. We performed a RCT assay using hamsters subjected to bile duct ligation (BDL) to clarify whether a transintestinal cholesterol efflux (TICE) pathway contributes to ezetimibe's enhancement of RCT. BDL markedly inhibited macrophage-derived (3)H-cholesterol excretion to feces and cancelled ezetimibe's stimulatory effect on RCT, suggesting that biliary cholesterol excretion is a major contributor in RCT promotion by ezetimibe but the contribution of the TICE pathway is minimal. In conclusions, ezetimibe exerts an additive anti-atherogenic property by enhancing RCT in hamsters. Our findings suggest that this property is independent of the TICE pathway., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The pharmacokinetics (PK) of fexofenadine (FEX) in pigs were investigated with the focus on exploring the interplay between hepatic transport and metabolism when administered intravenously (iv) alone or with verapamil. The in vivo pig model enabled simultaneous sampling from plasma (pre-liver, post-liver and peripheral), bile and urine. Each animal was administered FEX 35mg iv alone or with verapamil 35mg. Plasma, bile and urine were analyzed with liquid chromatography-tandem mass spectrometry. Non-compartmental analysis (NCA) was used to estimate traditional PK parameters. In addition, a physiologically based pharmacokinetic (PBPK) model consisting of 11 compartments (6 tissues +5 sample sites) was applied for mechanistic elucidation and estimation of individual PK parameters. FEX had a terminal half-life of 1.7h and a liver extraction of 3%. The fraction of the administered dose of unchanged FEX excreted into the bile was 25% and the bile exposure was more than 100 times higher than the portal vein total plasma exposure, indicating carrier-mediated (CM) disposition processes in the liver. 23% of the administered dose of FEX was excreted unchanged in the urine. An increase in FEX plasma exposure (+50%) and a decrease in renal clearance (-61%) were detected by NCA as a direct effect of concomitant administration of verapamil. However, analysis of the PBPK model also revealed that biliary clearance was significantly inhibited (-53%) by verapamil. In addition, PBPK analysis established that metabolism and CM uptake were important factors in the disposition of FEX in the liver. In conclusion, this study demonstrated that CM transport of FEX in both liver and kidneys was inhibited by a single dose of verapamil., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Avian bile is rich in matrix metalloproteinases (MMP), the enzymes that cleave extracellular matrix proteins such as collagens and proteoglycans. Changes in bile MMP expression have been correlated with hepatic and gall bladder pathologies, but the significance of their expression in normal, healthy bile is not understood. We hypothesized that the MMP in bile may aid the digestion of native collagens that are resistant to conventional gastric proteases. Hence, the objective of this study was to characterize the bile MMP and check its regulation in association with dietary factors. We used substrate zymography, azocoll protease assay, and gelatin affinity chromatography to identify and purify the MMP from chicken bile. Using zymography and SDS PAGE, 5 bands at 70, 64, 58, 50, and 42 kDa were detected. The bands corresponding to 64, 50, and 42 kDa were identified as MMP2 using trypsin in-gel digestion and matrix-assisted laser desorption time-of-flight mass spectrometry and peptide mass fingerprinting. Chickens fed diets containing gelatin supplements showed higher levels of MMP expression in the bile by both azocoll assay and zymography. We conclude that the bile MMP may be associated with the digestion of collagens and other extracellular matrix proteins in avian diets., (Poultry Science Association Inc.)

Previous studies demonstrated that L-Fabp KO mice are more susceptible to lithogenic diet (LD)-induced gallstones because of altered hepatic cholesterol metabolism and increased canalicular cholesterol secretion. Other studies demonstrated that liver-specific deletion of microsomal triglyceride transfer protein (Mttp-LKO) reduced LD-induced gallstone formation by increasing biliary phospholipid secretion. Here we show that mice with combined deletion (i.e., DKO mice) are protected from LD-induced gallstone formation. Following 2 weeks of LD feeding, 73% of WT and 100% of L-Fabp KO mice developed gallstones versus 18% of Mttp-LKO and 23% of DKO mice. This phenotype was recapitulated in both WT and L-Fabp KO mice treated with an Mttp antisense oligonucleotide (M-ASO). Biliary cholesterol secretion was increased in LD-fed L-Fabp KO mice and decreased in DKO mice. However, phospholipid secretion was unchanged in LD-fed Mttp-LKO and DKO mice as well as in M-ASO-treated mice. Expression of the canalicular export pump ABCG5/G8 was reduced in LD-fed DKO mice and in M-ASO-treated L-Fabp KO mice. We conclude that liver-specific Mttp deletion not only eliminates apical lipoprotein secretion from hepatocytes but also attenuates canalicular cholesterol secretion, which in turn decreases LD-induced gallstone susceptibility.