Treatment of primary sclerosing cholangitis.

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.

[Pathogens' distribution and changes of antimicrobial resistance in the bile of acute biliary tract infection patients].

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 χ2 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%; χ2=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.

Bile accelerates carcinogenic processes in pancreatic ductal adenocarcinoma cells through the overexpression of MUC4.

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.

Randomised clinical trial: significant biochemical and colonic transit effects of the farnesoid X receptor agonist tropifexor in patients with primary bile acid diarrhoea.

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.

Is gastrointestinal motility related to alkaloids of Charred Semen Arecae?

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.

Activation of Estrogen Receptor G Protein-Coupled Receptor 30 Enhances Cholesterol Cholelithogenesis in Female Mice.

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 ERß, 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.

Metformin Disrupts Bile Acid Efflux by Repressing Bile Salt Export Pump Expression.

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.

Misdiagnosis of CTX due to propofol: The interference of total intravenous propofol anaesthesia with bile acid profiling.

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-5ß-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.

The diversity of ursodeoxycholic acid precursors from bile waste of commercially available fishes, poultry and livestock in Indonesia

Ursodeoxycholic acid (UDCA), a secondary bile acid (BA), has been used as a drug to treat various liver diseases. UDCA is synthesised from cholic or chenodeoxycholic acid (CA/CDCA), two primary BAs frequently used as the starting materials. Nowadays, swine, cattle, and poultry bile are the main sources of those BAs. However, other commercial animals could be promising sources as well. We identified two livestock, two poultries, and eight fishes that are commercially cultivated in Indonesia. Four free BAs including CA, CDCA, deoxycholic acid (DCA), and lithocholic acid (LA) were identified for their occurrences using thin-layer chromatography and high-performance liquid chromatography. CA was detected in cow, duck, red tilapia, gourami, the common carp, and grouper, whereas CDCA was only detected in two poultries and the common carp. The occurrence of DCA was common and abundant in most tested animals. In contrast, the presence of LA was found to be very low in all samples. The biliary bile of tilapia has been found to contain a high abundance of free CA (43% of the total bile). A simple extraction was able to purify CA from biliary bile of tilapia. This is a new promising and competitive source of CA.

Discovery of ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl)ureidyl derivatives as selective non-steroidal agonists of the G-protein coupled bile acid receptor-1.

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, LXRß 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.

Green tea polyphenols modify gut-microbiota dependent metabolisms of energy, bile constituents and micronutrients in female Sprague-Dawley rats.

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.

Effect of prednisolone administration on gallbladder emptying rate and gallbladder bile composition in dogs.

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.

Luteolin-Enriched Artichoke Leaf Extract Alleviates the Metabolic Syndrome in Mice with High-Fat Diet-Induced Obesity.

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-1ß, 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 metabolic fate of fenclozic acid in chimeric mice with a humanized liver.

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.

Intravital Multiphoton Microscopy with Fluorescent Bile Salts in Rats as an In Vivo Biomarker for Hepatobiliary Transport Inhibition.

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.

Water-soluble quercetin modulates the choleresis and bile lipid ratio in rats.

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.

Model and methods to assess hepatic function from indocyanine green fluorescence dynamical measurements of liver tissue.

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.

In vitro and in vivo metabolic activation of rhein and characterization of glutathione conjugates derived from rhein.

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.

Metabolism of metofluthrin in rats: II. Excretion, distribution and amount of metabolites.

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-14C] 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.

Extra collagen overlay prolongs the differentiated phenotype in sandwich-cultured rat hepatocytes.

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.