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Objectives: Edaravone (EDR) is an effective neuroprotective agent in various neurological diseases; however, its use is restricted due to poor oral absorption. Bile salts are known for improving solubility and inhibiting drug crystallization in supersaturated conditions of the gastrointestinal tract (GIT). In our previous work, we prepared coamorphous dispersion (COAM) of EDR with sodium taurocholate (NaTC) using spray drying. The optimized EDR COAM exhibited superior in vitro performance compared to plain EDR. EDR is well absorbed in fasted-over-fed conditions., Methods: The present work, we conducted a pharmacokinetic study for EDR and EDR COAM in fasted and fed conditions to check effect of food on its oral absorption. The LC-MS/MS-based method was developed and validated to determine the amount of EDR in plasma., Results: The results suggested that EDR COAM did not show a significant difference in C max (P=0.3544) and AUC (P=0.1696) of fasted and fed states. On the other hand, plain EDR showed 2-fold and 3-fold reduced C max (P<0.0001) and AUC (P=0.0094) in the fed condition, respectively. The C max and AUC of EDR COAM were improved in fasted (AUC: 2.56-fold) and fed states (AUC: 5.74-fold) than plain EDR, suggesting better oral absorption of COAM than crystalline EDR without having the effect of food., Conclusions: The unique structural attributes of NaTC had the potential to inhibit the recrystallization of EDR in GIT, while concurrently reducing the impact of food on the oral absorption of EDR., (Copyright © 2024 Académie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.)

Objetive: To evaluate the enterohepatic circulation of 75-Selenium turoselecolic acid ( 75 Se-SeHCAT) during the first 3 h and its correlation with the abdominal retention at the 7th day (AR 7 ), as contribution to the clinical study of biliar acid malabsorption (BAM)., Materials and Methods: 38 patients with chronic diarrhea were retrospectively studied. Acquisition protocol included static abdominal images at 1st, 2nd and 3rd hour and the 7th day after oral administration of the radiopharmaceutical. Images of 1-3 h determined 5 patterns of enterohepatic circulation that, due to their location, were characterized as: 1) gallbladder 2-3 h, 2) gallbladder 3 h, 3) gallbladder-abdomen 2-3 h, 4) abdomen, 5) upper left abdomen. The association of these patterns with the AR 7 (Fisher, STATA) were investigated. Patients were classified as Non BAM (AR 7  > 15%), mild-BAM (AR 7 15-10%), moderate-BAM (AR 7 10-5%) or severe-BAM (AR 7  < 5%)., Results: 19 patients had an AR 7 diagnostic of BAM (7 mild-BAM, 5 moderate-BAM, 7 severe-BAM). The pattern "gallbladder at 2-3 h" was statistically associated with Non BAM (p 0,008), while "gallbladder-abdomen at 2-3 h" was correlated with having BAM (p 0,029)., Conclusion: Variations detected at the abdominal level in images during the first 3 h were associated with changes in intestinal absorption and the incorporation of the radiopharmaceutical into the pool of bile acids, so visual interpretation of the images at 2nd and 3rd hour could be useful in the final assessment of the study., (Copyright © 2021 Sociedad Española de Medicina Nuclear e Imagen Molecular. Published by Elsevier España, S.L.U. All rights reserved.)

An in vitro methodology for simulating the change in the pH and composition of gastrointestinal fluid associated with the transition of orally administered drugs from the stomach to the small intestine was developed (the stomach-to-intestine fluid changing system (the SIFC system)). This system was applied to in vitro sensitivity analysis on the dissolution of weakly basic drugs, and the obtained results were discussed in relation to the intrasubject variability in the plasma exposure in human bioequivalence (BE) study. Three types of protocols were employed (steep pH change: pH 1.6 FaSSGF → pH 6.5 FaSSIF, gradual pH change: pH 1.6 FaSSGF → pH 6.5 FaSSIF, and high gastric pH: pH 4.0 FaSSGF → pH 6.5 FaSSIF). Regardless of the protocols and the forms of drug applied in active pharmaceutical ingredient powder or formulation, dissolution profiles of pioglitazone after fluid shift were similar and the final concentrations in FaSSIF were approximately equal to the saturation solubility in FaSSIF, supporting its small intrasubject variance in human BE study. In contrast, dissolved concentration of terbinafine in the SIFC system became less than half in the high gastric pH protocol than that in other protocols, suggesting the fluctuation of gastric pH as one of the factors of high intrasubject variance of terbinafine in human. Plasma exposure of telmisartan was highly variable especially at the high dose. Although the dissolution of telmisartan in the SIFC system was greatly improved by formulation, it considerably fluctuated during fluid shift especially at the high dose, which corresponds well to in vivo results.

Aim: It has previously been proposed that entero-hepatic bile acid recycling frequency is a major determinant of whole-body retention (WBR) of SeHCAT. Hepatocyte to terminal ileum accounts for almost the entire cycle. The study aim was to test this hypothesis by comparing WBR with an estimate of speed of transit of bile acids using Tc-HIDA scintigraphy performed on a separate occasion., Methods: Using an un-collimated gamma camera and patient-to-camera distance of 1.5 m, WBR at 7 days after oral SeHCAT administration was measured in 14 patients with chronic diarrhoea, of whom 10 had previous cholecystectomy. The distance reached within the intestine of Tc-HIDA at 1 h (n = 14) and 2 h (n = 7) following iv injection was graded as follows: grades 1 and 2: small bowel on left and right sides of abdomen, respectively; and grade 3: colon. Relationships between WBR and grade were assessed using Spearman rank correlation., Results: Interval between studies ranged from 3 to 1219 (median 330) days. Grading correlated with WBR at 1 h (rs = -0.63; P = 0.02) and weakly at 2 h (rs = -0.68; P = 0.09) post-injection of Tc-HIDA. In nine patients in whom Tc-HIDA and SeHCAT scans were performed within 1 year of each other, the correlation remained significant at 1 h (rs = -0.73; P = 0.03). There was no difference in WBR or grading between patients with or without a gall bladder., Conclusion: Entero-hepatic bile acid recycling frequency is a major determinant of whole-body SeHCAT retention.

When first described in 1976, primary bile acid diarrhea (BAD Type 1) was regarded as a very rare cause of chronic diarrhea. Today, the incidence is estimated as > 1 %. Availability of diagnostic tools varies widely and, in Germany, there is no generally consented recommendation for their use. BAD is still widely underdiagnosed.Since the beginning of the '90 s, we have added a therapeutic trial with cholestyramine to our diagnostic approach of chronic diarrhea. Patients with a positive test were offered a Selenium-homocholic acid taurine (SeHCAT) test for confirmation of bile-acid-diarrhea (BAD), using a 7-day-retention of 20 % as cut-off value.From April 1991 to March 2017, after exclusion of other relevant causes for chronic diarrhea like IBD, celiac disease or microscopic colitis, 70 patients with a positive trial treatment of cholestyramine were identified for evaluation. Sixty of them had a SeHCAT test. Patients with BAD Type 1 and Type 3 were excluded, except for cholecystectomy.85 % (35/41) of patients with BAD Type 1 needed continued medical treatment (median follow-up time 8.3 (1 - 23.6) years). Among them, 68.6 % (24/35) took cholestyramine, 31.4 % (11/35) loperamide or another antidiarrheal treatment. 14.6 % (6/41) of patients reported a spontaneous remission after median 2.9 (0.7 - 5.7) years.In the group of patients with BAD after cholecystectomy, 82 % (8/11) still needed treatment after median 7.7 (1 - 24.5) years; 8 having taken cholestyramine, one patient nothing and two with spontaneous remissions.All (8/8) patients with a normal SeHCAT test (7-day retention > 20 %) had spontaneous relief after median 3.6 (1.2 - 6.3) months.Also, 70 % (7/10) of patients who had not been confirmed by the SeHCAT test still needed treatment after median 4.3 (3.7 - 18.3) years.Based on a trial treatment alone, diagnosis of BAD is possible but not assured. Due to its ubiquitous availability, it should be used consequently if other methods are not available. Despite the well-known shortcomings of cholestyramine, a therapeutic trial should be used more consequently. According to the current literature, using the SeHCAT test in the first place will give significantly better results and unnecessary follow-up examinations can be avoided. However, therapeutic consequences might be modest due to the well-known limitations of cholestyramine. A well-tolerated and licensed alternative to cholestyramine is urgently needed., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (© Georg Thieme Verlag KG Stuttgart · New York.)

It was the aim of this study to prepare trypsin decorated mucus permeating self-emulsifying drug delivery systems (SEDDS). Lipophilicity of enzyme was increased by hydrophobic ion pairing (HIP) with the anionic surfactants sodium dodecyl sulfate (SDS), sodium taurocholate (ST) and sodium deoxycholate (SDO) to facilitate its incorporation in SEDDS. Blank SEDDS and trypsin decorated SEDDS were characterized regarding droplet size, polydispersity index (PI), zeta potential and proteolytic activity using Nα-benzoyl-l-arginine ethyl ester (BAEE) assay. Log D SEDDS/release medium of each complex was determined to assess its affinity towards SEDDS oily droplet upon emulsification. Ability of trypsin decorated SEDDS to enhance mucus permeation was studied on mucus gel from porcine small intestine for the period of 4 h at 37 °C. Degree of enzyme precipitation via HIP was 94.5%, 85.7% and 48.2% for SDS, ST and SDO complex, respectively. SEDDS composed of 50% (w/w) cremophor EL, 20% (w/w) captex 300, and 30% (w/w) propylene glycol with a complex payload of 1% (w/w) exhibited a droplet size in the range of 29.92 ± 0.09 nm to 39.15 ± 0.37 nm, a polydispersity index of 0.116-0.265 and zeta potential in the range of -2.36 mv to -4.25 mv. The enzymatic activity of trypsin complexed with SDO, SDS and ST in SEDDS was 51.9%, 44.8%, and 40.7% respectively, of the corresponding activity of free trypsin. Log D values of trypsin, SDS, ST and SDO complex were -2.73, 1.97, 1.89 and 1.68, respectively, suggesting higher lipophilicity of trypsin complexes as compare to free trypsin and ability to reside on SEDDS droplets. Enzyme decorated SEDDS improved mucus permeation 1.6- to 2.6-fold in comparison to blank SEDDS. Results demonstrated that decorating SEDDS with trypsin can be a promising technique to improve their mucus permeating properties., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Targeting endogenous blood-brain barrier (BBB) transporters such as organic anion transporting polypeptide 1a4 (Oatp1a4) can facilitate drug delivery for treatment of neurological diseases. Advancement of Oatp targeting for optimization of CNS drug delivery requires characterization of sex-specific differences in BBB expression and/or activity of this transporter., Methods: In this study, we investigated sex differences in Oatp1a4 functional expression at the BBB in adult and prepubertal (i.e., 6-week-old) Sprague-Dawley rats. We also performed castration or ovariectomy surgeries to assess the role of gonadal hormones on Oatp1a4 protein expression and transport activity at the BBB. Slco1a4 (i.e., the gene encoding Oatp1a4) mRNA expression and Oatp1a4 protein expression in brain microvessels was determined using quantitative real-time PCR and western blot analysis, respectively. Oatp transport function at the BBB was determined via in situ brain perfusion using [ 3 H]taurocholate and [ 3 H]atorvastatin as probe substrates. Data were expressed as mean ± SD and analyzed via one-way ANOVA followed by the post hoc Bonferroni t-test., Results: Our results showed increased brain microvascular Slco1a4 mRNA and Oatp1a4 protein expression as well as increased brain uptake of [ 3 H]taurocholate and [ 3 H]atorvastatin in female rats as compared to males. Oatp1a4 expression at the BBB was enhanced in castrated male animals but was not affected by ovariectomy in female animals. In prepubertal rats, no sex-specific differences in brain microvascular Oatp1a4 expression were observed. Brain accumulation of [ 3 H]taurocholate in male rats was increased following castration as compared to controls. In contrast, there was no difference in [ 3 H]taurocholate brain uptake between ovariectomized and control female rats., Conclusions: These novel data confirm sex-specific differences in BBB Oatp1a4 functional expression, findings that have profound implications for treatment of CNS diseases. Studies are ongoing to fully characterize molecular pathways that regulate sex differences in Oatp1a4 expression and activity.

Angiopoietin-like 4 (ANGPTL4) raises plasma triglyceride levels by inhibiting lipoprotein lipase. A set of compounds that are able to reduce plasma triglyceride levels are bile acids (BA). Because BA have been shown to decrease ANGPTL4 secretion by intestinal cells, we hypothesized that BA lower plasma triglycerides (partly) via ANGPTL4. To test that hypothesis, wild-type and Angptl4 -/- mice were fed chow supplemented with taurocholic acid (TCA) for seven days. TCA supplementation effectively lowered plasma triglycerides in wild-type and Angptl4 -/- mice, indicating that ANGPTL4 is not required for plasma triglyceride-lowering by BA. Intriguingly, however, plasma and hepatic BA concentrations were significantly lower in TCA-supplemented Angptl4 -/- mice than in TCA-supplemented wild-type mice. These changes in the Angptl4 -/- mice were accompanied by lower BA levels in ileal scrapings and decreased expression of FXR-target genes in the ileum, including the BA transporter Slc10a2. By contrast, faecal excretion of specifically primary BA was higher in the Angptl4 -/- mice, suggesting that loss of ANGPTL4 impairs intestinal BA absorption. Since the gut microbiota converts primary BA into secondary BA, elevated excretion of primary BA in Angptl4 -/- mice may reflect differences in gut microbial composition and/or functionality. Indeed, colonic microbial composition was markedly different between Angptl4 -/- and wild-type mice. Suppression of the gut bacteria using antibiotics abolished differences in plasma, hepatic, and faecal BA levels between TCA-supplemented Angptl4 -/- and wild-type mice. In conclusion, 1) ANGPTL4 is not involved in the triglyceride-lowering effect of BA; 2) ANGPTL4 promotes BA absorption during TCA supplementation via a mechanism dependent on the gut microbiota., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Chronic diarrhoea is a common entity in daily clinical practice and it leads to a loss in these patients quality of life. It may be the main symptom of multiple ethiologies including bile acid malabsorption (BAM) which has a comparable prevalence to celiac disease. The BAM results from imbalances in the homeostasis of bile acids in the enterohepatic circulation. It can be a consequence of ileal disease or ileal dysfunction (BAM type i), it can be considered idiopathic or primary (BAM type ii) or associated with other gastrointestinal entities (BAM type iii). Among the different diagnostic methods available, 75 SeHCAT study is the primary current method due to its sensitivity, specificity, safety and low cost. The main disadvantage is that it's not available in all countries, so other diagnostic methods have appeared, such as serum measurement of FGF19 and C4, however they are significantly more complex and costly. The first-line treatment of bile acid diarrhoea is bile acid sequestrant, such as cholestyramine, which can be difficult to administer due to its poor tolerability and gastrointestinal side effects. These are less prominent with newer agents such as colesevelam. In summary, the BAM is a common entity underdiagnosed and undertreated, so it is essential to establish a diagnosis algorithm of chronic diarrhoea in which the 75 SeHCAT study would be first or second line in the differential diagnosis of these patients., (Copyright © 2016 Elsevier España, S.L.U. y SEMNIM. All rights reserved.)

The bile salt export pump (BSEP) is expressed on the canalicular membrane of hepatocytes regulating liver bile salt excretion, and impairment of BSEP function may lead to cholestasis in humans. This study explored drug biliary excretion, as well as serum chemistry, individual bile acid concentrations and liver transporter expressions, in the SAGE Bsep knockout (KO) rat model. It was observed that the Bsep protein in KO rats was decreased to 15% of that in the wild type (WT), as quantified using LC-MS/MS. While the levels of Ntcp and Mrp2 were not significantly altered, Mrp3 expression increased and Oatp1a1 decreased in KO animals. Compared with the WT rats, the KO rats had similar serum chemistry and showed normal liver transaminases. Although the total plasma bile salts and bile flow were not significantly changed in Bsep KO rats, individual bile acids in plasma and liver demonstrated variable changes, indicating the impact of Bsep KO. Following an intravenous dose of deuterium labeled taurocholic acid (D4-TCA, 2 mg/kg), the D4-TCA plasma exposure was higher and bile excretion was delayed by approximately 0.5 h in the KO rats. No differences were observed for the pravastatin plasma concentration-time profile or the biliary excretion after intravenous administration (1 mg/kg). Collectively, the results revealed that these rats have significantly lower Bsep expression, therefore affecting the biliary excretion of endogenous bile acids and Bsep substrates. However, these rats are able to maintain a relatively normal liver function through the remaining Bsep protein and via the regulation of other transporters. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

An LC-MS/MS method was developed and validated for the simultaneous quantification of chlorogenic acid (CGA) and taurocholic acid (TCA) in human plasma using hydrochlorothiazide as the internal standard. The chromatographic separation was achieved on a Hedera ODS-2 column with a gradient elution using 10 mmol·L(-1) of ammonium acetate buffer solution containing 0.5% of formic acid - acetonitrile as mobile phase at a flow rate of 300 μL·min(-1). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring in negative ESI mode. The method was fully validated over the concentration ranges of 0.1-10 ng·mL(-1) for CGA and 2-150 ng·mL(-1) for TCA. It was successfully applied to a pharmacokinetic study of CGA and TCA in healthy Chinese volunteers after oral administration of Shuanghua Baihe tablets (SBTs). In the single-dose study, the maximum plasma concentration (Cmax), time to reach Cmax (Tmax) and elimination half-life (t1/2) of CGA were (0.763 8 ± 0.542 0) ng·mL(-1), (1.0 ± 0.5) h, and (1.3 ± 0.6) h, respectively. In the multiple-dose study, the Cmax, Tmax and t1/2 of CGA were (0.663 7 ± 0.583 3) ng·mL(-1), (1.1 ± 0.5) h, and (1.4 ± 0.7) h, respectively. For TCA, no significant characteristic increasing plasma TCA concentration-time curve was found in the volunteers after oral administration of SBTs, indicating its complicated process in vivo as an endogenous ingredient., (Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

The bile salt export pump (BSEP) plays an important role in bile acid excretion. Impaired BSEP function may result in liver injury. Bile acids also undergo basolateral efflux, but the relative contributions of biliary (CLBile) versus basolateral efflux (CLBL) clearance to hepatocellular bile acid excretion have not been determined. In the present study, taurocholic acid (TCA; a model bile acid) disposition was characterized in human and rat sandwich-cultured hepatocytes (SCH) combined with pharmacokinetic modeling. In human SCH, biliary excretion of TCA predominated (CLBile = 0.14 ± 0.04 ml/min per g liver; CLBL = 0.042 ± 0.019 ml/min per g liver), whereas CLBile and CLBL contributed approximately equally to TCA hepatocellular excretion in rat SCH (CLBile = 0.34 ± 0.07 ml/min per g liver; CLBL = 0.26 ± 0.07 ml/min per g liver). Troglitazone decreased TCA uptake, CLBile, and CLBL; membrane vesicle assays revealed for the first time that the major metabolite, troglitazone sulfate, was a noncompetitive inhibitor of multidrug resistance-associated protein 4, a basolateral bile acid efflux transporter. Simulations revealed that decreased CLBile led to a greater increase in hepatic TCA exposure in human than in rat SCH. A decrease in both excretory pathways (CLBile and CLBL) exponentially increased hepatic TCA in both species, suggesting that 1) drugs that inhibit both pathways may have a greater risk for hepatotoxicity, and 2) impaired function of an alternate excretory pathway may predispose patients to hepatotoxicity when drugs that inhibit one pathway are administered. Simulations confirmed the protective role of uptake inhibition, suggesting that a drug's inhibitory effects on bile acid uptake also should be considered when evaluating hepatotoxic potential. Overall, the current study precisely characterized basolateral efflux of TCA, revealed species differences in hepatocellular TCA efflux pathways, and provided insights about altered hepatic bile acid exposure when multiple transport pathways are impaired., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Angiogenesis is a key feature of cancer development, thus it is a good target for cancer therapy. However, drugs that have been designed to block angiogenesis mainly capture growth factors in circulation, resulting not only in the transient inhibition of tumor progression but also in producing undesirable side effects. Nanoparticular drug delivery systems, on the other hand, may help overcome such drawbacks and improve the efficacy of anti-angiogenic therapies by altering the biodistribution and pharmacokinetics, improving tumor targeting ability, and reducing side effects. In this light, we propose a new approach of anti-angiogenic therapy that combines strategies of long circulating, passive tumor targeting, and anti-angiogenesis efficacy using a new polyelectrolyte complex system that combines LHT7, a previously developed heparin-based angiogenesis inhibitor, with a protamine to form a self-assembling nanocomplex with a mean diameter of 200nm, which is effective for anti-angiogenesis therapy. At first, LHT7 was modified with polyethylene glycol (PEG). We observed that PEG-LHT7/protamine nanocomplex was stable in buffer and slowly dissociated in plasma (9% dissociation for 24h). Compared to the free form of PEG-LHT7, the mean residence time of PEG-LHT7/protamine nanocomplex was found higher (15.9h) with its increased accumulation in tumor. Most importantly, PEG-LHT7/protamine nanocomplex was diffused and extravasated through the dense collagen matrix of the tumor. Thus, the study describes a successful application of functionalized PEG-LHT/protamine nanocomplex that can inhibit angiogenesis with long circulating, passive targeting, and tumor extravasating ability., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Gemcitabine-loaded solid lipid nanoparticles (SLNs) were produced by double emulsification technique using stearic acid as lipid, soy lecithin as surfactant and sodium taurocholate as cosurfactant. Prepared nanoparticles are characterized for particle size and surface morphology using scanning electron microscopy (SEM). Particle yield, entrapment efficiency and zeta potential were also determined. In-vitro release studies were performed in phosphate-buffered saline (PBS) pH 7.4 using metabolic shaker. The formulation F6 with maximum entrapment efficiency 72.42% and satisfactory in-vitro release was selected. In-vivo tissue distribution to liver, spleen, lung, heart and kidneys of optimized formulation followed by stability study under specific conditions were also determined. This investigation has shown preferential drug targeting to liver followed by spleen, lungs, kidneys and heart. Stability studies showed no significant change in the particle size followed with very slight decrease in entrapment efficiency at 25 ± 2 °C/60 ± 5% RH over a period of three months.

In our previous studies, taurocholic acid (TA)-conjugated low-molecular-weight heparin derivative (LHT7) has been proven to be a potent anti-angiogenic agent by demonstrated successful blockage capability of vascular endothelial growth factors (VEGF). Preliminary safety evaluations were conducted based on its mechanism of action and chemical behavior. For this purpose, acute toxicity study, and hematological and serological evaluations were carried out. Additionally, in order to evaluate mechanism-related side effects, both blood pressure and the occurrence of proteinuria were measured using a treatment regime of multiple high doses of LHT7 in a biodistribution study. LD50 values for LHT7 in female and male mice were 56.9 and 64.7 mg kg(-1) doses, respectively. There were no vital fluctuations in the serological and hematological parameters, except for the elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at 100 and 200 mg kg(-1) doses of LHT7, representing vital changes in the liver function. Moreover, the results of mechanism-related studies showed that blood pressure at 50 mg kg(-1) did not change but showed elevated levels of protein in urine. In the biodistribution study, a slight accumulation of LHT7 in the kidney and the liver were observed at the 50 mg kg(-1) repeated dose owing to the presence of bile acid. No fatal damage was observed in this study; most observations were related to the chemical composition or the mechanism of action of the material., (Copyright © 2014 John Wiley & Sons, Ltd.)

Deficiency of the phospholipid flippase ATPase, aminophospholipid transporter, class I, type 8B, member 1 (ATP8B1) causes progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1). Apart from cholestasis, many patients also suffer from diarrhea of yet unknown etiology. Here we have studied the hypothesis that intestinal ATP8B1 deficiency results in bile salt malabsorption as a possible cause of PFIC1/BRIC1 diarrhea. Bile salt transport was studied in ATP8B1-depleted intestinal Caco-2 cells. Apical membrane localization was studied by a biotinylation approach. Fecal bile salt and electrolyte contents were analyzed in stool samples of PFIC1 patients, of whom some had undergone biliary diversion or liver transplantation. Bile salt uptake by the apical sodium-dependent bile salt transporter solute carrier family 10 (sodium/bile acid cotransporter), member 2 (SLC10A2) was strongly impaired in ATP8B1-depleted Caco-2 cells. The reduced SLC10A2 activity coincided with strongly reduced apical membrane localization, which was caused by impaired apical membrane insertion of SLC10A2. Moreover, we show that endogenous ATP8B1 exists in a functional heterodimer with transmembrane protein 30A (CDC50A) in Caco-2 cells. Analyses of stool samples of post-transplant PFIC1 patients demonstrated that bile salt content was not changed, whereas sodium and chloride concentrations were elevated and potassium levels were decreased. The ATP8B1-CDC50A heterodimer is essential for the apical localization of SLC10A2 in Caco-2 cells. Diarrhea in PFIC1/BRIC1 patients has a secretory origin to which SLC10A2 deficiency may contribute. This results in elevated luminal bile salt concentrations and consequent enhanced electrolyte secretion and/or reduced electrolyte resorption., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This study aimed to characterize the in vitro hepatic transport mechanisms in primary rat and human hepatocytes of the fluorescent bile acid derivative N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2'-aminoethanesulfonate (tauro-nor-THCA-24-DBD), previously synthesized to study the activity of the bile salt export pump (BSEP). The fluorescent bile acid derivative exhibited saturable uptake kinetics in suspended rat hepatocytes. Hepatic uptake was inhibited in the presence of substrates/inhibitors of the organic anion transporting polypeptide (Oatp) family and Na(+) -taurocholate cotransporting peptide (Ntcp). Concentration-dependent uptake of the fluorescent bile acid was also saturable in Chinese hamster ovary cells transfected with rNtcp, hNTCP, OATP1B1, or OATP1B3. The fluorescent bile acid derivative was actively excreted in the bile canaliculi of sandwich-cultured rat and human hepatocytes (SCRH and SCHH), with a biliary excretion index (BEI) of 26% and 32%, respectively. In SCRH, cyclosporin A significantly decreased the BEI to 5%. Quantification by real-time confocal imaging further confirmed canalicular transport of the fluorescent bile acid derivative (BEI = 75%). We conclude that tauro-nor-THCA-24-DBD is a useful probe to study interference of drugs with NTCP/Ntcp- and BSEP/Bsep-mediated transport in fluorescence-based in vitro assays., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Oral delivery is the preferred route to deliver therapeutics via nanoparticles due to ease of administration and patient acceptance. Here, we report on the findings of the absorption pathway of taurocholic acid (TCA)-linked heparin and docetaxel (DTX) conjugate, which we refer to as HDTA. We studied the oral absorption of HDTA using a Caco-2 cell transport system and an animal model. We have also used other absorption enhancers, such as ethylene glycol tetraacetic acid (EGTA), or inhibitors, such as sodium azide, to compare the relative permeability of HDTA conjugates. In vivo comparative studies were conducted using free TCA as a pre-administration and exhibited the maximum absorption site of the organ after oral administration of HDTA conjugates. HDTA was found to be absorbed mainly in the ileum and Caco-2 cell monolayer through passive diffusion and bile acid transporters. High fluorescence intensity of HDTA in mice came from the ileum, and it was eliminated from the body through colon. This novel formulation could be further investigated by clinical trials to find the prospect of oral anti-cancer drug delivery through anti-angiogenic treatment strategies., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Bile acid diarrhoea is a common, under-diagnosed cause of chronic watery diarrhoea, responding to specific treatment with bile acid sequestrants. We previously showed patients with bile acid diarrhoea have lower median levels compared with healthy controls, of the ileal hormone fibroblast growth factor 19 (FGF19), which regulates bile acid synthesis., Aim: To measure serum FGF19 and SeHCAT retention prospectively in patients with chronic diarrhoea., Methods: One hundred and fifty-two consecutive patients were grouped according to (75) Se-homocholic acid taurine (SeHCAT) 7-day retention: normal (>15%) in 72 (47%) diarrhoea controls; ≤15% in 54 (36%) with primary bile acid diarrhoea, and in 26 (17%) with secondary bile acid diarrhoea. Fasting blood was assayed for FGF19, 7α-hydroxy-4-cholesten-3-one (C4) and total bile acids., Results: FGF19 was significantly lower in the primary bile acid diarrhoea group compared with the diarrhoea control group (median 147 vs. 225 pg/mL, P < 0.001), and also in the secondary group (P < 0.006). FGF19 and SeHCAT values were positively correlated (rs = 0.44, P < 0.001); both were inversely related to C4. Other significant relationships included SeHCAT and body mass index (BMI)(P = 0.02), and FGF19 with age (P < 0.01). The negative and positive predictive values of FGF19 ≤ 145 pg/mL for a SeHCAT <10% were 82% and 61%, respectively, and were generally improved in an index including BMI, age and C4. In a subset of 28 primary patients, limited data suggested that FGF19 could predict response to sequestrant therapy., Conclusions: Reduced fibroblast growth factor 19 is a feature of bile acid diarrhoea. Further studies will fully define its role in predicting the response of these patients to therapy., (© 2013 John Wiley & Sons Ltd.)

Measurement of the whole body retention of orally administered (75)SeHCAT is used to investigate patients with unexplained diarrhoea. Retention values of <15 % at 7 days post-administration are taken to indicate bile acid malabsorption (BAM). Whilst idiopathic BAM is frequently diagnosed with (75)SeHCAT, functional and morphological studies of the terminal ileum rarely show any abnormality, so the disorder may be more appropriately termed bile acid diarrhoea (BAD). In addition to malabsorption, excess bile acid may reach the colon, where the events leading to diarrhoea take place, as a result firstly of increased bile acid synthesis and secondly of an increased recycling rate of bile acids. Increased recycling has been largely ignored as a cause of BAD, but, as shown in this study, can readily result in excess bile acids reaching the colon even when ileal absorption efficiency is normal (i.e. 95-97 %). There needs to be a re-evaluation of the causes of BAD in patients without a history of previous intestinal resection or evidence of ileal pathology, such as Crohn's disease.

Human hepatocytes that had been cold-preserved in SureTran(TM) matrix (Abcellute Ltd, Cardiff, UK) were used for studies on cell viability, cytochrome P450 (CYP) 3A4, 2B6 and 1A2 induction and hepatic drug transporters. It has recently been shown that basal CYP activities are maintained in cold-preserved hepatocytes (Palmgren et al., 2012). After 5 d of cold preservation, the viability was still more than 70%, and after 8 d it was around 60%. In hepatocytes that had been cold-preserved for 3 d, the activity of CYP3A4 was induced around 15-fold upon treatment with 8 µM rifampicin for 72 h. For CYP2B6, the activity was induced 4- to 16-fold in hepatocytes that had been cold-preserved for 3 d and thereafter treated with 1 mM phenobarbital for 72 h. The activity of CYP1A2 was low and close to the limit of detection in non-treated cells that had been cold-preserved for up to 3 d, while the activity increased in cells treated with 0.3-25 µM β-naphthoflavone for 72 h. CYP3A4, 2B6 and 1A2 mRNA levels were only determined with hepatocytes from one donor and increased upon treatment with the inducers. Hepatic uptakes of estrone-3-sulfate, taurocholate, ipratropium and rosuvastatin were stable in human hepatocytes that had been cold-preserved for up to 2 d. In summary, cold-preserved human hepatocytes demonstrate retained viability and can advantageously be used for in vitro induction studies and for studies of hepatic uptake transporters.

TauroH-23-(Se) selena-25-homocholic acid retention values are used in the diagnosis of bile acid malabsorption. The standard method for measuring values is with an uncollimated gamma camera, which can create some logistic difficulties, with other background sources of activity, which are irrelevant when a collimator is used, becoming significant. In this study we compare the retention values obtained with a collimated and an uncollimated gamma camera in phantoms and in 23 patients. Bland-Altman plots were created using the data, which showed a mean bias in retention of 0.10% in the phantom study and 0.55% in the patient study between methods. A Wilcoxon signed-rank test with the null hypothesis of zero median difference between uncollimated and collimated methods was not statistically significant to P values less than 0.05 in the patient and phantom studies. In the patient study, on using a fixed boundary of retention (10%) between positive and negative status, the status of one patient was changed from negative (12%) to positive (9%). We conclude that measurement of retention with a collimated gamma camera is similar but not identical to that of uncollimated values. The clinical significance of this shift is unclear, as the threshold of significance and the method of integrating this measure with other clinical factors into management remain unclear.

Purpose: Intensity modulated radiation therapy (IMRT) is a significant therapeutic advance in prostate cancer, allowing increased tumor dose delivery and increased sparing of normal tissues. IMRT planning uses strict dose constraints to nearby organs to limit toxicity. Bile acid malabsorption (BAM) is a treatable disorder of the terminal ileum (TI) that presents with symptoms similar to radiation therapy toxicity. It has not been described in patients receiving RT for prostate cancer in the contemporary era. We describe new-onset BAM in men after IMRT for prostate cancer., Methods and Materials: Diagnosis of new-onset BAM was established after typical symptoms developed, selenium-75 homocholic acid taurine (SeHCAT) scanning showed 7-day retention of <15%, and patients' symptoms unequivocally responded to a bile acid sequestrant. The TI was identified on the original radiation therapy plan, and the radiation dose delivered was calculated and compared with accepted dose-volume constraints., Results: Five of 423 men treated in a prospective series of high-dose prostate and pelvic IMRT were identified with new onset BAM (median age, 65 years old). All reported having normal bowel habits before RT. The volume of TI ranged from 26-141 cc. The radiation dose received by the TI varied between 11.4 Gy and 62.1 Gy (uncorrected). Three of 5 patients had TI treated in excess of 45 Gy (equivalent dose calculated in 2-Gy fractions, using an α/β ratio of 3) with volumes ranging from 1.6 cc-49.0 cc. One patient had mild BAM (SeHCAT retention, 10%-15%), 2 had moderate BAM (SeHCAT retention, 5%-10%), and 2 had severe BAM (SeHCAT retention, <5%). The 3 patients whose TI received ≥45 Gy developed moderate to severe BAM, whereas those whose TI received <45 Gy had only mild to moderate BAM., Conclusions: Radiation delivered to the TI during IMRT may cause BAM. Identification of the TI from unenhanced RT planning computed tomography scans is difficult and may impede accurate dosimetric evaluation. Thorough toxicity assessment and close liaison between oncologist and gastroenterologist allow timely diagnosis and treatment., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The chemical conjugate of low molecular weight heparin with taurocholate (LHT7) was previously designed to offer anticancer activity while minimizing the anticoagulant activity. In the present study, we found that the systemic administration of LHT7 in nanolipoplex could substantially enhance tumor vasculature targeting and anticancer effects. Moreover, we found that co-delivery of LHT7 with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, in nanolipoplex could provide synergistic antitumor effect. LHT7/SAHA nanolipoplex was formulated by encapsulating SAHA inside cationic liposomes, followed by complexation of negatively charged LHT7 onto the cationic surfaces of SAHA-loaded liposomes (SAHA-L). LHT7/SAHA nanolipoplex was positively charged with a mean diameter of 117.6 nm, and stable in serum. The nanolipoplex form of LHT7 could alter its pharmacokinetics and biodistribution. Compared to the free form of LHT7, LHT7 in the nanolipoplex showed 1.9-fold higher mean residence time, and higher tumor vasculature accumulation after its intravenous administration. LHT7/SAHA nanolipoplex showed highest antitumor efficacy in SCC-bearing mice, compared to LHT7, SAHA-L and sequential co-administration of LHT7 and SAHA-L. Consistent with the enhanced antitumor effect, the reduction of abnormal vessels in the tumor site was also the highest in the LHT7/SAHA nanolipoplex-treated group. These results suggested the potential of LHT7/SAHA nanolipoplex for enhanced tumor vasculature targeting, and the importance of nanolipoplex-mediated co-delivery with a histone deacetylase inhibitor for maximal anticancer effect., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

TauroH-23-((75)Se)selena-25-homocholic acid (SeHCAT) retention has been used for the diagnosis of bile acid malabsorption since the early 1980s. Bile acid malabsorption has been increasingly recognized as an important cause of severe chronic diarroea with subsequent increase in the number of referrals for the SeHCAT test. In this study, we review and discuss the standard techniques for the measurement of SeHCAT retention and describe a simple and modified technique using a noncollimated whole-body scanner, suitable for most modern dual-headed cameras.

Aim: To evaluate the usefulness of the (75)SeHCAT abdominal retention (AR) measurement in the early diagnosis of diarrhea syndrome (DS)., Methods: Thirty-seven patients with diarrhea syndrome within the first month of evolution were prospectively evaluated. The (75)Se-SeHCAT abdominal retention was measured 4 and 7 days post-administration of 0.01 mCi of (75)SeHCAT. The test was performed prior to treatment and at 3 months when the baseline study was positive. The test was considered positive if the RA was <25% at 4(th) and <10% on the 7th day. The patients were visited at 3 months. Depending on the response, 3 groups were established: a) complete response: normalization of stool frequency, b) partial response, decrease of frequency or c) no response., Results: Group A: The AR of (75)Se-SEHCAT was normal in 21 patients. Six were diagnosed of colonic diverticulosis, 8 of irritable bowel syndrome, 1 of lymphocytic colitis, 1 of post-gastroenteritis syndrome, 1 of celiac disease and 1 of stenosis of the cardia. Four are still under study. Group B: The AR of (75)Se-SEHCAT decreased in 16 patients. All showed abnormal AR at day 7 and all but 1 at day 4. Following administration of cholestyramine resin, 8 (50%) presented partial response and 8 (50%) complete response. At 3 months, AR had increased at day 4 and 9 at day 7., Conclusion: The measurement of (75)SEHCAT abdominal retention allows the early diagnosis of bile acid malabsorption in 43% of the patients with DS. Measurement at 7 days seems more accurate than that at 4 days., (Copyright © 2010 Elsevier España, S.L. y SEMNIM. All rights reserved.)

Sandwich-cultured hepatocytes (SCH) are a useful tool for evaluating hepatobiliary drug transport in vitro. Some studies have investigated the in vitro-in vivo correlations of the biliary clearance of drugs using SCH. In most cases, the biliary clearance observed in vivo correlated well with the predicted clearance, but the predicted absolute values were underestimated when based on in vitro experiments with SCH. We hypothesized that the down-regulated function of uptake transporters is one of the causes of this underestimation. Therefore, the uptake of taurocholate, digoxin, pravastatin, and rosuvastatin was investigated in sandwich-cultured rat hepatocytes (SCRH) cultured for 5, 24, 48, and 96 h, and the predicted hepatic clearance from in vitro uptake clearance (CL(H, vitro)) was calculated with a dispersion model. In SCRH cultured for 96 h, the saturable uptake of taurocholate, digoxin, pravastatin, and rosuvastatin decreased to 7.5, 3.3, 64, and 23%, respectively, of their uptake in hepatocytes cultured for 5 h, and a better prediction of in vivo hepatic clearance (CL(H, vivo)) was achieved when based on CL(H, vitro) of 5-h-cultured hepatocytes. These results suggest that the uptake activity is considerably reduced in cell culture, even in a sandwich-culture format. In a similar study, we also examined taurocholate and rosuvastatin in sandwich-cultured human hepatocytes (SCHH). Unlike in SCRH, the saturable uptake of these compounds did not differ markedly in SCHH cultured for 5 or 96 h. Thus, the uptake activity in SCHH was maintained relatively well compared with that in SCRH.

Organic anion-transporting polypeptides (Oatp) 1a1 and 1a4 were deleted by homologous recombination, and mice were characterized for Oatp expression in liver and kidney, transport in isolated hepatocytes, in vivo disposition of substrates, and urinary metabolomic profiles. Oatp1a1 and Oatp1a4 proteins were undetected in liver, and both lines were viable and fertile. Hepatic constitutive messenger RNAs (mRNAs) for Oatp1a4, 1b2, or 2b1 were unchanged in Oatp1a1⁻/⁻ mice, whereas renal Oatp1a4 mRNA decreased approximately 50% (both sexes). In Oatp1a4⁻/⁻ mice, no changes in constitutive mRNAs for other Oatps were observed. Uptake of estradiol-17β-D-glucuronide and estrone-3-sulfate in primary hepatocytes decreased 95 and 75%, respectively, in Oatp1a1⁻/⁻ mice and by 60 and 30%, respectively, in Oatp1a4⁻/⁻ mice. Taurocholate uptake decreased by 20 and 50% in Oatp1a1⁻/⁻ and Oatp1a4⁻/⁻ mice, respectively, whereas digoxin was unaffected. Plasma area under the curve (AUC) for estradiol-17β-D-glucuronide increased 35 and 55% in male and female Oatp1a1⁻/⁻ mice, respectively, with a concurrent 50% reduction in liver-to-plasma ratios. In contrast, plasma AUC or tissue concentrations of estradiol-17β-D-glucuronide were unchanged in Oatp1a4⁻/⁻ mice. Plasma AUCs for dibromosulfophthalein increased nearly threefold in male Oatp1a1⁻/⁻ and Oatp1a4⁻/⁻ mice, increased by 40% in female Oatp1a4⁻/⁻ mice, and were unchanged in female Oatp1a1⁻/⁻ mice. In both lines, no changes in serum ALT, bilirubin, and cholesterol were noted. NMR analyses showed no generalized increase in urinary excretion of organic anions. However, urinary excretion of taurine decreased by 30-40% and was accompanied by increased excretion of isethionic acid, a taurine metabolite generated by intestinal bacteria, suggesting some perturbations in intestinal bacteria distribution.

The sodium-taurocholate (TC) cotransporting polypeptide (NTCP) facilitates bile formation by mediating sinusoidal Na(+)-TC cotransport. During sepsis-induced cholestasis, there is a decrease in NTCP-dependent uptake of bile acids and an increase in nitric oxide (NO) levels in hepatocytes. In rat hepatocytes NO inhibits Na(+)-dependent uptake of taurocholate. The aim of this study was to extend these findings to human NTCP and to further investigate the mechanism by which NO inhibits TC uptake. Using a human hepatoma cell line stably expressing NTCP (HuH-NTCP), we performed experiments with the NO donors sodium nitroprusside and S-nitrosocysteine and demonstrated that NO inhibits TC uptake in these cells. Kinetic analyses revealed that NO significantly decreased the V(max) but not the K(m) of TC uptake by NTCP, indicating noncompetitive inhibition. NO decreased the amount of NTCP in the plasma membrane, providing a molecular mechanism for the noncompetitive inhibition of TC uptake. One way that NO can modify protein function is through a posttranslational modification known as S-nitrosylation: the binding of NO to cysteine thiols. Using a biotin switch technique we observed that NTCP is S-nitrosylated under conditions in which NO inhibits TC uptake. Moreover, dithiothreitol reversed NO-mediated inhibition of TC uptake and S-nitrosylation of NTCP, indicating that NO inhibits TC uptake via modification of cysteine thiols. In addition, NO treatment led to a decrease in Ntcp phosphorylation. Taken together these results indicate that the inhibition of TC uptake by NO involves S-nitrosylation of NTCP.

It has been reported that the adjuvant-induced inflammation could affect drug metabolism in liver. Here we further investigated the effect of inflammation on drug transport in liver using taurocholate as a model drug. The hepatic disposition kinetics of [(3)H]taurocholate in perfused normal and adjuvant-treated rat livers were investigated by the multiple indicator dilution technique and data were analyzed by a previously reported hepatobiliary taurocholate transport model. Real-time RT-PCR was also performed to determine the mRNA expression of liver bile salt transporters in normal and diseased livers. The uptake and biliary excretion of taurocholate were impaired in the adjuvant-treated rats as shown by decreased influx rate constant k(in) (0.65 ± 0.09 vs. 2.12 ± 0.30) and elimination rate constant k(be) (0.09 ± 0.02 vs. 0.17 ± 0.04) compared with control rat group, whereas the efflux rate constant k(out) was greatly increased (0.07 ± 0.02 vs. 0.02 ± 0.01). The changes of mRNA expression of liver bile salt transporters were found in adjuvant-treated rats. Hepatic taurocholate extraction ratio in adjuvant-treated rats (0.86 ± 0.05, n = 6) was significantly reduced compared with 0.93 ± 0.05 (n = 6) in normal rats. Hepatic extraction was well correlated with altered hepatic ATP content (r(2) = 0.90). In conclusion, systemic inflammation greatly affects hepatic ATP content/production and associated transporter activities and causes an impairment of transporter-mediated solute trafficking and pharmacokinetics.

This study investigated the role of a multispecific organic anion transporter, Oatp1a4/Slco1a4, in drug transport across the blood-brain barrier. In vitro transport studies using human embryonic kidney 293 cells expressing mouse Oatp1a4 identified the following compounds as Oatp1a4 substrates: pitavastatin (K(m) = 8.3 microM), rosuvastatin (K(m) = 12 microM), pravastatin, taurocholate (K(m) = 40 microM), digoxin, ochratoxin A, and [d-penicillamine(2,5)]-enkephalin. Double immunohistochemical staining of Oatp1a4 with P-glycoprotein (P-gp) or glial fibrillary acidic protein demonstrated that Oatp1a4 signals colocalized with P-gp signals partly but not with glial fibrillary acidic protein, suggesting that Oatp1a4 is expressed in both the luminal and the abluminal membranes of mouse brain capillary endothelial cells. The brain-to-blood transport of pitavastatin, rosuvastatin, pravastatin, and taurocholate after microinjection into the cerebral cortex was significantly decreased in Oatp1a4(-/-) mice compared with that in wild-type mice. The blood-to-brain transport of pitavastatin, rosuvastatin, taurocholate, and ochratoxin A, determined by in situ brain perfusion, was significantly lower in Oatp1a4(-/-) mice than in wild-type mice, whereas transport of pravastatin and [D-penicillamine(2,5)]-enkephalin was unchanged. The blood-to-brain transport of digoxin was significantly lower in Oatp1a4(-/-) mice than in wild-type mice only when P-gp was inhibited by N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). Taken together, these results show that Oatp1a4 can mediate the brain-to-blood and blood-to-brain transport of its substrate drugs across the blood-brain barrier. The brain-to-plasma ratio of taurocholate, pitavastatin, and rosuvastatin was close to the capillary volume in wild-type mice, and it was not affected by Oatp1a4 dysfunction. Whether Oatp1a4 can deliver drugs from the blood to the brain remains controversial.

Hepatocyte uptake of phalloidin is carried out mainly by OATP1B1. We have used this compound as a prototypic substrate and assayed the ability to inhibit OATP-mediated phalloidin transport of four bile acid derivatives (BALU-1, BALU-2, BALU-3 and BALU-4) that showed positive results in preliminary screening. Using Xenopus laevis oocytes for heterologous expression of transporters, BALUs were found to inhibit taurocholic acid (TCA) transport by OATP1B1 (but not OATP1B3) as well as by rat Oatp1a1, Oatp1a4 and Oatp1b2. The study of their ability to inhibit sodium-dependent bile acid transporters revealed that the four BALUs induced an inhibition of rat Asbt-mediated TCA transport, which was similar to TCA-induced self-inhibition. Regarding human NTCP and rat Ntcp, BALU-1 differs from the other three BALUS in its lack of effect on TCA transport by these proteins. Using HPLC-MS/MS and CHO cells stably expressing OATP1B1 the ability of BALU-1 to inhibit the uptake of phalloidin itself by this transporter was confirmed. Kinetic analysis using X. laevis oocytes revealed that BALU-1-induced inhibition of OATP1B1 was mainly due to a competitive mechanism (Ki=8 microM). In conclusion, BALU-1 may be useful as a pharmacological tool to inhibit the uptake of compounds mainly taken up by OATP1B1 presumably without impairing bile acid uptake by the major carrier accounting for this process, i.e., NTCP.

1. The purpose of this study was to investigate the involvement of rat Mrp2 and human MRP2 in benzylpenicillin transport using canalicular liver plasma membrane (cLPM) vesicles isolated from Sprague-Dawley or Easai hyperbilirubinemic (EHBR) rats, and MDCKII cells overexpressing MRP2. 2. The adenosine triphosphate (ATP)-dependent uptake of benzylpenicillin and oestradiol-17beta-D-glucuronide (E(2)17betaG), a representative substrate for Mrp2, into EHBR-cLPM vesicles was decreased relative to that seen with control-cLPM vesicles, which may reflect the absence of Mrp2 in the EHBR. The ATP-dependent uptake of taurocholate, which is not a substrate for Mrp2, was similar in both control and EHBR-cLPM vesicles. The concentration dependence of ATP-dependent benzylpenicillin uptake was reflected in a K(m) of 44.0 microM and a V(max) of 508.4 pmol mg(-1) min(-1). Additional inhibition studies using E(2)17betaG and methotrexate as representative substrates for Mrp2/MRP2 demonstrated the involvement of rat Mrp2, but not human MRP2, in benzylpenicillin efflux. Benzylpenicillin appears not to be a substrate for or inhibitor of other human efflux transporters such as MDR1, MRP1, MRP3, or BCRP. 3. In conclusion, rat Mrp2, but not human MRP2, plays an important role in ATP-dependent benzylpenicillin uptake in the bile canalicular membrane, which may explain why biliary excretion of benzylpenicillin is high in the rat but negligible in humans.

The purpose of the present study was to evaluate the effects of bovine serum albumin (BSA) and essentially fatty acid-free BSA (BSA-FAF) on the biliary clearance of compounds in sandwich-cultured rat hepatocytes. Unbound fraction, biliary excretion index (BEI), and unbound intrinsic biliary clearance (intrinsic Clbiliary') were determined for digoxin, pravastatin, and taurocholate in the absence or presence of BSA or BSA-FAF. BSA had little effect on the BEI or intrinsic Clbiliary' of these compounds. Surprisingly, BSA-FAF decreased both BEI and intrinsic Clbiliary' for digoxin and pravastatin, which represent low and moderately bound compounds, respectively. The BEI and intrinsic Clbiliary' of taurocholate, a highly bound compound, were not altered significantly by BSA-FAF. Neither BSA nor BSA-FAF had a discernable effect on the bile canalicular networks based on carboxydichlorofluorescein retention. Neither the addition of physiological concentrations of calcium nor the addition of fatty acids to BSA-FAF was able to restore the BEI or intrinsic Clbiliary' of the model compounds to similar values in the absence or presence of BSA. Careful consideration is warranted when selecting the type of BSA for addition to in vitro systems such as sandwich-cultured rat hepatocytes.

Hepatocyte growth factor (HGF) ameliorates liver injuries in hepatectomized cholestatic rats. On the other hand, the protein level of organic anion-transporting polypeptide (Oatp1), which is responsible for the uptake of bile salts into hepatocytes, decreases in cholestatic humans and rats. However, the relationship between the ameliorative effects of HGF and the decrease in Oatp1 levels in cholestasis remains to be understood. Therefore, in order to investigate this relationship, we evaluated the effects of HGF on the function and protein level of Oatp1. HGF treatment significantly increased the uptake of radiolabeled estradiol 17beta-d-glucuronide ([(3)H]E(2)17betaG), a predominant Oatp1 substrate, in primary cultured rat hepatocytes. Additionally, there was an increase in the Oatp1 protein levels. The increased [(3)H]E(2)17betaG uptake was significantly inhibited by simultaneous incubation with the HGF receptor antibody and treatment with non-radiolabeled E(2)17betaG. However, inhibition by taurocholic acid, a Na(+)-taurocholate co-transporting polypeptide (Ntcp) substrate, was weaker than that caused by non-radiolabeled E(2)17betaG. Further, the increase was not altered by replacing Na(+) in the medium with Li(+). In the inhibition study, the increased [(3)H]E(2)17betaG uptake was inhibited by Oatp1 substrates, including bromosulfophthalein, ochratoxin A, and ouabain, but not by digoxin, which is an Oatp2-specific substrate. Furthermore, HGF did not alter the Oatp1 mRNA expression. In contrast, HGF treatment suppressed the ubiquitination of Oatp1 protein. In conclusion, this is the first report suggesting that HGF regulates Oatp1 protein level and that the ameliorative effects of HGF in cholestasis was induced, at least in part, by correcting the down-regulation of the Oatp1 protein level.

Polymorphisms in the SLCO1B1 gene encoding the human hepatocellular uptake transporter OATP1B1 can be associated with alterations in transporter properties and may affect the pharmacokinetics of drugs transported by OATP1B1. Therefore, it is of interest to investigate newly identified genetic variations on their impact on the pharmacokinetic of OATP1B1 substrates. We analyzed the allelic frequencies of five variations (c.452A>G, c.1007C>G, c.1454G>T, c.1628T>G, and c.1929A>C) in Caucasians and investigated the influence of SNP c.1929A>C which had an allelic frequency of 4.7%. None of the 285 Caucasian DNA donors were carriers of c.452A>G, c.1007C>G, c.1454G>T, c.1628T>G. Liver samples carrying SNP c.1929A>C were analyzed for OATP1B1 protein expression demonstrating no differences in expression levels compared to wild-type samples. Possible functional consequences were analyzed using HEK cells stably expressing the mutated OATP1B1 protein (OATP1B1-Leu643Phe). Uptake experiments with sulfobromophthalein, estradiol-17ssD-glucuronide, pravastatin, and taurocholic acid showed no significant difference in the uptake kinetics compared to wild-type OATP1B1. We showed that four variations frequent in the Asian population were not detected in Caucasians and demonstrated that the frequent SNP c.1929A>C had no effect on the hepatic OATP1B1 protein expression and on the transport properties. Therefore, it is unlikely that c.1929A>C contributes to interindividual variability in drug disposition.

Unlabelled: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by a mutation in the bile salt export pump (BSEP/ABCB11) gene. We previously reported that E297G and D482G BSEP, which are frequently found mutations in European patients, result in impaired membrane trafficking, whereas both mutants retain their transport function. The dysfunctional localization is probably attributable to the retention of BSEP in endoplasmic reticulum (ER) followed by proteasomal degradation. Because sodium 4-phenylbutyrate (4PBA) has been shown to restore the reduced cell surface expression of mutated plasma membrane proteins, in the current study, we investigated the effect of 4PBA treatment on E297G and D482G BSEP. Transcellular transport and cell surface biotinylation studies using Madin-Darby canine kidney (MDCK) II cells demonstrated that 4PBA treatment increased functional cell surface expression of wild-type (WT), E297G, and D482G BSEP. The prolonged half-life of cell surface-resident BSEP with 4PBA treatment was responsible for this result. Moreover, treatment of Sprague-Dawley rats with 4PBA resulted in an increase in BSEP expression at the canalicular membrane, which was accompanied by an increase in the biliary excretion of [(3)H]taurocholic acid (TC)., Conclusion: 4PBA treatment with a clinically achievable concentration enhances the cell surface expression and the transport capacity of WT, E297G, and D482G BSEP in MDCK II cells, and also induces functional BSEP expression at the canalicular membrane and bile acid transport via canalicular membrane in vivo. 4PBA is a potential pharmacological agent for treating not only PFIC2 patients with E297G and D482G mutations but also other cholestatic patients, in whom the BSEP expression at the canalicular membrane is reduced.

Aim: To determine if novel bile acid transporters may be expressed in human tissues., Methods: SLC10A1 (NTCP) was used as a probe to search the NCBI database for homology to previously uncharacterized ESTs. The homology search identified an EST (termed SLC10A4) that shares sequence identity with SLC10A1 and SLC10A2 (ASBT). We performed Northern blot analysis and RT-PCR to determine the tissue distribution of SLC10A4. SLC10A4 was cloned in frame with an epitope tag and overexpressed in CHO cells to determine cellular localization and functional analysis of bile acid uptake., Results: Northern analysis revealed that SLC10A4 mRNA is ubiquitously expressed in human tissues with the highest levels of mRNA expression in brain, placenta, and liver. In SLC10A4-transfected CHO cells, immunoblotting analysis and immunofluorescence staining demonstrated a 49-kDa protein that is expressed at the plasma membrane and intracellular compartments. Functional analysis of SLC10A4 showed no significant taurocholate uptake in the presence of sodium when compared to untransfected CHO cells., Conclusion: To date, we have shown that this protein has no capacity to transport taurocholate relative to SLC10A1; however, given its ubiquitous tissue distribution, it may play a more active role in transporting other endogenous organic anions.

Contrast-enhanced magnetic resonance imaging (CE-MRI) is a valuable technique for the diagnosis of liver diseases. As gadocoletic acid trisodium salt (B22956/1), a new contrast agent showing high biliary excretion, may be potentially advantageous in hepatobiliary imaging, the aim of the study was to investigate the molecular mechanisms of hepatic transport of the B22956 ion in a cellular model of hepatic tumor. B22956 ion uptake was measured in tumoral (HepG2) and nontumoral (Chang liver) hepatic cell lines. Absolute quantitative real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) analyses, using cloned PCR products as standards, were performed on total RNA of both cell lines and normal liver to evaluate the transcription of 12 transport genes: SLCO1A2, SLCO2B1, SLCO1B1, SLCO3A1, SLCO4A1, SLCO1B3, SLC22A7, SLC22A8, SLC22A1, SLC10A1, SLC15A1, and SLC15A2. B22956 transport was more efficient in Chang liver than in HepG2 cells and was inhibited by cholecystokinin-8, a specific substrate of OATP1B3. Real-time RT-PCR analyses revealed different transcription profiles in the tumoral and nontumoral cell lines. Compared with normal liver, the expression of SLCO1B1, SLCO3A1, and SLCO1B3 was greatly repressed in HepG2 cells, whereas SLCO2B1, SLC22A7, and SLC22A8 expression was either maintained or increased. On the contrary, in Chang liver cells, SLC22A7 and SLC22A8 genes were undetectable, whereas the expression of SLCO3A1, SLCO4A1, and SLCO1B3 was similar to normal liver. Transport studies and gene expression analyses indicated that B22956 ion is a good substrate to the liver-specific OATP1B3, reported to be poorly expressed or absent in human liver tumors. Therefore, B22956 may be helpful in detecting hepatic neoplastic lesions by CE-MRI.

The parallel artificial membrane permeability assay (PAMPA) system has promise to rapidly screen drug candidate passive permeability, but has been poorly described in terms of its lipid membrane structure and function. The objective was to investigate the role of PAMPA lipid composition on the permeability of five model compounds. PAMPA was used and employed individual phospholipids that varied in phosphate head group and acyl chain unsaturation. Transport of benzoic acid, taurocholic acid, metoprolol, sucrose, and mannitol was measured. Membrane fluidity was assessed by 1,3-diphenylhexatriene fluorescence anisotropy. Results indicate that compound permeability across PAMPA differed in their sensitivity to membrane lipid composition, where compounds with appreciable permeability (i.e. at least 0.2 x 10(-6)cm/s) were possibly sensitive to membrane fluidity and apparent ion pair effects. Benzoic acid permeability ranged 51-fold across membrane types, suggesting acyl chain effect on membrane fluidity. Mannitol, sucrose, and taurocholic acid permeabilities were low and independent of lipid composition. Metoprolol permeability ranged 17-fold and exhibited a markedly high permeability across 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] due to apparent ion pair-facilitated transport. Compound permeability was lowest across the phosphatidylcholines, which is consistent with phosphatidylcholine exhibiting relatively high membrane rigidity. In contrast to results from phosphatidylethanolamines and phosphatidylserines, acyl chain unsaturation had no effect on permeability across phosphatidylcholines. In conclusion, while much remains unknown about PAMPA structure and subsequent PAMPA permeability, results here from five solutes suggest that, for solutes with appreciable permeability, lipid composition modulated drug permeability through possible membrane fluidity and apparent ion pair influences.

We investigated the contribution of multidrug resistance associated protein 3 (Mrp3/ABCC3) to the transport of bile acids across the rat intestinal basolateral membrane using the everted sacs. The permeability-surface area (PS) products of taurocholate in the everted sacs of rat jejunum, ileum, and colon were determined in the absence or presence of inhibitors for Mrp3. The results were analyzed to determine the PS product for the uptake across the apical membrane (PS1) and that for the efflux across the basolateral membrane (PS3). The mucosal-to-serosal transport of taurocholate in the ileum was the highest. The concentration-dependent inhibitory effects by all inhibitors in the ileum were observed on both PS1 and PS3 for taurocholate. However, even in the presence of 1 mM of each inhibitor, the decrease of PS3 was low. Additionally, PS3 in the colon, where Mrp3 is expressed at a high level, was not inhibited by MK571 and taurolithocholate-3-sulfate. Unlike PS1, PS3 did not exhibit saturation at the concentration examined. These results suggest that Mrp3 makes only a minor contribution to the efflux of bile acids across the basolateral membrane. Ostalpha-Ostbeta heteromeric transporter is certainly one of the good candidates for such a transporter.

Hepatobiliary transporters are a major route for elimination of xenobiotics and endogenous products. In vitro hepatobiliary models have been reported for human and rat, but not for the other preclinical species used in safety evaluation. We have established methodologies for culturing dog and monkey hepatocytes with optimal bile canalicular formation and function, using a sandwich culture comprising rigid collagen substratum and gelled collagen overlay. Hepatic uptake utilizing sinusoidal transporters and biliary excretion through canalicular transporters were assessed using the bile salt taurocholate, salicylate (negative control), and the Bsep inhibitors cyclosporin A (CsA) and glyburide. There was significant taurocholate and salicylate canalicular efflux in dog and monkey hepatocytes, although the amount of salicylate transported was one thousandth that of taurocholate. Species differences were observed, as glyburide significantly inhibited taurocholate uptake in monkey (64% at 10 microM) but not dog hepatocytes, and inhibited taurocholate efflux in dog (100% at 10 microM) but not monkey hepatocytes. CsA did not inhibit bile salt uptake and significantly inhibited canalicular efflux in dog (at 0.1 microM) and monkey (at 1 and 10 microM) hepatocyte cultures. These results suggest that glyburide is a bile salt uptake inhibitor in monkey but not in dog hepatocytes and that CsA inhibits bile salt canalicular efflux but not basolateral uptake in these species. We have established dog and monkey hepatocytes in sandwich culture with intact bile canalicular formation and function. The differences observed in taurocholate transport between dog and monkey hepatocytes may be indicative of in vivo species differences.

The Isolated Perfused Liver (IPL) model is a widely used and appreciated in vitro method to demonstrate liver viability and metabolism. Reperfusion is performed in a controlled setting, however, via the portal vein only. To study transplant related questions concerning bile and transport of bile, the in vitro Isolated dual Perfused Liver model is revisited. The IdPL is an in vitro reperfusion model, using both portal vein and hepatic artery. Livers from 12 Wistar rats were flushed with University of Wisconsin-organ preservation solution, procured and reperfused in either the conventional IPL-model (n = 6) or the new IdPL-model (n = 6). Liver injury, assessed by the release of aspartate amino transferase and lactate dehydrogenase, showed similar levels during both IPL and I dPL reperfusion, only alanine amino transferase showed an improvement. Cumulative bile production showed an improvement: 176.3 +/- 8.4 in the IdPL compared to 126.1 +/- 12.2 microg/g-liver in the IPL (p < 0.05). Clearance of phenol red (PR) and taurocholic acid (TC) remained similar. At 90 minutes reperfusion the PR clearance showed 0.11 +/- 0.01 and 0.11 +/- 0.02 mg/30min/g-liver and the TC clearance 1.01 +/- 0.10 and 1.01 +/- 0.07 micromol/ml/30min/g-liver in the IPL and IdPL, respectively. Increasing the reperfusion time beyond the normally used 90 minutes resulted in a significant increase in transaminases and LDH and a decrease in bile production, liver morphology remained intact and glycogen content was appropriate. In conclusion, the IdPL-model showed similar or better results than the IPL-model, but the liver could not endure an extended reperfusion time using the IdPL.

Ursodeoxycholate (UDCA) is widely used for the treatment of cholestatic liver disease. After oral administration, UDCA is absorbed, taken up efficiently by hepatocytes, and conjugated mainly with glycine to form glycoursodeoxycholate (GUDC) or partly with taurine to form tauroursodeoxycholate (TUDC), which undergo enterohepatic circulation. In this study, to check whether three basolateral transporters--Na(+)-taurocholate cotransporting polypeptide (NTCP, SLC10A1), organic anion transporting polypeptide (OATP) 1B1 (OATP-C), and OATP1B3 (OATP8)-mediate uptake of UDCA, GUDC, and TUDC by human hepatocytes, we investigated their transport properties using transporter-expressing HEK293 cells and human cryopreserved hepatocytes. TUDC and GUDC could be taken up via human NTCP, OATP1B1, and OATP1B3, whereas UDCA could be transported significantly by NTCP, but not OATP1B1 and OATP1B3 in our expression systems. We observed a time-dependent and saturable uptake of UDCA and its conjugates by human cryopreserved hepatocytes, and more than half of the overall uptake involved a saturable component. Kinetic analyses revealed that the contribution of Na(+)-dependent and -independent pathways to the uptake of UDCA or TUDC was very similar, while the Na(+)-independent uptake of GUDC was predominant. These results suggest that UDCA and its conjugates are taken up by both multiple saturable transport systems and nonsaturable transport in human liver with different contributions. These results provide an explanation for the efficient hepatic clearance of UDCA and its conjugates in patients receiving UDCA therapy.

The bile salt export pump (BSEP) of hepatocyte secretes conjugated bile salts across the canalicular membrane in an ATP-dependent manner. The biliary bile salts of human differ from those of rat in containing a greater proportion of glycine conjugates and taurolithocholate 3-sulfate (TLC-S). In the present study, the transport properties of hBSEP and rBsep were investigated using membrane vesicles from HEK293 cells infected with recombinant adenoviruses containing hBSEP or rBsep cDNA. ATP-dependent uptake of radiolabeled glycine-, taurine-conjugated bile salts, and [(3)H]cholate was observed when hBSEP or rBsep was expressed. Comparison of initial uptake rates indicated that for both transporters, taurine-conjugated bile salts were transported more rapidly than glycine-conjugated bile salts, however, hBSEP transported glycine conjugates to an extent that was approximately 2-fold greater than rBsep. In addition, [(3)H]TLC-S was significantly transported by hBSEP, and hardly transported by rBsep. The mean K(m) value for the uptake of [(3)H]TLC-S by hBSEP was 9.5+/-1.5 microM, a value similar to that for hMRP2 (8.2+/-1.3 microM). In conclusion, both hBSEP and rBsep transport taurine-conjugated bile salts better than glycine-conjugated bile salts, but hBSEP transports glycine conjugates to a greater extent as compared to rBsep. TLC-S, which is present in human bile but not rodent bile, is more avidly transported by hBSEP compared with rBsep.

Background and Aims: alpha-Naphthylisothiocyanate (ANIT) is known to cause cholestasis due to injury of the bile duct epithelial cells. The aim of the present study was to examine the effect of a single dose of ANIT on the biliary excretion of various cholephilic compounds and on the amount of canalicular transporters., Methods: Twenty-four hours after the oral administration of ANIT (100 mg/kg), the biliary excretion of taurocholate, leukotriene C(4), pravastatin and vinblastine was studied. The protein levels of the bile salt export pump and multidrug resistance protein 2 and the immunostaining of multidrug resistance protein 2 in the liver were also examined., Results: The ANIT treatment markedly decreased the biliary excretion of tracer amounts of taurocholate, leukotriene C(4), pravastatin and vinblastine. The biliary excretory maximum of taurocholate was also markedly decreased after ANIT treatment. The ANIT treatment had no effect on the protein levels of bile salt export pump and multidrug resistance protein 2 and the immunostaining of multidrug resistance protein 2 in the liver., Conclusions: These findings support canalicular transporters having little effect on the marked impairment of biliary excretion of cholephilic compounds in ANIT-induced cholestasis., ((c) 2005 Blackwell Publishing Asia Pty Ltd.)

The bile acids filtered through the glomeruli nearly completely escape urinary excretion due to an efficient tubular reabsorption process. Reabsorption is mediated by the sodium-dependent bile acid transporter ASBT, which is localized in the brush border membranes of proximal tubular cells. The purpose of the present study was to assess whether tubular taurocholate transport is regulated by sex hormones. Clearance studies and studies on proximal tubular cells freshly isolated from rat kidneys were performed. The studies with the isolated proximal tubular cells revealed a cell to bath 3H-taurocholate accumulation ratio of 5.63+/-0.28 in male and of 3.67+/-0.43 in female rats (p<0.01). This difference in cellular taurocholate uptake was corroborated by the clearance studies, which showed a 3H-taurocholate clearance of 133.9+/-28.1 in male rats and of 262.0+/-45.4 microl/min x 100 g b.w. in female rats (p<0.05). Testosterone treatment of female rats did not significantly alter the cell to bath 3H-taurocholate accumulation ratio. However, the cellular taurocholate accumulation significantly decreased, by 61.6+/-10.1%, following ethinylestradiol treatment of male rats. Ovariectomy, chemical castration of female rats with buserelin or treatment of female rats with the estrogen receptor antagonist ICI 182780 did not affect taurocholate uptake, but treatment of ovariectomized rats with ethinylestradiol decreased the taurocholate accumulation ratio by 53.7+/-15.8%. By determination of serum bile acids the possibility was excluded that this change was an indirect effect of cholestasis induced by ethinylestradiol. This study demonstrates gender differences in the renal handling of taurocholate in rats that may be related to an inhibitory effect of estrogens on taurocholate transport in proximal tubular cells. Since the ASBT protein content of the proximal tubular cells was found not to be different between male and female rats, a nongenomic mechanism may underly this estrogen effect.

The role of the cholangiocyte apical Na(+)-dependent bile acid transporter (ASBT) in bile formation is unknown. Bile acid absorption by bile ducts results in cholehepatic shunting, a pathway that amplifies the canalicular osmotic effects of bile acids. We tested in isolated cholangiocytes if secretin enhances ASBT translocation to the apical membrane from latent preexisting intracellular stores. In vivo, in bile duct-ligated rats, we tested if increased ASBT activity (induced by secretin pretreatment) results in cholehepatic shunting of bile acids. We determined the increment in taurocholate-dependent bile flow and biliary lipid secretion and taurocholate (TC) biliary transit time during high ASBT activity. Secretin stimulated colchicine-sensitive ASBT translocation to the cholangiocyte plasma membrane and (3)H-TC uptake in purified cholangiocytes. Consistent with increased ASBT promoting cholehepatic shunting, with secretin pretreatment, we found TC induced greater-than-expected biliary lipid secretion and bile flow and there was a prolongation of the TC biliary transit time. Colchicine ablated secretin pretreatment-dependent bile acid-induced choleresis, increased biliary lipid secretion, and the prolongation of the TC biliary transit. In conclusion, secretin stimulates cholehepatic shunting of conjugated bile acids and is associated with increased cholangiocyte apical membrane ASBT. Bile acid transport by cholangiocyte ASBT can contribute to hepatobiliary secretion in vivo.

The disposition kinetics of [3H]taurocholate ([3H]TC) in perfused normal and cholestatic rat livers were studied using the multiple indicator dilution technique and several physiologically based pharmacokinetic models. The serum biochemistry levels, the outflow profiles and biliary recovery of [3H]TC were measured in three experimental groups: (i) control; (ii) 17 alpha-ethynylestradiol (EE)-treated (low dose); and (iii) EE-treated (high dose) rats. EE treatment caused cholestasis in a dose-dependent manner. A hepatobiliary TC transport model, which recognizes capillary mixing, active cellular uptake, and active efflux into bile and plasma described the disposition of [3H]TC in the normal and cholestatic livers better than the other pharmacokinetic models. An estimated five- and 18-fold decrease in biliary elimination rate constant, 1.7- and 2.7-fold increase in hepatocyte to plasma efflux rate constant, and 1.8- and 2.8-fold decrease in [3H]TC biliary recovery ratio was found in moderate and severe cholestasis, respectively, relative to normal. There were good correlations between the predicted and observed pharmacokinetic parameters of [3H]TC based on liver pathophysiology (e.g. serum bilirubin level and biliary excretion of [3H]TC). In conclusion, these results show that altered hepatic TC pharmacokinetics in cholestatic rat livers can be correlated with the relevant changes in liver pathophysiology in cholestasis.

Reports suggest that troglitazone, and to a lesser extent bosentan, may alter bile acid homeostasis by inhibiting the bile salt export pump. The present studies examined the hypothesis that these xenobiotics may modulate multiple hepatic bile acid transport mechanisms. In suspended rat hepatocytes, troglitazone (10 microM) decreased the initial rate of taurocholate uptake approximately 3-fold; the initial uptake rate of estradiol-17beta-D-glucuronide, a substrate of the organic anion transporting polypeptides, also was decreased approximately 4-fold. Bosentan (100 microM) decreased the initial uptake rate of taurocholate and estradiol-17beta-D-glucuronide by approximately 12- and approximately 7-fold, respectively. In sandwich-cultured rat hepatocytes, 10-min accumulation of taurocholate in cells + bile canaliculi (408 +/- 57 pmol/mg protein) was decreased significantly by troglitazone (157 +/- 17 pmol/mg protein, respectively) only in the presence of Na+, the driving force for the sodium taurocholate cotransporting polypeptide. A similar decrease with 10-fold higher concentrations of bosentan was noted. The biliary excretion index of taurocholate (55 +/- 8%) was decreased in the presence of 10 microM troglitazone (27 +/- 2%) and 100 microM bosentan (10 +/- 6%). In conclusion, xenobiotics may alter hepatic bile acid transport by inhibiting both hepatic uptake and biliary excretion.