Sandwich-cultured Rat Hepatocytes Research Articles

NADPH oxidase-generated reactive oxygen species are involved in estradiol 17ß-d-glucuronide-induced cholestasis

The endogenous metabolite of estradiol, estradiol 17β-D-glucuronide (E17G), is considered the main responsible of the intrahepatic cholestasis of pregnancy. E17G alters the activity of canalicular transporters through a signaling pathway-dependent cellular internalization, phenomenon that was attributed to oxidative stress in different cholestatic conditions. However, there are no reports involving oxidative stress in E17G-induced cholestasis, representing this the aim of our work. Using polarized hepatocyte cultures, we showed that antioxidant compounds prevented E17G-induced Mrp2 activity alteration, being this alteration equally prevented by the NADPH oxidase (NOX) inhibitor apocynin. The model antioxidant N-acetyl-cysteine prevented, in isolated and perfused rat livers, E17G-induced impairment of bile flow and Mrp2 activity, thus confirming the participation of reactive oxygen species (ROS) in this cholestasis. In primary cultured hepatocytes, pretreatment with specific inhibitors of ERK1/2 and p38MAPK impeded E17G-induced ROS production; contrarily, NOX inhibition did not affect ERK1/2 and p38MAPK phosphorylation. Both, knockdown of p47phox by siRNA and preincubation with apocynin in sandwich-cultured rat hepatocytes significantly prevented E17G-induced internalization of Mrp2, suggesting a crucial role for NOX in this phenomenon. Concluding, E17G-induced cholestasis is partially mediated by NOX-generated ROS through internalization of canalicular transporters like Mrp2, being ERK1/2 and p38MAPK necessary for NOX activation.

Identifying hepatoprotective mechanism and effective components of Yinchenzhufu decoction in chronic cholestatic liver injury using a comprehensive strategy based on metabolomics, molecular biology, pharmacokinetics, and cytology

Ethnopharmacological relevanceIn Traditional Chinese Medicine (TCM), cholestasis liver disease belongs to jaundice. Yinchenzhufu decoction (YCZFD) is a classic formula used for treating jaundice. Aim of the studyThis study was aimed to investigate the potential mechanism and effective components of YCZFD in chronic cholestatic liver injury (CCLI). Materials and methodsA chronic cholestatic mouse model induced by 3, 5-diethoxycarbonyl-1, 4-dihydroxychollidine was used to investigate the effect of YCZFD. Then, metabolomics was used to investigate the metabolites influenced by YCZFD. Serum and liver bile acid (BA) levels were measured using liquid chromatography coupled with triple quadruple mass spectrometry (LC-MS/MS), and the gene and protein expressions of BA transporters and metabolic enzymes were detected. Additionally, the pharmacokinetics of multiple components of YCZFD was explored to clarify the potential effective components. The effects of absorbed components of YCZFD on BA metabolism and transporter function, inflammation, and farnesoid X receptor (FXR) and pregnane X receptor (PXR) activation were analyzed using sandwich cultured rat hepatocytes, AML12 cells, and dual-luciferase receptor systems, respectively. ResultsYCZFD decreased the liver damage in chronic cholestatic mice. Serum metabolomics results indicated that the main pathways influenced by YCZFD involved primary BA biosynthesis and arachidonic acid metabolism. YCZFD upregulated the expression of FXR, PXR, and BA efflux transporters and the metabolic enzymes of liver tissues, promoting BA excretion and metabolism in cholestatic mice. Additionally, YCZFD downregulated the expression of genes and proteins of the toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway and decreased liver inflammation. The pharmacokinetic study indicated that multiple components showed different pharmacokinetic properties. Among the absorbed components of YCZFD, multiple components activated the transcription of FXR and PXR, regulated BA transporters and metabolic enzyme function, and reduced the gene expression of TLR4 and NF-κB1. ConclusionYCZFD can ameliorate CCLI by promoting the excretion and metabolism of BAs and inhibiting inflammation via the TLR4/NF-κB signaling pathway. The multiple components of YCZFD could act on BA homeostasis regulation and anti-inflammation, exhibiting a combined effect against CCLI.

2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucopyranoside enhances the hepatotoxicity of emodin in vitro and in vivo

Herb-induced liver injury results from the interplay between the herb and host with the herbal components serving as the major origin for hepatotoxicity. Although Polygoni Multiflori Radix (PMR) has been frequently reported to induce liver injury, contributions of its major components such as emodin, emodin-8-O-β-D-glucopyranoside, physcion and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucopyranoside (TSG) towards its hepatotoxicity have not been clearly identified. Our initial cytotoxicity screenings of the major PMR components using rat hepatocytes identified emodin as the most toxic. Subsequently, the bile acid homeostasis-related mechanisms of emodin and its combination treatment with TSG in PMR-associated liver injury were explored in sandwich-cultured rat hepatocytes (SCRH) and verified in rats. In SCRH, emodin was found to be able to induce total bile acid accumulation in a dose-dependent manner. In both SCRH and rats, the presence of TSG significantly enhanced the hepatotoxicity of emodin via i) increasing its hepatic exposure by inhibiting its glucuronidation mediated metabolism; ii) enhancing its disruption on bile acid homeostasis through amplifying its inhibition on bile acid efflux transporters and its up-regulation on bile acids synthesis enzymes; iii) enhancing its apoptosis. Our study for the first time demonstrated the critical role of the combination treatment with emodin and TSG in PMR-induced liver injury.

Mechanistic Study on the Species Differences in Excretion Pathway of HR011303 in Humans and Rats.

Excretion of [14C]HR011303-derived radioactivity showed significant species difference. Urine (81.50% of dose) was the main excretion route in healthy male subjects, whereas feces (87.16% of dose) was the main excretion route in rats. To further elucidate the underlying cause for excretion species differences of HR011303, studies were conducted to uncover its metabolism and excretion mechanism. M5, a glucuronide metabolite of HR011303, is the main metabolite in humans and rats. Results of a rat microsome incubation study suggested that HR011303 was metabolized to M5 in the rat liver. According to previous studies, M5 is produced in both human liver and kidney microsomes. We found that M5 in the human liver can be transported to the blood by multidrug resistance-associated protein (MRP) 3, and then the majority of M5 can be hydrolyzed to HR011303. HR011303 enters the human kidney or liver through passive diffusion, whereas M5 is taken up through organic anion transporter (OAT) 3, organic anion-transporting polypeptide (OATP) 1B1, and OATP1B3. When HR011303 alone is present, it can be metabolized to M5 in both sandwich-cultured rat hepatocytes (SCRH) and sandwich-cultured human hepatocytes (SCHH) and excreted into bile as M5 in SCRH. Using transporter inhibitors in sandwich-cultured model and membrane vesicles expressing MRP2 or Mrp2, we found that M5 was a substance of MRP2/Mrp2, and the bile efflux of M5 was mainly mediated by MRP2/Mrp2. Considering the significant role of MRP3/Mrp3 and MRP2/Mrp2 in the excretion of glucuronides, the competition between them for M5 was possibly the determinant for the different excretion routes in humans and rats. SIGNIFICANCE STATEMENT: Animal experiments are necessary to predict dosage and safety of candidate drugs prior to clinical trials. However, extrapolation results often differ from the actual situation. For HR011303, excretory pathways exhibited a complete reversal, through urine in humans and feces in rats. Such phenomena have been observed in several drugs, but no in-depth studies have been conducted to date. In the present study, the excretion species differences of HR011303 can be explained by the competition for M5 between MRP2/Mrp2 and MRP3/Mrp3.

Development of a Rat Sandwich-Cultured Hepatocytes Model Expressing Functional Human Organic Anion Transporting Polypeptide (OATP) 1B3: A Potential Screening Tool for Liver-Targeting Compounds.

Organic anion transporting polypeptide (OATP) 1B3 transports many clinically important drugs, including statins, from blood into the liver. It exclusively expresses in human liver under normal physiological conditions. There is no rodent ortholog of human OATP1B3. Tissue targeting of therapeutic molecules mediated by transporters, including liver-targeting via liver-specific OATPs, is an emerging area in drug development. Sandwich-cultured primary hepatocytes (SCH) are a well characterized in vitro model for assessment of hepatic drug uptake and biliary excretion. The current study was designed to develop a novel rat SCH model expressing human OATP1B3 to study the hepatic disposition of OATP1B3 substrates. Primary rat hepatocytes transduced with adenoviral vectors expressing FLAG-tagged OATP1B3 (Ad-OATP1B3), a control vector Ad-LacZ, or that were non-transduced were cultured in a sandwich configuration. FLAG immunoblot and immunofluorescence-staining determined expression and localization of OATP1B3. Uptake of [3H]-cholecystokinin octapeptide (CCK-8), a specific OATP1B3 substrate, was determined. Taurocholate (TC) is a substrate routinely used in SCH to assess biliary excretion via bile canaliculi (BC) and is also a substrate of OATP1B3. [3H]-TC accumulation in cells+BC, cells, biliary excretion index (BEI) and in vitro Clbiliary were determined using B-CLEAR® technology. OATP1B3 protein was extensively expressed and primarily localized on the plasma membrane in day 4 Ad-OATP1B3-transduced rat SCH. [3H]-CCK-8 accumulation in cells+BC was significantly greater (~5-13 folds, p<0.001) in day 4 SCH with vs. without Ad-OATP1B3-transduction. Expressing OATP1B3 in rat SCH significantly increased [3H]-TC accumulation in cells+BC and cells, without affecting BEI and in vitro Clbiliary. Rat SCH expressing human OATP1B3-is a novel in vitro model allowing simultaneous assessment of hepatic uptake, hepatocellular accumulation and biliary excretion process of a human OATP1B3 substrate. This model could be a potential tool for screening for liver-targeting compounds mediated by OATP1B3.

Triptolide and atorvastatin synergistically promote hepatotoxicity in cultured hepatocytes and female Sprague-Dawley rats by inhibiting pregnane X receptor-mediated transcriptional activation of CYP3A4

Triptolide (TP), an active component of Tripterygium wilfordii Hook. F, has been widely used in China for treating autoimmune and inflammatory diseases, and has also been validated by modern science and developed as a candidate anti-cancer treatment. However, liver toxicity of TP has seriously hindered its use and development, the clinical features and primary toxicological mechanism have been unclear. Considering the major target regulation mechanism of TP is the suppression of global transcription regulated by RNAPII, which is closed related with the detoxification of drugs. This paper tries to verify the synergistic liver injury and its mechanism of TP when co-administered with CYP3A4 substrate drug. The experiments showed that TP dose-dependently blocked transcriptional activation of CYP3A4 in both hPXR and hPXR-CYP3A4 reporter cell lines, lowered the mRNA and protein expression of PXR target genes such as CYP3A1, CYP2B1, and MDR1, and inhibited the functional activity of CYP3A in a time- and concentration-dependent manner in sandwich-cultured rat hepatocytes (SCRH) and female Sprague-Dawley (f-SD) rats. Furthermore, TP combined with atorvastatin (ATR), the substrate of CYP3A4, synergistically enhanced hepatotoxicity in cultured HepG2 and SCRH cells (CI is 0.38 and 0.29, respectively), as well as in f-SD rats, with higher exposure levels of both drugs. These results clearly indicate that TP inhibits PXR-mediated transcriptional activation of CYP3A4, leading to a blockade on the detoxification of itself and ATR, thereby greatly promoting liver injury. This study may implies the key cause of TP related liver injury and provides experimental data for the rational use of TP in a clinical scenario.

Numerical analysis of time-dependent inhibition kinetics: comparison between rat liver microsomes and rat hepatocyte data for mechanistic model fitting

Time-dependent inhibition (TDI) may confound drug interaction predictions. Recently, models were generated for an array of TDI kinetic schemes using numerical analysis of microsomal assays. Additionally, a distinct terminal inactivation step was identified for certain mechanism based inhibitors (MBI) following reversible metabolite intermediate complex (MIC) formation. Longer hepatocyte incubations potentially allow analysis of slow TDI and terminal inactivation. In the experiments presented here, we compared the quality of TDI parameterization by numerical analysis between hepatocyte and microsomal data. Rat liver microsomes (RLM), suspended rat hepatocytes (SRH) and sandwich-cultured rat hepatocytes (SCRH) were incubated with the prototypical CYP3A MBI troleandomycin and the substrate midazolam. Data from RLM provided a better model fit as compared to SRH. Increased CYP3A expression after dexamethasone (DEX) induction improved the fit for RLM and SRH. A novel sequential kinetic scheme, defining inhibitor metabolite production prior to MIC formation, improved the fit compared to direct MIC formation. Furthermore, terminal inactivation rate constants were parameterized for RLM and SRH samples with DEX-induced CYP3A. The low expression of CYP3A and experimental error in SCRH resulted in poor data for model fitting. Overall, RLM generated data better suited for elucidation of TDI mechanisms by numerical analysis.

Novel Mechanisms of Valproate Hepatotoxicity: Impaired Mrp2 Trafficking and Hepatocyte Depolarization.

Drug-induced liver injury (DILI) remains a major challenge in drug development. Although numerous mechanisms for DILI have been identified, few studies have focused on loss of hepatocyte polarization as a DILI mechanism. The current study investigated the effects of valproate (VPA), an antiepileptic drug with DILI risk, on the cellular mechanisms responsible for loss of hepatocyte polarization. Fully polarized collagen sandwich-cultured rat hepatocytes were treated with VPA (1-20 mM) for specified times (3-24 h). Hepatocyte viability was significantly decreased by 10 and 20 mM VPA. Valproate depolarized hepatocytes, even at noncytotoxic concentrations (≤5 mM). Depolarization was associated with significantly decreased canalicular levels of multidrug resistance-associated protein 2 (Mrp2) resulting in reduced canalicular excretion of the Mrp2 substrate carboxydichlorofluorescein. The decreased canalicular Mrp2 was associated with intracellular accumulation of Mrp2 in Rab11-positive recycling endosomes and early endosomes. Mechanistic studies suggested that VPA inhibited canalicular trafficking of Mrp2. This effect of VPA on Mrp2 appeared to be selective in that VPA had less impact on canalicular levels of the bile salt export pump (Bsep) and no detectable effect on P-glycoprotein (P-gp) canalicular levels. Treatment with VPA for 24 h also significantly downregulated levels of tight junction (TJ)-associated protein, zonula occludens 2 (ZO2), but appeared to have no effect on the levels of TJ proteins claudin 1, claudin 2, occludin, ZO1, and ZO3. These findings reveal that two novel mechanisms may contribute to VPA hepatotoxicity: impaired canalicular trafficking of Mrp2 and disruption of ZO2-associated hepatocyte polarization.

Baicalein Enhances the Oral Bioavailability and Hepatoprotective Effects of Silybin Through the Inhibition of Efflux Transporters BCRP and MRP2.

Although hepatoprotective properties of silybin are well documented, the clinical therapeutic efficacy is limited by its low bioavailability due to absorption rates, extensive phase II metabolism, and biliary excretion. As our previous study indicated that metabolic enzymes may have limited effects on the pharmacokinetic (PK) behavior of silymarin, here, we intended to increase the oral bioavailability and bio-efficacy of silybin through the inhibition of active efflux. In Caco-2 and transfected MDCKII cell models, flavone baicalein significantly inhibited the efflux of silybin as a BCRP and MRP2 inhibitor. In addition, baicalein reduced the biliary excretion index (BEI) and biliary clearance of silybin conjugates in the sandwich-cultured rat hepatocyte (SCH) model, indicating the inhibition of baicalein in biliary excretion of conjugated silybin metabolites. PK study demonstrated that baicalein significantly increased the area under the curve (AUC) and Cmax of silybin and its conjugates, suggesting enhanced absorption in vivo. Moreover, coadministration of silybin with baicalein boosted the liver protective, antioxidant, and anti-inflammatory effects of silybin in the carbon tetrachloride (CCl4)-induced liver injury model in comparison with silybin given alone. In summary, efflux transporters play a critical role in the low bioavailability of silybin, while inhibition of breast cancer resistance protein (BCRP) and multi-drug resistance protein 2 (MRP2) by baicalein can significantly increase the absorption and bio-efficacy of silybin, which provides a new combination therapeutic approach for the treatment of chronic liver diseases.

Piperine enhances the bioavailability of silybin via inhibition of efflux transporters BCRP and MRP2

BackgroundAlthough silybin serves as a well-known hepatoprotective agent with prominent anti-inflammatory, anti-oxidant and anti-fibrotic activities, its low bioavailability limits its application in the treatment of chronic liver diseases. However, novel formulation products with increased solubility were not sufficient to achieve pharmacologically meaningful concentrations of silybin in the clinical studies even used at high dosage. Hypothesis/PurposeWe hypothesized that inhibiting efflux transporter(s) and/or glucuronidation by piperine might enhance the bioavailability and efficacy of silybin. MethodsPharmacokinetics of silybin given alone or in-combination with piperine was determined by a validated LC-MS method. A CCl4 induced rat model of liver injury was prepared and verified for comparing the effects of silybin and combination treatment. To investigate the underlying mechanism, the inhibition effects of piperine on transportation of silybin were performed in Caco-2 and transfected MDCKII cell lines as well as sandwich–cultured rat hepatocytes (SCH). Human liver microsomes incubation was used for exploring the modulation effects of piperine on the phase-2 metabolism of silybin. ResultsIn the present study, we demonstrated for the first time that piperine as a bioenhancer increased the bioavailability of silybin (146%– 181%), contributing to a boosted therapeutic effect in CCl4-induced acute liver-injury rat model. The underlying mechanisms involved that piperine enhanced the absorption of silybin by inhibiting the efflux transporters including MRP2 and BCRP but not MDR1 in Caco-2 and transfected MDCKII cell lines. Moreover, piperine could inhibit the biliary excretion of silybin and conjugated metabolites in sandwich–cultured rat hepatocytes. Notably, we found that piperine did not affect the phase-2 metabolism of silybin. ConclusionEfflux transporters play an important role in the pharmacokinetic behavior of flavolignans, and modulating these transporters by bioenhancer such as piperine could enhance the in vivo absorption of silybin, leading to more effective treatments.

Mechanisms of canalicular transporter endocytosis in the cholestatic rat liver

Impaired canalicular secretion due to increased endocytosis and intracellular retention of canalicular transporters such as BSEP and MRP2 is a main, common pathomechanism of cholestasis. Nevertheless, the mechanisms governing this process are unknown. We characterized this process in estradiol 17 β-d-glucuronide (E17G)-induced cholestasis, an experimental model which partially mimics pregnancy-induced cholestasis. Inhibitors of clathrin-mediated endocytosis (CME) such as monodansylcadaverine (MDC) or K+ depletion, but not the caveolin-mediated endocytosis inhibitors filipin and genistein, prevented E17G-induced endocytosis of BSEP and MRP2, and the associated impairment of activity of these transporters in isolated rat hepatocyte couplets (IRHC). Immunofluorescence and confocal microscopy studies showed that, in E17G-treated IRHC, there was a significant increase in the colocalization of MRP2 with clathrin, AP2, and Rab5, three essential members of the CME machinery. Knockdown of AP2 by siRNA in sandwich-cultured rat hepatocytes completely prevented E17G-induced endocytosis of BSEP and MRP2. MDC significantly prevented this endocytosis, and the impairment of bile flow and biliary secretion of BSEP and MRP2 substrates, in isolated and perfused livers. BSEP and MRP2, which were mostly present in raft (caveolin-enriched) microdomains in control rats, were largely found in non-raft (clathrin-enriched) microdomains in livers from E17G-treated animals, from where they can be readily recruited for CME. In conclusion, our findings show that CME is the mechanism responsible for the internalization of the canalicular transporters BSEP and MRP2 in E17G-induced cholestasis. The shift of these transporters from raft to non-raft microdomains could be a prerequisite for the transporters to be endocytosed under cholestatic conditions.

Successful prediction of in vivo hepatobiliary clearance and hepatic concentrations of rosuvastatin using sandwich-cultured rat hepatocytes, transporter-expressing cell lines, and quantitative proteomics

Successful prediction of in vivo hepatobiliary clearance and hepatic concentrations of rosuvastatin using sandwich-cultured rat hepatocytes, transporter-expressing cell lines, and quantitative proteomics

Metabolism of the synthetic cannabinoid 5F-PY-PICA by human and rat hepatocytes and identification of biliary analytical targets by directional efflux in sandwich-cultured rat hepatocytes using UHPLC-HR-MS/MS

Analytical strategies for detecting drugs in biological samples rely on information on metabolism and elimination. 5F-PY-PICA belongs to the group of synthetic cannabinoids that are known to undergo excretion into the bile.The aims of this study were the in vitro identification of metabolites of 5F-PY-PICA and to determine which analytical targets are excreted into the bile and urine.Metabolites identified after incubation of 5F-PY-PICA with pooled human liver microsomes (pHLM), pooled human hepatocytes (pHH), or suspended and sandwich-cultured rat hepatocytes (SCRH). Rat hepatocytes were harvested following a two-step perfusion protocol and the SCRH were prepared between layers of rat-tail collagen. The biliary efflux of 5F-PY-PICA and its metabolites was determined in three-day–cultured SCRH by differential efflux into either standard buffer from intact bile canaliculi or standard buffer without divalent cations, which disrupts the bile canaliculi. The metabolites were identified using liquid chromatography–high resolution mass spectrometry/mass spectrometry (LC-HR-MS/MS).The main metabolites were the COOH-ω-metabolite (M4) in pHH, the defluoro-HO-ω-metabolite (M3) in pHLM, and the COOH-pyrrolidine-metabolite (M6) in rat hepatocytes.Efflux into standard buffer without divalent cations was significantly higher (p<0.050) for 5F-PY-PICA, M4, and the HO-indole-glucuronide-metabolite (M22). M6 did not undergo significant biliary efflux, indicating that basolateral efflux dominates for this metabolite.5F-PY-PICA, M4, and M22 are proposed as analytical targets for bile analysis in forensic screening protocols, whereas M6 should be one of the main urinary targets for 5F-PY-PICA analysis.

P-gp is involved in the intestinal absorption and biliary excretion of afatinib in vitro and in rats.

Afatinib is an irreversible multi-targeted TKI, used in the treatment with EGFR mutated non-small cell lung cancer (NSCLC). The purpose of this study is to explore the molecular pharmacokinetic mechanism underlying the effect of P-gp inhibitors on the intestinal absorption and biliary excretion and to understand how P-gp inhibitors affect afatinib pharmacokinetics. Pharmacokinetics in vivo, in situ intestinal perfusion, perfused rat liver in situ, Caco-2 cells, P-gp ATPase activity, sandwich-cultured rat hepatocytes (SCRH) and transfected-cell transport were used in the evaluation. P-gp inhibitor verapamil (Ver) markedly increased the plasma concentrations and significantly decreased the biliary excretion of afatinib in vivo. Ver increased the intestinal absorption and decreased biliary excretion of afatinib in situ single-pass intestinal perfusion studies and in situ perfused rat liver, respectively. The accumulation of afatinib in Caco-2 cells was enhanced by Ver and Cyclosporin A (CsA). The biliary excretion index (BEI) of afatinib in SCRH was decreased by Ver and CsA, respectively. The net efflux ratio of afatinib was 2.3 across vector-/MDR1-MDCKII cell monolayers and was decreased by P-gp inhibitor. The activity of P-gp ATPase was induced by afatinib and the Km and Vmax were 1.05μM and 59.88nmol ATP/mg hP-gp/min, respectively. At least partly P-gp is involved in increasing the intestinal absorption and decreasing the biliary excretion of afatinib in rats.

Successful Prediction of In Vivo Hepatobiliary Clearances and Hepatic Concentrations of Rosuvastatin Using Sandwich-Cultured Rat Hepatocytes, Transporter-Expressing Cell Lines, and Quantitative Proteomics.

We determined whether in vivo transporter-mediated hepatobiliary clearance (CL) and hepatic concentrations of rosuvastatin (RSV) in the rat could be predicted by transport activity in sandwich-cultured rat hepatocytes (SCRHs) and/or transporter-expressing cell lines scaled by differences in transporter protein expression between SCRHs, cell lines, and rat liver. The predicted hepatobiliary CLs and hepatic concentrations of RSV were compared with our previously published positron emission tomography imaging data. Sinusoidal uptake CL ([Formula: see text]) and efflux (canalicular and sinusoidal) CLs of [3H]-RSV in SCRHs were evaluated in the presence and absence of Ca2+ and in the absence and presence of 1 mM unlabeled RSV (to estimate passive diffusion CL). [Formula: see text] of RSV into cells expressing organic anion transporting polypeptide (Oatp) 1a1, 1a4, and 1b2 was also determined. Protein expression of Oatps in SCRHs and Oatp-expressing cells was quantified by liquid chromatography tandem mass spectrometry. SCRHs well predicted the in vivo RSV sinusoidal and canalicular efflux CLs but significantly underestimated in vivo [Formula: see text]. Oatp expression in SCRHs was significantly lower than that in the rat liver. [Formula: see text], based on RSV [Formula: see text] into Oatp-expressing cells (active transport) plus passive diffusion CL in SCRHs, scaled by the difference in protein expression in Oatp cells versus SCRH versus rat liver, was within 2-fold of that observed in SCRHs or in vivo. In vivo hepatic RSV concentrations were well predicted by Oatp-expressing cells after correcting [Formula: see text] for Oatp protein expression. This is the first demonstration of the successful prediction of in vivo hepatobiliary CLs and hepatic concentrations of RSV using transporter-expressing cells and SCRHs.

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

IntroductionSandwich-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. MethodsThe 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. ResultsIn 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. DiscussionThe 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.

Role of tangeretin as a potential bioavailability enhancer for silybin: Pharmacokinetic and pharmacological studies

Biological responses of a variety of naturally occurring compounds in vivo were restrained by their poor oral bioavailability. Silybin, as one of the active ingredients of silymarin, has presented promising bioactivity for the treatment of chronic liver diseases and cancer. However, its exposure in body was limited. In this study, silybin was demonstrated to be substrates of both BCRP and MRP2 by utilizing monolayer Caco-2 cell model and confirmed in MDCK cells overexpressing specific efflux transporter. Of all compounds screened, tangeretin, a potent inhibitor of efflux transporters of BCRP, MRP2 and P-gp, was able to enhance exposure of silybin by inhibiting functions of the barriers mediating transcellular transport. Moreover, study carried out in sandwich-cultured rat hepatocyte (SCH) model showed that the biliary excretion index (BEI) and in vitro biliary clearance of silybin decreased as levels of tangeretin increased, indicating efflux transporters mediating biliary excretion of silybin might be involved. Pharmacokinetic behaviors of silybin in rats were altered by co-administration of tangeretin, in terms of increased AUC and Cmax of silybin by comparing with that of silybin given alone. In addition, results coming from CCl4-induced acute liver injury rat model revealed that protection effect of silybin against liver damage in the presence of tangeretin was significantly enhanced. All these data were evident that efflux transporters play a critical role in transcellular transport of silybin and account for its low bioavailability. Enhanced bioavailability of silybin with co-administration of tangeretin by significantly inhibiting the efflux transporters further boost its bioactivity which is of particular importance in clinical use.

Understanding the Interplay Between Uptake and Efflux Transporters Within In Vitro Systems in Defining Hepatocellular Drug Concentrations

One of the most holistic in vitro systems for prediction of intracellular drug concentrations is sandwich-cultured hepatocytes (SCH); however, a comprehensive evaluation of the utility of SCH to estimate uptake and biliary clearances and the need for additional kinetic parameters has yet to be carried out. Toward this end, we have selected 9 compounds (rosuvastatin, valsartan, fexofenadine, pravastatin, repaglinide, telmisartan, atorvastatin, saquinavir, and quinidine) to provide a range of physicochemical and hepatic disposition properties. Uptake clearances were determined in SCH and compared with conventional monolayer and suspension hepatocyte systems, previously reported by our laboratory. CLuptake ranged from 1 to 41 μL/min/106 cells in SCH which were significantly lower (1%-10%) compared with the other hepatocyte models. The hepatocyte-to-media unbound concentration ratio (Kpu) has been assessed and ranged 0.7-59, lower compared with other hepatocyte systems (8-280). Estimates of in vitro biliary clearance (CLbile) for 4 drugs were determined and were scaled to predict in vivo values using both intracellular concentration and media drug concentrations. These studies demonstrate that reduced uptake in rat SCH may limit drug access to canalicular efflux transport proteins and highlight the importance of elucidating the interplay between these proteins for accurate prediction of hepatic clearance.

Involvement of Reactive Metabolites of Diclofenac in Cytotoxicity in Sandwich-Cultured Rat Hepatocytes.

Diclofenac (DIC) is metabolized to reactive metabolites such as diclofenac acyl-β-d-glucuronide (DIC-AG). It is possible that such reactive metabolites could cause tissue damage by formation of covalent protein adducts and other modification of cellular proteins or by induction of immune responses against its covalent protein adducts. However, the detailed mechanisms of idiosyncratic drug-induced liver injury (DILI) have been unclear. The objective is to clarify the involvement of DIC-AG and 4'hydroxydiclofenac (4'OH-DIC) in acute DILI. We examined the effects of inhibiting DIC-AG and 4'OH-DIC production on covalent protein adduct formation and lactate dehydrogenase leakage using sandwich-cultured rat hepatocytes (SCRHs). After pretreatment of SCRH with (-)-borneol (BOR, a uridine diphosphate (UDP)-glucuronosyltransferase inhibitor) or sulfaphenazole (SUL, a cytochrome P450 2C9 inhibitor) for 30 minutes, intracellular concentrations of DIC, DIC-AG, and 4'OH-DIC were determined after further treating cells with 300 μM DIC for 3 hours. The decreased levels of reactive metabolites caused by BOR or SUL pretreatment resulted in decreased lactate dehydrogenase leakage from SCRH, although the formation of covalent protein adducts was not affected. These results suggested that both DIC-AG and 4'OH-DIC may be involved in acute cytotoxicity by DIC.

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

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