Sandwich-cultured Hepatocyte Model Research Articles

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.

Dose-, treatment- and time-dependent toxicity of superparamagnetic iron oxide nanoparticles on primary rat hepatocytes.

As a first study in literature, to investigate concentration-dependent (0-400μg/ml) and exposure-dependent (single dosing vs cumulative dosing) effects of superparamagnetic iron oxide nanoparticles (d=10nm) on primary rat hepatocytes in a time-dependent manner. Sandwich-cultured hepatocyte model was used to evaluate viability, hepatocyte specific functions and reactive oxygen species level. In terms of all parameters, generally statistically more significant effects were observed in a concentration- and time-dependent manner. In terms of hepatocyte-specific functions, cumulative dosing caused significantly (p<0.05) more deleterious effects at 48th hour. A combination of various biomarkers should be employed for the evaluation of the effect of superparamagnetic iron oxide nanoparticles on liver, and each biomarker should be analyzed in a time- and exposure-dependent manner.

In vitro model systems to investigate bile salt export pump (BSEP) activity and drug interactions: A review

The bile salt export pump protein (BSEP), expressed on the canalicular membranes of hepatocytes, is primarily responsible for the biliary excretion of bile salts. The inhibition of BSEP transport activity can lead to an increase in intracellular bile salt levels and liver injury. This review discusses the various in vitro assays currently available for assessing the effect of drugs or other chemical entities to modulate BSEP transport activity. BSEP transporter assays use one of the following platforms: Xenopus laevis oocytes; canalicular membrane vesicles (CMV); BSEP-expressed membrane vesicles; cell lines expressing BSEP; sandwich cultured hepatocytes (SCH); and hepatocytes in suspension. Two of these, BSEP-expressed insect membrane vesicles and sandwich cultured hepatocytes, are the most commonly used assays. BSEP membrane vesicles prepared from transfected insect cells are useful for assessing BSEP inhibition or substrate specificity and exploring mechanisms of BSEP-associated genetic diseases. This model can be applied in a high-throughput format for discovery-drug screening. However, experimental results from use of membrane vesicles may lack physiological relevance and the model does not allow for investigation of in situ metabolism in modulation of BSEP activity. Hepatocyte-based assays that use the SCH format provide results that are generally more physiologically relevant than membrane assays. The SCH model is useful in detailed studies of the biliary excretion of drugs and BSEP inhibition, but due to the complexity of SCH preparation, this model is used primarily for determining biliary clearance and BSEP inhibition in a limited number of compounds. The newly developed hepatocyte in suspension assay avoids many of the complexities of the SCH method. The use of pooled cryopreserved hepatocytes in suspension minimizes genetic variance and individual differences in BSEP activity and also provides the opportunity for higher throughput screening and cross-species comparisons.

Mechanism-based pharmacokinetic modeling to evaluate transporter-enzyme interplay in drug interactions and pharmacogenetics of glyburide.

The purpose of this study is to characterize the involvement of hepato-biliary transport and cytochrome-P450 (CYP)-mediated metabolism in the disposition of glyburide and predict its pharmacokinetic variability due to drug interactions and genetic variations. Comprehensive in vitro studies suggested that glyburide is a highly permeable drug with substrate affinity to multiple efflux pumps and to organic anion transporting polypeptide (OATP)1B1 and OATP2B1. Active hepatic uptake was found to be significantly higher than the passive uptake clearance (15.8 versus 5.3 μL/min/10(6)-hepatocytes), using the sandwich-cultured hepatocyte model. In vitro, glyburide is metabolized (intrinsic clearance, 52.9 μL/min/mg-microsomal protein) by CYP3A4, CYP2C9, and CYP2C8 with fraction metabolism of 0.53, 0.36, and 0.11, respectively. Using these in vitro data, physiologically based pharmacokinetic models, assuming rapid-equilibrium between blood and liver compartments or permeability-limited hepatic disposition, were built to describe pharmacokinetics and evaluate drug interactions. Permeability-limited model successfully predicted glyburide interactions with rifampicin and other perpetrator drugs. Conversely, model assuming rapid-equilibrium mispredicted glyburide interactions, overall, suggesting hepatic uptake as the primary rate-determining process in the systemic clearance of glyburide. Further modeling and simulations indicated that the impairment of CYP2C9 function has a minimal effect on the systemic exposure, implying discrepancy in the contribution of CYP2C9 to glyburide clearance.

Role of Hepatic Efflux Transporters in Regulating Systemic and Hepatocyte Exposure to Xenobiotics

Hepatic efflux transporters include numerous well-known and emerging proteins localized to the canalicular or basolateral membrane of the hepatocyte that are responsible for the excretion of drugs into the bile or blood, respectively. Altered function of hepatic efflux transporters due to drug-drug interactions, genetic variation, and/or disease states may lead to changes in xenobiotic exposure in the hepatocyte and/or systemic circulation. This review focuses on transport proteins involved in the hepatocellular efflux of drugs and metabolites, discusses mechanisms of altered transporter function as well as the interplay between multiple transport pathways, and highlights the importance of considering intracellular unbound concentrations of transporter substrates and/or inhibitors. Methods to evaluate hepatic efflux transport and predict the effects of impaired transporter function on systemic and hepatocyte exposure are discussed, and the sandwich-cultured hepatocyte model to evaluate comprehensively the role of hepatic efflux in the hepatobiliary disposition of xenobiotics is characterized.

Determination of Intracellular Unbound Concentrations and Subcellular Localization of Drugs in Rat Sandwich-Cultured Hepatocytes Compared with Liver Tissue

Prediction of clinical efficacy, toxicity, and drug-drug interactions may be improved by accounting for the intracellular unbound drug concentration (C(unbound)) in vitro and in vivo. Furthermore, subcellular drug distribution may aid in predicting efficacy, toxicity, and risk assessment. The present study was designed to quantify the intracellular C(unbound) and subcellular localization of drugs in rat sandwich-cultured hepatocytes (SCH) compared with rat isolated perfused liver (IPL) tissue. Probe drugs with distinct mechanisms of hepatocellular uptake and accumulation were selected for investigation. Following drug treatment, SCH and IPL tissues were homogenized and fractionated by differential centrifugation to enrich for subcellular compartments. Binding in crude lysate and cytosol was determined by equilibrium dialysis; the C(unbound) and intracellular-to-extracellular C(unbound) ratio (K(pu,u)) were used to describe accumulation of unbound drug. Total accumulation (K(pobserved)) in whole tissue was well predicted by the SCH model (within 2- to 3-fold) for the selected drugs. Ritonavir (K(pu,u) ∼1) was evenly distributed among cellular compartments, but highly bound, which explained the observed accumulation within liver tissue. Rosuvastatin was recovered primarily in the cytosolic fraction, but did not exhibit extensive binding, resulting in a K(pu,u) >1 in liver tissue and SCH, consistent with efficient hepatic uptake. Despite extensive binding and sequestration of furamidine within liver tissue, a significant portion of cellular accumulation was attributed to unbound drug (K(pu,u) >16), as expected for a charged, hepatically derived metabolite. Data demonstrate the utility of SCH to predict quantitatively total tissue accumulation and elucidate mechanisms of hepatocellular drug accumulation such as active uptake versus binding/sequestration.

Comparison of In Vitro-In Vivo Extrapolation of Biliary Clearance Using an Empirical Scaling Factor Versus Transport-Based Scaling Factors in Sandwich-Cultured Rat Hepatocytes

Biliary clearance (CLb ) is often underestimated by in vitro-in vivo extrapolation from sandwich-cultured hepatocytes (SCHs). The objective of this study was to compare the performance of a universal correction factor with transporter-based correction factors in correcting underestimation of CLb . The apparent in vitro CLb of a training set of 21 compounds was determined using the SCH model and extrapolated to apparent in vivo CLb (CLb, app ). A universal correction factor (10.2) was obtained by a linear regression of the predicted CLb, app and observed in vivo CLb of training set compounds and applied to an independent test set (n = 20); the corrected CLb predictions of 13 compounds were within twofold error of observed values. Furthermore, two transporter-based correction factors (Organic anion transporting polypeptides/multidrug-resistance-related protein 2 and diffusion/P-glycoprotein) were estimated by linear regression analysis of training set compounds. The applications of the two correction factors to the test set resulted in improved prediction precision. In conclusion, both the universal correction factor and transporter-based correction factors provided reasonable corrections of CLb values, which are often underestimated by the SCH model. The use of transporter-based correction factors resulted in an even greater improvement of predictions for compounds with intermediate-to-high CLb values.

Sandwich-cultured hepatocytes: utility for in vitro exploration of hepatobiliary drug disposition and drug-induced hepatotoxicity

Introduction: The sandwich-cultured hepatocyte (SCH) model has become an invaluable in vitro tool for studying hepatic drug transport, metabolism, biliary excretion and toxicity. The relevant expression of many hepatocyte-specific functions together with the in vivo-like morphology favor SCHs over other preclinical models for evaluating hepatobiliary drug disposition and drug-induced hepatotoxicity.Areas covered: In this review, the authors highlight recommended procedures required for reproducibly culturing hepatocytes in sandwich configuration. It also provides an overview of the SCH model characteristics as a function of culture time. Lastly, the article presents a summary of the most prominent applications of the SCH model, including hepatic drug clearance prediction, drug–drug interaction potential and drug-induced hepatotoxicity.Expert opinion: When human (cryopreserved) hepatocytes are used to establish sandwich cultures, the model appears particularly valuable to quantitatively investigate clinically relevant mechanisms related to in vivo hepatobiliary drug disposition and hepatotoxicity. Nonetheless, the SCH model would largely benefit from better insight into the fundamental cell signaling mechanisms that are critical for long-term in vitro maintenance of the hepatocytic phenotype. Studies systematically exploring improved cell culture conditions (e.g., co-cultures or extracellular matrix modifications), as well as in vitro work identifying key transcription factors involved in hepatocyte differentiation are currently emerging.

Assessment of Biliary Clearance in Early Drug Discovery using Sandwich-cultured Hepatocyte Model

It is challenging to predict biliary clearance (CL(b) ) for new chemical entities (NCEs) in early drug discovery. Although sandwich-cultured hepatocyte (SCH) model has offered a valuable tool for characterizing hepatobiliary disposition and drug-drug interaction potential of NCEs, no comprehensive study was reported to project in vivo biliary clearance (in vivo CL(b,observed) ) potential using in vitro SCH model during the drug discovery stage. In this study, the CL(b) of 110 discovery compounds was evaluated using rat SCH model. Parallel artificial membrane permeability assay, Caco-2, and rat liver microsomes were employed in parallel to explore the interplay of biliary excretion with cellular permeability and liver metabolism. Selected compounds were further tested in bile-duct-cannulated rats, confirming the value of the SCH model for ranking and predicting in vivo CL(b,observed) during drug discovery. For compounds with extremely low passive permeability and metabolism, rat SCH may underestimate in vivo CL(b,observed) . The combination of passive permeability, metabolic intrinsic clearance, and the SCH model could serve as an initial screening platform for biliary excretion potential as well as a means for improving compound liabilities and properties. A preliminary evaluation strategy was proposed to highlight biliary excretion risk evaluation during the drug discovery process.

Evaluation of 99mTechnetium-Mebrofenin and 99mTechnetium-Sestamibi as Specific Probes for Hepatic Transport Protein Function in Rat and Human Hepatocytes

This study characterized 99mTc-Mebrofenin (MEB) and 99mTc-Sestamibi (MIBI) hepatic transport and preferential efflux routes (canalicular vs. basolateral) in rat and human sandwich-cultured hepatocytes (SCH). 99mTc-MEB and 99mTc-MIBI disposition was determined in suspended hepatocytes and in SCH in the presence and absence of inhibitors and genetic knockdown of breast cancer resistance protein (Bcrp). The general organic anion transporting polypeptide (Oatp/OATP) inhibitor rifamycin SV reduced initial 99mTc-MEB uptake in rat and human suspended hepatocytes. Initial 99mTc-MIBI uptake in suspended rat hepatocytes was not Na+-dependent or influenced by inhibitors. Multidrug resistance-associated protein (Mrp2/MRP2) inhibitors decreased 99mTc-MEB canalicular efflux in rat and human SCH. 99mTc-MEB efflux in human SCH was predominantly canalicular (45.8 +/- 8.6%) and approximately 3-fold greater than in rat SCH. 99mTc-MIBI canalicular efflux was similar in human and rat SCH; basolateral efflux was 37% greater in human than rat SCH. 99mTc-MIBI cellular accumulation, biliary excretion index and in vitro biliary clearance in rat SCH were unaffected by Bcrp knockdown. 99mTc-MEB hepatic uptake is predominantly Oatp-mediated with biliary excretion by Mrp2. 99mTc-MIBI appears to passively diffuse into hepatocytes; biliary excretion is mediated by P-gp. The SCH model is useful to investigate factors that may alter the route and/or extent of hepatic basolateral and canalicular efflux of substrates.

Understanding the clinical pharmacokinetics of a GABAA partial agonist by application of in vitro tools

4-Oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxylic acid (4-methylaminomethyl-phenyl)-amide (1), developed for general anxiety disorder, was discontinued from clinical development due to unsuitable oral pharmacokinetics.In humans, (1) demonstrated an unacceptable high apparent oral clearance (Clp/F) that also demonstrated a supraproportional dose-exposure relationship. Secondary peaks in the plasma concentration-time profile suggested possible enterohepatic recirculation of (1). A combination of in vitro mechanistic tools was applied to better understand the processes underlying these complex clinical pharmacokinetic profiles of (1).In metabolism experiments, (1) was shown to be a substrate of monoamine oxidase A (MAO-A) as well as being metabolized by cytochrome P450. The former appeared to be a high KM process with a high capacity, while the latter showed saturation between 1 and 10 μM, consistent with the supraproportional dose-exposure relationship.In a sandwich-cultured hepatocyte model, (1) was shown to be a substrate for both uptake and efflux into the canicular space, which is consistent with the observation of pharmacokinetics suggestive of enterohepatic recirculation. Finally, in human epithelial colon adenocarcinoma cell line (Caco-2) and Madin-Darby canine kidney cells transwell flux experiments, (1) was shown to have relatively low permeability and a basolateral-to-apical flux ratio consistent with the activity of P-glycoprotein.In combination, a compounding of the contributions of MAO-A, hepatic uptake and efflux transporters, and P-glycoprotein to the disposition of (1) may underlie the low oral exposure, saturable clearance, and aberrant concentration versus time profiles observed for this compound in humans.

Use of Sandwich-Cultured Hepatocytes To Evaluate Impaired Bile Acid Transport as a Mechanism of Drug-Induced Hepatotoxicity

Drug-induced liver toxicity is a significant problem in drug development and clinical practice, yet its mechanisms are not well understood. Growing evidence suggests that inhibition of bile acid transport may be one mechanism of hepatotoxicity. A number of hepatic transporters work in concert to transport bile acids and xenobiotics from blood to bile, and many drugs have been shown to perturb this process with detrimental consequences. Hepatocytes cultured in a sandwich configuration maintain transporter activity and liver-specific metabolic functions; thus, the sandwich-cultured hepatocyte model represents a useful tool for evaluating hepatotoxicity caused by interference with hepatic transporters. As an example, the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist troglitazone is one such drug that has been shown to inhibit bile acid transport in vitro. Data presented in this manuscript indicate that troglitazone inhibits both basolateral uptake and canalicular excretion of taurocholate in a concentration-dependent manner in both sandwich-cultured and suspended human and rat hepatocytes. These data confirm both the interaction of troglitazone with bile acid transporters in hepatocytes and the utility of the sandwich-cultured hepatocyte model to study such interactions.

Hepatobiliary Disposition of a Drug/Metabolite Pair: Comprehensive Pharmacokinetic Modeling in Sandwich-Cultured Rat Hepatocytes

The hepatobiliary disposition of xenobiotics may involve passive and/or active uptake, metabolism by cytochromes P450, and excretion of the parent compound and/or metabolite(s) into bile. Although in vitro systems have been used to evaluate these individual processes discretely, mechanistic in vitro studies of the sequential processes of uptake, metabolism, and biliary or basolateral excretion are limited. The current studies used sandwich-cultured (SC) rat hepatocytes combined with a comprehensive pharmacokinetic modeling approach to investigate the hepatobiliary disposition of terfenadine and fexofenadine, a model drug/metabolite pair. The metabolism of terfenadine and the biliary excretion of terfenadine and fexofenadine were determined in control and dexamethasone-treated SC rat hepatocytes. Dexamethasone (DEX) treatment increased the formation rates of the terfenadine metabolites azacyclonol and fexofenadine approximately 20- and 2-fold, respectively. The biliary excretion index (BEI) of fexofenadine, when generated by terfenadine metabolism, was not significantly different from the BEI of preformed fexofenadine (15 +/- 2% versus 19 +/- 2%, respectively). Pharmacokinetic modeling revealed that the rate constant for hepatocyte uptake was faster for terfenadine compared with preformed fexofenadine (2.5 versus 0.08 h(-1), respectively), whereas the biliary excretion rate constant for preformed fexofenadine exceeded that of terfenadine (0.44 versus 0.039 h(-1), respectively). Interestingly, the rate constants for basolateral excretion of terfenadine and fexofenadine were comparable (3.2 versus 1.9 h(-1), respectively) and increased only slightly with DEX treatment. These studies demonstrate the utility of the SC hepatocyte model, coupled with pharmacokinetic modeling, to evaluate the hepatobiliary disposition of generated metabolites.