Sandwich-cultured Human Hepatocytes Research Articles

Mathematical modeling analysis of hepatic uric acid disposition using human sandwich-cultured hepatocytes

Uric acid is biosynthesized from purine by xanthine oxidase (XO) mainly in the liver and is excreted into urine and feces. Although several transporters responsible for renal and intestinal handling of uric acid have been reported, information on hepatic transporters is limited. In the present study, we studied quantitative contribution of transporters for hepatic handling of uric acid by mathematical modeling analysis in human sandwich-cultured hepatocytes (hSCH). Stable isotope-labeled hypoxanthine, hypoxanthine-13C2,15N (HX), was incubated with hSCH and formed 13C2,15N-labeled xanthine (XA) and uric acid (UA) were measured by LC-MS/MS time dependently. Rate constants for metabolism and efflux and uptake transport across sinusoidal and bile canalicular membranes of HX, XA and UA were estimated in the presence of inhibitors of XO and uric acid transporters. An XO inhibitor allopurinol significantly decreased metabolisms of HX and XA. Efflux into bile canalicular lumen was negligible and sinusoidal efflux was considered main efflux pathway of formed UA. Transporter inhibition study highlighted that GLUT9 strongly and MRP4 intermediately contribute to the sinusoidal efflux of UA with minor contribution of NPT1/4. Modeling analysis developed in the present study should be useful for quantitative prediction of uric acid disposition in liver.

Role of Organic Solute Transporter Alpha/Beta in Hepatotoxic Bile Acid Transport and Drug Interactions.

Organic solute transporter (OST) α/β is a key bile acid transporter expressed in various organs, including the liver under cholestatic conditions. However, little is known about the involvement of OSTα/β in bile acid-mediated drug-induced liver injury (DILI), a major safety concern in drug development. This study investigated whether OSTα/β preferentially transports more hepatotoxic, conjugated, primary bile acids and to what extent xenobiotics inhibit this transport. Kinetic studies with OSTα/β-overexpressing cells revealed that OSTα/β preferentially transported bile acids in the following order: taurochenodeoxycholate > glycochenodeoxycholate > taurocholate > glycocholate. The apparent half-maximal inhibitory concentrations for OSTα/β-mediated bile acid (5 µM) transport inhibition by fidaxomicin, troglitazone sulfate, and ethinyl estradiol were: 210, 334, and 1050 µM, respectively, for taurochenodeoxycholate; 97.6, 333, and 337 µM, respectively, for glycochenodeoxycholate; 140, 265, and 527 µM, respectively, for taurocholate; 59.8, 102, and 117 µM, respectively, for glycocholate. The potential role of OSTα/β in hepatocellular glycine-conjugated bile acid accumulation and cholestatic DILI was evaluated using sandwich-cultured human hepatocytes (SCHH). Treatment of SCHH with the farnesoid X receptor agonist chenodeoxycholate (100 µM) resulted in substantial OSTα/β induction, among other proteomic alterations, reducing glycochenodeoxycholate and glycocholate accumulation in cells+bile 4.0- and 4.5-fold, respectively. Treatment of SCHH with troglitazone and fidaxomicin together under cholestatic conditions resulted in increased hepatocellular toxicity compared with either compound alone, suggesting that OSTα/β inhibition may accentuate DILI. In conclusion, this study provides insights into the role of OSTα/β in preferential disposition of bile acids associated with hepatotoxicity, the impact of xenobiotics on OSTα/β-mediated bile acid transport, and the role of this transporter in SCHH and cholestatic DILI.

Hepatic Transporter Alterations by Nuclear Receptor Agonist T0901317 in Sandwich-Cultured Human Hepatocytes: Proteomic Analysis and PBPK Modeling to Evaluate Drug-Drug Interaction Risk

In vitro approaches for predicting drug-drug interactions (DDIs) caused by alterations in transporter protein regulation are not well established. However, reports of transporter regulation via nuclear receptor (NR) modulation by drugs are increasing. This study examined alterations in transporter protein levels in sandwich-cultured human hepatocytes (SCHH; n = 3 donors) measured by liquid chromatography-tandem mass spectrometry-based proteomic analysis after treatment with N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide (T0901317), the first described synthetic liver X receptor agonist. T0901317 treatment (10 μM, 48 hours) decreased the levels of organic cation transporter (OCT) 1 (0.22-, 0.43-, and 0.71-fold of control) and organic anion transporter (OAT) 2 (0.38-, 0.38-, and 0.53-fold of control) and increased multidrug resistance protein (MDR) 1 (1.37-, 1.48-, and 1.59-fold of control). The induction of NR downstream gene expression supports the hypothesis that T0901317 off-target effects on farnesoid X receptor and pregnane X receptor activation are responsible for the unexpected changes in OCT1, OAT2, and MDR1. Uptake of the OCT1 substrate metformin in SCHH was decreased by T0901317 treatment. Effects of decreased OCT1 levels on metformin were simulated using a physiologically-based pharmacokinetic (PBPK) model. Simulations showed a clear decrease in metformin hepatic exposure resulting in a decreased pharmacodynamic effect. This DDI would not be predicted by the modest changes in simulated metformin plasma concentrations. Altogether, the current study demonstrated that an approach combining SCHH, proteomic analysis, and PBPK modeling is useful for revealing tissue concentration-based DDIs caused by unexpected regulation of hepatic transporters by NR modulators. SIGNIFICANCE STATEMENT: This study utilized an approach combining sandwich-cultured human hepatocytes, proteomic analysis, and physiologically based pharmacokinetic modeling to evaluate alterations in pharmacokinetics (PK) and pharmacodynamics (PD) caused by transporter regulation by nuclear receptor modulators. The importance of this approach from a mechanistic and clinically relevant perspective is that it can reveal drug-drug interactions (DDIs) caused by unexpected regulation of hepatic transporters and enable prediction of altered PK and PD changes, especially for tissue concentration-based DDIs.

Probe Cocktail to Assess Transporter Function in Sandwich-Cultured Human Hepatocytes.

Probe substrates are used routinely to assess transporter function in vitro. Administration of multiple probe substrates together as a "cocktail" in sandwich-cultured human hepatocytes (SCHH) could increase the throughput of transporter function assessment in a physiologically-relevant in vitro system. This study was designed to compare transporter function between cocktail and single agent administration in SCHH. Rosuvastatin, digoxin, and metformin were selected as probe substrates of hepatic transporters OATP1B1, OATP1B3, BCRP, P-gp, and OCT1. Total accumulation (Cells+Bile) and biliary excretion index (BEI) values derived from administration of the cocktail were compared to values obtained after administration of single agents in the absence and presence of a model inhibitor, erythromycin estolate. For rosuvastatin and metformin accumulation, the ratio of means [90% confidence interval (CI)] for cocktail to single agent administration was 100% [94%, 106%] and 90% [82%, 99%], respectively. Therefore, the cocktail and single-agent mode of administration were deemed equivalent per standard equivalence criterion of 80-120% for rosuvastatin and metformin accumulation, but not for digoxin accumulation (77% [62%, 92%]). The ratio of means [90% CI] for rosuvastatin BEI values between the two administration modes (105% [97%, 114%]) also was deemed equivalent. The ratio for digoxin BEI values between the two administration modes was 99% [78%, 120%]. In the presence of erythromycin estolate, the two administration modes were deemed equivalent for evaluation of rosuvastatin, digoxin, and metformin accumulation; the ratio of means [90% CI] was 104% [94%, 115%], 94% [82%, 105%], and 100% [88%, 111%], respectively. However, rosuvastatin and digoxin BEI values were low and quite variable in the presence of the inhibitor, so the BEI results were inconclusive. These data suggest that rosuvastatin and metformin can be administered as a cocktail to evaluate the function of OATP1B1, OATP1B3, BCRP, and OCT1 in SCHH, and that digoxin may not be an ideal component of such a cocktail.

Mechanistic Modeling of the Hepatic Disposition of Estradiol-17β-Glucuronide in Sandwich-Cultured Human Hepatocytes.

Estradiol-17β-glucuronide (E217G) is an estrogen metabolite that has cholestatic properties. In humans, circulating E217G is transported into hepatocytes by organic anion transporting polypeptides (OATPs) and is excreted into bile by multidrug-resistance associated protein 2 (MRP2). E217G is also a substrate of the basolateral efflux transporters MRP3 and MRP4, which translocate E217G from hepatocytes to blood. However, the contribution of basolateral efflux to hepatocyte disposition of E217G has not been evaluated previously. To address this question, E217G disposition was studied in sandwich-cultured human hepatocytes and mechanistic modeling was applied to calculate clearance values (mean ± S.D.) for uptake, intrinsic biliary excretion (CLint,bile) and intrinsic basolateral efflux (CLint,BL). The biliary excretion index of E217G was 45% ± 6%. The CLint,BL of E217G [0.18 ± 0.03 (ml/min)/g liver) was 1.6-fold higher than CLint,bile [0.11 ± 0.06 (ml/min)/g liver]. Simulations were performed to study the effects of increased CLint,BL and a concomitant decrease in CLint,bile on hepatic E217G exposure. Results demonstrated that increased CLint,BL can effectively reduce hepatocellular and biliary exposure to this potent cholestatic agent. Simulations also revealed that basolateral efflux can compensate for impaired biliary excretion and, vice versa, to avoid accumulation of E217G in hepatocytes. However, when both clearance processes are impaired by 90%, hepatocyte E217G exposure increases up to 10-fold. These data highlight the contribution of basolateral efflux transport, in addition to MRP2-mediated biliary excretion, to E217G disposition in human hepatocytes. This elimination route could be important, especially in cases where basolateral efflux is induced, such as cholestasis. SIGNIFICANCE STATEMENT: The disposition of the cholestatic estrogen metabolite estradiol-17β-glucuronide (E217G) was characterized in sandwich-cultured human hepatocytes. The intrinsic basolateral efflux clearance was estimated to be 1.6-fold higher than the intrinsic biliary excretion clearance, emphasizing the contribution of basolateral elimination in addition to biliary excretion. Simulations highlight how hepatocytes can effectively cope with increased E217G through the regulation of both basolateral and biliary transporters.

Organic Anion-Transporting Polypeptide Genes Are Not Induced by the Pregnane X Receptor Activator Rifampin: Studies in Hepatocytes In Vitro and in Monkeys In Vivo.

Induction potentials of the pregnane X receptor (PXR) activator rifampin (RIF) on transporter genes [e.g., organic anion-transporting polypeptides (OATPs)] are still in its infancy or remain controversial in the field. The present investigations characterized changes in transporter gene expression by RIF in sandwich-cultured hepatocytes from multiple donors of human and cynomolgus monkey using real-time quantitative reverse transcription polymerase chain reaction method. Three-day treatment of RIF significantly induced CYP3A4 (∼60-fold induction), but not CYP1A2 and CYP2D6 genes. SLC51B was the most highly induced uptake transporter gene (>10-fold) in both human and monkey hepatocytes. A greater induction of CYP2C9 was observed in monkey hepatocytes than that in humans. ATP-binding cassette (ABC)B1 and ABCC2 were induced slightly above 2-fold in human and monkey hepatocytes and appeared to be dose-dependent. The induction of OATP and other transporter genes was generally less than 2-fold and considered not clinically relevant. SLCO2B1 was not detectable in monkey hepatocytes. To investigate in vivo OATP induction, RIF (18 mg/kg per day) was orally dosed to cynomolgus monkeys for 7 days. Pitavastatin and antipyrine were intravenously dosed before and after RIF treatment as exogenous probes of OATP and CYP activities, respectively. Plasma coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) were measured as OATP endogenous biomarkers. Although a significant increase of antipyrine clearance (CL) was observed after RIF treatment, the plasma exposures of pitavastatin, CP-I, and CP-III remained unchanged, suggesting that OATP function was not significantly altered. The results suggested that OATP transporters were not significantly induced by PXR ligand RIF. The data are consistent with current regulatory guidances that the in vitro characterization of transporter induction during drug development is not required. SIGNIFICANCE STATEMENT: Organic anion-transporting polypeptide (OATP) genes were not induced by rifampin in sandwich-cultured human and monkey hepatocytes OATP functions measured by OATP probe pitavastatin and endogenous marker coproporphyrins were not altered in monkeys in vivo by 7-day rifampin treatment. The data suggested that OATP transporters are unlikely induced by the pregnane X receptor ligand rifampin, which are consistent with current regulatory guidances that the in vitro characterization of OATP1B induction during drug development is not required.

Mechanisms of CYP3A Induction During Pregnancy: Studies in HepaRG Cells.

Activity of CYP3A, an enzyme responsible for metabolism of many marketed drugs, is induced by ~ 2-fold in pregnant women. Through studies in sandwich-cultured human hepatocytes (SCHH) and HepaRG cells, our laboratory has shown that this induction is likely mediated by the increase in cortisol plasma concentrations during pregnancy. Cortisol, at plasma concentrations observed during the third trimester (~ 800nM), either alone or in combination with other pregnancy-related hormones, induces CYP3A activity in SCHH and HepaRG cells when cultured in dexamethasone-free media. To determine the mechanism(s) by which cortisol induces CYP3A activity, HepaRG cells were pre-incubated in dexamethasone-free medium and then incubated for 72h with cortisol (798nM). Glucocorticoid receptor (GR), pregnane X receptor (PXR), and CYP3A4 or CYP3A5 were knocked down using siRNA, and mRNA expression of these genes was measured. CYP3A4, and not CYP3A5, was found to be the dominant contributor to total CYP3A activity in control- and cortisol-treated HepaRG cells. Constitutive mRNA expression of CYP3A4 in HepaRG cells was regulated by both PXR and GR whereas constitutive expression of CYP3A5 in HepaRG cells was regulated by GR alone. Cortisol-mediated CYP3A4 induction in HepaRG cells was primarily mediated by GR-dependent PXR induction pathway and to a smaller extent via a PXR-independent pathway. Cortisol-mediated CYP3A5 induction was regulated by GR-dependent PXR-independent pathway. These data indicate that PXR plays a central role in cortisol-mediated induction of CYP3A activity during pregnancy and suggests that other enzymes and transporters, such as CYP2B6 and P-glycoprotein, may also be induced during pregnancy via the same mechanism(s).

Mechanistic Evaluation of the Complex Drug-Drug Interactions of Maraviroc: Contribution of Cytochrome P450 3A, P-Glycoprotein and Organic Anion Transporting Polypeptide 1B1

The aim of the present study was to quantitatively evaluate the drug-drug interactions (DDIs) of maraviroc (MVC) with various perpetrator drugs, including telaprevir (TVR), using an in vitro data-informed physiologically based pharmacokinetic (PBPK) model. MVC showed significant active uptake and biliary excretion in sandwich-cultured human hepatocytes, and biphasic organic anion transporting polypeptide (OATP)1B1-mediated uptake kinetics in transfected cells (high-affinity K m ∼5 µM). No measureable active uptake was noted in OATP1B3- and OATP2B1-transfceted cells. TVR inhibited OATP1B1-mediated MVC transport in vitro, and also exhibited CYP3A time-dependent inhibition in human hepatocytes (inactivation constant, K I = 2.24 µM, and maximum inactivation rate constant, k inact = 0.0112 minute-1). The inactivation efficiency (k inact/K I) was approximately 34-fold lower in human hepatocytes compared with liver microsomes. A PBPK model accounting for interactions involving CYP3A, P-glycoprotein (P-gp), and OATP1B1 was developed based on in vitro mechanistic data. MVC DDIs with ketoconazole (inhibition of CYP3A and P-gp), ritonavir (inhibition of CYP3A and P-gp), efavirenz (induction of CYP3A), rifampicin (induction of CYP3A and P-gp; inhibition of OATP1B1), and TVR (inhibition of CYP3A, P-gp, and OATP1B1) were well described by the PBPK model with optimized transporter K i values implying that OATP1B1-mediated uptake along with CYP3A metabolism determines the hepatic clearance of MVC, and P-gp-mediated efflux limits its intestinal absorption. In summary, MVC disposition involves intestinal P-gp/CYP3A and hepatic OATP1B1/CYP3A interplay, and TVR simultaneously inhibits these multiple mechanisms leading to a strong DDI-about 9.5-fold increase in MVC oral exposure.

Detection of Drug-Induced Cholestasis Potential in Sandwich-Cultured Human Hepatocytes.

Drug-induced cholestasis poses a major hurdle for the pharmaceutical industry as it is one the primary mechanisms of drug-induced liver injury. Hence, detection of drug-induced cholestasis during the early stages of drug development is of utmost importance. The most commonly used in vitro models rely on the extent of inhibition of bile salt export pump-mediated taurocholic acid transport, thereby assuming that drug-induced cholestasis mechanisms are merely restricted to the interaction with this sole hepatic transporter. Sandwich-cultured human hepatocytes represent a more holistic in vitro tool to investigate drug-induced cholestasis as they preserve all relevant disposition pathways and cellular functions involved in bile acid homeostasis. We developed and validated a sandwich-cultured human hepatocytes-based in vitro assay which is able to identify compounds causing cholestasis by altering bile acid disposition. The in vitro cholestatic potential is expressed by calculating a drug-induced cholestasis index value, which reflects the relative residual urea formation of sandwich-cultured human hepatocytes co-incubated with bile acids and test compound as compared to sandwich-cultured human hepatocytes treated with test compound alone. In addition, a safety margin can be calculated to determine the in vivo risk for cholestasis based on the determination of the drug-induced cholestasis index at various concentrations and the peak plasma concentration of the drug candidate. This chapter outlines the various steps involved in performing our sandwich-cultured human hepatocytes-based in vitro assay.

An integrated in vitro screen using sandwich-cultured human hepatocytes for prediction of cholestatic hepatotoxicity

An integrated in vitro screen using sandwich-cultured human hepatocytes for prediction of cholestatic hepatotoxicity

Cholestatic Drug Induced Liver Injury: A Function of Bile Salt Export Pump Inhibition and Farnesoid X Receptor Antagonism

Introduction:In vitro bile salt export pump (BSEP) inhibition alone does not accurately predict in vivo drug-induced liver injury in humans, suggesting that other mechanisms may be involved. Inhibition of BSEP has been shown to increase the hepatocellular concentrations of bile acids (BAs), and hepatobiliary disposition of BAs is tightly regulated by the farnesoid X receptor (FXR). Activation of FXR leads to decreased synthesis of BAs and increased expression of BA efflux transporters, BSEP, and organic solute and steroid transporter (OSTα/β). The link between BSEP inhibition and activation of the BA compensatory mechanism has not been clearly demonstrated. Materials and Methods: Therefore, we utilized BSEP inhibitors, cyclosporine A (CsA) and troglitazone (Trog), to explore this “triggering” event using Transporter Certified™ sandwich-cultured human hepatocytes (SCHH). Results: Biliary excretion of glycine-cholic acid (GCA) as a percentage of total GCA accumulation was reduced in SCHH treated with either CsA or Trog. Within 12 hours, CsA treatment concomitantly increased intracellular concentrations (ICCs) of GCA and FGF19 mRNA content, an FXR-target gene. Separately, a synergistic 76.6-fold increase of OSTβ mRNA was observed following CsA and chenodeoxycholic acid co-exposure. In contrast, Trog exposure prevented the synergistic increase of OSTβ, a component of the basolateral BA efflux transporter, under the same conditions. Finally, BA toxicity potency was increased in SCHH exposed to Trog but not CsA. Discussion and Conclusions: Overall, these results suggested that BSEP inhibition activates the BA compensatory mechanism reducing ICCs of BAs. However, Trog possesses both BSEP inhibition and FXR antagonist properties blocking the activation of the compensatory mechanism resulting in BA-induced hepatotoxicity.

Evaluation of Drug Biliary Excretion Using Sandwich-Cultured Human Hepatocytes.

Evaluation of hepatobiliary transport of drugs is an important challenge, notably during the development of new molecular identities. In this context, sandwich-cultured human hepatocytes (SCHH) have been proposed as an interesting and integrated tool for predicting in vitro biliary excretion of drugs. The present review was therefore designed to summarize key findings about SCHH, including their establishment, their main functional features and their use for the determination of canalicular transport and the prediction of in vivo biliary clearance and hepatobiliary excretion-related drug-drug interactions. Reviewed data highlight the fact that SCHH represent an original and probably unique holistic in vitro approach to predict biliary clearance in humans, through taking into account sinusoidal drug uptake, passive drug diffusion, drug metabolism and sinusoidal and canalicular drug efflux. Limits and proposed refinements for SCHH-based analysis of drug biliary excretion, as well as putative human alternative in vitro models to SCHH are also discussed.

Total hepatocellular disposition profiling of rosuvastatin and pitavastatin in sandwich-cultured human hepatocytes

This study describes the total disposition profiling of rosuvastatin (RSV) and pitavastatin (PTV) using a single systematic procedure called D-PREX (Disposition Profile Exploration) in sandwich-cultured human hepatocytes (SCHH). The biliary excretion fractions of both statins were clearly observed, which were significantly decreased dependent on the concentration of Ko143, an inhibitor for breast cancer resistance protein (BCRP). Ko143 also decreased the basolateral efflux fraction of RSV, whereas that of PTV was not significantly affected. To understand these phenomena, effects of Ko143 on biliary excretion (BCRP and multidrug resistance-associated protein (MRP) 2) and basolateral efflux (MRP3 and MRP4) transporters were examined using transporter-expressing membrane vesicles. BCRP, MRP3 and MRP4-mediated transport of RSV was observed, and Ko143 inhibited these transporters except MRP3. BCRP and MRP4 also mediated the transport of PTV, but the Ko143-mediated inhibition was only clear for BCRP. These results might explain the Ko143-mediated complete and partial inhibition of the biliary excretion and the basolateral efflux of RSV, respectively, in SCHH. In conclusion, D-PREX with sequential sampling of supernatants prior to cell lysis enables the evaluation of total drug disposition profiles resulting from complex interplays of intracellular pathways, which would provide high-throughput evaluation of drug disposition during drug discovery.

Glycine Formation Drives Bile Acid Toxicity in Sandwich‐Cultured Human Hepatocyes

Disturbances in bile acid (BA) disposition and consequent intracellular accumulation of bile acids (BAs) are hypothesized to be associated with toxicity observed in cholestatic disease. The aim of the current study was to assess the link between toxicity and disposition of both chenodeoxycholic acid (CDCA) and its glycine conjugate glycochenodeoxycholic acid (GCDCA) in sandwich‐cultured human hepatocytes (SCHH). Biochemical integrity of the hepatocytes was assessed on multiple days of culture (day‐2 and day‐6) by determining the urea formation capacity of the cultured cells. CDCA exerted culture‐time dependent toxicity, as reflected by a more prominent reduction in relative urea formation in day‐2 SCHH, compared to day‐6 SCHH. To further explore the observed culture time‐dependent toxicity, in vitro disposition data were obtained by treating day‐2 and day‐6 SCHH with either 2.5, 10 or 25 μM CDCA. Media containing CDCA and GCDCA were collected, after which cells were lysed and intracellular accumulation was determined. Additionally, amounts excreted into the bile canaliculi were calculated. Concentration‐time profiles were fitted to a mechanistic ten‐compartment model using NONMEM v7.3.0. The model includes compartments representing the media, cells, bile and cells+bile with compound distributed among them through linear processes. Culture time (day‐2 and day‐6) was implemented as a covariate in the dataset and its effect on kinetic parameter estimations was assessed by the difference in successive objective function values. Covariate analysis showed a significant effect (p < 0.01) of culture time on both sinusoidal efflux clearance of GCDCA (day‐2: 0.6 (± 1%) μL/min/106 cells, day‐6: 1.7 (± 2%) μL/min/106 cells) and metabolic clearance (day‐2: 17.7 (± 3%) μL/min/106 cells, day‐6: 7.2 (± 11%) μL/min/106 cells), indicating that the lower toxicity of CDCA in day‐6 SCHH can be explained by decreased intracellular levels of GCDCA due to reduced formation and increased sinusoidal efflux of GCDCA. Interestingly, culture time‐dependent changes in mRNA and protein levels of the conjugating enzyme bile acid‐coenzyme A:amino acid N‐acetyltransferase (BAAT) did not correlate with the observed culture time‐dependent decrease in GCDCA formation. In conclusion, our results indicate that CDCA exerts culture‐time dependent toxicity in SCHH which seems to be related to changes in both sinusoidal efflux clearance of GCDCA and metabolic clearance of CDCA.Support or Funding InformationFinancial support for this research was provided by the Research Foundation ‐ Flanders (FWO)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MRP2 Inhibition by HIV Protease Inhibitors in Rat and Human Hepatocytes: A Quantitative Confocal Microscopy Study.

Hepatic drug transporters play a pivotal role in the excretion of drugs from the body, in drug-drug interactions, as well as in drug-induced liver toxicity. Hepatocytes cultured in sandwich configuration are an advantageous model to investigate the interactions of drug candidates with apical efflux transporters in a biorelevant manner. However, the commonly used "offline" assays (i.e., that rely on measuring intracellular accumulated amounts after cell lysis) are time- and resource-consuming, and the data output is often highly variable. In the present study, we used confocal microscopy to investigate the inhibitory effect of all marketed HIV protease inhibitors (10 μM) on the apical efflux transporter multidrug resistance-associated protein 2 (MRP2; ABCC2) by visualizing the biliary accumulation of the fluorescent substrate 5(6)-carboxy-2',7'-dichlorofluorescein (CDF). This method was applied with sandwich-cultured human and rat hepatocytes. Alterations in the biliary excretion index of CDF were calculated on the basis of quantitative analysis of fluorescence intensities in the confocal images. In human hepatocytes, lopinavir followed by tipranavir, saquinavir, atazanavir, and darunavir were the most potent inhibitors of MRP2-mediated efflux of CDF. In rat hepatocytes, tipranavir inhibited Mrp2-mediated CDF efflux most potently, followed by lopinavir and nelfinavir. In conclusion, a comparison of these findings with previously published data generated in offline transporter inhibition assays indicates that this microscopy-based approach enables investigation of the inhibitory effect of drugs on efflux transporters in a very sensitive but nondestructive manner.

Farnesoid X Receptor Agonists Obeticholic Acid and Chenodeoxycholic Acid Increase Bile Acid Efflux in Sandwich-Cultured Human Hepatocytes: Functional Evidence and Mechanisms.

The farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in bile acid homeostasis. FXR agonists, obeticholic acid (OCA) and chenodeoxycholic acid (CDCA), increase mRNA expression of efflux transporters in sandwich-cultured human hepatocytes (SCHH). This study evaluated the effects of OCA and CDCA treatment on the uptake, basolateral efflux, and biliary excretion of a model bile acid, taurocholate (TCA), in SCHH. In addition, changes in the protein expression of TCA uptake and efflux transporters were investigated. SCHH were treated with 1 µM OCA, 100 µM CDCA, or vehicle control for 72 hours followed by quantification of deuterated TCA uptake and efflux over time in Ca2+-containing and Ca2+-free conditions (n = 3 donors). A mechanistic pharmacokinetic model was fit to the TCA mass-time data to obtain estimates for total uptake clearance (CLUptake), total intrinsic basolateral efflux clearance (CLint,BL), and total intrinsic biliary clearance (CLint,Bile). Modeling results revealed that FXR agonists significantly increased CLint,BL by >6-fold and significantly increased CLint,Bile by 2-fold, with minimal effect on CLUptake Immunoblotting showed that protein levels of the basolateral transporter subunits organic solute transporter α and β (OSTα and OSTβ) in FXR agonist-treated SCHH were significantly induced by >2.5- and 10-fold, respectively. FXR agonist-mediated changes in the expression of other TCA transporters in SCHH were modest. In conclusion, this is the first report demonstrating that OCA and CDCA increased TCA efflux in SCHH, which contributed to reduced intracellular TCA concentrations. Increased basolateral efflux of TCA was consistent with increased OSTα/β protein expression in OCA- and CDCA-treated SCHH.

Hepatic Transport of 25-Hydroxyvitamin D3 Conjugates: A Mechanism of 25-Hydroxyvitamin D3 Delivery to the Intestinal Tract.

Vitamin D3 is an important prohormone critical for maintaining calcium and phosphate homeostasis in the body and regulating drug-metabolizing enzymes and transporters. 25-Hydroxyvitamin D3 (25OHD3), the most abundant circulating metabolite of vitamin D3, is further transformed to the biologically active metabolite 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) by CYP27B1 in the kidney and extrarenal tissues, and to nonactive metabolites by other cytochrome P450 enzymes. In addition, 25OHD3 undergoes sulfation and glucuronidation in the liver, forming two major conjugative metabolites, 25OHD3-3-O-sulfate (25OHD3-S) and 25OHD3-3-O-glucuronide (25OHD3-G), both of which were detected in human blood and bile. Considering that the conjugates excreted into the bile may be circulated to and reabsorbed from the intestinal lumen, deconjugated to 25OHD3, and then converted to 1α,25-(OH)2D3, exerting local intestinal cellular effects, it is crucial to characterize enterohepatic transport mechanisms of 25OHD3-S and 25OHD3-G, and thereby understand and predict mechanisms of interindividual variability in mineral homeostasis. In the present study, with plasma membrane vesicle and cell-based transport studies, we showed that 25OHD3-G is a substrate of multidrug resistance proteins 2 and 3, OATP1B1, and OATP1B3, and that 25OHD3-S is probably a substrate of breast cancer resistance protein, OATP2B1, and OATP1B3. We also demonstrated sinusoidal and canalicular efflux of both conjugates using sandwich-cultured human hepatocytes. Given substantial expression of these transporters in liver hepatocytes and intestinal enterocytes, this study demonstrates for the first time that transporters could play important roles in the enterohepatic circulation of 25OHD3 conjugates, providing an alternative pathway of 25OHD3 delivery to the intestinal tract, which could be critical for vitamin D receptor-dependent gene regulation in enterocytes.

LXR/FXR agonist alters transporter expression in sandwich-cultured human hepatocytes; proteomics-driven PBPK modeling implicates a drug–drug interaction with metformin

LXR/FXR agonist alters transporter expression in sandwich-cultured human hepatocytes; proteomics-driven PBPK modeling implicates a drug–drug interaction with metformin

Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner.

OATP1B1 and OATP1B3 mediate hepatic uptake of many drugs (e.g., statins) and can mediate transporter-mediated drug-drug-interactions (DDIs). Bortezomib is the first-in-class proteasome inhibitor drug approved by the U. S. Food and Drug Administration for the treatment of multiple myeloma. The potential of bortezomib to cause OATP-mediated DDIs has not been assessed. The current study investigated the involvement of the ubiquitin-proteasome system (UPS) in OATP1B1 and OATP1B3 degradation and determined the effects of proteasome inhibitors on OATP1B1- and OATP1B3-mediated transport. Co-immunoprecipitation of FLAG-OATP1B1/1B3 and HA-ubiquitin was observed in human embryonic kidney (HEK) 293 cells co-transfected with FLAG-tagged OATP1B1/OATP1B3 and hemagglutinin (HA)-tagged ubiquitin, suggesting that OATP1B1 and OATP1B3 can be ubiquitin-modified. Although blocking proteasome activity by bortezomib treatment (50 nM, 7 h) increased the endogenous ubiquitin-conjugated FLAG-OATP1B1 and FLAG-OATP1B3 in HEK293-FLAG-OATP1B1 and–OATP1B3 cells, such treatment did not affect the total protein levels of OATP1B1 and OATP1B3, suggesting that the UPS plays a minor role in degradation of OATP1B1 and OATP1B3 under current constitutive conditions. Pretreatment with bortezomib (50–250 nM, 2–7 h) significantly decreased transport of [3H]CCK-8, a specific OATP1B3 substrate, in HEK293-OATP1B3 and human sandwich-cultured hepatocytes (SCH). However, bortezomib pretreatment had negligible effects on the transport of [3H]E217βG and [3H]pitavastatin, dual substrates of OATP1B1 and OATP1B3, in HEK293-OATP1B1/1B3 cells and/or human SCH. Compared with vehicle control treatment, bortezomib pretreatment significantly decreased the maximal transport velocity (Vmax) of OATP1B3-mediated transport of CCK-8 (92.25 ± 14.2 vs. 133.95 ± 15.5 pmol/mg protein/min) without affecting the affinity constant (Km) values. Treatment with other proteasome inhibitors MG132, epoxomicin, and carfilzomib also significantly decreased OATP1B3-mediated [3H]CCK-8 transport. In summary, the current studies for the first time report ubiquitination of OATP1B1 and OATP1B3 and the apparent substrate-dependent inhibitory effect of bortezomib on OATP1B3-mediated transport. The data suggest that bortezomib has a low risk of causing OATP-mediated DDIs.

Transient Changes in Hepatic Physiology That Alter Bilirubin and Bile Acid Transport May Explain Elevations in Liver Chemistries Observed in Clinical Trials of GGF2 (Cimaglermin Alfa).

GGF2 is a recombinant human neuregulin-1β in development for chronic heart failure. Phase 1 clinical trials of GGF2 were put on hold when transient elevations in serum aminotransferases and total bilirubin were observed in 2 of 43 subjects who received single doses of GGF2 at 1.5 or 0.378 mg/kg. However, aminotransferase elevations were modest and not typical of liver injury sufficient to result in elevated serum bilirubin. Cynomolgus monkeys administered a single 15 mg/kg dose of GGF2 had similar transient elevations in serum aminotransferases and bilirubin as well as transient elevations in serum bile acids. However, no hepatocellular necrosis was observed in liver biopsies obtained during peak elevations. When sandwich-cultured human hepatocytes were treated with GGF2 for up to 72 h at concentrations approximately 0.8-fold average plasma Cmax for the 0.378 mg/kg dose, no cytotoxicity was observed. Gene expression profiling identified approximately 50% reductions in mRNAs coding for bilirubin transporters and bile acid conjugating enzymes, as well as changes in expression of additional genes mimicking the interleukin-6-mediated acute phase response. Similar gene expression changes were observed in GGF2-treated HepG2 cells and primary monkey hepatocytes. Additional studies conducted in sandwich-cultured human hepatocytes revealed a transient and GGF2 concentration-dependent decrease in hepatocyte bile acid content and biliary clearance of taurocholate without affecting biliary taurocholate efflux. Taken together, these data suggest that GGF2 does not cause significant hepatocellular death, but transiently modifies hepatic handling of bilirubin and bile acids, effects that may account for the elevations in serum bilirubin observed in the clinical trial subjects.