Sandwich-cultured Hepatocytes Research Articles

Transporter Gene Regulation in Sandwich Cultured Human Hepatocytes Through the Activation of Constitutive Androstane Receptor (CAR) or Aryl Hydrocarbon Receptor (AhR).

The induction potentials of ligand-activated nuclear receptors on metabolizing enzyme genes are routinely tested for new chemical entities. However, regulations of drug transporter genes by the nuclear receptor ligands are underappreciated, especially in differentiated human hepatocyte cultures. In this study, gene induction by the ligands of constitutive androstane receptor (CAR) and aryl hydrocarbon receptor (AhR) was characterized in sandwich-cultured human hepatocytes (SCHH) from multiple donors. The cells were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), omeprazole (OP), 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) and phenobarbital (PB) for three days. RNA samples were analyzed by qRT-PCR method. As expected, CITCO, the direct activator, and PB, the indirect activator of CAR, induced CYP3A4 (31 and 40-fold), CYP2B6 (24 and 28-fold) and UGT1A1 (2.9 and 4.2-fold), respectively. Conversely, TCDD and OP, the activators of AhR, induced CYP1A1 (38 and 37-fold), and UGT1A1 (4.3 and 5.0-fold), respectively. In addition, OP but not TCDD induced CY3A4 by about 61-fold. Twenty-four hepatic drug transporter genes were characterized, and of those, SLC51B was induced the most by PB and OP by about 3.3 and 6.5 fold, respectively. Marginal inductions (about 2-fold) of SLC47A1 and SLCO4C1 genes by PB, and ABCG2 gene by TCDD were observed. In contrast, SLC10A1 gene was suppressed about 2-fold by TCDD and CITCO. While clinical relevance of SLC51B gene induction or SLC10A1 gene suppression warrants further investigation, the results verified that the assessment of transporter gene inductions are not required for new drug entities, when a drug does not remarkably induce metabolizing enzyme genes by CAR and AhR activation.

Function and Expression of Bile Salt Export Pump in Suspension Human Hepatocytes.

The mechanistic understanding of bile salt disposition is not well established in suspension human hepatocytes (SHH) because of the limited information on the expression and function of bile salt export protein (BSEP) in this system. We investigated the transport function of BSEP in SHH using a method involving in situ biosynthesis of bile salts from their precursor bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). Our data indicated that glycine- and taurine-conjugated CA and CDCA were generated efficiently and transported out of hepatocytes in a concentration- and time-dependent manner. We also observed that the membrane protein abundance of BSEP was similar between SHH and sandwich-cultured human hepatocytes. Furthermore, known cholestatic agents significantly inhibited G-CA and G-CDCA efflux in SHH. Interestingly, cyclosporine A, troglitazone, itraconazole, loratadine, and lovastatin inhibited G-CA efflux more potently than G-CDCA efflux (3- to 5-fold). Because of these significant differential effects on G-CA and G-CDCA efflux inhibition, we determined the IC50 values of troglitazone for G-CA (9.9 µM) and for G-CDCA (43.1 µM) efflux. The observed discrepancy in the IC50 was attributed to the fact that troglitazone also inhibits organic anion transporting polypeptides and Na+/taurocholate cotransporting polypeptide in addition to BSEP. The hepatocyte uptake study suggested that both active uptake and passive diffusion contribute to the liver uptake of CA, whereas CDCA primarily undergoes passive diffusion into the liver. In summary, these data demonstrated the expression and function of BSEP and its major role in transport of bile salts in cryopreserved SHH. SIGNIFICANCE STATEMENT: BSEP transport function and protein abundance was evident in SHH in the present study. The membrane abundance of BSEP protein was similar between SHH and sandwich-cultured human hepatocytes. The study also illustrated the major role of BSEP relative to basolateral MRP3 and MRP4 in transport of bile salts in SHH. Understanding of BSEP function in SHH may bolster the utility of this platform in mechanistic understanding of bile salt disposition and potentially in the assessment of drugs for BSEP inhibition.

Prediction of Transporter-Mediated Drug-Drug Interactions and Phenotyping of Hepatobiliary Transporters Involved in the Clearance of E7766, a Novel Macrocycle-Bridged Dinucleotide.

E7766 represents a novel class of macrocycle-bridged dinucleotides and is under clinical development for immuno-oncology. In this report, we identified mechanism of systemic clearance E7766 and investigated the hepatobiliary transporters involved in the disposition of E7766 and potential drug interactions of E7766 as a victim of organic anion-transporting polypeptide (OATP) inhibitors. In bile-duct cannulated rats and dogs, E7766 was mainly excreted unchanged in bile (>80%) and to a lesser extent in urine (<20%). Sandwich-cultured human hepatocytes (SCHHs), transfected cells, and vesicles were used to phenotype the hepatobiliary transporters involved in the clearance of E7766. SCHH data showed temperature-dependent uptake of E7766 followed by active biliary secretion. In vitro transport assays using transfected cells and membrane vesicles confirmed that E7766 was a substrate of OATP1B1, OATP1B3, and multidrug resistance-associated protein 2. Phenotyping studies suggested predominant contribution of OATP1B3 over OATP1B1 in the hepatic uptake of E7766. Studies in OATP1B1/1B3 humanized mice showed that plasma exposure of E7766 increased 4.5-fold when coadministered with Rifampicin. Physiologically based pharmacokinetic models built upon two independent bottom-up approaches predicted elevation of E7766 plasma exposure when administered with Rifampicin, a clinical OATP inhibitor. In conclusion, we demonstrate that OATP-mediated hepatic uptake is the major contributor to the clearance of E7766, and inhibition of OATP1B may increase its systemic exposure. Predominant contribution of OATP1B3 in the hepatic uptake of E7766 was observed, suggesting polymorphisms in OATP1B1 would be unlikely to cause variability in the exposure of E7766. SIGNIFICANCE STATEMENT: Understanding the clearance mechanisms of new chemical entities is critical to predicting human pharmacokinetics and drug interactions. A physiologically based pharmacokinetic model that incorporated parameters from mechanistic in vitro and in vivo experiments was used to predict pharmacokinetics and drug interactions of E7766, a novel dinucleotide drug. The findings highlighted here may shed a light on the pharmacokinetic profile and transporter-mediated drug interaction propensity of other dinucleotide drugs.

Tanshinone ⅡA may alleviate rifampin-induced cholestasis by regulating the expression and function of NTCP.

The Na+-taurocholate cotransporting polypeptide (NTCP) acts as the major hepatic basolateral uptake system, and plays a key role in balancing bile flow. The anti-tuberculosis drugs rifampin (RFP) can affect bile flow causing liver injury, while tanshinone IIA (TAN IIA) has the effect of protecting liver. This study aimed to investigate the effects of RFP and TAN IIA on the NTCP expression and activity, and explore the potential connections. Herein, we established sandwich-cultured primary rat hepatocytes, and quantified mRNA and protein levels of NRF2 and NTCP after treatment with RFP (10, 25, or 50 μM) or co-treatment with TAN IIA (5, 10, or 20 μM) for 12, 24, 48 h (n = 3). NTCP activity was assessed by measuring the initial uptake rates of known substrates taurocholate (TCA) (n = 3) after treatment with different concentrations of RFP, TAN ⅡA for 12, 24 and 48 h. We found that RFP had inhibition effects on NRF2, NTCP mRNA and protein expression, and co-administration of TAN IIA could reverse RFP inhibition. TCA cellular accumulation was significantly decreased by RFP (39.1%), and TAN IIA could significantly induce TCA uptake of NTCP (2.9-fold at 48 h). The TCA uptake activity was correlated with the NTCP mRNA expression, confirming the role of RFP or TAN IIA on NTCP expression and activity is synchronous, and we can predict NTCP activity by detecting its mRNA expression. In conclusion, our work will enrich the significance of NTCP in the liver protection, and provide theoretical basis for TAN IIA to prevent RFP induced cholestatic liver injury.

Prediction of Transporter-Mediated Rosuvastatin Hepatic Uptake Clearance and Drug Interaction in Humans Using Proteomics-Informed REF Approach.

Suspended, plated, or sandwich-cultured human hepatocytes are routinely used for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated hepatic clearance (CL) of drugs. However, these hepatocyte models have been reported to underpredict transporter-mediated in vivo hepatic uptake CL (CL uptake,in vivo ) of some drugs. Therefore, we determined whether transporter-expressing cells (TECs) can accurately predict the CL uptake,in vivo of drugs. To do so, we determined the uptake CL (CL int,uptake,cells ) of rosuvastatin (RSV) by TECs (organic anion transporting polypeptides/Na+-taurocholate cotransporting polypeptide) and then scaled it to that in vivo by relative expression factor (REF) (the ratio of transporter abundance in human livers and TEC) determined by liquid chromatography tandem mass spectrometry-based quantitative proteomics. Both the TEC and hepatocyte models did not meet our predefined success criteria of predicting within 2-fold the RSV CL uptake,in vivo value obtained from our positron emission tomography (PET) imaging. However, the TEC performed better than the hepatocyte models. Interestingly, using REF, TECs successfully predicted RSV CL int,uptake,hep obtained by the hepatocyte models, suggesting that the underprediction of RSV CL uptake,in vivo by TECs and hepatocytes is due to endogenous factor(s) not present in these in vitro models. Therefore, we determined whether inclusion of plasma (or albumin) in TEC uptake studies improved IVIVE of RSV CL uptake,in vivo It did, and our predictions were close to or just fell above our lower 2-fold acceptance boundary. Despite this success, additional studies are needed to improve transporter-mediated IVIVE of hepatic uptake CL of drugs. However, using REF and TEC, we successfully predicted the magnitude of PET-imaged inhibition of RSV CL uptake,in vivo by cyclosporine A. SIGNIFICANCE STATEMENT: We showed that the in vivo transporter-mediated hepatic uptake CL of rosuvastatin, determined by PET imaging, can be predicted (within 2-fold) from in vitro studies in transporter-expressing cells (TECs) (scaled using REF), but only when plasma proteins were included in the in vitro studies. This conclusion did not hold when plasma proteins were absent in the TEC or human hepatocyte studies. Thus, additional studies are needed to improve in vitro to in vivo extrapolation of transporter-mediated drug CL.

Pregnancy-Related Hormones Increase Nifedipine Metabolism in Human Hepatocytes by Inducing CYP3A4 Expression

Pregnancy-related hormones (PRH) have emerged as key regulators of hepatic cytochrome P450 (CYP) enzyme expression and function. The impact of PRH on protein levels of CYP3A4 and other key CYP enzymes, and the metabolism of nifedipine (a CYP3A4 substrate commonly prescribed during pregnancy), was evaluated in primary human hepatocytes. Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to PRH (estradiol, estriol, estetrol, progesterone, and cortisol), individually or in combination as a cocktail. Absolute protein concentrations of twelve CYP isoforms in SCHH membrane fractions were quantified by nanoLC-MS/MS, and metabolism of nifedipine to dehydronifedipine in SCHH was evaluated. PRH significantly increased CYP3A4 protein concentrations and nifedipine metabolism to dehydronifedipine in a concentration-dependent manner. CYP3A4 mRNA levels in hepatocyte-derived exosomes positively correlated with CYP3A4 protein levels and dehydronifedipine formation in SCHH. PRH also increased CYP2B6, CYP2C8 and CYP2A6 levels. Our findings demonstrate that PRH increase nifedipine metabolism in SCHH by inducing CYP3A4 expression and alter expression of other key CYP proteins in an isoform-specific manner, and suggest that hepatocyte-derived exosomes warrant further investigation as biomarkers of hepatic CYP3A4 metabolism. Together, these results offer mechanistic insight into the increases in nifedipine metabolism and clearance observed in pregnant women.

Quantitative Systems Toxicology Modeling Predicts that Reduced Biliary Efflux Contributes to Tolvaptan Hepatotoxicity

Patients with autosomal dominant polycystic kidney disease (ADPKD) exhibit enhanced susceptibility to tolvaptan hepatotoxicity relative to other patient populations. In a rodent model of ADPKD, the expression and function of the biliary efflux transporter Mrp2 was reduced, and biliary excretion of a major tolvaptan metabolite (DM-4103) was decreased. The current study investigated whether reduced biliary efflux could contribute to increased susceptibility to tolvaptan-associated hepatotoxicity using a quantitative systems toxicology (QST) model (DILIsym). QST simulations revealed that decreased biliary excretion of DM-4103, but not tolvaptan, resulted in substantial hepatic accumulation of bile acids, decreased electron transport chain activity, reduced hepatic adenosine triphosphate concentrations, and an increased incidence of hepatotoxicity. In vitro experiments (C-DILI) with sandwich-cultured human hepatocytes and HepaRG cells were performed to assess tolvaptan-associated hepatotoxic effects when MRP2 was impaired by chemical inhibition (MK571, 50µM) or genetic knockout, respectively. Tolvaptan (64µM, 24-hour) treatment of these cells increased cytotoxicity markers up to 27.9-fold and 1.6-fold, respectively, when MRP2 was impaired, indicating that MRP2 dysfunction may be involved in tolvaptan-associated cytotoxicity. In conclusion, QST modeling supported the hypothesis that reduced biliary efflux of tolvaptan and/or DM-4103 could account for increased susceptibility to tolvaptan-associated hepatotoxicity; in vitro experiments implicated MRP2 dysfunction as a key factor in susceptibility. QST simulations revealed that DM-4103 may contribute to hepatotoxicity more than the parent compound. ADPKD progression and gradual reduction in MRP2 activity may explain why acute liver events can occur well after oneyear of tolvaptan treatment.

Pitfalls in Predicting Hepatobiliary Drug Transport Using Human Sandwich-Cultured Hepatocytes.

During drug development, in vivo human biliary drug clearances (CL) are usually predicted using human sandwich-cultured hepatocytes (SCH). To do so, SCH are pre-incubated with Ca2+-containing or Ca2+-free buffer to maintain or disrupt canalicular tight junctions (CTJ), respectively. Drug uptake into SCH is then conducted in the presence of Ca2+ (up to 20min). Under this standard protocol, two key assumptions are made: first, that the CTJ are not reformed during the uptake phase when Ca2+ is repleted, and second, disruption of CTJ by the Ca2+-free buffer does not affect the activity of any of the transporters present in the sinusoidal or canalicular membrane. Here we investigated the validity of these assumptions using rosuvastatin (RSV) and taurocholic acid (TCA) as our model drugs. In human SCH, the disrupted CTJ were "reformed" with just 10-min Ca2+ repletion as reflected in a significant increase in TCA cell accumulation. To avoid CTJ reformation and cell toxicity, the standard SCH protocol was modified by conducting the uptake in the absence of Ca2+ for 10min. Surprisingly, using this protocol, RSV uptake into SCH, plated hepatocytes, and transporter-expressing cells confirmed that Ca2+ depletion substantially decreased NTCP and not OATP1B1 activity. Collectively, this study provides the first evidence of reformation of CTJ in human SCH with 20-min Ca2+ repletion, whereas Ca2+ depletion, during the uptake phase, leads to a significant reduction in NTCP uptake. Thus, the entire SCH protocol needs to be re-examined and optimized to correctly estimate hepatobiliary CL of drugs including those that are NTCP substrates.

Centella asiatica Water Extract Shows Low Potential for Cytochrome P450-Mediated Drug Interactions.

Centella asiatica (CA) shows considerable promise for development as a botanical drug for cognitive decline. Its primary bioactive components include triterpene glycosides asiaticoside and madecassoside and their corresponding aglycones asiatic acid and madecassic acid. Exploration of the bioactivity of CA's caffeoylquinic acids is ongoing. In this study, an aqueous extract of CA (CAW-R61J) was evaluated for drug interaction potential through inhibition or induction of P450 enzymes, as required by the US Food and Drug Administration. CAW-R61J was assessed for induction potential of CYP1A2, CYP2B6, and CYP3A4 using transporter-certified cryopreserved human hepatocytes in sandwich culture. Gene expression of these target P450s was quantified, and enzyme activities were determined to confirm gene expression results. No induction was observed up to 16.7 µg/ml CAW-R61J (equivalent to 1.1 µM asiaticoside, 0.8 µM madecassoside, 0.09 µM asiatic acid, and 0.12 µM madecassic acid). Reversible and time-dependent inhibitory effects of CAW-R61J on CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5 were evaluated using human liver microsomes. CAW-R61J showed weak reversible inhibition of most of the P450 forms tested, with the strongest being CYP2C9 (IC50 of 330 µg/ml). CAW-R61J (≤1000 µg/ml) was not a time-dependent inhibitor of any of these P450 enzymes. In summary, CAW-R61J had no, or only a weak impact, on P450 induction and inhibition in vitro. The clinical relevance of these results will depend on the in vivo concentration of CAW-R61J components achieved in humans. Plasma triterpene concentrations measured in our recent clinical studies suggest minimal risk of P450-mediated drug interactions by these components. SIGNIFICANCE STATEMENT: A preparation of Centella asiatica is currently under clinical development for the prevention or treatment of cognitive decline. The US Food and Drug Administration required an evaluation of its potential for drug interactions mediated through drug-metabolizing enzymes. This in vitro study revealed minimal induction or inhibition of a range of P450 enzymes, including CYP3A4, by the C. asiatica extract, suggesting a low potential for drug interactions modulated by P450 metabolism.

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.

ERRATUM.

Testing drugs in isogenic rodent strains to satisfy regulatory requirements is insufficient for derisking organ toxicity in genetically diverse human populations; in contrast, advances in mouse genetics can help mitigate these limitations. Compared to the expensive and slower in vivo testing, in vitro cultures enable the testing of large compound libraries toward prioritizing lead compounds and selecting an animal model with human-like response to a compound. In the case of the liver, a leading cause of drug attrition, isolated primary mouse hepatocytes (PMHs) rapidly decline in function within current culture platforms, which restricts their use for assessing the effects of longer-term compound exposure. Here we addressed this challenge by fabricating mouse micropatterned cocultures (mMPCC) containing PMHs and 3T3-J2 murine embryonic fibroblasts that displayed 4 weeks of functions; mMPCCs created from either C57Bl/6J or CD-1 PMHs outperformed collagen/Matrigel™ sandwich-cultured hepatocyte monocultures by ∼143-fold, 413-fold, and 10-fold for albumin secretion, urea synthesis, and cytochrome P450 activities, respectively. Such functional longevity of mMPCCs enabled in vivo relevant comparisons across strains for CYP induction and hepatotoxicity following exposure to 14 compounds with subsequent comparison to responses in primary human hepatocytes (PHHs). In conclusion, mMPCCs display high levels of major liver functions for several weeks and can be used to assess strain- and species-specific compound effects when used in conjunction with responses in PHHs. Ultimately, mMPCCs can be used to leverage the power of mouse genetics for characterizing subpopulations sensitive to compounds, characterizing the degree of interindividual variability, and elucidating genetic determinants of severe hepatotoxicity in humans.

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.

Functional Comparison of HepaRG Cells and Primary Human Hepatocytes in Sandwich and Spheroid Culture as Repeated-Exposure Models for Hepatotoxicity

Introduction: Reliable and effective predictions of hepatotoxicity resulting from repeated exposure to chemicals are of critical importance for the safety assessment of chemicals; however, in vitro...

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.

Multiple cellular pathways regulate lipid droplet homeostasis for the establishment of polarity in collagen sandwich-cultured hepatocytes.

Hepatocyte polarization is energy dependent. The establishment of polarization in collagen sandwich culture of hepatocytes requires utilization of lipid droplets and mitochondrial β-oxidation to supply ATP. Multiple cellular pathways are involved in lipid droplet homeostasis; however, mechanistic insights of how hepatocytes utilize lipid droplets during polarization remain elusive. The current study investigated the effects of various pathways involved in lipid droplet homeostasis on bioenergetics during hepatocyte polarization. The results showed that hepatocytes were dependent on lipolysis of lipid droplets to release fatty acids for β-oxidation. Inhibition of lipolysis significantly decreased cellular fatty acid and ATP levels and inhibited hepatocyte polarization, revealing that lipolysis was an important mechanism for providing energy for hepatocyte polarization. The results also demonstrated that autophagic degradation of lipid droplets (lipophagy) was not essential for breaking down lipid droplets. Conversely, autophagy contributed to lipid droplet formation and played a key role in sustaining lipid droplet stores for energy production. In addition, cholesterol biosynthesis/cholesterol esterification and de novo fatty acid synthesis also contributed to maintaining lipid droplet stores for bioenergetics during hepatocyte polarization. In summary, multiple cellular pathways are coordinated to maintain lipid droplet homeostasis and sustain fatty acid β-oxidation during hepatocyte polarization.

Long-Term Engineered Cultures of Primary Mouse Hepatocytes for Strain and Species Comparison Studies During Drug Development.

Testing drugs in isogenic rodent strains to satisfy regulatory requirements is insufficient for derisking organ toxicity in genetically diverse human populations; in contrast, advances in mouse genetics can help mitigate these limitations. Compared to the expensive and slower in vivo testing, in vitro cultures enable the testing of large compound libraries toward prioritizing lead compounds and selecting an animal model with human-like response to a compound. In the case of the liver, a leading cause of drug attrition, isolated primary mouse hepatocytes (PMHs) rapidly decline in function within current culture platforms, which restricts their use for assessing the effects of longer-term compound exposure. Here we addressed this challenge by fabricating mouse micropatterned cocultures (mMPCC) containing PMHs and 3T3-J2 murine embryonic fibroblasts that displayed 4 weeks of functions; mMPCCs created from either C57Bl/6J or CD-1 PMHs outperformed collagen/Matrigel™ sandwich-cultured hepatocyte monocultures by ∼143-fold, 413-fold, and 10-fold for albumin secretion, urea synthesis, and cytochrome P450 activities, respectively. Such functional longevity of mMPCCs enabled in vivo relevant comparisons across strains for CYP induction and hepatotoxicity following exposure to 14 compounds with subsequent comparison to responses in primary human hepatocytes (PHHs). In conclusion, mMPCCs display high levels of major liver functions for several weeks and can be used to assess strain- and species-specific compound effects when used in conjunction with responses in PHHs. Ultimately, mMPCCs can be used to leverage the power of mouse genetics for characterizing subpopulations sensitive to compounds, characterizing the degree of interindividual variability, and elucidating genetic determinants of severe hepatotoxicity in humans.

A Novel MicroRNA Signature for Cholestatic Drugs in Human Hepatocytes and Its Translation into Novel Circulating Biomarkers for Drug-Induced Liver Injury Patients.

Drug-induced liver injury (DILI) diagnosis and classification (hepatocellular, cholestatic, and mixed) relies on traditional clinical biomarkers (eg ALT and ALP), despite limitations such as extrahepatic interferences, narrow dynamic ranges, and low mechanistic value. microRNAs may be very useful for complementing traditional DILI biomarkers but most studies in this direction have considered only paracetamol poisoning. Thus the value of microRNAs (miRNAs) as biomarkers for idiosyncratic DILI has not yet been demonstrated. In this study, we first examined the effect of model cholestatic drugs on the human hepatocyte miRNome by RNAseq and RT-qPCR. Results demonstrated that chlorpromazine, cyclosporin A, and ANIT induced (miR-21-3p, -21-5p, -22-3p, -27a-5p, -1260b, -34a-5p, and -98-5p) and repressed (-122-5p, -192-5p, -30c-5p, -424-5p, and -16-5p) specific miRNAs in sandwich-cultured upcyte hepatocytes. However, no common signature was found for cholestatic drugs. Next we investigated the levels of these miRNA in human serum and found that most were also significantly altered in cholestatic/mixed DILI patients upon hospital/ambulatory admission. However, miR-122-5p, -192-5p, -34a-5p, and -22-3p demonstrated a much more significant induction in patients with hepatocellular DILI, thus revealing better specificity for hepatocellular damage. Time-course analyses demonstrated that -1260b and -146 had a very similar profile to ALP, but with wider dynamic ranges, while -16-5p and -451a showed a negative correlation. Conversely, -122-5p and -192-5p correlated with ALT but with wider dynamic ranges and faster recoveries. Finally, the 122/451a and 122/16 ratios showed excellent prediction performances in both the study [area under the receiver operating characteristic curve (AUROC) >0.93] and the validation cohort (AUROC > 0.82), and can, therefore, be postulated for the first time as circulating miRNA biomarkers for idiosyncratic DILI.