Combining the novel all-human co-cultured hepatocytes system with physiologically based pharmacokinetic modeling to assess the translatability of cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase induction data.

The isolation of hepatocytes from intact liver involves collagenase digestion of the tissue, resulting in loss of cell polarization and functional vectorial excretion. These studies examined repolarization, localization of P-glycoprotein (P-gp) to the canalicular domain of the hepatocyte, and re-establishment of vectorial transport in sandwich-cultured (SC) rat and human primary hepatocytes. Protein localization and expression were determined in SC hepatocytes by confocal microscopy and Western blotting, respectively. Transporter function was evaluated by measuring [D-penicillamine2,5]enkephalin (3H-DPDPE) and 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF) biliary excretion in SC hepatocytes. P-gp and the canalicular marker protein dipeptidyl peptidase IV (DPPIV) co-localized by Day 3 and Day 6 in SC rat hepatocytes and SC human hepatocytes, respectively, consistent with canalicular network formation visualized by light microscopy. Co-localization of multidrug resistance associated protein 2 (MRP2) and P-gp in SC human hepatocytes was observed on Day 6 in culture. Expression levels of P-gp increased slightly in both species over days in culture; similar expression was observed for MRP2 in SC human hepatocytes. Oatp1a1 expression in SC rat hepatocytes was maintained over days in culture, whereas Oatp1a4 expression decreased. OATP1B1 expression decreased slightly on Day 3 in SC human hepatocytes. OATP1B3 expression was constant in SC human hepatocytes. In vitro biliary excretion of the opioid peptide 3H-DPDPE correlated with the proper localization of canalicular proteins in both species. Excretion of CDF in SC human hepatocytes confirmed network formation and MRP2 function. These studies indicate that SC hepatocytes repolarize and traffic functional canalicular transport proteins to the appropriate cellular domain.

Pre-incubation with OATP1B1 and OATP1B3 inhibitors potentiates inhibitory effects in physiologically relevant sandwich-cultured primary human hepatocytes

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.

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.

In the present study MRP2/ABCC2 and BSEP/ABCB11 expression were investigated in sandwich cultured (SC) human and rat hepatocytes exposed to the proinflammatory cytokines. The investigation was also done in lipopolysaccharide (LPS)-treated rats. In SC human hepatocytes, both absolute protein and mRNA levels of MRP2/ABCC2 were significantly down-regulated by TNF-α, IL-6, or IL-1β. In contrast to mRNA decrease, which was observed for BSEP/ABCB11, the protein amount was significantly increased by IL-6 or IL-1β. A discrepancy between the change in BSEP/ABCB11 mRNA and protein levels was encountered in SC human hepatocytes treated with proinflammatory cytokines. In SC rat hepatocytes, Mrp2/Abcc2 mRNA was down-regulated by TNF-α and IL-6, whereas the protein level was decreased by all three cytokines. Down-regulations of both Bsep/Abcb11 mRNA and protein levels were found in SC rat hepatocytes exposed to TNF-α or IL-1β. Administration of LPS triggered the release of the proinflammatory cytokines and caused the decrease of Mrp2/Abcc2 and Bsep/Abcb11 protein in liver at 24 h post-treatment; however, the Mrp2 and Bsep protein levels rebounded at 48 h post-LPS treatment. In total, our results indicate that proinflammatory cytokines regulate the expression of MRP2/Mrp2 and BSEP/Bsep and for the first time demonstrate the differential effects on BSEP/Bsep expression between SC human and rat hepatocytes. Furthermore, the agreement between transporter regulation in vitro in SC rat hepatocytes and in vivo in LPS-treated rats during the acute response phase demonstrates the utility of in vitro SC hepatocyte models for predicting in vivo effects.

Pregnancy-related hormones (PRH) are recognized as important regulators of hepatic cytochrome P450 enzyme expression and function. However, the impact of PRH on the hepatic expression and function of uridine diphosphate glucuronosyltransferases (UGTs) remains unclear. Using primary human hepatocytes, we evaluated the effect of PRH exposure on mRNA levels and protein concentrations of UGT1A1, UGT2B7, and other key UGT enzymes, and on the metabolism of labetalol (a UGT1A1 and UGT2B7 substrate commonly prescribed to treat hypertensive disorders of pregnancy). Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to the PRH estradiol, estriol, estetrol, progesterone, and cortisol individually or in combination. We quantified protein concentrations of UGT1A1, UGT2B7, and four additional UGT1A isoforms in SCHH membrane fractions and evaluated the metabolism of labetalol to its glucuronide metabolites in SCHH. PRH exposure increased mRNA levels and protein concentrations of UGT1A1 and UGT1A4 in SCHH. PRH exposure also significantly increased labetalol metabolism to its UGT1A1-derived glucuronide metabolite in a concentration-dependent manner, which positively correlated with PRH-induced changes in UGT1A1 protein concentrations. In contrast, PRH did not alter UGT2B7 mRNA levels or protein concentrations in SCHH, and formation of the UGT2B7-derived labetalol glucuronide metabolite was decreased following PRH exposure. Our findings demonstrate that PRH alter expression and function of UGT proteins in an isoform-specific manner and increase UGT1A1-mediated labetalol metabolism in human hepatocytes by inducing UGT1A1 protein concentrations. These results provide mechanistic insight into the increases in labetalol clearance observed in pregnant individuals.

This was the first study to construct a physiologically-based pharmacokinetic (PBPK) model for mirabegron which incorporates the overall elimination pathways of metabolism by cytochrome P450 (CYP) 3A4, uridine 5'-diphosphate-glucuronosyltransferase (UGT) 2B7, and butyrylcholinesterase (BChE) and renal excretion. The objective was to assess the risk of drug-drug interactions (DDIs) by estimating the contribution of each elimination pathway and simulating the magnitude of the DDIs with UGT2B7 inhibitors.A PBPK model for mirabegron was constructed to reproduce the plasma concentration-time curves from a phase 1 study and the magnitude of the DDI with ketoconazole taking into account the overall elimination pathways. The PBPK model was subsequently verified using data from other DDI studies.The constructed PBPK model estimated the contribution for each elimination pathway: 44% and 29% for CYP3A4 and UGT2B7 in the liver, 1.6% for UGT2B7 in the kidney, 3.2% for BChE in plasma, and 22% for renal excretion.Co-administration of probenecid (an UGT2B7 inhibitor) or fluconazole (an UGT2B7 and CYP3A4 inhibitor) was predicted to increase area under the curve for mirabegron to 115% or 174%, respectively.In conclusion, PBPK modeling and simulation revealed a low DDI risk for mirabegron following co-administration with BChE or UGT2B7 inhibitors.

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.

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

Introduction Vericiguat is a once daily, novel oral stimulator of soluble guanylate cyclase (sGC) that showed clinical benefit in the Phase III VICTORIA study in heart failure patients with reduced ejection fraction (HFrEF, NCT02861534). Nonclinical and clinical studies demonstrated that the primary route of elimination of vericiguat was glucuronidation to an inactive metabolite M-1 (N-glucuronide). This glucuronidation was catalyzed by uridine 5'-diphospho-glucuronosyltransferases (UGT)1A9 as well as UGT1A1, thus vericiguat may have a potential for victim drug-drug interaction (DDI) when co-administered with potent UGT inhibitors. Purpose In a clinical DDI study with mefenamic acid as an UGT1A9 inhibitor no clinically relevant increase in vericiguat exposure in healthy subjects was observed (EudraCT 2014–000764–17). This analysis aims to prospectively investigate as extrinsic factors the DDI potential with atazanavir as a selective UGT1A1 inhibitor via full dynamic physiologically-based pharmacokinetic (PBPK) modelling. Methods A PBPK model for vericiguat and M-1 in healthy adults was built with PK-Sim (PBPK platform as part of the Open Systems Pharmacology Suite) by integrating physicochemical, in vitro metabolism and transporter data as well as PK data from clinical pharmacology studies in order to assess the victim DDI potential of vericiguat when co-administered with UGT inhibitors. First, PBPK models for mefenamic acid and atazanavir were separately developed and verified using published literature data. The PBPK model for vericiguat was then verified with regard to its fraction of metabolism by UGTs by comparing simulated and observed data of the clinical mefenamic acid DDI study. Finally, the UGT1A1 DDI potential of vericiguat was prospectively predicted by simulating an in silico study between the UGT1A1 inhibitor atazanavir and vericiguat. Results In line with the results of the clinical DDI study with mefenamic acid, an increase in total vericiguat exposure by 14% (area under the concentration time curve ratio (AUCR) of 1.14 (geoCV 5.3%; 90% population interval: 1.06 to 1.25) and peak exposure increase by 6% (CmaxR of 1.06; geoCV 5.9%; 90% population interval: 1.01 to 1.20) was simulated using the PBPK model. A prospective prediction of a virtual DDI trial between the UGT1A1 inhibitor atazanavir yielded an AUCR of 1.12 (geoCV 2.9%; 90% population interval: 1.07 to 1.17) and a CmaxR of 1.04 (geoCV 1.1%; 90% population interval: 1.03 to 1.06). The proposed population intervals for AUCR and CmaxR for both DDI studies lie within the default no-effect boundary of 0.80 to 1.25 according to the to January 2020 FDA DDI guideline. Conclusion(s) Results of UGT1A9-DDI simulations were consistent with those of the clinical study-The prospective UGT1A1-DDI simulation results suggest a low potential for vericiguat to be subject to DDI when co-administered with UGT1A1 inhibitors. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Funding for this research was provided by Bayer and Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA

Due to cholestasis or adverse drug effects, the excretion of bilirubin conjugates can decrease; therefore, the level of bilirubin (B) and bilirubin glucuronides (BGs) increases in the serum with the concomitant shift of bilirubin diversus monoglucuronide (BDG/BMG) equilibrium. The aim of this study was to utilize the collagen-sandwich culture of hepatocytes as an in vitro model for studying B conjugation and canalicular versus sinusoidal disposition of BGs. Canalicular and sinusoidal efflux of BMG and BDG obtained in sandwich-cultured rat primary hepatocytes was compared with that measured in human hepatocyte cultures. The BMG and BDG were separated by high-performance liquid chromatography and identified by mass spectrometry. The biliary excretion index (BEI) was estimated by measuring disposition of BGs into standard and Ca(2+), Mg(2+)-free medium. Significantly more BGs were excreted into the canalicular networks than into the medium in 96-h sandwich culture of both human and rat hepatocytes (BEI, 62.5 and 80.6, respectively). The BDG/BMG ratio in the medium versus that in the canalicular networks was 0.55/1.48, which is similar to the serum/bile values (0.6/1.5) observed in vivo by Mesa et al. [Mesa VA, De Vos R, and Fevery J (1997) J Hepatol 27:912-916]. In contrast, the BEI for p-nitrophenol glucuronide was 5.2. The low BEI value is in agreement with empirical observations, which suggest that molecules with low molecular weight are preferably excreted by the kidney. In conclusion, sandwich-cultured primary hepatocytes provide a useful in vitro method to differentiate between sinusoidal and canalicular disposition of BGs. Since the normal BDG/BMG ratio changes in hyperbilirubinemia, this model could be used to predict drug effects leading to hyperbilirubinemia.

Opioid Metabolism

Use of Primary Rat and Human Hepatocyte Sandwich Cultures for Activation of Indirect Carcinogens: Monitoring of DNA Strand Breaks and Gene Mutations in Co-cultured Cells

We previously reported that both the concentrative (hCNT) and equilibrative (hENT) nucleoside transporters are expressed in the human liver (21). Here we report a study that investigated the expression of these transporters (transcripts and proteins) and their role in the hepatobiliary transport of nucleosides/nucleoside drugs using sandwich-cultured human hepatocytes. In the hepatic tissue, the rank order of the mRNA expression of the transporters was hCNT1 approximately hENT1>hENT2 approximately hCNT2>hCNT3. In sandwich-cultured hepatocytes, the mRNA expression of hCNT2 and hENT2 was comparable to that in hepatic tissue, whereas the expression of corresponding transporters in the two-dimensional hepatocyte cultures was lower. Colocalization studies demonstrated predominant localization of these transporters at the sinusoidal membrane and of hENT1, hCNT1, and hCNT2 at the canalicular membrane. In the sandwich-cultured hepatocytes, ENTs were the major contributors to the transport of thymidine (hENT1, 63%; hENT2, 23%) or guanosine (hENT1, 53%; hENT2, 24%) into the hepatocytes followed by hCNT1 (10%) for thymidine or hCNT2 (23%) for guanosine. Although ribavirin was predominately transported (89%) into the hepatocytes by hENT1, fialuridine (FIAU) was transported by both hENT1 (30%) and hCNTs (61%). The extensively metabolized natural nucleosides were not effluxed into the bile, whereas significant biliary-efflux was observed of FIAU (19%), ribavirin (30%), and formycin B (35%). We conclude that the hepatic activity of hENT1 and hCNT1/2 transporters will determine the in vivo hepatic distribution and therefore the efficacy and/or toxicity of nucleoside drugs used to treat hepatic diseases.

Since the substrate specificities of OATP1B1, 1B3, and 2B1 are broad and overlapping, the contribution of each isoform to the overall hepatic uptake is of concern when assessing transporter-mediated drug-drug interactions (DDIs) or genetic polymorphism impact in the clinic. Herein, we quantitatively measured OATP proteins in cryopreserved hepatocytes, sandwich-cultured human hepatocytes (SCHH), and the liver, and examined the relationship with functional uptake of OATP substrates in an effort to identify the OATP isoform(s) contributing to the hepatic uptake of pitavastatin. The modulation of OATP expression in SCHH was found to be lot-dependent. However, OATP protein measurements averaged from 5 lots of SCHH were comparable to that of suspended hepatocytes. All three OATP transporters in suspended hepatocytes and SCHH were significantly lower than those in the liver. In SCHH, the uptake of CCK-8 and pravastatin was found to be associated with the expression of OATP1B3 and OATP1B1, respectively. In suspended hepatocytes, OATP1B1 appeared to show a positive trend with respect to the uptake of pitavastatin, which suggests a selective contribution of OATP1B1 to pitavastatin transport and thus an OATP quantitative protein expression-activity relationship. While the passive diffusion of rosuvastatin in SCHH was consistent across hepatocyte lots, the passive diffusion of pitavastatin varied over a broad range (>4-fold) in suspended hepatocytes and was inversely correlated with transporter-mediated uptake, presumably due to cell membrane alterations imparted by cryopreservation. Collectively, SCHH maintains OATP protein expression and membrane integrity and, if feasible when considering research goals, would be considered a superior tool for the characterization of in vitro transport parameters without the complication of membrane leakage.