Intestinal organoids as an in vitro platform to characterize disposition, metabolism, and safety profile of small molecules.

Intestinal organoids derived from LGR5+ adult stem cells allow for long-term culturing, more closely resemble human physiology than traditional intestinal models, like Caco-2, and have been established for several species. Here we evaluated intestinal organoids for drug disposition, metabolism, and safety applications. Enterocyte-enriched human duodenal organoids were cultured as monolayers to enable bidirectional transport studies. 3D enterocyte-enriched human duodenal and colonic organoids were incubated with probe substrates of major intestinal drug metabolizing enzymes (DMEs). To distinguish human intestinal toxic (high incidence of diarrhea in clinical trials and/or black box warning related to intestinal side effects) from non-intestinal toxic compounds, ATP-based cell viability was used as a readout, and compounds were ranked based on their IC50 values in relation to their 30-times maximal total plasma concentration (Cmax). To assess if rat and dog organoids reproduced the respective in vivo intestinal safety profiles, ATP-based viability was assessed in rat and dog organoids and compared to in vivo intestinal findings when available. Human duodenal monolayers discriminated high and low permeable compounds and demonstrated functional activity for the main efflux transporters Multi drug resistant protein 1 (MDR1, P-glycoprotein P-gp) and Breast cancer resistant protein (BCRP). Human 3D duodenal and colonic organoids also showed metabolic activity for the main intestinal phase I and II DMEs. Organoids derived from specific intestinal segments showed activity differences in line with reported DMEs expression. Undifferentiated human organoids accurately distinguished all but one compound from the test set of non-toxic and toxic drugs. Cytotoxicity in rat and dog organoids correlated with preclinical toxicity findings and observed species sensitivity differences between human, rat, and dog organoids. In conclusion, the data suggest intestinal organoids are suitable in vitro tools for drug disposition, metabolism, and intestinal toxicity endpoints. The possibility to use organoids from different species, and intestinal segment holds great potential for cross-species and regional comparisons.

Unlocking the Mechanisms of Cutaneous Adverse Drug Reactions: Activation of the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway by EGFR Inhibitors Triggers Keratinocyte Differentiation and Polarization of Epidermal Immune Responses.

EGFR inhibitors used in oncology therapy modify the keratinocyte differentiation processes, impairing proper skin barrier formation and leading to cutaneous adverse drug reactions. To uncover the molecular signatures associated with cutaneous adverse drug reactions, we applied phosphoproteomic and transcriptomic assays on reconstructed human epidermis tissues exposed to a therapeutically relevant concentration of afatinib, a second-generation EGFR inhibitor. After drug exposure, we observed activation of the phosphatidylinositol 3-kinase/protein kinase B pathway associated with an increased expression of gene families involved in keratinocyte differentiation, senescence, oxidative stress, and alterations in the epidermal immune-related markers. Furthermore, our results show that afatinib may interfere with vitamin D3 metabolism, acting via CYP27A1 and CYP24A1 to regulate calcium concentration through the phosphatidylinositol 3-kinase/protein kinase B pathway. Consequently, basal layer keratinocytes switch from a pro-proliferating to a prodifferentiative program, characterized by upregulation of biomarkers associated with increased keratinization, cornification, T helper type 2 response, and decreased innate immunity. Such effects may increase skin susceptibility to cutaneous penetration of irritants and pathogens. Taken together, these findings demonstrate a molecular mechanism of EGFR inhibitor-induced cutaneous adverse drug reactions.

Prediction of the drug-drug interaction potential of the α1-acid glycoprotein bound, CYP3A4/CYP2C9 metabolized oncology drug, erdafitinib.

Erdafitinib is a potent oral pan-fibroblast growth factor receptor inhibitor being developed as oncology drug for patients with alterations in the fibroblast growth factor receptor pathway. Erdafitinib binds preferentially to α1-acid glycoprotein (AGP) and is primarily metabolized by cytochrome P450 (CYP) 2C9 and 3A4. This article describes a physiologically based pharmacokinetic (PBPK) model for erdafitinib to assess the drug-drug interaction (DDI) potential of CYP3A4 and CYP2C9 inhibitors and CYP3A4/CYP2C9 inducers on erdafitinib pharmacokinetics (PK) in patients with cancer exhibiting higher AGP levels and in populations with different CYP2C9 genotypes. Erdafitinib's DDI potential as a perpetrator for transporter inhibition and for time-dependent inhibition and/or induction of CYP3A was also evaluated. The PBPK model incorporated input parameters from various in vitro and clinical PK studies, and the model was verified using a clinical DDI study with itraconazole and fluconazole. Erdafitinib clearance in the PBPK model consisted of multiple pathways (CYP2C9/3A4, renal, intestinal; additional hepatic clearance), making the compound less susceptible to DDIs. In poor-metabolizing CYP2C9 populations carrying the CYP2C9*3/*3 genotype, simulations shown clinically relevant increase in erdafitinib plasma concentrations. Simulated luminal and enterocyte concentration showed potential risk of P-glycoprotein inhibition with erdafitinib in the first 5 h after dosing, and simulations showed this interaction can be avoided by staggering erdafitinib and digoxin dosing. Other than a simulated ~ 60% exposure reduction with strong CYP3A/2C inducers such as rifampicin, other DDI liabilities were minimal and considered not clinically relevant.

Clinical Investigation on Endogenous Biomarkers to Predict Strong OAT-Mediated Drug-Drug Interactions.

BACKGROUND: Endogenous biomarkers are promising tools to assess transporter-mediated drug-drug interactions early in humans. METHODS: We evaluated on a common and validated in vitro system the selectivity of 4-pyridoxic acid (PDA), homovanillic acid (HVA), glycochenodeoxycholate-3-sulphate (GCDCA-S) and taurine towards different renal transporters, including multidrug resistance-associated protein, and assessed the in vivo biomarker sensitivity towards the strong organic anion transporter (OAT) inhibitor probenecid at 500 mg every 6 h to reach close to complete OAT inhibition. RESULTS: PDA and HVA were substrates of the OAT1/2/3, OAT4 (PDA only) and multidrug resistance-associated protein 4; GCDCA-S was more selective, having affinity only towards OAT3 and multidrug resistance-associated protein 2. Taurine was not a substrate of any of the investigated transporters under the in vitro conditions tested. Plasma exposure of PDA and HVA significantly increased and the renal clearance of GCDCA-S, PDA and HVA decreased; the magnitude of these changes was comparable to those of known clinical OAT probe substrates. PDA and GCDCA-S were the most promising endogenous biomarkers of the OAT pathway activity: PDA plasma exposure was the most sensitive to probenecid inhibition, and, in contrast, GCDCA-S was the most sensitive OAT biomarker based on renal clearance, with higher selectivity towards the OAT3 transporter. CONCLUSIONS: The current findings illustrate a clear benefit of measuring PDA plasma exposure during phase I studies when a clinical drug candidate is suspected to be an OAT inhibitor based on in vitro data. Subsequently, combined monitoring of PDA and GCDCA-S in both urine and plasma is recommended to tease out the involvement of OAT1/3 in the inhibition interaction. CLINICAL TRIAL REGISTRATION: EudraCT number: 2016-003923-49.

EGFR inhibitors switch keratinocytes from a proliferative to a differentiative phenotype affecting epidermal development and barrier function.

BACKGROUND: Cutaneous adverse drug reactions (CADR) associated with oncology therapy involve 45-100% of patients receiving kinase inhibitors. Such adverse reactions may include skin inflammation, infection, pruritus and dryness, symptoms that can significantly affect the patient's quality of life. To prevent severe skin damages dose adjustment or drug discontinuation is often required, interfering with the prescribed oncology treatment protocol. This is particularly the case of Epidermal Growth Factor Receptor inhibitors (EGFRi) targeting carcinomas. Since the EGFR pathway is pivotal for epidermal keratinocytes, it is reasonable to hypothesize that EGFRi also affect these cells and therefore interfere with the epidermal structure formation and skin barrier function. METHODS: To test this hypothesis, the effects of EGFRi and Vascular Endothelial Growth Factor Receptor inhibitors (VEGFRi) at therapeutically relevant concentrations (3, 10, 30, 100 nM) were assessed on proliferation and differentiation markers of human keratinocytes in a novel 3D micro-epidermis tissue culture model. RESULTS: EGFRi directly affect basal keratinocyte growth, leading to tissue size reduction and switching keratinocytes from a proliferative to a differentiative phenotype, as evidenced by decreased Ki67 staining and increased filaggrin, desmoglein-1 and involucrin expression compared to control. These effects lead to skin barrier impairment, which can be observed in a reconstructed human epidermis model showing a decrease in trans-epidermal water loss rates. On the other hand, pan-kinase inhibitors mainly targeting VEGFR barely affect keratinocyte differentiation and rather promote a proliferative phenotype. CONCLUSIONS: This study contributes to the mechanistic understanding of the clinically observed CADR during therapy with EGFRi. These in vitro results suggest a specific mode of action of EGFRi by directly affecting keratinocyte growth and barrier function.

The utility of a differentiated preclinical liver model, HepaRG cells, in investigating delayed toxicity via inhibition of mitochondrial-replication induced by fialuridine.

During its clinical development fialuridine caused liver toxicity and the death of five patients. This case remains relevant due to the continued development of mechanistically-related compounds against a back-drop of simple in vitro models which remain limited for the preclinical detection of such delayed toxicity. Here, proteomic investigation of a differentiated, HepaRG, and proliferating, HepG2 cell model was utilised to confirm the presence of the hENT1 transporter, thymidine kinase-1 and -2 (TK1, TK2) and thymidylate kinase, all essential in order to reproduce the cellular activation and disposition of fialuridine in the clinic. Acute metabolic modification assays could only identify mitochondrial toxicity in HepaRG cells following extended dosing, 2 weeks. Toxic effects were observed around 10 µM, which is within a range of 10-15 X approximate Cmax. HepaRG cell death was accompanied by a significant decrease in mitochondrial DNA content, indicative of inhibition of mitochondrial replication, and a subsequent reduction in mitochondrial respiration and the activity of mitochondrial respiratory complexes, not replicated in HepG2 cells. The structural epimer of fialuridine, included as a pharmacological negative control, was shown to have no cytotoxic effects in HepaRG cells up to 4 weeks. Overall, these comparative studies demonstrate the HepaRG model has translational relevance for fialuridine toxicity and therefore may have potential in investigating the inhibition of mitochondrial replication over prolonged exposure for other toxicants.

Pharmacokinetic Drug-Drug Interaction of Apalutamide, Part 2: Investigating Interaction Potential Using a Physiologically Based Pharmacokinetic Model.

BACKGROUND: Apalutamide is predominantly metabolized via cytochrome P450 (CYP) 2C8 and CYP3A4, whose contributions change due to autoinduction with repeated dosing. OBJECTIVES: We aimed to predict CYP3A4 and CYP2C8 inhibitor/inducer effects on the steady-state pharmacokinetics of apalutamide and total potency-adjusted pharmacologically active moieties, and simulated drug-drug interaction (DDI) between single-dose and repeated-dose apalutamide coadministered with known inhibitors/inducers. METHODS: We applied physiologically based pharmacokinetic modeling for our predictions, and simulated DDI between single-dose and repeated-dose apalutamide 240 mg coadministered with ketoconazole, gemfibrozil, or rifampicin. RESULTS: The estimated contribution of CYP2C8 and CYP3A4 to apalutamide metabolism is 58% and 13%, respectively, after single dosing, and 40% and 37%, respectively, at steady-state. Apalutamide exposure is predicted to increase with ketoconazole (maximum observed concentration at steady-state [Cmax,ss] 38%, area under the plasma concentration-time curve at steady-state [AUCss] 51% [pharmacologically active moieties, Cmax,ss 23%, AUCss 28%]) and gemfibrozil (Cmax,ss 32%, AUCss 44% [pharmacologically active moieties, Cmax,ss 19%, AUCss 23%]). Rifampicin exposure is predicted to decrease apalutamide (Cmax,ss 25%, AUCss 34% [pharmacologically active moieties, Cmax,ss 15%, AUCss 19%]). CONCLUSIONS: Based on our simulations, no major changes in the pharmacokinetics of apalutamide or pharmacologically active moieties are expected with strong CYP3A4/CYP2C8 inhibitors/inducers. This observation supports the existing recommendations that no dose adjustments are needed during coadministration of apalutamide and the known inhibitors or inducers of CYP2C8 or CYP3A4.

Amino acid levels determine metabolism and CYP450 function of hepatocytes and hepatoma cell lines.

Predicting drug-induced liver injury in a preclinical setting remains challenging, as cultured primary human hepatocytes (PHHs), pluripotent stem cell-derived hepatocyte-like cells (HLCs), and hepatoma cells exhibit poor drug biotransformation capacity. We here demonstrate that hepatic functionality depends more on cellular metabolism and extracellular nutrients than on developmental regulators. Specifically, we demonstrate that increasing extracellular amino acids beyond the nutritional need of HLCs and HepG2 cells induces glucose independence, mitochondrial function, and the acquisition of a transcriptional profile that is closer to PHHs. Moreover, we show that these high levels of amino acids are sufficient to drive HLC and HepG2 drug biotransformation and liver-toxin sensitivity to levels similar to those in PHHs. In conclusion, we provide data indicating that extracellular nutrient levels represent a major determinant of cellular maturity and can be utilized to guide stem cell differentiation to the hepatic lineage.

Acute Metabolic Switch Assay Using Glucose/Galactose Medium in HepaRG Cells to Detect Mitochondrial Toxicity.

Using galactose instead of glucose in the culture medium of hepatoma cell lines, such as HepG2 cells, has been utilized for a decade to unmask the mitochondrial liability of chemical compounds. A modified glucose-galactose assay on HepG2 cells, reducing the experimental period for screening of mitochondrial toxicity to 2 to 4 hr, has been previously reported. HepaRG cells are one of the few cell lines that retain some of the important characteristics of human hepatocytes, offering advantages of working with a cell line, therefore, are considered an alternative for HepG2 cells in drug toxicity screening. A method is described here using HepaRG cells in an acute metabolic switch assay utilizing specific glucose/galactose media, a combined ATP-protein-LDH assay measuring three endpoints from one 96-well plate, and a criteria to label a compound as a mitochondrial toxin. © 2019 by John Wiley & Sons, Inc.

Characterization of Healthy Donor-Derived T-Cell Responses Specific to Telaprevir Diastereomers.

Telaprevir, a protease inhibitor, was used alongside PEGylated interferon-α and ribavirin to treat hepatitis C viral infections. The triple regimen proved successful; however, the appearance of severe skin reactions alongside competition from newer drugs restricted its use. Skin reactions presented with a delayed onset indicative of a T-cell mediated reaction. Thus, the aim of this study was to investigate whether telaprevir and/or its diastereomer, which is generated in humans, activates T-cells. Telaprevir in its S-configured therapeutic form and the R-diastereomer were cultured directly with peripheral blood mononuclear cells from healthy donors prior to the generation of T-cell clones by serial dilution. Drug-specific CD4+ and CD8+ T-cell clones responsive to telaprevir and the R-diastereomer were generated and characterized in terms of phenotype and function. The clones proliferated with telaprevir and diastereomer concentrations of 5-20 µM and secreted IFN-γ, IL-13, and granzyme B. In contrast, the telaprevir M11 metabolite did not stimulate T-cells. The CD8+ T-cell response was MHC I-restricted and dependent on the presence of soluble drug. Flow cytometric analysis showed that clones expressed chemokine receptors CCR4 (skin homing) and CXCR3 (migration to peripheral tissue) and 1 of 3 distinct TCR Vßs; TCR Vß 2, 5.1, or 22. These data show the propensity of both R- and S-forms of telaprevir to generate skin-homing cytotoxic T-cells that may induce the adverse reactions observed in human patients.

Effect of Plasma Protein Binding on the Anti-Hepatitis B Virus Activity and Pharmacokinetic Properties of NVR 3-778.

High plasma protein binding (PPB) levels not only affect drug-target engagement but can also impact exposure of hepatocytes to antivirals and thereby affect antiviral activity. In this study, we assessed the effect of PPB on the antiviral activity of NVR 3-778, a sulfamoylbenzamide capsid assembly modulator (CAM). To this end, primary human hepatocyte (PHH) medium was spiked with plasma proteins. First, the effect of plasma proteins on the hepatitis B virus (HBV) infection assay was evaluated. The addition of plasma proteins neither decreased cell viability nor affected HBV DNA secretion or intracellular HBV RNA accumulation. In contrast, the secretion and intracellular amount of HBV proteins were induced with increasing amounts of plasma proteins. Next, the antiviral activity of NVR 3-778 was demonstrated by multiple assays while PPB and the time-dependent disappearance of the parent drug were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma proteins strongly decreased the free fraction of NVR 3-778, resulting in a physiologically relevant in vitro hepatocyte exposure. NVR 3-778 displayed a high PPB level, while the antiviral activity was reduced approximately only 4-fold. The disconnect between the high PPB level and the only moderate shift of the antiviral activity was explained by the rapid hepatic clearance of NVR 3-778 in the absence of plasma proteins. This study highlights the use of PHHs as a model to accurately determine the antiviral activity by capturing PPB, clearance, and liver distribution. It is advantageous to consider both pharmacokinetics and pharmacodynamics for selection of HBV antiviral drug candidates and for successful extrapolation of in vitro data to clinical studies.

Finding synergies for 3Rs - Toxicokinetics and read-across: Report from an EPAA partners' Forum.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a Partners' Forum Toxicokinetics and Read-Across to provide an overview on research activities to develop in vitro toxicokinetics methods and physiologically-based kinetic (PBK) models and to find synergies to enhance use of toxicokinetic data to strengthen read-across. Currently, lacking toxicokinetic data often prevent the application of read-across. Preferably, toxicokinetic data should be generated using in vitro and in silico tools and anchored towards human relevance. In certain sectors, PBK modelling is being used for risk assessment, but less so in others. Specific activities were identified to facilitate the use of in vitro and in silico toxicokinetic data to support read-across: The collation of available tools indicating the parameters and applicability domains covered; endpoint-specific guidance on toxicokinetics parameters required for read-across; case studies exemplifying how toxicokinetic data help support read-across. Activities to enhance the scientific robustness of read-across include the further user-friendly combination of read-across tools and formal guidance by the authorities specifying the minimum information requirements to justify read-across for a given toxicity endpoint. The EPAA was invited to continue dissemination activities and to explore possibilities to collate a contemporaneous list of open toxicokinetics tools that assist risk assessment.

Establishing a systematic framework to characterise in vitro methods for human hepatic metabolic clearance.

Hepatic metabolic clearance is one of the most important factors driving the overall kinetics of chemicals including substances used in various product categories such as pesticides, biocides, pharmaceuticals, and cosmetics. A large number of in vitro systems from purified isozymes and subcellular organelles to hepatocytes in simple cultures and in complex scaffold setups are available for measuring hepatic metabolic clearance for different applications. However, there is currently no approach for systematically characterising and comparing these in vitro methods in terms of their design, applicability and performance. To address this, existing knowledge in the field of in vitro human hepatic metabolic clearance methods was gathered and analysed in order to establish a framework to systematically characterise methods based on a set of relevant components. An analogous framework would be also applicable for non-human in vitro systems. The components are associated with the biological test systems used (e.g. subcellular or cells), the in vitro method (e.g. number of cells, test item solubility), related analytical techniques, data interpretation methods (based on substrate depletion/metabolite formation), and performance assessments (precision and accuracy of clearance measurements). To facilitate the regulatory acceptance of this class of methods, it is intended that the framework provide the basis of harmonisation work within the OECD.

The utility of HepaRG cells for bioenergetic investigation and detection of drug-induced mitochondrial toxicity.

The importance of mitochondrial toxicity in drug-induced liver injury is well established. The bioenergetic phenotype of the HepaRG cell line was defined in order to assess their suitability as a model of mitochondrial hepatotoxicity. Bioenergetic phenotyping categorised the HepaRG cells as less metabolically active when measured beside the more energetic HepG2 cells. However, inhibition of mitochondrial ATP synthase induced an increase in glycolytic activity of both HepaRG and HepG2 cells suggesting an active Crabtree Effect in both cell lines. The suitability of HepaRG cells for the acute metabolic modification assay as a screen for mitotoxicity was confirmed using a panel of compounds, including both positive and negative mitotoxic compounds. Seahorse respirometry studies demonstrated that a statistically significant decrease in spare respiratory capacity is the first indication of mitochondrial dysfunction. Furthermore, based upon comparing changes in respiratory parameters to those of the positive controls, rotenone and carbonyl cyanide m-chlorophenyl hydrazone, compounds were categorised into two mechanistic groups; inhibitors or uncouplers of the electron transport chain. Overall, the findings from this study have demonstrated that HepaRG cells, despite having different resting bioenergetic phenotype to HepG2 cells are a suitable model to detect drug-induced mitochondrial toxicity with similar detection rates to HepG2 cells.

Clinical Investigation of Coproporphyrins as Sensitive Biomarkers to Predict Mild to Strong OATP1B-Mediated Drug-Drug Interactions.

INTRODUCTION: Coproporphyrin (CP) I and III have recently been proposed as endogenous clinical biomarkers to predict organic anion-transporting polypeptide 1B (OATP1B)-mediated drug-drug interactions (DDIs). In the present study, we first investigated the in vitro selectivity of CPI and CPIII towards drug uptake and efflux transporters. We then assessed the in vivo biomarker sensitivity towards OATP1B inhibition. METHODS: To assess transporter selectivity, incubations with CPI and CPIII were performed in vitro, using single transporter-expressing and control systems. Furthermore, CPI and CPIII plasma concentrations were determined from participants of three independent clinical trials who were administered with either a strong, moderate, or mild clinical OATP1B inhibitor. RESULTS: Our results show that CPI and CPIII are substrates of OATP1B1, OATP1B3, the multidrug resistance-associated protein (MRP) 2, and MRP3. No substrate interaction was shown for other prominent drug transporters that have been associated with clinical DDIs. Results from clinical studies demonstrated that changes in CPI and CPIII plasma levels were predictive for moderate (two to threefold area under the concentration-time curve [AUC] increase) and strong (≥ fivefold increases) clinical OATP1B inhibition. Furthermore, CPI, but not CPIII, concentration changes were predictive for a mild clinically observed DDI where CPI AUC increases of 1.4-fold were comparable with those observed for pitavastatin as victim drug (AUC increases of 1.5-fold). CONCLUSION: Our results demonstrate the selectivity of CPI and CPIII towards the OATP1B/MRP pathway, and the herein reported data further underline the potential of CPI and CPIII as selective and sensitive clinical biomarkers to quantify OATP1B-mediated DDIs.

Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture.

The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) is of great interest for applications in pharmacological research. However, full maturation of hiPSC-derived hepatocytes has not yet been achieved in vitro. To improve hepatic differentiation, co-cultivation of hiPSC with human umbilical vein endothelial cells (HUVEC) during hepatic differentiation was investigated in this study. In the first step, different culture media variations based on hepatocyte culture medium (HCM) were tested in HUVEC mono-cultures to establish a suitable culture medium for co-culture experiments. Based on the results, two media variants were selected to differentiate hiPSC-derived definitive endodermal (DE) cells into mature hepatocytes with or without HUVEC addition. DE cells differentiated in mono-cultures in the presence of those media variants showed a significant increase (p < 0.05) in secretion of α-fetoprotein and in activities of cytochrome P450 (CYP) isoenzymes CYP2B6 and CYP3A4 as compared with cells differentiated in unmodified HCM used as control. Co-cultivation with HUVEC did not further improve the differentiation outcome. Thus, it can be concluded that the effect of the used medium outweighed the effect of HUVEC co-culture, emphasizing the importance of the culture medium composition for hiPSC differentiation.

Elucidating the Plasma and Liver Pharmacokinetics of Simeprevir in Special Populations Using Physiologically Based Pharmacokinetic Modelling.

The disposition of simeprevir (SMV) in humans is characterised by cytochrome P450 3A4 metabolism and hepatic uptake by organic anion transporting polypeptide 1B1/3 (OATP1B1/3). This study was designed to investigate SMV plasma and liver exposure upon oral administration in subjects infected with hepatitis C virus (HCV), in subjects of Japanese or Chinese origin, subjects with organ impairment and subjects with OATP genetic polymorphisms, using physiologically based pharmacokinetic modelling. Simulations showed that compared with healthy Caucasian subjects, SMV plasma exposure was 2.4-, 1.7-, 2.2- and 2.0-fold higher, respectively, in HCV-infected Caucasian subjects, in healthy Japanese, healthy Chinese and subjects with severe renal impairment. Further simulations showed that compared with HCV-infected Caucasian subjects, SMV plasma exposure was 1.6-fold higher in HCV-infected Japanese subjects. In subjects with OATP1B1 genetic polymorphisms, no noteworthy changes in SMV pharmacokinetics were observed. Simulations suggested that liver concentrations in Caucasians with HCV are 18 times higher than plasma concentrations.

Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor.

The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 10(6) hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP), a marker for DE, was significantly (p < 0.05) higher in 2D cultures, while secretion of albumin, a typical characteristic for mature hepatocytes, was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2, CYP2B6, and CYP3A4 in both groups, although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p < 0.05) higher in 3D bioreactors compared with 2D cultures, which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin, cytokeratin 18 (CK18), and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition, cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture systems in stem cell differentiation approaches for improved formation of differentiated tissue structures.

In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System.

Accurate prediction of the potential hepatotoxic nature of new pharmaceuticals remains highly challenging. Therefore, novel in vitro models with improved external validity are needed to investigate hepatic metabolism and timely identify any toxicity of drugs in humans. In this study, we examined the effects of diclofenac, as a model substance with a known risk of hepatotoxicity in vivo, in a dynamic multi-compartment bioreactor using primary human liver cells. Biotransformation pathways of the drug and possible effects on metabolic activities, morphology and cell transcriptome were evaluated. Formation rates of diclofenac metabolites were relatively stable over the application period of seven days in bioreactors exposed to 300 µM diclofenac (300 µM bioreactors (300 µM BR)), while in bioreactors exposed to 1000 µM diclofenac (1000 µM BR) metabolite concentrations declined drastically. The biochemical data showed a significant decrease in lactate production and for the higher dose a significant increase in ammonia secretion, indicating a dose-dependent effect of diclofenac application. The microarray analyses performed revealed a stable hepatic phenotype of the cells over time and the observed transcriptional changes were in line with functional readouts of the system. In conclusion, the data highlight the suitability of the bioreactor technology for studying the hepatotoxicity of drugs in vitro.