Comparison of Human Long-Term Liver Models for Clearance Prediction of Slowly Metabolized Compounds.

The accurate prediction of human clearance is an important task during drug development. The proportion of low clearance compounds has increased in drug development pipelines across the industry since such compounds may be dosed in lower amounts and at lower frequency. Such compounds present new challenges to in vitro systems used for clearance extrapolation. In this study we compared the accuracy of clearance predictions of suspension culture to four different long-term stable in vitro liver models, including HepaRG sandwich culture, the Hµrel stochastic co-culture, the Hepatopac micropatterned co-culture (MPCC) and a micro-array spheroid culture. Hepatocytes in long-term stable systems remained viable and active over several days of incubation. Although intrinsic clearance values were generally high in suspension culture, clearance of low turnover compounds could frequently not be determined using this method. Metabolic activity and intrinsic clearance values from HepaRG cultures were low and, consequently, many compounds with low turnover did not show significant decline despite long incubation times. Similarly, stochastic co-cultures occasionally failed to show significant turnover for multiple low and medium turnover compounds. Among the different methods, MPCCs and spheroids provided the most consistent measurements. Notably, all culture methods resulted in underprediction of clearance, this could however be compensated for by regression correction. Combined, the results indicate that spheroid culture as well as the MPCC system provide adequate in vitro tools for human extrapolation for compounds with low metabolic turnover. Significance Statement In this study, we compared suspension cultures, HepaRG sandwich cultures, the Hµrel liver stochastic co-cultures, the Hepatopac micropatterned co-cultures (MPCC) and micro-array spheroid cultures for low clearance determination and prediction. Overall, HepaRG and suspension cultures showed modest value for the low determination and prediction of clearance compounds. The micro-array spheroid culture resulted in the most robust clearance measurements, whereas using the MPCC resulted in the most accurate prediction for low clearance compounds.

Compound Absorption in Polymer Devices Impairs the Translatability of Preclinical Safety Assessments.

Organotypic and microphysiological systems (MPS) that can emulate the molecular phenotype and function of human tissues, such as liver, are increasingly used in preclinical drug development. However, despite their improved predictivity, drug development success rates have remained low with most compounds failing in clinical phases despite promising preclinical data. Here, it is tested whether absorption of small molecules to polymers commonly used for MPS fabrication can impact preclinical pharmacological and toxicological assessments and contribute to the high clinical failure rates. To this end, identical devices are fabricated from eight different MPS polymers and absorption of prototypic compounds with different physicochemical properties are analyzed. It is found that overall absorption is primarily driven by compound hydrophobicity and the number of rotatable bonds. However, absorption can differ by >1000-fold between polymers with polydimethyl siloxane (PDMS) being most absorptive, whereas polytetrafluoroethylene (PTFE) and thiol-ene epoxy (TEE) absorbed the least. Strikingly, organotypic primary human liver cultures successfully flagged hydrophobic hepatotoxins in lowly absorbing TEE devices at therapeutically relevant concentrations, whereas isogenic cultures in PDMS devices are resistant, resulting in false negative safety signals. Combined, these results can guide the selection of MPS materials and facilitate the development of preclinical assays with improved translatability.

3D microperfusion of mesoscale human microphysiological liver models improves functionality and recapitulates hepatic zonation.

Hepatic in vitro models that accurately replicate phenotypes and functionality of the human liver are needed for applications in toxicology, pharmacology and biomedicine. Notably, it has become clear that liver function can only be sustained in 3D culture systems at physiologically relevant cell densities. Additionally, drug metabolism and drug-induced cellular toxicity often follow distinct spatial micropatterns of the metabolic zones in the liver acinus, calling for models that capture this zonation. We demonstrate the manufacture of accurate liver microphysiological systems (MPS) via engineering of 3D stereolithography printed hydrogel chips with arrays of diffusion open synthetic vasculature channels at spacings approaching in vivo capillary distances. Chip designs are compatible with seeding of cell suspensions or preformed liver cell spheroids. Importantly, primary human hepatocytes (PHH) and hiPSC-derived hepatocyte-like cells remain viable, exhibit improved molecular phenotypes compared to isogenic monolayer and static spheroid cultures and form interconnected tissue structures over the course of multiple weeks in perfused culture. 3D optical oxygen mapping of embedded sensor beads shows that the liver MPS recapitulates oxygen gradients found in the acini, which translates into zone-specific acet-ami-no-phen toxicity patterns. Zonation, here naturally generated by high cell densities and associated oxygen and nutrient utilization along the flow path, is also documented by spatial proteomics showing increased concentration of periportal- versus perivenous-associated proteins at the inlet region and vice versa at the outlet region. The presented microperfused liver MPS provides a promising platform for the mesoscale culture of human liver cells at phenotypically relevant densities and oxygen exposures. STATEMENT OF SIGNIFICANCE: A full 3D tissue culture platform is presented, enabled by massively parallel arrays of high-resolution 3D printed microperfusion hydrogel channels that functionally mimics tissue vasculature. The platform supports long-term culture of liver models with dimensions of several millimeters at physiologically relevant cell densities, which is difficult to achieve with other methods. Human liver models are generated from seeded primary human hepatocytes (PHHs) cultured for two weeks, and from seeded spheroids of hiPSC-derived human liver-like cells cultured for two months. Both model types show improved functionality over state-of-the-art 3D spheroid suspensions cultured in parallel. The platform can generate physiologically relevant oxygen gradients driven by consumption rather than supply, which was validated by visualization of embedded oxygen-sensitive microbeads, which is exploited to demonstrate zonation-specific toxicity in PHH liver models.

Bioengineered Pancreas-Liver Crosstalk in a Microfluidic Coculture Chip Identifies Human Metabolic Response Signatures in Prediabetic Hyperglycemia.

Aberrant glucose homeostasis is the most common metabolic disturbance affecting one in ten adults worldwide. Prediabetic hyperglycemia due to dysfunctional interactions between different human tissues, including pancreas and liver, constitutes the largest risk factor for the development of type 2 diabetes. However, this early stage of metabolic disease has received relatively little attention. Microphysiological tissue models that emulate tissue crosstalk offer emerging opportunities to study metabolic interactions. Here, a novel modular multitissue organ-on-a-chip device is presented that allows for integrated and reciprocal communication between different 3D primary human tissue cultures. Precisely controlled heterologous perfusion of each tissue chamber is achieved through a microfluidic single "synthetic heart" pneumatic actuation unit connected to multiple tissue chambers via specific configuration of microchannel resistances. On-chip coculture experiments of organotypic primary human liver spheroids and intact primary human islets demonstrate insulin secretion and hepatic insulin response dynamics at physiological timescales upon glucose challenge. Integration of transcriptomic analyses with promoter motif activity data of 503 transcription factors reveals tissue-specific interacting molecular networks that underlie ß-cell stress in prediabetic hyperglycemia. Interestingly, liver and islet cultures show surprising counter-regulation of transcriptional programs, emphasizing the power of microphysiological coculture to elucidate the systems biology of metabolic crosstalk.

Multi-Well Array Culture of Primary Human Hepatocyte Spheroids for Clearance Extrapolation of Slowly Metabolized Compounds.

Accurate prediction of human pharmacokinetics using in vitro tools is an important task during drug development. Albeit, currently used in vitro systems for clearance extrapolation such as microsomes and primary human hepatocytes in suspension culture show reproducible turnover, the utility of these systems is limited by a rapid decline of activity of drug metabolizing enzymes. In this study, a multi-well array culture of primary human hepatocyte spheroids was compared to suspension and single spheroid cultures from the same donor. Multi-well spheroids remained viable and functional over the incubation time of 3 days, showing physiological excretion of albumin and α-AGP. Their metabolic activity was similar compared to suspension and single spheroid cultures. This physiological activity, the high cell concentration, and the prolonged incubation time resulted in significant turnover of all tested low clearance compounds (n = 8). In stark contrast, only one or none of the compounds showed significant turnover when single spheroid or suspension cultures were used. Using multi-well spheroids and a regression offset approach (log(CLint) = 1.1 × + 0.85), clearance was predicted within 3-fold for 93% (13/14) of the tested compounds. Thus, multi-well spheroids represent a novel and valuable addition to the ADME in vitro tool kit for the determination of low clearance and overall clearance prediction. Graphical Abstract.

Organotypic and Microphysiological Human Tissue Models for Drug Discovery and Development-Current State-of-the-Art and Future Perspectives.

The number of successful drug development projects has been stagnant for decades despite major breakthroughs in chemistry, molecular biology, and genetics. Unreliable target identification and poor translatability of preclinical models have been identified as major causes of failure. To improve predictions of clinical efficacy and safety, interest has shifted to three-dimensional culture methods in which human cells can retain many physiologically and functionally relevant phenotypes for extended periods of time. Here, we review the state of the art of available organotypic culture techniques and critically review emerging models of human tissues with key importance for pharmacokinetics, pharmacodynamics, and toxicity. In addition, developments in bioprinting and microfluidic multiorgan cultures to emulate systemic drug disposition are summarized. We close by highlighting important trends regarding the fabrication of organotypic culture platforms and the choice of platform material to limit drug absorption and polymer leaching while supporting the phenotypic maintenance of cultured cells and allowing for scalable device fabrication. We conclude that organotypic and microphysiological human tissue models constitute promising systems to promote drug discovery and development by facilitating drug target identification and improving the preclinical evaluation of drug toxicity and pharmacokinetics. There is, however, a critical need for further validation, benchmarking, and consolidation efforts ideally conducted in intersectoral multicenter settings to accelerate acceptance of these novel models as reliable tools for translational pharmacology and toxicology. SIGNIFICANCE STATEMENT: Organotypic and microphysiological culture of human cells has emerged as a promising tool for preclinical drug discovery and development that might be able to narrow the translation gap. This review discusses recent technological and methodological advancements and the use of these systems for hit discovery and the evaluation of toxicity, clearance, and absorption of lead compounds.

Deconvolution of Cytochrome P450 Induction Mechanisms in HepaRG Nuclear Hormone Receptor Knockout Cells.

Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and PXR/CAR knockout (KO) HepaRG cells, as well as a PXR reporter gene assay, were used to investigate the mechanism of CYP3A4 and CYP2B6 induction by prototypical substrates and a group of compounds from the Merck KGaA oncology drug discovery pipeline. The basal and inducible gene expression of CYP3A4 and CYP2B6 of nuclear hormone receptor (NHR) KO HepaRG relative to control HepaRG was characterized. The basal expression of CYP3A4 was markedly higher in the PXR (10-fold) and CAR (11-fold) KO cell lines compared with control HepaRG, whereas inducibility was substantially lower. Inversely, basal expression of CYP3A4 in PXR/CAR double KO (dKO) was low (10-fold reduction). Basal CYP2B6 expression was high in PXR KO (9-fold) cells which showed low inducibility, whereas the basal expression remained unchanged in CAR and dKO cell lines compared with control cells. Most of the test compounds induced CYP3A4 and CYP2B6 via PXR and, to a lesser extent, via CAR. Furthermore, other non-NHR-driven induction mechanisms were implicated, either alone or in addition to NHRs. Notably, 5 of the 16 compounds (31%) that were PXR inducers in HepaRG did not activate PXR in the reporter gene assay, illustrating the limitations of this system. This study indicates that HepaRG is a highly sensitive system fit for early screening of cytochrome P450 (P450) induction in drug discovery. Furthermore, it shows the applicability of HepaRG NHR KO cells as tools to deconvolute mechanisms of P450 induction using novel compounds representative for oncology drug discovery. SIGNIFICANCE STATEMENT: This work describes the identification of induction mechanisms of CYP3A4 and CYP2B6 for an assembly of oncology drug candidates using HepaRG nuclear hormone receptor knockout and displays its advantages compared to a pregnane X receptor reporter gene assay. With this study, risk assessment of drug candidates in early drug development can be improved.

Bacterial microbiota diversity and composition in red and white wines correlate with plant-derived DNA contributions and botrytis infection.

Wine is a globally produced, marketed and consumed alcoholic beverage, which is valued for its aromatic and qualitative complexity and variation. These properties are partially attributable to the bacterial involvement in the fermentation process. However, the organizational principles and dynamic changes of the bacterial wine microbiota remain poorly understood, especially in the context of red and white wine variations and environmental stress factors. Here, we determined relative and absolute bacterial microbiota compositions from six distinct cultivars during the first week of fermentation by quantitative and qualitative 16S rRNA gene amplification and amplicon sequencing. All wines harboured complex and variable bacterial communities, with Tatumella as the most abundant genus across all batches, but red wines were characterized by higher bacterial diversity and increased relative and absolute abundance of lactic and acetic acid bacteria (LAB/AAB) and bacterial taxa of predicted environmental origin. Microbial diversity was positively correlated with plant-derived DNA concentrations in the wine and Botrytis cinerea infection before harvest. Our findings suggest that exogenous factors, such as procedural differences between red and white wine production and environmental stress on grape integrity, can increase bacterial diversity and specific bacterial taxa in wine, with potential consequences for wine quality and aroma.