Investigative Toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, Investigative Toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adhering to the 3R (Replacement, Reduction and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, Investigative Toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of Investigative Toxicology is improving preclinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded due to the use of inappropriate animal models. Progress in Investigative Toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an “Investigative Toxicology Think-Tank”, which aimed to enhance the interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights Investigative Toxicology’s position by identifying key challenges and perspectives.
Bioprinted 3D primary human intestinal tissues model aspects of native physiology and ADME/Tox functions
The human intestinal mucosa is a critical site for absorption, distribution, metabolism, and excretion (ADME)/Tox studies in drug development and is difficult to recapitulate in vitro. Using bioprinting, we generated three-dimensional (3D) intestinal tissue composed of human primary intestinal epithelial cells and myofibroblasts with architecture and function to model the native intestine. The 3D intestinal tissue demonstrates a polarized epithelium with tight junctions and specialized epithelial cell types and expresses functional and inducible CYP450 enzymes. The 3D intestinal tissues develop physiological barrier function, distinguish between high- and low-permeability compounds, and have functional P-gp and BCRP transporters. Biochemical and histological characterization demonstrate that 3D intestinal tissues can generate an injury response to compound-induced toxicity and inflammation. This model is compatible with existing preclinical assays and may be implemented as an additional bridge to clinical trials by enhancing safety and efficacy prediction in drug development.
Utilization of the ExVive human liver tissue model to assess drug-induced liver injury across a diverse set of chemical classes
One of the key challenges in the drug development process continues to be the early identification of compounds with adverse and potentially dose-limiting liver toxicity. Traditionally hepatotoxicity prediction has relied on two-dimensional hepatocyte monolayers, sandwich culture assays and non-human animal models. These systems are limited in their ability to accurately reflect in vivo human biology and lack of the cellular complexity required to model tissue level outcomes of toxicity. In this study, drug-induced liver injury (DILI) was assessed in 3D-bioprinted human liver tissues comprised of primary hepatocytes, hepatic stellate cells, and endothelial cells (ExVive™ Human Liver Tissues) treated with known high and low DILI risk compounds. Tissue response to compounds was evaluated using a range of biochemical, cytokine secretion, gene expression and histologic analyses. The high DILI risk compounds tolcapone, benzbromarone, danazol and tamoxifen were evaluated using a 28 day dosing regimen and compared to safer compounds entacapone, phentolamine, betahistine, nifedipine and chloramphenicol. Tissues treated with the known toxicants exhibited evidence of toxicity in at least two assays. A comparison of the clinically related compounds tolcapone and entacapone at concentrations of 1x, 3x and 10x Cmax revealed clear differences in their impact on the bioprinted tissues; tolcapone resulted in a dose dependent decrease in tissue viability at 10x Cmax while entacapone resulted in no significant changes in viability. Significant reductions in albumin secretion were seen at 3x Cmax with tolcapone treatment, vs. 10x Cmax with entacapone. Treatment of the 3D liver tissues with 5x and 20x Cmax concentrations of benzbromarone for 28 days resulted in decreased tissue viability with the highest concentration along with time and dose dependent decreases in albumin production beginning at treatment day 7 with the 5x and 20x Cmax concentrations. Histologic assessment of these tissues revealed significant loss of tissue and disruption of cellular cohesion at the 20x Cmax dose. These results suggest 3D bioprinted liver tissues are well suited to differentiate high risk from low risk DILI compounds and utilize both biochemical and histologic endpoints to assess multiple mechanisms of DILI in vitro, providing a comprehensive means of examining tissue injury.
3D proximal tubule tissues recapitulate key aspects of renal physiology to enable nephrotoxicity testing
Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule is a primary site of nephrotoxicity and resulting attrition in the drug development pipeline. Current preclinical methods using 2D cell cultures and animal models are unable to fully recapitulate clinical drug responses due to limited in vitro functional lifespan, or species-specific differences. Using Organovo’s proprietary 3D bioprinting platform, we have developed a fully cellular human in vitro model of the proximal tubule interstitial interface comprising renal fibroblasts, endothelial cells, and primary human renal proximal tubule epithelial cells to enable more accurate prediction of tissue-level clinical outcomes. Histological characterization demonstrated formation of extensive microvascular networks supported by endogenous extracellular matrix deposition. The epithelial cells of the 3D proximal tubule tissues demonstrated tight junction formation and expression of renal uptake and efflux transporters; the polarized localization and function of P-gp and SGLT2 were confirmed. Treatment of 3D proximal tubule tissues with the nephrotoxin cisplatin induced loss of tissue viability and epithelial cells in a dose-dependent fashion, and cimetidine rescued these effects, confirming the role of the OCT2 transporter in cisplatin-induced nephrotoxicity. The tissues also demonstrated a fibrotic response to TGFβ as assessed by an increase in gene expression associated with human fibrosis and histological verification of excess extracellular matrix deposition. Together, these results suggest that the bioprinted 3D proximal tubule model can serve as a test bed for the mechanistic assessment of human nephrotoxicity and the development of pathogenic states involving epithelial-interstitial interactions, making them an important adjunct to animal studies.
Monocrotaline toxicity in ExVive 3D bioprinted human liver tissue
Monocrotaline (MCT), a pyrrolizidine alkaloid causes liver injury in animals similar to that of hepatic venoocclusive disorder in humans. MCT induced liver injury occurs through a complex set of cellular insults involving multiple cell types which can ultimately lead to fibrotic changes. In the study, we evaluated the effects of MCT in 3D-bioprinted human liver tissue comprising of primary hepatocytes, hepatic stellate cells, and endothelial cells (ExVive™ Human Liver Tissue). The bioprinted tissues were treated with MCT for fourteen days. MCT treatment led to time- and dose-dependent decreases in tissue health as measured by LDH leakage and albumin synthesis and by histopathologic changes in the tissues, as well as increases in the production of the pro-inflammatory cytokines IL-1β, IL-4, IL-8 and IL-10. Histologic assessment of formalin-fixed, paraffin-embedded tissue revealed signs of tissue damage, including dissociation of the network of hepatocytes and reduced cellularity within the tissues. Immunohistochemical analyses revealed a dose-dependent increase in CD31+ cells and a marked increase in the appearance of large, CD31+ bright cells that co-expressed smooth muscle actin (α-SMA), often forming clusters or complex multicellular structures. Changes in organization of CD31 expressing endothelial cells and appearance of α-SMA expressing cells are indicative of remodeling and initiation of fibrotic events. Observations which emerged from this study capture the spectrum of changes induced by MCT ranging from reduced hepatocellular function and vascular remodeling, which may involve endothelial cell migration, organization, proliferation, apoptosis, and endothelial-to-mesenchymal transformation to early fibrotic events.
A 3D bioprinted model of the renal proximal tubule for evaluation of drug-induced nephrotoxicity
The kidney proximal tubule (PT) is a primary site of nephrotoxicity and resulting drug attrition in the development pipeline. Using Organovo’s proprietary bioprinting platform, we have developed a fully-human in vitro model of the PT to potentially enable more accurate prediction of tissue-level clinical outcomes. ExVive™ Human Kidney Tissue is created in a standard 24-well Transwell® plate by spatially-controlled deposition of cell aggregates in the absence of exogenous scaffold. Tissues are composed of an interstitial layer of renal fibroblasts and endothelial cells supporting a monolayer of human primary PT epithelial cells.
Bioprinted human tissues for toxicology and disease modeling
The high rate of attrition among clinical-stage therapies highlights the need for in vitro models that generate data which translates into the clinic. Fully human bioprinted tissues with spatially-controlled architecture enable biochemical, genetic, and histologic interrogation following exposure to modulators of interest, making them valuable in vitro tools for toxicology and disease modeling. We have generated bioprinted human liver tissues exhibiting histological and functional similarity to native liver, with sustained viability (ATP, albumin) and CYP3A4 activity over 4 weeks. The liver tissues display hallmark biochemical and histologic responses to hepatotoxicants such as valproic acid, exemplified by decreases in ATP and GSH and the accumulation of cytoplasmic vacuoles within the hepatocytes reflective of a steatotic disease phenotype. Characterization of bioprinted human tissues that mimic the kidney proximal tubule reveal a well-organized tubulointerstitial interface, with CD31+ endothelial cell networks throughout the interstitium, formation of a polarized layer of renal epithelium on top of the interstitium, and a basal lamina. Finally, a tissue model for the breast tumor microenvironment has been developed to better predict drug efficacy on both cancer cells and the surrounding stroma.
A 3D bioprinted model of the renal proximal tubulointerstitial interface for evaluation of drug-induced toxicity
Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule (PT) is a primary site of nephrotoxicity, a leading cause of attrition in the drug development pipeline. Current preclinical methods using 2D cell cultures and animal models are unable to fully recapitulate clinical drug responses due to limited in vitro functional lifespan, or species-specific differences. Our NovoGen Bioprinter® Technology offers an opportunity to build in vitro tissue models to enable more accurate prediction of clinical outcomes.
Bioprinted 3D primary liver tissues allow assessment of organ-level response to clinical drug induced toxicity in vitro
Modeling clinically relevant tissue responses using cell models poses a significant challenge for drug development, in particular for drug induced liver injury (DILI). This is mainly because existing liver models lack longevity and tissue-level complexity which limits their utility in predictive toxicology. In this study, we established and characterized novel bioprinted human liver tissue mimetics comprised of patient-derived hepatocytes and non-parenchymal cells in a defined architecture. Scaffold-free assembly of different cell types in an in vivo-relevant architecture allowed for histologic analysis that revealed distinct intercellular hepatocyte junctions, CD31+ endothelial networks, and desmin positive, smooth muscle actin negative quiescent stellates. Unlike what was seen in 2D hepatocyte cultures, the tissues maintained levels of ATP, Albumin as well as expression and drug-induced enzyme activity of Cytochrome P450s over 4 weeks in culture. To assess the ability of the 3D liver cultures to model tissue-level DILI, dose responses of Trovafloxacin, a drug whose hepatotoxic potential could not be assessed by standard preclinical models, were compared to the structurally related non-toxic drug Levofloxacin. Trovafloxacin induced significant, dose-dependent toxicity at clinically relevant doses (≤ 4 μM). Interestingly, Trovafloxacin toxicity was observed without lipopolysaccharide stimulation and in the absence of resident macrophages in contrast to earlier reports. Together, these results demonstrate that 3D bioprinted liver tissues can both effectively model DILI and distinguish between highly related compounds with differential profile. Thus, the combination of patient-derived primary cells with bioprinting technology here for the first time demonstrates superior performance in terms of mimicking human drug response in a known target organ at the tissue level.
Modeling drug-induced hepatic fibrosis in vitro using three-dimensional liver tissue constructs
While the major precipitating factors underlying drug- and chemical-induced fibrosis have been gleaned from animal models, the key initiating and series of adaptive events that perpetuate this response, especially in humans, are still not well understood. Regardless of etiology, progressive fibrotic liver injury is orchestrated by complex intercellular interactions among hepatocytes (HCs), endothelial cells (ECs), hepatic stellate cells (HSCs), Kupffer cells (KCs) and recruited bone marrow derived cells. This interplay between resident and recruited cell types results in the appearance and progression of disease features that are best detected and interpreted in the context of a three-dimensional (3D) tissue environment, including inflammation, fibrogenesis, tissue remodeling, and compensatory hepatocellular regeneration. The recent availability of human liver tissue models that incorporate both parenchymal (i.e., HCs) and non-parenchymal cells (i.e., HSCs and ECs) in a three-dimensional context has created the opportunity to examine progressive liver injury in response to known pro-fibrotic modulators. Here we demonstrate the utility of 3D bioprinted tissues to perform more in-depth evaluation of compound-induced liver fibrosis in an in vitro setting.
