Taking the leap toward human-specific nonanimal methodologies: The need for harmonizing global policies for microphysiological systems.

With a recent amendment, India joined other countries that have removed the legislative barrier toward the use of human-relevant methods in drug development. Here, global stakeholders weigh in on the urgent need to globally harmonize the guidelines toward the standardization of microphysiological systems. We discuss a possible framework for establishing scientific confidence and regulatory approval of these methods.

New approach methodologies in human regulatory toxicology - Not if, but how and when!

The predominantly animal-centric approach of chemical safety assessment has increasingly come under pressure. Society is questioning overall performance, sustainability, continued relevance for human health risk assessment and ethics of this system, demanding a change of paradigm. At the same time, the scientific toolbox used for risk assessment is continuously enriched by the development of "New Approach Methodologies" (NAMs). While this term does not define the age or the state of readiness of the innovation, it covers a wide range of methods, including quantitative structure-activity relationship (QSAR) predictions, high-throughput screening (HTS) bioassays, omics applications, cell cultures, organoids, microphysiological systems (MPS), machine learning models and artificial intelligence (AI). In addition to promising faster and more efficient toxicity testing, NAMs have the potential to fundamentally transform today's regulatory work by allowing more human-relevant decision-making in terms of both hazard and exposure assessment. Yet, several obstacles hamper a broader application of NAMs in current regulatory risk assessment. Constraints in addressing repeated-dose toxicity, with particular reference to the chronic toxicity, and hesitance from relevant stakeholders, are major challenges for the implementation of NAMs in a broader context. Moreover, issues regarding predictivity, reproducibility and quantification need to be addressed and regulatory and legislative frameworks need to be adapted to NAMs. The conceptual perspective presented here has its focus on hazard assessment and is grounded on the main findings and conclusions from a symposium and workshop held in Berlin in November 2021. It intends to provide further insights into how NAMs can be gradually integrated into chemical risk assessment aimed at protection of human health, until eventually the current paradigm is replaced by an animal-free "Next Generation Risk Assessment" (NGRA).

The evolving role of investigative toxicology in the pharmaceutical industry.

For decades, preclinical toxicology was essentially a descriptive discipline in which treatment-related effects were carefully reported and used as a basis to calculate safety margins for drug candidates. In recent years, however, technological advances have increasingly enabled researchers to gain insights into toxicity mechanisms, supporting greater understanding of species relevance and translatability to humans, prediction of safety events, mitigation of side effects and development of safety biomarkers. Consequently, investigative (or mechanistic) toxicology has been gaining momentum and is now a key capability in the pharmaceutical industry. Here, we provide an overview of the current status of the field using case studies and discuss the potential impact of ongoing technological developments, based on a survey of investigative toxicologists from 14 European-based medium-sized to large pharmaceutical companies.

High-speed videography of transparent media using illumination-based multiplexed schlieren.

Schlieren photography is widely used for visualizing phenomena within transparent media. The technique, which comes in a variety of configurations, is based on detecting or extracting the degree to which light is deflected whilst propagating through a sample. To date, high-speed schlieren videography can only be achieved using high-speed cameras, thus limiting the frame rate of such configurations to the capabilities of the camera. Here we demonstrate, for the first time, optically multiplexed schlieren videography, a concept that allows such hardware limitations to be bypassed, opening up for, in principle, an unlimited frame rate. By illuminating the sample with a rapid burst of uniquely spatially modulated light pulses, a temporally resolved sequence can be captured in a single photograph. The refractive index variations are thereafter measured by quantifying the local phase shift of the superimposed intensity modulations. The presented results demonstrate the ability to acquire a series of images of flame structures at frame rates up to 1 Mfps using a standard 50 fps sCMOS camera.

Human microphysiological systems for drug development.

Organs-on-chips could be used to assess drug efficacy and support personalized medicine.

Challenging the pipeline.

This commentary presents a thought experiment seeking to answer the key question: "If you were to put aside all the traditional drug discovery processes and start a new drug discovery program that places the highest priority on human and disease-relevant models throughout the entire process, how could it be done?"

Human immunocompetent Organ-on-Chip platforms allow safety profiling of tumor-targeted T-cell bispecific antibodies.

Traditional drug safety assessment often fails to predict complications in humans, especially when the drug targets the immune system. Here, we show the unprecedented capability of two human Organs-on-Chips to evaluate the safety profile of T-cell bispecific antibodies (TCBs) targeting tumor antigens. Although promising for cancer immunotherapy, TCBs are associated with an on-target, off-tumor risk due to low levels of expression of tumor antigens in healthy tissues. We leveraged in vivo target expression and toxicity data of TCBs targeting folate receptor 1 (FOLR1) or carcinoembryonic antigen (CEA) to design and validate human immunocompetent Organs-on-Chips safety platforms. We discovered that the Lung-Chip and Intestine-Chip could reproduce and predict target-dependent TCB safety liabilities, based on sensitivity to key determinants thereof, such as target expression and antibody affinity. These novel tools broaden the research options available for mechanistic understandings of engineered therapeutic antibodies and assessing safety in tissues susceptible to adverse events.

A Model-Based Workflow to Benchmark the Clinical Cholestasis Risk of Drugs.

We present a generic workflow combining physiology-based computational modeling and in vitro data to assess the clinical cholestatic risk of different drugs systematically. Changes in expression levels of genes involved in the enterohepatic circulation of bile acids were obtained from an in vitro assay mimicking 14 days of repeated drug administration for 10 marketed drugs. These changes in gene expression over time were contextualized in a physiology-based bile acid model of glycochenodeoxycholic acid. The simulated drug-induced response in bile acid concentrations was then scaled with the applied drug doses to calculate the cholestatic potential for each compound. A ranking of the cholestatic potential correlated very well with the clinical cholestasis risk obtained from medical literature. The proposed workflow allows benchmarking the cholestatic risk of novel drug candidates. We expect the application of our workflow to significantly contribute to the stratification of the cholestatic potential of new drugs and to support animal-free testing in future drug development.

Translational precision medicine: an industry perspective.

In the era of precision medicine, digital technologies and artificial intelligence, drug discovery and development face unprecedented opportunities for product and business model innovation, fundamentally changing the traditional approach of how drugs are discovered, developed and marketed. Critical to this transformation is the adoption of new technologies in the drug development process, catalyzing the transition from serendipity-driven to data-driven medicine. This paradigm shift comes with a need for both translation and precision, leading to a modern Translational Precision Medicine approach to drug discovery and development. Key components of Translational Precision Medicine are multi-omics profiling, digital biomarkers, model-based data integration, artificial intelligence, biomarker-guided trial designs and patient-centric companion diagnostics. In this review, we summarize and critically discuss the potential and challenges of Translational Precision Medicine from a cross-industry perspective.

Implementation of a Human Renal Proximal Tubule on a Chip for Nephrotoxicity and Drug Interaction Studies.

Proximal tubule epithelial cells (PTEC) are susceptible to drug-induced kidney injury (DIKI). Cell-based, two-dimensional (2D) in vitro PTEC models are often poor predictors of DIKI, probably due to the lack of physiological architecture and flow. Here, we assessed a high throughput, 3D microfluidic platform (Nephroscreen) for the detection of DIKI in pharmaceutical development. This system was established with four model nephrotoxic drugs (cisplatin, tenofovir, tobramycin and cyclosporin A) and tested with eight pharmaceutical compounds. Measured parameters included cell viability, release of lactate dehydrogenase (LDH) and N-acetyl-ß-d-glucosaminidase (NAG), barrier integrity, release of specific miRNAs, and gene expression of toxicity markers. Drug-transporter interactions for P-gp and MRP2/4 were also determined. The most predictive read outs for DIKI were a combination of cell viability, LDH and miRNA release. In conclusion, Nephroscreen detected DIKI in a robust manner, is compatible with automated pipetting, proved to be amenable to long-term experiments, and was easily transferred between laboratories. This proof-of-concept-study demonstrated the usability and reproducibility of Nephroscreen for the detection of DIKI and drug-transporter interactions. Nephroscreen it represents a valuable tool towards replacing animal testing and supporting the 3Rs (Reduce, Refine and Replace animal experimentation).

FuSe: a tool to move RNA-Seq analyses from chromosomal/gene loci to functional grouping of mRNA transcripts.

SUMMARY: Typical RNA sequencing (RNA-Seq) analyses are performed either at the gene level by summing all reads from the same locus, assuming that all transcripts from a gene make a protein or at the transcript level, assuming that each transcript displays unique function. However, these assumptions are flawed, as a gene can code for different types of transcripts and different transcripts are capable of synthesizing similar, different or no protein. As a consequence, functional changes are not well illustrated by either gene or transcript analyses. We propose to improve RNA-Seq analyses by grouping the transcripts based on their similar functions. We developed FuSe to predict functional similarities using the primary and secondary structure of proteins. To estimate the likelihood of proteins with similar functions, FuSe computes two confidence scores: knowledge (KS) and discovery (DS) for protein pairs. Overlapping protein pairs exhibiting high confidence are grouped to form 'similar function protein groups' and expression is calculated for each functional group. The impact of using FuSe is demonstrated on in vitro cells exposed to paracetamol, which highlight genes responsible for cell adhesion and glycogen regulation which were earlier shown to be not differentially expressed with traditional analysis methods. AVAILABILITY AND IMPLEMENTATION: The source code is available at https://github.com/rajinder4489/FuSe. Data for APAP exposure are available in the BioStudies database (http://www.ebi.ac.uk/biostudies) under accession numbers S-HECA143, S-HECA(158) and S-HECA139. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Network integration and modelling of dynamic drug responses at multi-omics levels.

Uncovering cellular responses from heterogeneous genomic data is crucial for molecular medicine in particular for drug safety. This can be realized by integrating the molecular activities in networks of interacting proteins. As proof-of-concept we challenge network modeling with time-resolved proteome, transcriptome and methylome measurements in iPSC-derived human 3D cardiac microtissues to elucidate adverse mechanisms of anthracycline cardiotoxicity measured with four different drugs (doxorubicin, epirubicin, idarubicin and daunorubicin). Dynamic molecular analysis at in vivo drug exposure levels reveal a network of 175 disease-associated proteins and identify common modules of anthracycline cardiotoxicity in vitro, related to mitochondrial and sarcomere function as well as remodeling of extracellular matrix. These in vitro-identified modules are transferable and are evaluated with biopsies of cardiomyopathy patients. This to our knowledge most comprehensive study on anthracycline cardiotoxicity demonstrates a reproducible workflow for molecular medicine and serves as a template for detecting adverse drug responses from complex omics data.

Neutrophilic infiltration in organ-on-a-chip model of tissue inflammation.

The multiphasic etiology of tissue inflammation and the fundamental immunological differences between species render inflammatory pathologies difficult to recapitulate in animal models, and account for the paucity of therapies that are successfully translated from rodents to humans. Here, we present a human-relevant organ-on-a-chip platform for experimental inflammatory diseases. We created an immunocompetent in vitro gut model by incorporating intestinal epithelial and immune cells into microfluidic chambers that permit cell movement across an extracellular matrix (ECM) and fluidic channels. This is the first model that integrates a mucosal barrier, a three-dimensional ECM, resident and infiltrating immune cells, and simulates a functional crosstalk that ultimately triggers cellular processes representative of inflammation. Under homeostatic conditions, enterocytes form a tight epithelium and subepithelial macrophages are non-activated. Introduction of pro-inflammatory mediators triggers macrophage activation and inflammation-induced intestinal barrier leakiness. Neutrophils in a parallel, matrix-separated non-epithelial channel are attracted by such a pro-inflammatory microenvironment and migrate through the extracellular matrix, further exacerbating tissue inflammation and damage. With this model, we provide the foundations to recapitulate and investigate the onset of tissue inflammation in a controlled, human-relevant system.

Characterization of a long-term mouse primary liver 3D tissue model recapitulating innate-immune responses and drug-induced liver toxicity.

Three-dimensional liver in vitro systems have recently attracted a lot of attention in drug development. These systems help to gain unprecedented insights into drug-induced liver injury (DILI), as they more closely reproduce liver biology, and as drug effects can be studied in isolated and controllable microenvironments. Many groups established human-based in vitro models but so far neglected the animal equivalent, although the availability of both models would be desirable. Animal in vitro models enable back- and forward translation of in vitro and in vivo findings, bridge the gap between rodent in vivo and human in vitro scenarios, and ultimately support the interpretation of data generated with preclinical species and humans. Since mice are often used in drug development and physiologically relevant in vitro systems are lacking, we established, for the first time, a mouse liver model that encompasses primary parenchymal and non-parenchymal cells with preserved viability and functionality over three weeks. Using our three-dimensional liver spheroids, we were able to predict the toxicity of known DILI compounds, demonstrated the interaction cascades between the different cell types and showed evidence of drug-induced steatosis and cholestasis. In summary, our mouse liver spheroids represent a valuable in vitro model that can be applied to study DILI findings, reported from mouse studies, and offers the potential to detect immune-mediated drug-induced liver toxicity.

Differential Monocyte Actuation in a Three-Organ Functional Innate Immune System-on-a-Chip.

A functional, human, multiorgan, pumpless, immune system-on-a-chip featuring recirculating THP-1 immune cells with cardiomyocytes, skeletal muscle, and liver in separate compartments in a serum-free medium is developed. This in vitro platform can emulate both a targeted immune response to tissue-specific damage, and holistic proinflammatory immune response to proinflammatory compound exposure. The targeted response features fluorescently labeled THP-1 monocytes selectively infiltrating into an amiodarone-damaged cardiac module and changes in contractile force measurements without immune-activated damage to the other organ modules. In contrast to the targeted immune response, general proinflammatory treatment of immune human-on-a-chip systems with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) causes nonselective damage to cells in all three-organ compartments. Biomarker analysis indicates upregulation of the proinflammation cytokines TNF-α, IL-6, IL-10, MIP-1, MCP-1, and RANTES in response to LPS + IFN-γ treatment indicative of the M1 macrophage phenotype, whereas amiodarone treatment only leads to an increase in the restorative cytokine IL-6 which is a marker for the M2 phenotype. This system can be used as an alternative to humanized animal models to determine direct immunological effects of biological therapeutics including monoclonal antibodies, vaccines, and gene therapies, and the indirect effects caused by cytokine release from target tissues in response to a drug's pharmacokinetics (PK)/pharmacodynamics (PD) profile.

Snapshot 3D reconstruction of liquid surfaces.

In contrast to static objects, liquid structures such as drops, blobs, as well as waves and ripples on water surfaces are challenging to image in 3D due to two main reasons: first, the transient nature of those phenomena requires snapshot imaging that is fast enough to freeze the motion of the liquid. Second, the transparency of liquids and the specular reflections from their surfaces induce complex image artefacts. In this article we present a novel imaging approach to reconstruct in 3D the surface of irregular liquid structures that only requires a single snapshot. The technique is named Fringe Projection - Laser Induced Fluorescence (FP-LIF) and uses a high concentration of fluorescent dye in the probed liquid. By exciting this dye with a fringe projection structured laser beam, fluorescence is generated primarily at the liquid surface and imaged at a backward angle. By analysing the deformation of the initial projected fringes using phase-demodulation image post-processing, the 3D coordinates of the liquid surface are deduced. In this article, the approach is first numerically tested by considering a simulated pending drop, in order to analyse its performance. Then, FP-LIF is applied for two experimental cases: a quasi-static pending drop as well as a transient liquid sheet. We demonstrate reconstruction RMS errors of 1.4% and 6.1% for the simulated and experimental cases respectively. The technique presented here demonstrates, for the first time, a fringe projection approach based on LIF detection to reconstruct liquid surfaces in 3D. FP-LIF is promising for the study of more complex liquid structures and is paving the way for high-speed 3D videography of liquid surfaces.

Combining In Vivo and Organotypic In Vitro Approaches to Assess the Human Relevance of Basimglurant (RG7090), a Potential CAR Activator.

Basimglurant (RG7090), a small molecule under development to treat certain forms of depression, demonstrated foci of altered hepatocytes in a long-term rodent-toxicity study. Additional evidence pointed toward the activation of the constitutive androstane receptor (CAR), an established promoter of nongenotoxic and rodent-specific hepatic tumors. This mode of action and the potential human relevance was explored in vivo using rodent and cynomolgus monkey models and in vitro using murine and human liver spheroids. Wild type (WT) and CAR/pregnane X receptor (PXR) knockout mice (CAR/PXR KO) were exposed to RG7090 for 8 consecutive days. Analysis of liver lysates revealed induction of Cyp2b mRNA and enzyme activity, a known activation marker of CAR, in WT but not in CAR/PXR KO animals. A series of proliferative genes were upregulated in WT mice only, and immunohistochemistry data showed increased cell proliferation exclusively in WT mice. In addition, primary mouse liver spheroids were challenged with RG7090 in the presence or absence of modified antisense oligonucleotides inhibiting CAR and/or PXR mRNA, showing a concentration-dependent Cyp2b mRNA induction only if CAR was not repressed. On the contrary, neither human liver spheroids nor cynomolgus monkeys exposed to RG7090 triggered CYP2B mRNA upregulation. Our data suggested RG7090 to be a rodent-specific CAR activator, and that CAR activation and its downstream processes were involved in the foci of altered hepatocytes formation detected in vivo. Furthermore, we demonstrated the potential of a new in vitro approach using liver spheroids and antisense oligonucleotides for CAR knockdown experiments, which could eventually replace in vivo investigations using CAR/PXR KO mice.