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.

Magnesium enhances cardiomyocyte proliferation and suppresses cardiac fibrosis induced by chronic ACTH exposure in rats.

Chronic stress has been implicated in the development and progression of heart disease. In the past decade, a link between chronic stress and cardiac fibrosis has been described. Here, we focused on investigating the effects of one of the key molecular effectors of the stress response-adrenocorticotropic hormone (ACTH) on cardiac histopathology. More importantly, as the literature data support interplay between magnesium (Mg) and the hypothalamo-pituitary-adrenal (HPA) stress system, we explored potential cardioprotective effects of Mg supplementation in a rat model of ACTH-induced cardiac remodeling. Protracted ACTH exposure in rats resulted in a prominent increase in proliferation of fibroblasts and excessive collagen deposition in the heart, accompanied by enhanced proliferation of cardiomyocytes and vascular endothelial cells. Our results show, for the first time, that administration of Mg in rats was effective in ameliorating the development of ACTH-evoked cardiac fibrosis, while facilitating cardiomyocyte proliferation. Furthermore, we propose that Mg supplementation attenuates ACTH-induced HPA axis hyperactivity, as one of the underlying plausible mechanisms, which may contribute to its cardioprotective effects.

JAK inhibition reduces SARS-CoV-2 liver infectivity and modulates inflammatory responses to reduce morbidity and mortality.

Using AI, we identified baricitinib as having antiviral and anticytokine efficacy. We now show a 71% (95% CI 0.15 to 0.58) mortality benefit in 83 patients with moderate-severe SARS-CoV-2 pneumonia with few drug-induced adverse events, including a large elderly cohort (median age, 81 years). An additional 48 cases with mild-moderate pneumonia recovered uneventfully. Using organotypic 3D cultures of primary human liver cells, we demonstrate that interferon-α2 increases ACE2 expression and SARS-CoV-2 infectivity in parenchymal cells by greater than fivefold. RNA-seq reveals gene response signatures associated with platelet activation, fully inhibited by baricitinib. Using viral load quantifications and superresolution microscopy, we found that baricitinib exerts activity rapidly through the inhibition of host proteins (numb-associated kinases), uniquely among antivirals. This reveals mechanistic actions of a Janus kinase-1/2 inhibitor targeting viral entry, replication, and the cytokine storm and is associated with beneficial outcomes including in severely ill elderly patients, data that incentivize further randomized controlled trials.

Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients.

Baricitinib is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI) algorithms, to be useful for COVID-19 infection via proposed anti-cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID-19 infection. We validated the AI-predicted biochemical inhibitory effects of baricitinib on human numb-associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID-19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS-CoV-2 viral load, inflammatory markers, and IL-6 levels. Collectively, these data support further evaluation of the anti-cytokine and anti-viral activity of baricitinib and support its assessment in randomized trials in hospitalized COVID-19 patients.

Clinically Relevant Cytochrome P450 3A4 Induction Mechanisms and Drug Screening in Three-Dimensional Spheroid Cultures of Primary Human Hepatocytes.

Cytochrome P450 (CYP) 3A4 induction is an important cause of drug-drug interactions, making early identification of drug candidates with CYP3A4 induction liability in drug development a prerequisite. Here, we present three-dimensional (3D) spheroid cultures of primary human hepatocytes (PHHs) as a novel CYP3A4 induction screening model. Screening of 25 drugs (12 known CYP3A4 inducers in vivo and 13 negative controls) at physiologically relevant concentrations revealed a 100% sensitivity and 100% specificity of the system. Three of the in vivo CYP3A4 inducers displayed much higher CYP3A4 induction capacity in 3D spheroid cultures as compared with in two-dimensional (2D) monolayer cultures. Among those, we identified AZD1208, a proviral integration site for Moloney murine leukemia virus (PIM) kinase inhibitor terminated in phase I of development due to unexpected CYP3A4 autoinduction, as a CYP3A4 inducer only active in 3D spheroids but not in 2D monolayer cultures. Gene knockdown experiments revealed that AZD1208 requires pregnane X receptor (PXR) to induce CYP3A4. Rifampicin requires solely PXR to induce CYP3A4 and CYP2B6, while phenobarbital-mediated induction of these CYPs did not show absolute dependency on either PXR or constitutive androstane receptor (CAR), suggesting its ability to switch nuclear receptor activation. Mechanistic studies into AZD1208 uncovered an involvement of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway in CYP3A4 induction that is sensitive to the culture format used, as revealed by its inhibition of ERK1/2 Tyrosine 204 phosphorylation and sensitivity to epidermal growth factor (EGF) pressure. In line, we also identified lapatinib, a dual epidermal growth factor receptor/human epidermal growth factor receptor 2 (EGFR/HER2) inhibitor, as another CYP3A4 inducer only active in 3D spheroid culture. Our findings offer insights into the pathways involved in CYP3A4 induction and suggest PHH spheroids for preclinical CYP3A4 induction screening.

Organotypic and Microphysiological Models of Liver, Gut, and Kidney for Studies of Drug Metabolism, Pharmacokinetics, and Toxicity.

Despite extensive breakthroughs in chemistry, molecular biology, and genetics in the last decades, the success rates of drug development projects remain low. To improve predictions of clinical efficacy and safety of new compounds, a plethora of 3D culture methods of human cells have been developed in which the cultured cells retain physiologically and functionally relevant phenotypes for multiple weeks. Here, we critically review current paradigms for organotypic cultures of human liver, gut, and kidney such as perfused microchips, spheroids, and hollow fiber bioreactors and discuss their utility for understanding drug pharmacokinetics, metabolism, and toxicity. Furthermore, bioprinting and the microfluidic integration of different tissue models to mimic systemic drug effects are highlighted as promising technological trends. In the last part of the review, we discuss important considerations regarding the choice of culture substratum material to limit adverse effects such as drug absorption while facilitating the phenotypic maintenance of cultured cells. We conclude that recent advances in organotypic and microphysiological culture models of human tissues can improve drug development and contribute to an amelioration of clinical attrition rates. However, further validation, benchmarking, and consolidation efforts are needed to achieve more widespread dissemination and eventually regulatory acceptance of these novel tools.

Human hepatic 3D spheroids as a model for steatosis and insulin resistance.

Non-alcoholic fatty liver disease (NAFLD) has emerged as a public health concern as reflected in its widespread distribution in the general population. Yet, treatment options are scarce which is at least in part due to lack of reliable human in vitro disease models. Here, we report a human hepatic 3D spheroid system cultured under defined chemical conditions that has the potential to mimic steatotic conditions in a reversible manner, useful for identification of novel drug treatment conditions. Primary human hepatocytes (PHH) from different donors were cultured as spheroid microtissues in physiological in vivo -like culture conditions. Hepatic steatosis was induced over the course of three weeks in culture by supplementing the culture medium with pathophysiological concentrations of free fatty acids, carbohydrates and insulin. Effects of steatosis in the 3D system were evaluated on transcriptional, metabolomic and lipidomic levels. Free fatty acids on one hand as well as a combination of insulin and monosaccharides, promoted lipid accumulation in hepatocytes and increased expression of lipogenic genes, such as fatty acid synthase. This milieu also promoted development of insulin resistance within 2 weeks as manifested by an increase in gluconeogenic and insulin resistance markers, which are observed in type 2 diabetes mellitus and metabolic syndrome. Induced steatosis was reversible after withdrawal of lipogenic substrates and a further reduction in cellular fat content was observed following treatment with different antisteatotic compounds, such as metformin, glucagon, olaparib and antioxidants. Taken together, these results demonstrate that the 3D hepatic spheroids can serve as a valuable, HTS compatible model for the study of liver steatosis and facilitate translational discovery of novel drug targets.

Prediction of Drug-Induced Hepatotoxicity Using Long-Term Stable Primary Hepatic 3D Spheroid Cultures in Chemically Defined Conditions.

High failure rates of drug candidates in the clinics, restricted-use warnings as well as withdrawals of drugs in postmarketing stages are of substantial concern for the pharmaceutical industry and drug-induced liver injury (DILI) constitutes one of the most frequent reasons for such safety failures. Importantly, as DILI cannot be accurately predicted using animal models, animal safety tests are commonly complemented with assessments in human in vitro systems. 3D spheroid cultures of primary human hepatocytes in chemically defined conditions, hereafter termed CD-spheroids, have recently emerged as a microphysiological model system in which hepatocytes retain their molecular phenotypes and hepatic functions for multiple weeks in culture. However, their predictive power for the detection of hepatotoxic liabilities has not been systematically assessed. Therefore, we here evaluated the hepatotoxicity of 123 drugs with or without direct implication in clinical DILI events. Importantly, using ATP quantifications as the single endpoint, the model accurately distinguished between hepatotoxic and nontoxic structural analogues and exceeded both sensitivity and specificity of all previously published in vitro assays at substantially lower exposure levels, successfully detecting 69% of all hepatotoxic compounds without producing any false positive results (100% specificity). Furthermore, the platform supports the culture of spheroids of primary hepatocytes from preclinical animal models, thereby allowing the identification of animal-specific toxicity events. We anticipate that CD-spheroids represent a powerful and versatile tool in drug discovery and preclinical drug development that can reliably flag hepatotoxic drug candidates and provide guidance for the selection of the most suitable animal models.

Application of Microphysiological Systems to Enhance Safety Assessment in Drug Discovery.

Enhancing the early detection of new therapies that are likely to carry a safety liability in the context of the intended patient population would provide a major advance in drug discovery. Microphysiological systems (MPS) technology offers an opportunity to support enhanced preclinical to clinical translation through the generation of higher-quality preclinical physiological data. In this review, we highlight this technological opportunity by focusing on key target organs associated with drug safety and metabolism. By focusing on MPS models that have been developed for these organs, alongside other relevant in vitro models, we review the current state of the art and the challenges that still need to be overcome to ensure application of this technology in enhancing drug discovery.