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Metabolic dysfunction-associated steatotic liver disease (MASLD) is a worldwide health epidemic with a global occurrence of approximately 30%. The pathogenesis of MASLD is a complex, multisystem disorder driven by multiple factors including genetics, lifestyle, and the environment. Patient heterogeneity presents challenges for developing MASLD therapeutics, creation of patient cohorts for clinical trials and optimization of therapeutic strategies for specific patient cohorts. Implementing pre-clinical experimental models for drug development creates a significant challenge as simple in vitro systems and animal models do not fully recapitulate critical steps in the pathogenesis and the complexity of MASLD progression. To address this, we implemented a precision medicine strategy that couples the use of our liver acinus microphysiology system (LAMPS) constructed with patient-derived primary cells. We investigated the MASLD-associated genetic variant PNPLA3 rs738409 (I148M variant) in primary hepatocytes, as it is associated with MASLD progression. We constructed LAMPS with genotyped wild type and variant PNPLA3 hepatocytes together with key non-parenchymal cells and quantified the reproducibility of the model. We altered media components to mimic blood chemistries, including insulin, glucose, free fatty acids, and immune activating molecules to reflect normal fasting (NF), early metabolic syndrome (EMS) and late metabolic syndrome (LMS) conditions. Finally, we investigated the response to treatment with resmetirom, an approved drug for metabolic syndrome-associated steatohepatitis (MASH), the progressive form of MASLD. This study using primary cells serves as a benchmark for studies using patient biomimetic twins constructed with patient iPSC-derived liver cells using a panel of reproducible metrics. We observed increased steatosis, immune activation, stellate cell activation and secretion of pro-fibrotic markers in the PNPLA3 GG variant compared to wild type CC LAMPS, consistent with the clinical characterization of this variant. We also observed greater resmetirom efficacy in PNPLA3 wild type CC LAMPS compared to the GG variant in multiple MASLD metrics including steatosis, stellate cell activation and the secretion of pro-fibrotic markers. In conclusion, our study demonstrates the capability of the LAMPS platform for the development of MASLD precision therapeutics, enrichment of patient cohorts for clinical trials, and optimization of therapeutic strategies for patient subgroups with different clinical traits and disease stages.
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Biologics address a range of unmet clinical needs, but the occurrence of biologics-induced liver injury remains a major challenge. Development of cimaglermin alfa (GGF2) was terminated due to transient elevations in serum aminotransferases and total bilirubin. Tocilizumab has been reported to induce transient aminotransferase elevations, requiring frequent monitoring. To evaluate the clinical risk of biologics-induced liver injury, a novel quantitative systems toxicology modeling platform, BIOLOGXsym™, representing relevant liver biochemistry and the mechanistic effects of biologics on liver pathophysiology, was developed in conjunction with clinically relevant data from a human biomimetic liver microphysiology system. Phenotypic and mechanistic toxicity data and metabolomics analysis from the Liver Acinus Microphysiology System showed that tocilizumab and GGF2 increased high mobility group box 1, indicating hepatic injury and stress. Tocilizumab exposure was associated with increased oxidative stress and extracellular/tissue remodeling, and GGF2 decreased bile acid secretion. BIOLOGXsym simulations, leveraging the in vivo exposure predicted by physiologically-based pharmacokinetic modeling and mechanistic toxicity data from the Liver Acinus Microphysiology System, reproduced the clinically observed liver signals of tocilizumab and GGF2, demonstrating that mechanistic toxicity data from microphysiology systems can be successfully integrated into a quantitative systems toxicology model to identify liabilities of biologics-induced liver injury and provide mechanistic insights into observed liver safety signals.
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Productos Biológicos , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Enfermedad Hepática Inducida por Sustancias y Drogas , Humanos , Productos Biológicos/farmacología , Biomimética , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , HígadoRESUMEN
Non-alcoholic fatty liver disease (NAFLD) has a high global prevalence with a heterogeneous and complex pathophysiology that presents barriers to traditional targeted therapeutic approaches. We describe an integrated quantitative systems pharmacology (QSP) platform that comprehensively and unbiasedly defines disease states, in contrast to just individual genes or pathways, that promote NAFLD progression. The QSP platform can be used to predict drugs that normalize these disease states and experimentally test predictions in a human liver acinus microphysiology system (LAMPS) that recapitulates key aspects of NAFLD. Analysis of a 182 patient-derived hepatic RNA-sequencing dataset generated 12 gene signatures mirroring these states. Screening against the LINCS L1000 database led to the identification of drugs predicted to revert these signatures and corresponding disease states. A proof-of-concept study in LAMPS demonstrated mitigation of steatosis, inflammation, and fibrosis, especially with drug combinations. Mechanistically, several structurally diverse drugs were predicted to interact with a subnetwork of nuclear receptors, including pregnane X receptor (PXR; NR1I2), that has evolved to respond to both xenobiotic and endogenous ligands and is intrinsic to NAFLD-associated transcription dysregulation. In conjunction with iPSC-derived cells, this platform has the potential for developing personalized NAFLD therapeutic strategies, informing disease mechanisms, and defining optimal cohorts of patients for clinical trials.
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BACKGROUND: Hemolysis occurs in many injury settings and can trigger disease processes. In the kidney, extracellular hemoglobin can induce damage via several mechanisms. These include oxidative stress, mitochondrial dysfunction, and inflammation, which promote fibrosis and chronic kidney disease. Understanding the pathophysiology of these injury pathways offers opportunities to develop new therapeutic strategies. METHODS: To model hemolysis-induced kidney injury, human kidney organoids were treated with hemin, an iron-containing porphyrin, that generates reactive oxygen species. In addition, we developed an induced pluripotent stem cell line expressing the biosensor, CytochromeC-GFP (CytoC-GFP), which provides a real-time readout of mitochondrial morphology, health, and early apoptotic events. RESULTS: We found that hemin-treated kidney organoids show oxidative damage, increased expression of injury markers, impaired functionality of organic anion and cation transport and undergo fibrosis. Injury could be detected in live CytoC-GFP organoids by cytoplasmic localization of fluorescence. Finally, we show that 4-(phenylthio)butanoic acid, an HDAC inhibitor with anti-fibrotic effects in vivo, reduces hemin-induced human kidney organoid fibrosis. CONCLUSION: This work establishes a hemin-induced model of kidney organoid injury. This platform provides a new tool to study the injury and repair response pathways in human kidney tissue and will assist in the development of new therapeutics.
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Células Madre Pluripotentes , Insuficiencia Renal Crónica , Humanos , Riñón/metabolismo , Organoides/metabolismo , Estrés Oxidativo , Insuficiencia Renal Crónica/metabolismoRESUMEN
Predicting human hepatic clearance remains a fundamental challenge in both pharmaceutical drug development and toxicological assessments of environmental chemicals, with concerns about both accuracy and precision of in vitro-derived estimates. Suggested sources of these issues have included differences in experimental protocols, differences in cell sourcing, and use of a single cell type, liver parenchymal cells (hepatocytes). Here we investigate the ability of human microfluidic four-cell liver acinus microphysiology system (LAMPS) to make predictions as to hepatic clearance for seven representative compounds: Caffeine, Pioglitazone, Rosiglitazone, Terfenadine, Tolcapone, Troglitazone, and Trovafloxacin. The model, whose reproducibility was recently confirmed in an inter-lab comparison, was constructed using primary human hepatocytes or human induced pluripotent stem cell (iPSC)-derived hepatocytes and 3 human cell lines for the endothelial, Kupffer and stellate cells. We calculated hepatic clearance estimates derived from experiments using LAMPS or traditional 2D cultures and compared the outcomes with both in vivo human clinical study-derived and in vitro human hepatocyte suspension culture-derived values reported in the literature. We found that, compared to in vivo clinically-derived values, the LAMPS model with iPSC-derived hepatocytes had higher precision as compared to primary cells in suspension or 2D culture, but, consistent with previous studies in other microphysiological systems, tended to underestimate in vivo clearance. Overall, these results suggest that use of LAMPS and iPSC-derived hepatocytes together with an empirical scaling factor warrants additional study with a larger set of compounds, as it has the potential to provide more accurate and precise estimates of hepatic clearance.
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Células Acinares/metabolismo , Hígado/metabolismo , Modelos Biológicos , Preparaciones Farmacéuticas/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Microfluídica/métodos , Reproducibilidad de los ResultadosRESUMEN
A human microfluidic four-cell liver acinus microphysiology system (LAMPS), was evaluated for reproducibility and robustness as a model for drug pharmacokinetics and toxicology. The model was constructed using primary human hepatocytes or human induced pluripotent stem cell (iPSC)-derived hepatocytes and 3 human cell lines for the endothelial, Kupffer and stellate cells. The model was tested in two laboratories and demonstrated to be reproducible in terms of basal function of hepatocytes, Terfenadine metabolism, and effects of Tolcapone (88 µM), Troglitazone (150 µM), and caffeine (600 µM) over 9 days in culture. Additional experiments compared basal outputs of albumin, urea, lactate dehydrogenase (LDH) and tumor necrosis factor (TNF)α, as well as drug metabolism and toxicity in the LAMPS model, and in 2D cultures seeded with either primary hepatocytes or iPSC-hepatocytes. Further experiments to study the effects of Terfenadine (10 µM), Tolcapone (88 µM), Trovafloxacin (150 µM with or without 1 µg/mL lipopolysaccharide), Troglitazone (28 µM), Rosiglitazone (0.8 µM), Pioglitazone (3 µM), and caffeine (600 µM) were carried out over 10 days. We found that both primary human hepatocytes and iPSC-derived hepatocytes in 3D culture maintained excellent basal liver function and Terfenadine metabolism over 10 days compared the same cells in 2D cultures. In 2D, non-overlay monolayer cultures, both cell types lost hepatocyte phenotypes after 48 h. With respect to drug effects, both cell types demonstrated comparable and more human-relevant effects in LAMPS, as compared to 2D cultures. Overall, these studies show that LAMPS is a robust and reproducible in vitro liver model, comparable in performance when seeded with either primary human hepatocytes or iPSC-derived hepatocytes, and more physiologically and clinically relevant than 2D monolayer cultures.
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Células Acinares/efectos de los fármacos , Células Acinares/metabolismo , Técnicas de Cultivo de Célula/métodos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Microfluídica/métodos , Células Acinares/patología , Hepatocitos/patología , Antagonistas de los Receptores Histamínicos H1 no Sedantes/toxicidad , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Terfenadina/toxicidadRESUMEN
We report the development, automation and validation of a 3D, microfluidic liver-on-a-chip for high throughput hepatotoxicity screening, the OrganoPlate LiverTox™. The model is comprised of aggregates of induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep) seeded in an extracellular matrix in the organ channel and co-cultured with endothelial cells and THP-1 monoblasts differentiated to macrophages seeded in the vascular channel of the 96 well Mimetas OrganoPlate 2-lane. A key component of high throughput screening is automation and we report a protocol to seed, dose, collect and replenish media and add assay reagents in the OrganoPlate 2-lane using a standard laboratory liquid handling robot. A combination of secretome measurements and image-based analysis was used to demonstrate stable 15 day cell viability, albumin and urea secretion. Over the same time-period, CYP3A4 activity increased and alpha-fetoprotein secretion decreased suggesting further maturation of the iHeps. Troglitazone, a clinical hepatotoxin, was chosen as a control compound for validation studies. Albumin, urea, hepatocyte nuclear size and viability staining provided Robust Z'factors > 0.2 in plates treated 72 h with 180 µM troglitazone compared with a vehicle control. The viability assay provided the most robust statistic for a Robust Z' factor = 0.6. A small library of 159 compounds with known liver effects was added to the OrganoPlate LiverTox model for 72 h at 50 µM and the Toxicological Prioritization scores were calculated. A follow up dose-response evaluation of select hits revealed the albumin assay to be the most sensitive in calculating TC50 values. This platform provides a robust, novel model which can be used for high throughput hepatotoxicity screening.
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Técnicas de Cultivo de Célula/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Hígado/efectos de los fármacos , Microfluídica/métodos , Pruebas de Toxicidad/métodos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Citocromo P-450 CYP3A/metabolismo , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Hepatocitos/fisiología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/fisiología , Hígado/citología , Hígado/fisiología , Troglitazona/toxicidadRESUMEN
Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGFß1 is a key upstream transcriptome regulator in AH and induces the use of HNF4α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4α-dependent gene expression. We conclude that targeting TGFß1 and epigenetic drivers that modulate HNF4α-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH.
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Hepatitis Alcohólica/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/patología , Hígado/patología , Factor de Crecimiento Transformador beta1/metabolismo , Adulto , Anciano , Animales , Biopsia , Ensamble y Desensamble de Cromatina , Metilación de ADN , Progresión de la Enfermedad , Epigénesis Genética , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hepatitis Alcohólica/patología , Factor Nuclear 4 del Hepatocito/genética , Humanos , Hígado/citología , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Análisis de Secuencia de ARN , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
The establishment of metabolic zonation within a hepatic lobule ascribes specific functions to hepatocytes based on unique, location-dependent gene expression patterns. Recently, there have been significant developments in the field of metabolic liver zonation. A little over a decade ago, the role of ß-catenin signaling was identified as a key regulator of gene expression and function in pericentral hepatocytes. Since then, additional molecules have been identified that regulate the pattern of Wnt/ß-catenin signaling within a lobule and determine gene expression and function in other hepatic zones. Currently, the molecular basis of metabolic zonation in the liver appears to be a 'push and pull' between signaling pathways. Such compartmentalization not only provides an efficient assembly line for hepatocyte functions but also can account for restricting the initial hepatic damage and pathology from some hepatotoxic drugs to specific zones, possibly enabling effective regeneration and restitution responses from unaffected cells. Careful analysis and experimentation have also revealed that many pathological conditions in the liver lobule are spatially heterogeneous. We will review current research efforts that have focused on examination of the role and regulation of such mechanisms of hepatocyte adaptation and repair. We will discuss how the pathological organ-specific microenvironment affects cell signaling and metabolic liver zonation, especially in steatosis, viral hepatitis, and hepatocellular carcinoma. We will discuss how the use of new human microphysiological platforms will lead to a better understanding of liver disease progression, diagnosis, and therapies. In conclusion, we aim to provide insights into the role and regulation of metabolic zonation and function using traditional and innovative approaches. Impact statement Liver zonation of oxygen tension along the liver sinusoids has been identified as a critical liver microenvironment that impacts specific liver functions such as intermediary metabolism of amino acids, lipids, and carbohydrates, detoxification of xenobiotics and as sites for initiation of liver diseases. To date, most information on the role of zonation in liver disease including, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC) have been obtained from animal models. It is now possible to complement animal studies with human liver, microphysiology systems (MPS) containing induced pluripotent stem cells engineered to create disease models where it is also possible to control the in vitro liver oxygen microenvironment to define the role of zonation on the mechanism(s) of disease progression. The field now has the tools to investigate human liver disease progression, diagnosis, and therapeutic development.
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Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Hígado/metabolismo , Hígado/patología , Microfluídica/métodos , Vía de Señalización Wnt/fisiología , Animales , Carcinoma Hepatocelular/metabolismo , Células Cultivadas , Hígado Graso/metabolismo , Expresión Génica , Hepatitis/metabolismo , Hepatitis/virología , Humanos , Neoplasias Hepáticas/metabolismo , Ratones , Ratas , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
This article describes our next generation human Liver Acinus MicroPhysiology System (LAMPS). The key demonstration of this study was that Zone 1 and Zone 3 microenvironments can be established by controlling the oxygen tension in individual devices over the range of ca. 3 to 13%. The oxygen tension was computationally modeled using input on the microfluidic device dimensions, numbers of cells, oxygen consumption rates of hepatocytes, the diffusion coefficients of oxygen in different materials and the flow rate of media in the MicroPhysiology System (MPS). In addition, the oxygen tension was measured using a ratiometric imaging method with the oxygen sensitive dye, Tris(2,2'-bipyridyl) dichlororuthenium(II) hexahydrate (RTDP) and the oxygen insensitive dye, Alexa 488. The Zone 1 biased functions of oxidative phosphorylation, albumin and urea secretion and Zone 3 biased functions of glycolysis, α1AT secretion, Cyp2E1 expression and acetaminophen toxicity were demonstrated in the respective Zone 1 and Zone 3 MicroPhysiology System. Further improvements in the Liver Acinus MicroPhysiology System included improved performance of selected nonparenchymal cells, the inclusion of a porcine liver extracellular matrix to model the Space of Disse, as well as an improved media to support both hepatocytes and non-parenchymal cells. In its current form, the Liver Acinus MicroPhysiology System is most amenable to low to medium throughput, acute through chronic studies, including liver disease models, prioritizing compounds for preclinical studies, optimizing chemistry in structure activity relationship (SAR) projects, as well as in rising dose studies for initial dose ranging. Impact statement Oxygen zonation is a critical aspect of liver functions. A human microphysiology system is needed to investigate the impact of zonation on a wide range of liver functions that can be experimentally manipulated. Because oxygen zonation has such diverse physiological effects in the liver, we developed and present a method for computationally modeling and measuring oxygen that can easily be implemented in all MPS models. We have applied this method in a liver MPS in which we are then able to control oxygenation in separate devices and demonstrate that zonation-dependent hepatocyte functions in the MPS recapitulate what is known about in vivo liver physiology. We believe that this advance allows a deep experimental investigation on the role of zonation in liver metabolism and disease. In addition, modeling and measuring oxygen tension will be required as investigators migrate from PDMS to plastic and glass devices.
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Hepatocitos/metabolismo , Hígado/metabolismo , Procedimientos Analíticos en Microchip/métodos , Microfluídica/métodos , Consumo de Oxígeno/fisiología , Oxígeno/metabolismo , Acetaminofén/toxicidad , Línea Celular , Hígado Graso/patología , Glucosa/metabolismo , Glucólisis/fisiología , Humanos , Interleucina-6/metabolismo , Dispositivos Laboratorio en un Chip , Lipopolisacáridos , Macrófagos/citología , Monocitos/citología , Fosforilación Oxidativa , Factor de Necrosis Tumoral alfa/metabolismo , Células U937RESUMEN
Dual specificity mitogen-activated protein kinase (MAPK) phosphatases [dual specificity phosphatase/MAP kinase phosphatase (DUSP-MKP)] have been hypothesized to maintain cancer cell survival by buffering excessive MAPK signaling caused by upstream activating oncogenic products. A large and diverse body of literature suggests that genetic depletion of DUSP-MKPs can reduce tumorigenicity, suggesting that hyperactivating MAPK signaling by DUSP-MKP inhibitors could be a novel strategy to selectively affect the transformed phenotype. Through in vivo structure-activity relationship studies in transgenic zebrafish we recently identified a hyperactivator of fibroblast growth factor signaling [(E)-2-benzylidene-5-bromo-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI-215)] that is devoid of developmental toxicity and restores defective MAPK activity caused by overexpression of DUSP1 and DUSP6 in mammalian cells. Here, we hypothesized that BCI-215 could selectively affect survival of transformed cells. In MDA-MB-231 human breast cancer cells, BCI-215 inhibited cell motility, caused apoptosis but not primary necrosis, and sensitized cells to lymphokine-activated killer cell activity. Mechanistically, BCI-215 induced rapid and sustained phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) in the absence of reactive oxygen species, and its toxicity was partially rescued by inhibition of p38 but not JNK or ERK. BCI-215 also hyperactivated MKK4/SEK1, suggesting activation of stress responses. Kinase phosphorylation profiling documented BCI-215 selectively activated MAPKs and their downstream substrates, but not receptor tyrosine kinases, SRC family kinases, AKT, mTOR, or DNA damage pathways. Our findings support the hypothesis that BCI-215 causes selective cancer cell cytotoxicity in part through non-redox-mediated activation of MAPK signaling, and the findings also identify an intersection with immune cell killing that is worthy of further exploration.
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Neoplasias de la Mama/metabolismo , Inhibidores Enzimáticos/farmacología , Células Asesinas Activadas por Linfocinas/efectos de los fármacos , Células Asesinas Activadas por Linfocinas/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/antagonistas & inhibidores , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Animales , Animales Modificados Genéticamente , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/uso terapéutico , Femenino , Células HeLa , Hepatocitos/efectos de los fármacos , Hepatocitos/inmunología , Hepatocitos/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/inmunología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Células Asesinas Activadas por Linfocinas/inmunología , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/inmunología , Ratas , Pez CebraRESUMEN
In this article, we review the past applications of in vitro models in identifying human hepatotoxins and then focus on the use of multiscale experimental models in drug development, including the use of zebrafish and human cell-based, 3-dimensional, microfluidic systems of liver functions as key components in applying Quantitative Systems Pharmacology (QSP). We have implemented QSP as a platform to improve the rate of success in the process of drug discovery and development of therapeutics.
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Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Hígado/efectos de los fármacos , Animales , Descubrimiento de Drogas , Evolución Química , Humanos , Mamíferos , Modelos Animales , Valor Predictivo de las Pruebas , Medición de RiesgoRESUMEN
This paper describes the development and characterization of a microphysiology platform for drug safety and efficacy in liver models of disease that includes a human, 3D, microfluidic, four-cell, sequentially layered, self-assembly liver model (SQL-SAL); fluorescent protein biosensors for mechanistic readouts; as well as a microphysiology system database (MPS-Db) to manage, analyze, and model data. The goal of our approach is to create the simplest design in terms of cells, matrix materials, and microfluidic device parameters that will support a physiologically relevant liver model that is robust and reproducible for at least 28 days for stand-alone liver studies and microfluidic integration with other organs-on-chips. The current SQL-SAL uses primary human hepatocytes along with human endothelial (EA.hy926), immune (U937) and stellate (LX-2) cells in physiological ratios and is viable for at least 28 days under continuous flow. Approximately, 20% of primary hepatocytes and/or stellate cells contain fluorescent protein biosensors (called sentinel cells) to measure apoptosis, reactive oxygen species (ROS) and/or cell location by high content analysis (HCA). In addition, drugs, drug metabolites, albumin, urea and lactate dehydrogenase (LDH) are monitored in the efflux media. Exposure to 180 µM troglitazone or 210 µM nimesulide produced acute toxicity within 2-4 days, whereas 28 µM troglitazone produced a gradual and much delayed toxic response over 21 days, concordant with known mechanisms of toxicity, while 600 µM caffeine had no effect. Immune-mediated toxicity was demonstrated with trovafloxacin with lipopolysaccharide (LPS), but not levofloxacin with LPS. The SQL-SAL exhibited early fibrotic activation in response to 30 nM methotrexate, indicated by increased stellate cell migration, expression of alpha-smooth muscle actin and collagen, type 1, alpha 2. Data collected from the in vitro model can be integrated into a database with access to related chemical, bioactivity, preclinical and clinical information uploaded from external databases for constructing predictive models.
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Biomarcadores Farmacológicos , Evaluación Preclínica de Medicamentos/métodos , Hígado Artificial , Hígado/efectos de los fármacos , Hígado/fisiología , Microfluídica/métodos , Técnicas de Cultivo de Órganos/métodos , Técnicas Biosensibles/métodos , Supervivencia Celular , Humanos , Proteínas Luminiscentes/análisis , Factores de TiempoRESUMEN
No therapies have been shown to accelerate recovery or prevent fibrosis after acute kidney injury (AKI). In part, this is because most therapeutic candidates have to be given at the time of injury and the diagnosis of AKI is usually made too late for drugs to be efficacious. Strategies to enhance post-AKI repair represent an attractive approach to address this. Using a phenotypic screen in zebrafish, we identified 4-(phenylthio)butanoic acid (PTBA), which promotes proliferation of embryonic kidney progenitor cells (EKPCs), and the PTBA methyl ester UPHD25, which also increases postinjury repair in ischemia-reperfusion and aristolochic acid-induced AKI in mice. In these studies, a new panel of PTBA analogs was evaluated. Initial screening was performed in zebrafish EKPC assays followed by survival assays in a gentamicin-induced AKI larvae zebrafish model. Using this approach, we identified UPHD186, which in contrast to UPHD25, accelerates recovery and reduces fibrosis when administered several days after ischemia-reperfusion AKI and reduces fibrosis after unilateral ureteric obstruction in mice. UPHD25 and 186 are efficiently metabolized to the active analog PTBA in liver and kidney microsome assays, indicating both compounds may act as PTBA prodrugs in vivo. UPHD186 persists longer in the circulation than UPHD25, suggesting that sustained levels of UPHD186 may increase efficacy by acting as a reservoir for renal metabolism to PTBA. These findings validate use of zebrafish EKPC and AKI assays as a drug discovery strategy for molecules that reduce fibrosis in multiple AKI models and can be administered days after initiation of injury.
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Lesión Renal Aguda/tratamiento farmacológico , Butiratos/uso terapéutico , Riñón/efectos de los fármacos , Sulfuros/uso terapéutico , Lesión Renal Aguda/patología , Animales , Butiratos/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Fibrosis/tratamiento farmacológico , Fibrosis/patología , Riñón/patología , Masculino , Ratones , Sulfuros/farmacología , Pez CebraRESUMEN
One of the greatest challenges in biomedical research, drug discovery and diagnostics is understanding how seemingly identical cells can respond differently to perturbagens including drugs for disease treatment. Although heterogeneity has become an accepted characteristic of a population of cells, in drug discovery it is not routinely evaluated or reported. The standard practice for cell-based, high content assays has been to assume a normal distribution and to report a well-to-well average value with a standard deviation. To address this important issue we sought to define a method that could be readily implemented to identify, quantify and characterize heterogeneity in cellular and small organism assays to guide decisions during drug discovery and experimental cell/tissue profiling. Our study revealed that heterogeneity can be effectively identified and quantified with three indices that indicate diversity, non-normality and percent outliers. The indices were evaluated using the induction and inhibition of STAT3 activation in five cell lines where the systems response including sample preparation and instrument performance were well characterized and controlled. These heterogeneity indices provide a standardized method that can easily be integrated into small and large scale screening or profiling projects to guide interpretation of the biology, as well as the development of therapeutics and diagnostics. Understanding the heterogeneity in the response to perturbagens will become a critical factor in designing strategies for the development of therapeutics including targeted polypharmacology.
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Descubrimiento de Drogas/métodos , Línea Celular Tumoral , Humanos , Células MCF-7 , Factor de Transcripción STAT3/metabolismoRESUMEN
The integration of high-content screening (HCS) readers with organ-specific cell models, panels of functional biomarkers, and advanced informatics is a powerful approach to identifying the toxic liabilities of compounds early in the development process and forms the basis of "early safety assessment." This cellular systems biology (CSB) approach (CellCiphr profile) has been used to integrate rodent and human cellular hepatic models with panels of functional biomarkers measured at multiple time points to profile both the potency and specificity of the cellular toxicological response. These profiles also provide initial insights on the mechanism of the toxic response. The authors describe here mechanistic assay profiles designed to further dissect the toxic mechanisms of action and elucidate subtle effects apparent in subpopulations of cells. They measured 8 key mechanisms of toxicity with multiple biomarker feature measurements made simultaneously in populations of living primary hepatocytes and HepG2 cells. Mining the cell population response from these mechanistic profiles revealed the concentration dependence and nature of the heterogeneity of the response, as well as relationships between the functional responses. These more detailed mechanistic profiles define differences in compound activities that are not apparent in the average population response. Because cells and tissues encounter wide ranges of drug doses in space and time, these mechanistic profiles build on the CellCiphr profile and better reflect the complexity of the response in vivo.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Bibliotecas de Moléculas Pequeñas/toxicidad , Biología de Sistemas/métodos , Animales , Bioensayo , Biomarcadores/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Hep G2 , Hepatocitos/citología , Humanos , Lisosomas/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/análisis , Factores de TiempoRESUMEN
ABSTRACT Phospholipidosis is the excessive intralysosomal accumulation of phospholipids and is induced in humans and animals by the chronic administration of cationic amphiphilic drugs. To identify compounds that may induce phospholipidosis early in the discovery process, we have developed a predictive fluorescent cell-based assay amenable to automated high content screening using the 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (ss-BODIPY C(12)-HPC) dye and primary rat hepatocytes. ss-BODIPY C(12)-HPC localized to lysosomes that accumulate phospholipids and not to lipid droplets, indicating the selectivity for phospholipid-containing granules. Accumulation of ss-BODIPY C(12)-HPC was monitored in primary rat hepatocytes plated onto 96-well plates and 24 h after exposure to increasing concentrations of 13 drugs known to induce phospholipidosis and four negative compounds. Fluorescent images were captured and analyzed using the Discovery-1 automated cellular imaging system. Eleven out of the 12 selected positive compounds and all negative compounds were properly assigned as positive and negative inducers of phospholipidosis, respectively, indicating the high degree of sensitivity and specificity of this assay. The ability of ss-BODIPY C(12)-HPC to detect and quantify phospholipidosis is similar to that of the well-established probe, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dipalmitoylphosphatidylethanolamine (NBD-PE).