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Human-on-a-chip systems are rapidly advancing due to the availability of human stem cells from a variety of tissues, but publications have utilized mostly simple methods of biochemical analysis. Here, we apply mass spectrometry to a sophisticated multiorgan human-on-a-chip system for the comprehensive study of tolcapone metabolite profiling and metabolomics. The developed human-on-a-chip includes seven interacting microphysiological systems (MPSs), brain, pancreas, liver, lung, heart, gut, and endometrium, with a mixer chamber for systemic circulation and tolcapone dose. We investigated tolcapone metabolism by analyzing the circulating medium using mass spectrometry. Twelve tolcapone metabolites were identified, three of which are newly reported. These metabolites demonstrated that oxidation, reduction, and conjugation reactions were the most important routes of tolcapone metabolism. In parallel, metabolomics in brain MPS evaluated the tolcapone influences on endogenous pathways in human brain. Untargeted metabolomics identified 18 key biomarkers significantly changed in human brain MPS after tolcapone dosing, which were mainly associated with perturbation of tryptophan and phenylalanine metabolism (BH4 cycle), glycerophospholipid metabolism, energy metabolism, and aspartate metabolism. This is the first example of successfully combining drug metabolism, metabolomics, and cell engineering to capture complex human physiology and the multiorgan interactions; the results we present here could be a step toward using analytical chemistry to advance the utilization of human-on-a-chip for testing both drug efficacy and toxicity in a single system.
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Biomarcadores/metabolismo , Encéfalo/metabolismo , Hígado/metabolismo , Espectrometría de Masas/métodos , Metaboloma , Microtecnología/métodos , Tolcapona/metabolismo , Metabolismo Energético , Humanos , Metabolismo de los Lípidos , Microtecnología/instrumentaciónRESUMEN
In vitro hepatocyte culture systems have inherent limitations in capturing known human drug toxicities that arise from complex immune responses. Therefore, we established and characterized a liver immunocompetent coculture model and evaluated diclofenac (DCF) metabolic profiles, in vitro-in vivo clearance correlations, toxicological responses, and acute phase responses using liquid chromatography-tandem mass spectrometry. DCF biotransformation was assessed after 48 hours of culture, and the major phase I and II metabolites were similar to the in vivo DCF metabolism profile in humans. Further characterization of secreted bile acids in the medium revealed that a glycine-conjugated bile acid was a sensitive marker of dose-dependent toxicity in this three-dimensional liver microphysiological system. Protein markers were significantly elevated in the culture medium at high micromolar doses of DCF, which were also observed previously for acute drug-induced toxicity in humans. In this immunocompetent model, lipopolysaccharide treatment evoked an inflammatory response that resulted in a marked increase in the overall number of acute phase proteins. Kupffer cell-mediated cytokine release recapitulated an in vivo proinflammatory response exemplified by a cohort of 11 cytokines that were differentially regulated after lipopolysaccharide induction, including interleukin (IL)-1ß, IL-1Ra, IL-6, IL-8, IP-10, tumor necrosis factor-α, RANTES (regulated on activation normal T cell expressed and secreted), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, macrophage inflammatory protein-1ß, and IL-5. In summary, our findings indicate that three-dimensional liver microphysiological systems may serve as preclinical investigational platforms from the perspective of the discovery of a set of clinically relevant biomarkers including potential reactive metabolites, endogenous bile acids, excreted proteins, and cytokines to predict early drug-induced liver toxicity in humans.
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Proteínas de Fase Aguda/metabolismo , Antiinflamatorios no Esteroideos , Citocinas/inmunología , Diclofenaco , Hígado/efectos de los fármacos , Modelos Biológicos , Antiinflamatorios no Esteroideos/farmacocinética , Antiinflamatorios no Esteroideos/toxicidad , Biotransformación , Técnicas de Cocultivo , Diclofenaco/farmacocinética , Diclofenaco/toxicidad , Relación Dosis-Respuesta a Droga , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Inflamación , Macrófagos del Hígado/citología , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/metabolismo , Lipopolisacáridos/toxicidad , Hígado/inmunología , Hígado/metabolismo , Unión Proteica , ProteómicaRESUMEN
A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut-liver tissue interactions under normal and inflammatory contexts, via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover, significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/ß/γ signaling during inflammatory gut-liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. Biotechnol. Bioeng. 2017;114: 2648-2659. © 2017 Wiley Periodicals, Inc.
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Comunicación Celular/inmunología , Colon/inmunología , Hepatocitos/inmunología , Factores Inmunológicos/inmunología , Inflamación/inmunología , Macrófagos del Hígado/inmunología , Dispositivos Laboratorio en un Chip , Células CACO-2 , Células Cultivadas , Técnicas de Cocultivo/instrumentación , Citocinas/inmunología , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Inmunoensayo/instrumentación , Hígado/inmunología , Miniaturización , Integración de SistemasRESUMEN
The extracellular signal-regulated kinase (ERK) signaling pathway controls cell proliferation and differentiation in metazoans. Two hallmarks of its dynamics are adaptation of ERK phosphorylation, which has been linked to negative feedback, and nucleocytoplasmic shuttling, which allows active ERK to phosphorylate protein substrates in the nucleus and cytosol. To integrate these complex features, we acquired quantitative biochemical and live-cell microscopy data to reconcile phosphorylation, localization, and activity states of ERK. While maximal growth factor stimulation elicits transient ERK phosphorylation and nuclear translocation responses, ERK activities available to phosphorylate substrates in the cytosol and nuclei show relatively little or no adaptation. Free ERK activity in the nucleus temporally lags the peak in nuclear translocation, indicating a slow process. Additional experiments, guided by kinetic modeling, show that this process is consistent with ERK's modification of and release from nuclear substrate anchors. Thus, adaptation of whole-cell ERK phosphorylation is a by-product of transient protection from phosphatases. Consistent with this interpretation, predictions concerning the dose-dependence of the pathway response and its interruption by inhibition of MEK were experimentally confirmed.
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Transporte Activo de Núcleo Celular , Quinasas MAP Reguladas por Señal Extracelular/química , Retroalimentación Fisiológica , Modelos Teóricos , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Cinética , Ratones , Células 3T3 NIH , Fosforilación , Transporte de Proteínas/genética , Receptores de Factores de Crecimiento/genética , Transducción de Señal/genéticaRESUMEN
Polypharmacy-related drug-drug interactions (DDIs) are a significant and growing healthcare concern. Increasing number of therapeutic drugs on the market underscores the necessity to accurately assess the new drug combinations during pre-clinical evaluation for DDIs. In vitro primary human hepatocyte (PHH)-only models are commonly used only for perpetrator DDI studies due to their rapid loss of metabolic function. But co-culturing non-human cells with human PHHs can stabilize metabolic activity and be utilized for both perpetrator and victim studies, though this raises concerns about human specificity for accurate clinical assessment. In this study, we evaluated Liver Tissue Chip (LTC) with PHH-only liver microphysiological system (MPS) for DDI induction studies. Liver MPS from three individual donors maintained their functional and metabolic activity for up to 4 weeks demonstrating suitability for long-term pharmacokinetics (PK) studies. The responses to rifampicin induction of three PHH donors were assessed using CYP activity and mRNA changes. Additionally, victim PK studies were conducted with midazolam (high clearance) and alprazolam (low clearance) following rifampicin-mediated induction which resulted in a 2-fold and a 2.6-fold increase in midazolam and alprazolam intrinsic clearance values respectively compared to the untreated liver MPS. We also investigated the induction effects of different dosing regimens of the perpetrator drug (rifampicin) on CYP activity levels, showing minimal variation in the intrinsic clearance of the victim drug (midazolam). This study demonstrates the utility of the LTC for in vitro liver-specific DDI induction studies, providing a translational experimental system to predict clinical clearance values of both perpetrator and victim drugs.
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Signal transduction networks in mammalian cells, comprising a limited set of interacting biochemical pathways, are accessed by various growth factor and cytokine receptors to elicit distinct cell responses. This raises the question as to how specificity of the stimulus-response relationship is encoded at the molecular level. It has been proposed that specificity arises not only from the activation of unique signalling pathways, but also from quantitative differences in the activation and regulation of shared receptor-proximal signalling proteins. To address such hypotheses, data sets with greater precision and coverage of experimental conditions will need to be acquired, and rigorous frameworks that codify and parameterize the inherently non-linear relationships among signalling activities will need to be developed. In the present study we apply a systematic approach combining quantitative measurements and mathematical modelling to compare the signalling networks accessed by FGF (fibroblast growth factor) and PDGF (platelet-derived growth factor) receptors in mouse fibroblasts, in which the ERK (extracellular-signal-regulated kinase) cascade is activated by Ras- and PI3K (phosphoinositide 3-kinase)-dependent pathways. We show that, whereas the FGF stimulation of PI3K signalling is relatively weak, this deficiency is compensated for by a more potent Ras-dependent activation of ERK. Thus, as the modelling would predict, the ERK pathway is activated to a greater extent in cells co-stimulated with FGF and PDGF, relative to the saturated levels achieved with either ligand alone. It is envisaged that similar approaches will prove valuable in the elucidation of quantitative differences among other closely related receptor signalling networks.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Becaplermina , Simulación por Computador , Quinasas MAP Reguladas por Señal Extracelular/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Regulación Enzimológica de la Expresión Génica , Humanos , Cinética , Ratones , Modelos Biológicos , Células 3T3 NIH , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-sis/genética , Proteínas Proto-Oncogénicas c-sis/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento de Fibroblastos/genética , Transducción de SeñalRESUMEN
A crucial step in lead selection during drug development is accurate estimation and optimization of hepatic clearance using in vitro methods. However, current methods are limited by factors such as lack of physiological relevance, short culture/incubation times that are not consistent with drug exposure patterns in patients, use of drug absorbing materials, and evaporation during long-term incubation. To address these technological needs, we developed a novel milli-fluidic human liver tissue chip (LTC) that was designed with continuous media recirculation and optimized for hepatic cultures using human primary hepatocytes. Here, we characterized the LTC using a series of physiologically relevant metrics and test compounds to demonstrate that we could accurately predict the PK of both low- and high-clearance compounds. The non-biological characterization indicated that the cyclic olefin copolymer (COC)-based LTC exhibited negligible evaporation and minimal non-specific binding of drugs of varying ionic states and lipophilicity. Biologically, the LTC exhibited functional and polarized hepatic culture with sustained metabolic CYP activity for at least 15 days. This long-term culture was then used for drug clearance studies for low- and high-clearance compounds for at least 12 days, and clearance was estimated for a range of compounds with high in vitro-in vivo correlation (IVIVC). We also demonstrated that LTC can be induced by rifampicin, and the culture age had insignificant effect on depletion kinetic and predicted clearance value. Thus, we used advances in bioengineering to develop a novel purpose-built platform with high reproducibility and minimal variability to address unmet needs for PK applications.
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Hepatocitos , Hígado , Humanos , Reproducibilidad de los Resultados , Tasa de Depuración Metabólica , Hígado/metabolismo , Hepatocitos/metabolismo , Modelos Biológicos , FarmacocinéticaRESUMEN
Ras is a key signal transduction protein in the cell. Mutants of Gly(12) and Gln(61) impair GTPase activity and are found prominently in cancers. In wild type Ras-GTP, an allosteric switch promotes disorder to order transition in switch II, placing Gln(61) in the active site. We show that the "on" and "off" conformations of the allosteric switch can also be attained in RasG12V and RasQ61L. Although both mutants have similarly impaired active sites in the on state, RasQ61L stabilizes an anti-catalytic conformation of switch II in the off state of the allosteric switch when bound to Raf. This translates into more potent activation of the MAPK pathway involving Ras, Raf kinase, MEK, and ERK (Ras/Raf/MEK/ERK) in cells transfected with RasQ61L relative to RasG12V. This differential is not observed in the Raf-independent pathway involving Ras, phosphoinositide 3-kinase (PI3K), and Akt (Ras/PI3K/Akt). Using a combination of structural analysis, hydrolysis rates, and experiments in NIH-3T3 cells, we link the allosteric switch to the control of signaling in the Ras/Raf/MEK/ERK pathway, supporting a GTPase-activating protein-independent model for duration of the Ras-Raf complex.
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Regulación Alostérica , Guanosina Trifosfato/metabolismo , Transducción de Señal , Quinasas raf/metabolismo , Proteínas ras/metabolismo , Sitio Alostérico , Animales , Cinética , Ratones , Mutación Missense , Células 3T3 NIH , Conformación Proteica , Estabilidad Proteica , Proteínas ras/genéticaRESUMEN
INTRODUCTION: The present study was designed to evaluate inhaler techniques and patient satisfaction with fixed-combination budesonide/formoterol dry-powder inhaler chronic obstructive pulmonary disease (COPD) in Turkey in real-life clinical practice. PATIENTS AND METHODS: A total of 442 patients with COPD [mean (SD) age: 63.2 (10.6) years, 76.5% were males] were included in this cross-sectional study conducted at 25 outpatient clinics across Turkey. Data on socio-demographic characteristics, characteristics of COPD, inhaler technique and satisfaction with dry-powder inhaler were recorded at a single crosssectional visit performed at the study enrolment. RESULTS: Patients were characterized by prominence of moderate to severe (78.1%) COPD, high rate of regular use of overall COPD medications (89.4%) and Turbuhaler® for an average of 33.7 months, predominance of males (76.5%), primary education (85.7%), urban location (68.3), ex-smokers (61.1%) and spending time outdoors for ≥ 4 hour/day (62.0%). Use of correct techniques was evident in majority of patients (≥ 94%), whereas inhalation maneuvers including breathing out gently away from mouthpiece without blowing into it (71.9%) and holding the breath for 5-10 seconds (78.3%) were performed correctly by lesser percent of patients especially in the older group (≥ 65 years, p< 0.05). Overall percent of patients with the feeling that she/he used the inhaler very/fairly correctly was 73.3%, while 86% of patients identified that they were very/fairly satisfied with the inhaler, irrespective of age and educational status. CONCLUSION: In conclusion, our findings revealed the majority of patients are able to use Turbuhaler® correctly regardless of the educational status, while older age was associated with higher rate of errors in inhalation maneuvers in the real clinical practice in Turkey. Majority of our patients identified Turbuhaler® to be very/fairly convenient regarding ease of use, portability, and usability with an overall self-confidence in using the inhaler correctly among 73% and the satisfaction rate of 86%; irrespective of age and educational level.
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Broncodilatadores/uso terapéutico , Inhaladores de Polvo Seco/métodos , Satisfacción del Paciente , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Administración por Inhalación , Factores de Edad , Anciano , Broncodilatadores/administración & dosificación , Budesonida/administración & dosificación , Budesonida/uso terapéutico , Estudios Transversales , Combinación de Medicamentos , Inhaladores de Polvo Seco/instrumentación , Escolaridad , Etanolaminas/administración & dosificación , Etanolaminas/uso terapéutico , Fumarato de Formoterol , Humanos , Masculino , Persona de Mediana Edad , Resultado del Tratamiento , TurquíaRESUMEN
BACKGROUND: Traumatic knee injuries in humans trigger an immediate increase in synovial fluid levels of inflammatory cytokines that accompany impact damage to joint tissues. We developed a human in vitro cartilage-bone-synovium (CBS) coculture model to study the role of mechanical injury and inflammation in the initiation of post-traumatic osteoarthritis (PTOA)-like disease. METHODS: Osteochondral plugs (cartilage-bone, CB) along with joint capsule synovium explants (S) were harvested from 25 cadaveric distal femurs from 16 human donors (Collin's grade 0-2, 23-83years). Two-week monocultures (cartilage (C), bone (B), synovium (S)) and cocultures (CB, CBS) were established. A PTOA-like disease group was initiated via coculture of synovium explants with mechanically impacted osteochondral plugs (CBS+INJ, peak stress 5MPa) with non-impacted CB as controls. Disease-like progression was assessed through analyses of changes in cell viability, inflammatory cytokines released to media (10-plex ELISA), tissue matrix degradation, and metabolomics profile. RESULTS: Immediate increases in concentrations of a panel of inflammatory cytokines occurred in CBS+INJ and CBS cocultures and cultures with S alone (IL-1, IL-6, IL-8, and TNF-α among others). CBS+INJ and CBS also showed increased chondrocyte death compared to uninjured CB. The release of sulfated glycosaminoglycans (sGAG) and associated ARGS-aggrecan neoepitope fragments to the medium was significantly increased in CBS and CBS+INJ groups. Distinct metabolomics profiles were observed for C, B, and S monocultures, and metabolites related to inflammatory response in CBS versus CB (e.g., kynurenine, 1-methylnicotinamide, and hypoxanthine) were identified. CONCLUSION: CBS and CBS+INJ models showed distinct cellular, inflammatory, and matrix-related alterations relevant to PTOA-like initiation/progression. The use of human knee tissues from donors that had no prior history of OA disease suggests the relevance of this model in highlighting the role of injury and inflammation in earliest stages of PTOA progression.
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Cartílago Articular , Osteoartritis , Cartílago Articular/metabolismo , Citocinas/metabolismo , Humanos , Inflamación/metabolismo , Osteoartritis/etiología , Osteoartritis/metabolismo , Membrana Sinovial/metabolismoRESUMEN
Cell responses are actuated by tightly controlled signal transduction pathways. Although the concept of an integrated signaling network replete with interpathway cross-talk and feedback regulation is broadly appreciated, kinetic data of the type needed to characterize such interactions in conjunction with mathematical models are lacking. In mammalian cells, the Ras/ERK pathway controls cell proliferation and other responses stimulated by growth factors, and several cross-talk and feedback mechanisms affecting its activation have been identified. In this work, we take a systematic approach to parse the magnitudes of multiple regulatory mechanisms that attenuate ERK activation through canonical (Ras-dependent) and non-canonical (PI3K-dependent) pathways. In addition to regulation of receptor and ligand levels, we consider three layers of ERK-dependent feedback: desensitization of Ras activation, negative regulation of MEK kinase (e.g. Raf) activities, and up-regulation of dual-specificity ERK phosphatases. Our results establish the second of these as the dominant mode of ERK self-regulation in mouse fibroblasts. We further demonstrate that kinetic models of signaling networks, trained on a sufficient diversity of quantitative data, can be reasonably comprehensive, accurate, and predictive in the dynamical sense.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Retroalimentación Fisiológica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal , Proteínas ras/metabolismo , Animales , Humanos , Immunoblotting , Cinética , Ratones , Células 3T3 NIH , FosforilaciónRESUMEN
Productive cell migration requires the spatiotemporal coordination of cell adhesion, membrane protrusion, and actomyosin-mediated contraction. Integrins, engaged by the extracellular matrix (ECM), nucleate the formation of adhesive contacts at the cell's leading edge(s), and maturation of nascent adhesions to form stable focal adhesions constitutes a functional switch between protrusive and contractile activities. To shed additional light on the coupling between integrin-mediated adhesion and membrane protrusion, we have formulated a quantitative model of leading edge dynamics combining mechanistic and phenomenological elements and studied its features through classical bifurcation analysis and stochastic simulation. The model describes in mathematical terms the feedback loops driving, on the one hand, Rac-mediated membrane protrusion and rapid turnover of nascent adhesions, and on the other, myosin-dependent maturation of adhesions that inhibit protrusion at high ECM density. Our results show that the qualitative behavior of the model is most sensitive to parameters characterizing the influence of stable adhesions and myosin. The major predictions of the model, which we subsequently confirmed, are that persistent leading edge protrusion is optimal at an intermediate ECM density, whereas depletion of myosin IIA relieves the repression of protrusion at higher ECM density.
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Adhesión Celular/fisiología , Movimiento Celular/fisiología , Matriz Extracelular/metabolismo , Modelos Biológicos , Procesos Estocásticos , Animales , Células CHO , Extensiones de la Superficie Celular/metabolismo , Biología Computacional/métodos , Simulación por Computador , Cricetinae , Cricetulus , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Integrinas/metabolismo , Miosinas/metabolismo , Paxillin/genética , Paxillin/metabolismo , Transducción de Señal/fisiología , Proteínas de Unión al GTP rac/metabolismoRESUMEN
The development and application of quantitative systems pharmacology models in neuroscience have been modest relative to other fields, such as oncology and immunology, which may reflect the complexity of the brain. Technological and methodological advancements have enhanced the quantitative understanding of brain physiology and pathophysiology and the effects of pharmacological interventions. To maximize the knowledge gained from these novel data types, pharmacometrics modelers may need to expand their toolbox to include additional mathematical and statistical frameworks. A session was held at the 10th annual American Conference on Pharmacometrics (ACoP10) to highlight several recent advancements in quantitative and systems neuroscience. In this mini-review, we provide a brief overview of technological and methodological advancements in the neuroscience therapeutic area that were discussed during the session and how these can be leveraged with quantitative systems pharmacology modeling to enhance our understanding of neurological diseases. Microphysiological systems using human induced pluripotent stem cells (IPSCs), digital biomarkers, and large-scale imaging offer more clinically relevant experimental datasets, enhanced granularity, and a plethora of data to potentially improve the preclinical-to-clinical translation of therapeutics. Network neuroscience methodologies combined with quantitative systems models of neurodegenerative disease could help bridge the gap between cellular and molecular alterations and clinical end points through the integration of information on neural connectomics. Additional topics, such as the neuroimmune system, microbiome, single-cell transcriptomic technologies, and digital device biomarkers, are discussed in brief.
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Encéfalo/metabolismo , Descubrimiento de Drogas , Modelos Biológicos , Farmacología en Red , Enfermedades Neurodegenerativas/tratamiento farmacológico , Congresos como Asunto , HumanosRESUMEN
Although it is appreciated that canonical signal-transduction pathways represent dominant modes of regulation embedded in larger interaction networks, relatively little has been done to quantify pathway cross-talk in such networks. Through quantitative measurements that systematically canvas an array of stimulation and molecular perturbation conditions, together with computational modeling and analysis, we have elucidated cross-talk mechanisms in the platelet-derived growth factor (PDGF) receptor signaling network, in which phosphoinositide 3-kinase (PI3K) and Ras/extracellular signal-regulated kinase (Erk) pathways are prominently activated. We show that, while PI3K signaling is insulated from cross-talk, PI3K enhances Erk activation at points both upstream and downstream of Ras. The magnitudes of these effects depend strongly on the stimulation conditions, subject to saturation effects in the respective pathways and negative feedback loops. Motivated by those dynamics, a kinetic model of the network was formulated and used to precisely quantify the relative contributions of PI3K-dependent and -independent modes of Ras/Erk activation.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor Cross-Talk , Proteínas ras/metabolismo , Biología Computacional , Humanos , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de SeñalRESUMEN
The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.
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Alternativas a las Pruebas en Animales , Bienestar del Animal , Desarrollo de Medicamentos , Evaluación Preclínica de Medicamentos/métodos , Dispositivos Laboratorio en un Chip , Animales , Industria Farmacéutica , Humanos , Modelos BiológicosRESUMEN
Drug-induced kidney injury, a major cause of acute kidney injury, results in progressive kidney disease and is linked to increased mortality in hospitalized patients. Primary injury sites of drug-induced kidney injury are proximal tubules. Clinically, kidney injury molecule-1, an established tubule-specific biomarker, is monitored to assess the presence and progression of injury. The ability to accurately predict drug-related nephrotoxicity preclinically would reduce patient burden and drug attrition rates, yet state-of-the-art in vitro and animal models fail to do so. In this study, we demonstrate the use of kidney injury molecule-1 measurement in the kidney microphysiological system as a preclinical model for drug toxicity assessment. To show clinical relevance, we use quantitative systems pharmacology computational models for in vitro-in vivo translation of the experimental results and to identify favorable dosing regimens for one of the tested drugs.
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Cisplatino/efectos adversos , Gentamicinas/efectos adversos , Receptor Celular 1 del Virus de la Hepatitis A/metabolismo , Necrosis Tubular Aguda/inducido químicamente , Rifampin/efectos adversos , Biomarcadores/metabolismo , Línea Celular , Cisplatino/farmacocinética , Humanos , Necrosis Tubular Aguda/metabolismo , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Modelos Teóricos , Rifampin/farmacocinética , Investigación Biomédica TraslacionalRESUMEN
Early assessment of adverse drug effects in humans is critical to avoid long-lasting harm. However, current approaches for early detection of adverse effects still lack predictive and organ-specific biomarkers to evaluate undesired responses in humans. Microphysiological systems (MPSs) are in vitro representations of human tissues and provide organ-specific translational insights for physiological processes. In this study, a brain MPS was utilized to assess molecular signatures of neurotoxic and non-neurotoxic compounds using targeted and untargeted molecular approaches. The brain MPS comprising of human embryonic stem (ES) cell-derived neural progenitor cells seeded on three-dimensional (3D), chemically defined, polyethylene glycol hydrogels was treated with the neurotoxic drug, bortezomib and the non-neurotoxic drug, tamoxifen over 14-days. Possible toxic effects were monitored with human N-acetylaspartic acid (NAA) kinetics, which correlates the neuronal function/health and DJ-1/PARK7, an oxidative stress biomarker. Changes in NAA levels were observed as early as 2-days post-bortezomib treatment, while onset detection of oxidative stress (DJ-1) was delayed until 4-days post-treatment. Separately, the untargeted extracellular metabolomics approach revealed distinct fingerprints 2-days post-bortezomib treatment as perturbations in cysteine and glycerophospholipid metabolic pathways. These results suggest accumulation of reactive oxygen species associated with oxidative stress, and disruption of membrane structure and integrity. The NAA response was strongly correlated with changes in a subset of the detected metabolites at the same time point 2-days post-treatment. Moreover, these metabolite changes correlated strongly with DJ-1 levels measured at the later time point (4-days post-treatment). This suggests that early cellular metabolic dysfunction leads to later DJ-1 leakage and cell death, and that early measurement of this subset of metabolites could predict the later occurrence of cell death. While the approach demonstrated here provides an individual case study for proof of concept, we suggest that this approach can be extended for preclinical toxicity screening and biomarker discovery studies.
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Microphysiological systems (MPS) hold promise for improving therapeutic drug approval rates by providing more physiological, human-based, in vitro assays for preclinical drug development activities compared to traditional in vitro and animal models. Here, we first summarize why MPSs are needed in pharmaceutical development, and examine how MPS technologies can be utilized to improve preclinical efforts. We then provide the perspective that the full impact of MPS technologies will be realized only when robust approaches for in vitro-in vivo (MPS-to-human) translation are developed and utilized, and explain how the burgeoning field of quantitative systems pharmacology (QSP) can fill that need.
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Evaluación Preclínica de Medicamentos , Dispositivos Laboratorio en un Chip , Procedimientos Analíticos en Microchip , Humanos , Modelos BiológicosRESUMEN
Microphysiological systems (MPS), consisting of tissue constructs, biomaterials, and culture media, aim to recapitulate relevant organ functions in vitro. MPS components are housed in fluidic hardware with operational protocols, such as periodic complete media replacement. Such batch-like operations provide relevant nutrients and remove waste products but also reset cell-secreted mediators (e.g. cytokines, hormones) and potentially limit exposure to drugs (and metabolites). While each component plays an essential role for tissue functionality, MPS-specific nutrient needs are not yet well-characterized nor utilized to operate MPSs at more physiologically-relevant conditions. MPS-specific nutrient needs for gut (immortalized cancer cells), liver (human primary hepatocytes) and cardiac (iPSC-derived cardiomyocytes) MPSs were experimentally quantified. In a long-term study of the gut MPS (10 days), this knowledge was used to design operational protocols to maintain glucose and lactate at desired levels. This quasi-steady state operation was experimentally validated by monitoring glucose and lactate as well as MPS functionality. In a theoretical study, nutrient needs of an integrated multi-MPS platform (gut, liver, cardiac MPSs) were computationally simulated to identify long-term quasi-steady state operations. This integrative experimental and computational approach demonstrates the utilization of quantitative multi-scale characterization of MPSs and incorporating MPS-specific information to establish more physiologically-relevant experimental operations.