RESUMEN
Here is reported the design and synthesis of a series of highly potent and selective α2C antagonists using benzodioxine methyl piperazine as a central scaffold by pharmacophoric analysis to improve the pharmacokinetics of suboptimal clinical candidate molecules.
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Receptores Adrenérgicos alfa 2RESUMEN
In this manuscript, we report a series of benzodioxine methyl piperidine derivatives as highly potent and selective α2C antagonists by ligand design to improve the pharmacokinetics of a previous candidate molecule.
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Dioxinas , Receptores Adrenérgicos alfa 2 , Piperidinas/farmacologíaRESUMEN
Drug-induced liver injury (DILI) is a patient-specific, temporal, multifactorial pathophysiological process that cannot yet be recapitulated in a single in vitro model. Current preclinical testing regimes for the detection of human DILI thus remain inadequate. A systematic and concerted research effort is required to address the deficiencies in current models and to present a defined approach towards the development of new or adapted model systems for DILI prediction. This Perspective defines the current status of available models and the mechanistic understanding of DILI, and proposes our vision of a roadmap for the development of predictive preclinical models of human DILI.
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Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Modelos Animales de Enfermedad , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/prevención & control , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Humanos , Valor Predictivo de las PruebasRESUMEN
Primary human hepatocytes (PHHs) are commonly used for in vitro studies of drug-induced liver injury. However, when cultured as 2D monolayers, PHH lose crucial hepatic functions within hours. This dedifferentiation can be ameliorated when PHHs are cultured in sandwich configuration (2Dsw), particularly when cultures are regularly re-overlaid with extracellular matrix, or as 3D spheroids. In this study, the 6 participating laboratories evaluated the robustness of these 2 model systems made from cryopreserved PHH from the same donors considering both inter-donor and inter-laboratory variability and compared their suitability for use in repeated-dose toxicity studies using 5 different hepatotoxins with different toxicity mechanisms. We found that expression levels of proteins involved in drug absorption, distribution, metabolism, and excretion, as well as catalytic activities of 5 different CYPs, were significantly higher in 3D spheroid cultures, potentially affecting the exposure of the cells to drugs and their metabolites. Furthermore, global proteomic analyses revealed that PHH in 3D spheroid configuration were temporally stable whereas proteomes from the same donors in 2Dsw cultures showed substantial alterations in protein expression patterns over the 14 days in culture. Overall, spheroid cultures were more sensitive to the hepatotoxic compounds investigated, particularly upon long-term exposures, across testing sites with little inter-laboratory or inter-donor variability. The data presented here suggest that repeated-dosing regimens improve the predictivity of in vitro toxicity assays, and that PHH spheroids provide a sensitive and robust system for long-term mechanistic studies of drug-induced hepatotoxicity, whereas the 2Dsw system has a more dedifferentiated phenotype and lower sensitivity to detect hepatotoxicity.
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Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hepatocitos/efectos de los fármacos , Preparaciones Farmacéuticas/administración & dosificación , Esferoides Celulares/efectos de los fármacos , Pruebas de Toxicidad/métodos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Criopreservación , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Valor Predictivo de las Pruebas , Cultivo Primario de Células , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Factores de Tiempo , Pruebas de Toxicidad/normasRESUMEN
INTRODUCTION: The liver is an important target for drug-induced toxicities. Early detection of hepatotoxic drugs requires use of well-characterized test systems, yet current knowledge, gaps and limitations of tests employed remains an important issue for drug development. Areas Covered: The current state of the science, understanding and application of test systems in use for the detection of drug-induced cytotoxicity, mitochondrial toxicity, cholestasis and inflammation is summarized. The test systems highlighted herein cover mostly in vitro and some in vivo models and endpoint measurements used in the assessment of small molecule toxic liabilities. Opportunities for research efforts in areas necessitating the development of specific tests and improved mechanistic understanding are highlighted. Expert Opinion: Use of in vitro test systems for safety optimization will remain a core activity in drug discovery. Substantial inroads have been made with a number of assays established for human Drug-induced Liver Injury. There nevertheless remain significant gaps with a need for improved in vitro tools and novel tests to address specific mechanisms of human Drug-Induced Liver Injury. Progress in these areas will necessitate not only models fit for application, but also mechanistic understanding of how chemical insult on the liver occurs in order to identify translational and quantifiable readouts for decision-making.
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Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Descubrimiento de Drogas/métodos , Pruebas de Toxicidad/métodos , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Diseño de Fármacos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Humanos , Modelos Biológicos , Medición de Riesgo/métodosRESUMEN
Current preclinical drug testing does not predict some forms of adverse drug reactions in humans. Efforts at improving predictability of drug-induced tissue injury in humans include using stem cell technology to generate human cells for screening for adverse effects of drugs in humans. The advent of induced pluripotent stem cells means that it may ultimately be possible to develop personalized toxicology to determine interindividual susceptibility to adverse drug reactions. However, the complexity of idiosyncratic drug-induced liver injury means that no current single-cell model, whether of primary liver tissue origin, from liver cell lines, or derived from stem cells, adequately emulates what is believed to occur during human drug-induced liver injury. Nevertheless, a single-cell model of a human hepatocyte which emulates key features of a hepatocyte is likely to be valuable in assessing potential chemical risk; furthermore, understanding how to generate a relevant hepatocyte will also be critical to efforts to build complex multicellular models of the liver. Currently, hepatocyte-like cells differentiated from stem cells still fall short of recapitulating the full mature hepatocellular phenotype. Therefore, we convened a number of experts from the areas of preclinical and clinical hepatotoxicity and safety assessment, from industry, academia, and regulatory bodies, to specifically explore the application of stem cells in hepatotoxicity safety assessment and to make recommendations for the way forward. In this short review, we particularly discuss the importance of benchmarking stem cell-derived hepatocyte-like cells to their terminally differentiated human counterparts using defined phenotyping, to make sure the cells are relevant and comparable between labs, and outline why this process is essential before the cells are introduced into chemical safety assessment. (Hepatology 2017;65:710-721).
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Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Hepatocitos/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Pruebas de Toxicidad , Células Cultivadas/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Técnicas In Vitro , Células Madre Pluripotentes/metabolismo , Valor Predictivo de las Pruebas , Sensibilidad y EspecificidadRESUMEN
The current test systems employed by pharmaceutical industry are poorly predictive for drug-induced liver injury (DILI). The 'MIP-DILI' project addresses this situation by the development of innovative preclinical test systems which are both mechanism-based and of physiological, pharmacological and pathological relevance to DILI in humans. An iterative, tiered approach with respect to test compounds, test systems, bioanalysis and systems analysis is adopted to evaluate existing models and develop new models that can provide validated test systems with respect to the prediction of specific forms of DILI and further elucidation of mechanisms. An essential component of this effort is the choice of compound training set that will be used to inform refinement and/or development of new model systems that allow prediction based on knowledge of mechanisms, in a tiered fashion. In this review, we focus on the selection of MIP-DILI training compounds for mechanism-based evaluation of non-clinical prediction of DILI. The selected compounds address both hepatocellular and cholestatic DILI patterns in man, covering a broad range of pharmacologies and chemistries, and taking into account available data on potential DILI mechanisms (e.g. mitochondrial injury, reactive metabolites, biliary transport inhibition, and immune responses). Known mechanisms by which these compounds are believed to cause liver injury have been described, where many if not all drugs in this review appear to exhibit multiple toxicological mechanisms. Thus, the training compounds selection offered a valuable tool to profile DILI mechanisms and to interrogate existing and novel in vitro systems for the prediction of human DILI.
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Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Biología Computacional/métodos , Drogas en Investigación/efectos adversos , Medicina Basada en la Evidencia , Sistemas Especialistas , Hígado/efectos de los fármacos , Modelos Biológicos , Inmunidad Adaptativa/efectos de los fármacos , Animales , Inteligencia Artificial , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Drogas en Investigación/química , Drogas en Investigación/clasificación , Drogas en Investigación/farmacología , Eliminación Hepatobiliar/efectos de los fármacos , Humanos , Hígado/inmunología , Hígado/metabolismo , Hígado/fisiopatología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Estructura Molecular , Estrés Oxidativo/efectos de los fármacos , Índice de Severidad de la EnfermedadRESUMEN
Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug.