RESUMEN
Drug-induced kidney injury (DIKI) refers to kidney damage resulting from the administration of medications. The aim of this project was to identify reliable urinary microRNA (miRNAs) biomarkers that can be used as potential predictors of DIKI before disease diagnosis. This study quantified a panel of six miRNAs (miRs-210-3p, 423-5p, 143-3p, 130b-3p, 486-5p, 193a-3p) across multiple time points using urinary samples from a previous investigation evaluating effects of a nephrotoxicant in cynomolgus monkeys. Exosome-associated miRNA exhibited distinctive trends when compared to miRNAs quantified in whole urine, which may reflect a different urinary excretion mechanism of miRNAs than those released passively into the urine. Although further research and mechanistic studies are required to elucidate how these miRNAs regulate signaling in disease pathways, we present, for the first time, data that several miRNAs displayed strong correlations with histopathology scores, thus indicating their potential use as biomarkers to predict the development of DIKI in preclinical studies and clinical trials. Also, these findings can potentially be translated into other non-clinical species or human for the detection of DIKI.
Asunto(s)
Biomarcadores , Macaca fascicularis , MicroARNs , Animales , MicroARNs/orina , MicroARNs/genética , Biomarcadores/orina , Masculino , Riñón/efectos de los fármacos , Riñón/patología , Riñón/metabolismo , Exosomas/genéticaRESUMEN
Cardiovascular toxicity is an important cause of drug failures in the later stages of drug development, early clinical safety assessment, and even postmarket withdrawals. Early-stage in vitro assessment of potential cardiovascular liabilities in the pharmaceutical industry involves assessment of interactions with cardiac ion channels, as well as induced pluripotent stem cell-derived cardiomyocyte-based functional assays, such as calcium flux and multielectrode-array assays. These methods are appropriate for the identification of acute functional cardiotoxicity but structural cardiotoxicity, which manifests effects after chronic exposure, is often only captured in vivo. CardioMotion is a novel, label-free, high throughput, in vitro assay and analysis pipeline which records and assesses the spontaneous beating of cardiomyocytes and identifies compounds which impact beating. This is achieved through the acquisition of brightfield images at a high framerate, combined with an optical flow-based python analysis pipeline which transforms the images into waveform data which are then parameterized. Validation of this assay with a large dataset showed that cardioactive compounds with diverse known direct functional and structural mechanisms-of-action on cardiomyocytes are identified (sensitivity = 72.9%), importantly, known structural cardiotoxins also disrupt cardiomyocyte beating (sensitivity = 86%) in this method. Furthermore, the CardioMotion method presents a high specificity of 82.5%.
Asunto(s)
Cardiotoxicidad , Células Madre Pluripotentes Inducidas , Humanos , Cardiotoxicidad/etiología , Células Cultivadas , Miocitos CardíacosRESUMEN
Use of molecular data in human and ecological health risk assessments of industrial chemicals and agrochemicals has been anticipated by the scientific community for many years; however, these data are rarely used for risk assessment. Here, a logic framework is proposed to explore the feasibility and future development of transcriptomic methods to refine and replace the current apical endpoint-based regulatory toxicity testing paradigm. Four foundational principles are outlined and discussed that would need to be accepted by stakeholders prior to this transformative vision being realized. Well-supported by current knowledge, the first principle is that transcriptomics is a reliable tool for detecting alterations in gene expression that result from endogenous or exogenous influences on the test organism. The second principle states that alterations in gene expression are indicators of adverse or adaptive biological responses to stressors in an organism. Principle 3 is that transcriptomics can be employed to establish a benchmark dose-based point of departure (POD) from short-term, in vivo studies at a dose level below which a concerted molecular change (CMC) is not expected. Finally, Principle 4 states that the use of a transcriptomic POD (set at the CMC dose level) will support a human health-protective risk assessment. If all four principles are substantiated, this vision is expected to transform aspects of the industrial chemical and agrochemical risk assessment process that are focused on establishing safe exposure levels for mammals across numerous toxicological contexts resulting in a significant reduction in animal use while providing equal or greater protection of human health. Importantly, these principles and approaches are also generally applicable for ecological safety assessment.
Asunto(s)
Pruebas de Toxicidad , Transcriptoma , Animales , Humanos , Medición de Riesgo/métodos , Benchmarking , MamíferosRESUMEN
Activation of MrgX2, an orphan G protein-coupled receptor expressed on mast cells, leads to degranulation and histamine release. Human MrgX2 binds promiscuously to structurally diverse peptides and small molecules that tend to have basic properties (basic secretagogues), resulting in acute histamine-like adverse drug reactions of injected therapeutic agents. We set out to identify MrgX2 orthologues from other mammalian species used in nonclinical stages of drug development. Previously, the only known orthologue of human MrgX2 was from mouse, encoded by Mrgprb2. MrgX2 genes of rat, dog (beagle), minipig, pig, and Rhesus and cynomolgus monkey were identified by bioinformatic approaches and verified by their ability to mediate calcium mobilization in transfected cells in response to the classical MrgX2 agonist, compound 48/80. The peptide GSK3212448 is an inhibitor of the PRC2 epigenetic regulator that caused profound anaphylactoid reactions upon intravenous infusion to rat. We showed GSK3212448 to be a potent MrgX2 agonist particularly at rat MrgX2. We screened sets of drug-like molecules and peptides to confirm the highly promiscuous nature of MrgX2. Approximately 20% of drug-like molecules activated MrgX2 (pEC50 ranging from 4.5 to 6), with the principle determinant being basicity. All peptides tested of net charge +3 or greater exhibited agonist activity, including the cell penetrating peptides polyarginine (acetyl-Arg9-amide) and TAT (49-60), a fragment of HIV-1 TAT protein. Finally, we showed that the glycopeptide antibiotic vancomycin, which is associated with clinical pseudo-allergic reactions known as red man syndrome, is an agonist of MrgX2.
Asunto(s)
Anafilaxia/inducido químicamente , Mastocitos/efectos de los fármacos , Proteínas del Tejido Nervioso/agonistas , Fragmentos de Péptidos/efectos adversos , Receptores Acoplados a Proteínas G/agonistas , Receptores de Neuropéptido/agonistas , Vancomicina/efectos adversos , Anafilaxia/inmunología , Animales , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/inmunología , Línea Celular Tumoral , Péptidos de Penetración Celular/administración & dosificación , Péptidos de Penetración Celular/efectos adversos , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/efectos adversos , Células HEK293 , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Humanos , Mastocitos/inmunología , Mastocitos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/metabolismo , Fragmentos de Péptidos/administración & dosificación , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/inmunología , Receptores de Neuropéptido/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Síndrome , Vancomicina/administración & dosificación , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
Induction of IFNα in the upper airways via activation of TLR7 represents a novel immunomodulatory approach to the treatment of allergic asthma. Exploration of 8-oxoadenine derivatives bearing saturated oxygen or nitrogen heterocycles in the N-9 substituent has revealed a remarkable selective enhancement in IFNα inducing potency in the nitrogen series. Further potency enhancement was achieved with the novel (S)-pentyloxy substitution at C-2 leading to the selection of GSK2245035 (32) as an intranasal development candidate. In human cell cultures, compound 32 resulted in suppression of Th2 cytokine responses to allergens, while in vivo intranasal administration at very low doses led to local upregulation of TLR7-mediated cytokines (IP-10). Target engagement was confirmed in humans following single intranasal doses of 32 of ≥20 ng, and reproducible pharmacological response was demonstrated following repeat intranasal dosing at weekly intervals.
Asunto(s)
Adenina/análogos & derivados , Asma/tratamiento farmacológico , Descubrimiento de Drogas , Piperidinas/administración & dosificación , Piperidinas/farmacología , Receptor Toll-Like 7/agonistas , Adenina/administración & dosificación , Adenina/química , Adenina/farmacología , Administración Intranasal , Asma/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Piperidinas/química , Relación Estructura-ActividadRESUMEN
An αVß3 receptor antagonist (SB-273005) induced unique vascular lesions in the aorta of mice, but not other pharmacologically responsive species. Vascular smooth muscle cell (VSMC) necrosis was observed ~6 h postdose followed by VSMC loss with no evidence of hemorrhage/thrombosis, inflammation or damage to endothelium. Since direct drug-induced vascular toxicity is uncommon, involvement of VSMC-endothelial cell (EC) interactions was hypothesized. In vitro model systems of murine aortic VSMC and EC monocultures and cocultures were established and used to investigate the mechanism of toxicity. Incubation of cultures with SB-273005 within a dose range and timeframe comparable to in vivo studies, showed a concentration-dependent decrease in viability with increases in cytotoxicity for monocultures and VSMC/EC cocultures; however, VSMC monocultures responded at lower doses (were most sensitive) suggesting a direct effect on VSMC which is not mediated or enhanced through EC/VSMC interactions. Further studies revealed increased caspase-9 and caspase-3/7 activation in VSMC beginning as early as 0.5 and 1h following treatment, respectively. These findings suggest SB-273005 causes direct chemical vascular toxicity in murine VSMC which involves apoptosis mediated through the intrinsic (mitochondrial) apoptotic pathway. To our knowledge, this is the first report to provide a link between VSMC apoptosis and treatment with an αVß3 receptor antagonist.