RESUMEN
Preventing clinical drug-induced liver injury (DILI) remains a major challenge, because DILI develops via multifactorial mechanisms. Immune and inflammatory reactions are considered important mechanisms of DILI; however, biomarkers from in vitro systems using immune cells have not been comprehensively studied. The aims of this study were (1) to identify promising biomarker genes for predicting DILI in an in vitro coculture model of peripheral blood mononuclear cells (PBMCs) with a human liver cell line, and (2) to evaluate these genes as predictors of DILI using a panel of drugs with different clinical DILI risk. Transcriptome-wide analysis of PBMCs cocultured with HepG2 or differentiated HepaRG cells that were treated with several drugs revealed an appropriate separation of DILI-positive and DILI-negative drugs, from which 12 putative biomarker genes were selected. To evaluate the predictive performance of these genes, PBMCs cocultured with HepG2 cells were exposed to 77 different drugs, and gene expression levels in PBMCs were determined. The MET proto-oncogene receptor tyrosine kinase (MET) showed the highest area under the receiver-operating characteristic curve (AUC) value of 0.81 among the 12 genes with a high sensitivity/specificity (85/66%). However, a stepwise logistic regression model using the 12 identified genes showed the highest AUC value of 0.94 with a high sensitivity/specificity (93/86%). Taken together, we established a coculture system using PBMCs and HepG2 cells and selected biomarkers that can predict DILI risk. The established model would be useful in detecting the DILI potential of compounds, in particular those that involve an immune mechanism.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hepatocitos/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Supervivencia Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Técnicas de Cocultivo , Perfilación de la Expresión Génica , Marcadores Genéticos , Células Hep G2 , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proto-Oncogenes Mas , Medición de RiesgoRESUMEN
Glyburide is widely used for the treatment of type 2 diabetes. We studied the mechanisms involved in the disposition of glyburide and its pharmacologically active hydroxy metabolites M1 and M2b and evaluated their clinical pharmacokinetics and the potential role in glyburide-induced cholestasis employing physiologically based pharmacokinetic (PBPK) modeling. Transport studies of parent and metabolites in human hepatocytes and transfected cell systems imply hepatic uptake mediated by organic anion-transporting polypeptides. Metabolites are also subjected to basolateral and biliary efflux by P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated proteins, and are substrates to renal organic anion transporter 3. A PBPK model in combination with a Bayesian approach was developed considering the identified disposition mechanisms. The model reasonably described plasma concentration time profiles and urinary recoveries of glyburide and the metabolites, implying the role of multiple transport processes in their pharmacokinetics. Predicted free liver concentrations of the parent (â¼30-fold) and metabolites (â¼4-fold) were higher than their free plasma concentrations. Finally, all three compounds showed bile salt export pump inhibition in vitro; however, significant in vivo inhibition was not apparent for any compound on the basis of a predicted unbound liver exposure-response effect model using measured in vitro IC50 values. In conclusion, this study demonstrates the important role of multiple drug transporters in the disposition of glyburide and its active metabolites, suggesting that variability in the function of these processes may lead to pharmacokinetic variability in the parent and the metabolites, potentially translating to pharmacodynamic variability.
Asunto(s)
Transporte Biológico/fisiología , Colestasis/metabolismo , Gliburida/metabolismo , Gliburida/farmacocinética , Transportadoras de Casetes de Unión a ATP/metabolismo , Teorema de Bayes , Línea Celular , Células HEK293 , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transportadores de Anión Orgánico/metabolismoRESUMEN
Conjugated hyperbilirubinemia accompanied by cholestasis is a frequent side effect during chronic treatment with the antimicrobial agent fusidic acid. Previous studies from our laboratory, addressing mechanisms of musculoskeletal toxicity arising from coadministration of fusidic acid with statins, demonstrated the ability of fusidic acid to potently inhibit human organic anion transporting polypeptides OATP1B1 (IC50 = 1.6 µM) and OATP1B3 (IC50 = 2.5 µM), which are responsible for the uptake-limited clearance of statins as well as bilirubin glucuronide conjugates. In the present work, inhibitory effects of fusidic acid were characterized against additional human hepatobiliary transporters [Na+/taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BSEP), and multidrug resistance-associated proteins MRP2 and MRP3] as well as uridine glucuronosyl transferase (UGT1A1), which mediate the disposition of bile acids and bilirubin (and its conjugated metabolites). Fusidic acid demonstrated concentration-dependent inhibition of human NTCP- and BSEP-mediated taurocholic acid transport with IC50 values of 44 and 3.8 µM, respectively. Inhibition of BSEP activity by fusidic acid was also consistent with the potent disruption of cellular biliary flux (AC50 = 11 µM) in the hepatocyte imaging assay technology assay, with minimal impact on other toxicity end points (e.g., cytotoxicity, mitochondrial membrane potential, reactive oxygen species generation, glutathione depletion, etc.). Fusidic acid also inhibited UGT1A1-catalyzed ß-estradiol glucuronidation activity in human liver microsomes with an IC50 value of 16 µM. Fusidic acid did not demonstrate any significant inhibition of ATP-dependent LTC4 transport (IC50's > 300 µM) in human MRP2 or MRP3 vesicles. R values, which reflect maximal in vivo inhibition, were estimated from a static mathematical model by taking into consideration the IC50 values generated in the various in vitro assays and clinically efficacious unbound fusidic acid concentrations. The magnitudes of in vivo interaction (R values) resulting from the inhibition of OATP1B1, UGT1A1, NTCP, and BSEP transport were â¼1.9-2.6, 1.1-1.2, 1.0-1.1, and 1.4-1.7, respectively, which are indicative of some degree of inherent toxicity risk, particularly via inhibition of OATP and BSEP. Collectively, these observations indicate that inhibition of human BSEP by fusidic acid could affect bile acid homeostasis, resulting in cholestatic hepatotoxicity in the clinic. Lack of direct inhibitory effects on MRP2 transport by fusidic acid suggests that conjugated hyperbilirubinemia does not arise via interference in MRP2-mediated biliary disposition of bilirubin glucuronides. Instead, it is possible that elevation in the level of bilirubin conjugates in blood is mediated through inhibition of hepatic OATPs, which are responsible for their reuptake and/or downregulation of MRP2 transporter as a consequence of cholestatic injury.
RESUMEN
Many highly proliferative cells generate almost all ATP via glycolysis despite abundant O(2) and a normal complement of fully functional mitochondria, a circumstance known as the Crabtree effect. Such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, such as the inhibitors rotenone, antimycin, oligomycin, and compounds like carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that uncouple the respiratory electron transfer system from phosphorylation. These cells are also resistant to the toxicity of many drugs whose deleterious side effect profiles are either caused, or exacerbated, by impairment of mitochondrial function. Drug-induced mitochondrial toxicity is shown by members of important drug classes, including the thiazolidinediones, statins, fibrates, antivirals, antibiotics, and anticancer agents. To increase detection of drug-induced mitochondrial effects in a preclinical cell-based assay, HepG2 cells were forced to rely on mitochondrial oxidative phosphorylation rather than glycolysis by substituting galactose for glucose in the growth media. Oxygen consumption doubles in galactose-grown HepG2 cells and their susceptibility to canonical mitochondrial toxicants correspondingly increases. Similarly, toxicity of several drugs with known mitochondrial liabilities is more readily apparent in aerobically poised HepG2 cells compared to glucose-grown cells. Some drugs were equally toxic to both glucose- and galactose-grown cells, suggesting that mitochondrial impairment is likely secondary to other cytotoxic mechanisms.
Asunto(s)
Medios de Cultivo/química , Galactosa/metabolismo , Galactosa/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Glucólisis/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Pruebas de Toxicidad/métodos , Adenosina Trifosfato/metabolismo , Recuento de Células , Línea Celular , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/toxicidad , Humanos , Consumo de Oxígeno/efectos de los fármacos , Desacopladores/toxicidadRESUMEN
The bile salt export pump (BSEP, ABCB11) belongs to the ATP-binding-cassette superfamily of transporters and is predominately found in the liver. BSEP is an efflux transporter that plays a critical role in the secretion of bile salts into the bile. Inhibition of BSEP function by drugs can result in the buildup of bile salts in the liver and eventually leads to cholestasis and drug-induced liver injury (DILI). DILI is a major cause of withdrawal of drugs from the pharmaceutical market and accounts for >50% of acute liver failures. Therefore, early detection of BSEP inhibition by drugs can help to mitigate the possibility of BSEP-associated liver injury. This unit describes two assays that investigate the relationship between drug interference with BSEP function and liver injury using membrane vesicles prepared from Hi5 insect cells transfected with human BSEP. Comprehensive protocols for assessing BSEP inhibition in a 384-well format using radiolabeled and liquid chromatography/mass spectrometry (LC/MS)-based detection methods are described. © 2017 by John Wiley & Sons, Inc.
Asunto(s)
Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/antagonistas & inhibidores , Cromatografía Líquida de Alta Presión/métodos , Radiometría/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas , Humanos , Concentración 50 Inhibidora , Insectos , Límite de Detección , Pruebas de ToxicidadRESUMEN
Mitochondrial dysfunction is a common mechanism of drug-induced toxicity. Early identification of new chemical entities (NCEs) that perturb mitochondrial function is of significant importance to avoid attrition in later stages of drug development. One of the most informative ways of assessing mitochondrial dysfunction is by measuring mitochondrial oxygen consumption. However, the conventional polarographic method of measuring oxygen consumption is not amenable to high sample throughput or automation. We present an alternative, low-bulk, high-throughput approach to the analysis of isolated-mitochondrial oxygen consumption using luminescent oxygen-sensitive probes. These probes are dispensable and are analyzed in standard microtitre plates on a fluorescence plate reader. Respiratory substrate and adenosine diphosphate (ADP) dependencies of mitochondrial oxygen consumption were assessed using the fluorescence-based method, and results compared favourably to conventional polarographic analysis. To assess assay performance, the method was then applied to the analysis of a panel of classical modulators of oxidative phosphorylation. The effect of uncoupler concentration was analyzed in detail to identify factors which would be important in applying this method to large scale NCE screening and mechanistic investigations. Results demonstrate that the 96-well format can accommodate up to approximately 200 compounds/day at a single concentration or alternatively IC(50) values can be generated for approximately 25 compounds. Throughput may be increased by moving to a 384-well plate format.
Asunto(s)
Mitocondrias Hepáticas/efectos de los fármacos , Sondas Moleculares , Oxígeno/química , Pruebas de Toxicidad , Animales , Fluorescencia , Masculino , Consumo de Oxígeno , Polarografía , Ratas , Ratas Sprague-DawleyRESUMEN
The Bcr-abl tyrosine kinase inhibitor imatinib mesylate is the frontline therapy for chronic myeloid leukemia. Imatinib has been reported to cause congestive heart failure and left ventricular contractile dysfunction in patients and cardiomyopathy in rodents, findings proposed to be associated with its pharmacological activity. To investigate the specific role of Abelson oncogene 1 (c-Abl) in imatinib-induced cardiac toxicity, we performed targeted gene inhibition of c-Abl by RNA interference in neonatal cardiomyocytes (NCMs). Suppression of c-Abl did not lead to cytotoxicity or induction of endoplasmic reticulum (ER) stress. To further dis associate c-Abl from imatinib-induced cardiac toxicity, we designed imatinib structural analogs that do not have appreciable c-Abl inhibition in NCMs. The c-Abl inactive analogs induced cytotoxicity and ER stress, at similar or greater potencies and magnitudes as imatinib. Furthermore, combining c-Abl gene silencing with imatinib and analogs treatment did not significantly shift the cytotoxicity dose response curves. Imatinib and analogs were shown to accumulate in lysosomes, likely due to their physicochemical properties, and disrupt autophagy. The toxicity induced by imatinib and analogs can be rescued by bafilomycin A pretreatment, demonstrating the involvement of lysosomal accumulation in cardiac toxicity. The results from our studies strongly suggest that imatinib induces cardiomyocyte dysfunction through disruption of autophagy and induction of ER stress, independent of c-Abl inhibition.
Asunto(s)
Antineoplásicos/toxicidad , Corazón/efectos de los fármacos , Piperazinas/toxicidad , Proteínas Quinasas/metabolismo , Pirimidinas/toxicidad , Animales , Secuencia de Bases , Benzamidas , Cartilla de ADN , Mesilato de Imatinib , Interferencia de ARN , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Clinical trials of selective RAF inhibitors in patients with melanoma tumors harboring activated BRAFV600E have produced very promising results, and a RAF inhibitor has been approved for treatment of advanced melanoma. However, about a third of patients developed resectable skin tumors during the course of trials. This is likely related to observations that RAF inhibitors activate extracellular signal-regulated kinase (ERK) signaling, stimulate proliferation, and induce epithelial hyperplasia in preclinical models. Because these findings raise safety concerns about RAF inhibitor development, we further investigated the underlying mechanisms. We showed that the RAF inhibitor PF-04880594 induces ERK phosphorylation and RAF dimerization in those epithelial tissues that undergo hyperplasia. Hyperplasia and ERK hyperphosphorylation are prevented by treatment with the mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD-0325901 at exposures that extrapolate to clinically well-tolerated doses. To facilitate mechanistic and toxicologic studies, we developed a three-dimensional cell culture model of epithelial layering that recapitulated the RAF inhibitor-induced hyperplasia and reversal by MEK inhibitor in vitro. We also showed that PF-04880594 stimulates production of the inflammatory cytokine interleukin 8 in HL-60 cells, suggesting a possible mechanism for the skin flushing observed in dogs. The complete inhibition of hyperplasia by MEK inhibitor in epithelial tissues does not seem to reduce RAF inhibitor efficacy and, in fact, allows doubling of the PF-04880594 dose without toxicity usually associated with such doses. These findings indicated that combination treatment with MEK inhibitors might greatly increase the safety and therapeutic index of RAF inhibitors for the treatment of melanoma and other cancers.
Asunto(s)
Benzamidas/administración & dosificación , Benzamidas/farmacología , Difenilamina/análogos & derivados , Epitelio/patología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Benzamidas/química , Difenilamina/administración & dosificación , Difenilamina/química , Difenilamina/farmacología , Perros , Relación Dosis-Respuesta a Droga , Epidermis/efectos de los fármacos , Epidermis/patología , Epitelio/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HL-60 , Humanos , Hiperplasia , Interleucina-8/metabolismo , Ratones , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Biológicos , Fosforilación , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-raf/metabolismo , Pirazoles/administración & dosificación , Pirazoles/química , Pirimidinas/administración & dosificación , Pirimidinas/químicaRESUMEN
Mitochondrial toxicity is increasingly implicated in a host of drug-induced organ toxicities, including hepatotoxicity. Nefazodone was withdrawn from the U.S. market in 2004 due to hepatotoxicity. Accordingly, we evaluated nefazodone, another triazolopyridine trazodone, plus the azaspirodecanedione buspirone, for cytotoxicity and effects on mitochondrial function. In accord with its clinical disposition, nefazodone was the most toxic compound of the three, trazodone had relatively modest effects, whereas buspirone showed the least toxicity. Nefazodone profoundly inhibited mitochondrial respiration in isolated rat liver mitochondria and in intact HepG2 cells where this was accompanied by simultaneous acceleration of glycolysis. Using immunocaptured oxidative phosphorylation (OXPHOS) complexes, we identified Complex 1, and to a lesser amount Complex IV, as the targets of nefazodone toxicity. No inhibition was found for trazodone, and buspirone showed 3.4-fold less inhibition of OXPHOS Complex 1 than nefazodone. In human hepatocytes that express cytochrome P450, isoform 3A4, after 24 h exposure, nefazodone and trazodone collapsed mitochondrial membrane potential, and imposed oxidative stress, as detected via glutathione depletion, leading to cell death. Our results suggest that the mitochondrial impairment imposed by nefazodone is profound and likely contributes to its hepatotoxicity, especially in patients cotreated with other drugs with mitochondrial liabilities.
Asunto(s)
Ansiolíticos/toxicidad , Antidepresivos de Segunda Generación/toxicidad , Buspirona/toxicidad , Hepatocitos/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Trazodona/toxicidad , Triazoles/toxicidad , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Respiración de la Célula/efectos de los fármacos , Respiración de la Célula/fisiología , Supervivencia Celular/efectos de los fármacos , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , Hepatocitos/enzimología , Hepatocitos/patología , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Redes y Vías Metabólicas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Piperazinas , Ratas , Ratas Sprague-DawleyRESUMEN
Cardiovascular disease has recently been suggested to be a significant complication of cancer treatment with several kinase inhibitors. In some cases, the mechanisms leading to cardiotoxicity are postulated to include mitochondrial dysfunction, either as a primary or secondary effect. Detecting direct effects on mitochondrial function, such as uncoupling of oxidative phosphorylation or inhibition of electron transport chain components, as well as identifying targets within the mitochondrial electron transport chain, can be accomplished in vitro. Here, we examined the effects of the tyrosine kinase inhibitor drugs imatinib, dasatinib, sunitinib, and sorafenib on ATP content in H9c2 cells grown under conditions where cells are either glycolytically or aerobically poised. Furthermore, we measured respiratory capacity of isolated rat heart mitochondria in the presence of the four kinase inhibitors and examined their effect on each of the oxidative phosphorylation complexes. Of the four kinase inhibitors examined, only sorafenib directly impaired mitochondrial function at clinically relevant concentrations, potentially contributing to the cytotoxic effect of the drug. For the other three kinase inhibitors lacking direct mitochondrial effects, altered kinase and other signaling pathways, are a more reasonable explanation for potential toxicity.
Asunto(s)
Bencenosulfonatos/toxicidad , Indoles/toxicidad , Mitocondrias Cardíacas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Piperazinas/toxicidad , Inhibidores de Proteínas Quinasas/toxicidad , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridinas/toxicidad , Pirimidinas/toxicidad , Pirroles/toxicidad , Tiazoles/toxicidad , Adenosina Trifosfato/metabolismo , Animales , Benzamidas , Supervivencia Celular/efectos de los fármacos , Dasatinib , Relación Dosis-Respuesta a Droga , Transporte de Electrón/efectos de los fármacos , Galactosa/metabolismo , Glucosa/metabolismo , Mesilato de Imatinib , Masculino , Mitocondrias Cardíacas/enzimología , Proteínas de Transporte de Membrana Mitocondrial/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Miocitos Cardíacos/enzimología , Niacinamida/análogos & derivados , Fosforilación Oxidativa/efectos de los fármacos , Compuestos de Fenilurea , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Sorafenib , SunitinibRESUMEN
Mitochondrial dysfunction is increasingly implicated in the etiology of drug-induced toxicities and negative side-effect profiles. Early identification of mitochondrial liabilities for new chemical entities is therefore crucial for avoiding late-stage attrition during drug development. Limitations of traditional methods for assessing mitochondrial dysfunction have discouraged routine evaluation of mitochondrial liabilities. To circumvent this bottleneck, a high-throughput screen has been developed that measures oxygen consumption; one of the most informative parameters for the assessment of mitochondrial status. This technique has revealed that some, but not all, members of many major drug classes have mitochondrial liabilities. This dichotomy encourages optimism that efficacy can be disassociated from mitochondrial toxicity, resulting in safer drugs in the future.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Animales , Interacciones Farmacológicas , Humanos , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins that specifically target insects and nematodes and are used around the world to kill insect pests. To better understand how pore-forming toxins interact with their host, we have screened for Caenorhabditis elegans mutants that resist Cry protein intoxication. We find that Cry toxin resistance involves the loss of two glycosyltransferase genes, bre-2 and bre-4. These glycosyltransferases function in the intestine to confer susceptibility to toxin. Furthermore, they are required for the interaction of active toxin with intestinal cells, suggesting they make an oligosaccharide receptor for toxin. Similarly, the bre-3 resistance gene is also required for toxin interaction with intestinal cells. Cloning of the bre-3 gene indicates it is the C. elegans homologue of the Drosophila egghead (egh) gene. This identification is striking given that the previously identified bre-5 has homology to Drosophila brainiac (brn) and that egh-brn likely function as consecutive glycosyltransferases in Drosophila epithelial cells. We find that, like in Drosophila, bre-3 and bre-5 act in a single pathway in C. elegans. bre-2 and bre-4 are also part of this pathway, thereby extending it. Consistent with its homology to brn, we demonstrate that C. elegans bre-5 rescues the Drosophila brn mutant and that BRE-5 encodes the dominant UDP-GlcNAc:Man GlcNAc transferase activity in C. elegans. Resistance to Cry toxins has uncovered a four component glycosylation pathway that is functionally conserved between nematodes and insects and that provides the basis of the dominant mechanism of resistance in C. elegans.