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1.
Drug Metab Rev ; 44(1): 127-40, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22242931

RESUMEN

Drug-induced liver injury (DILI) is a major cause of attrition during both the early and later stages of the drug development and marketing process. Reducing or eliminating drug-induced severe liver injury, especially those that lead to liver transplants or death, would be tremendously beneficial for patients. Therefore, developing new pharmaceuticals that have the highest margins and attributes of hepatic safety would be a great accomplishment. Given the current low productivity of pharmaceutical companies and the high costs of bringing new medicines to market, any early screening assay(s) to identify and eliminate pharmaceuticals with the potential to cause severe liver injury in humans would be of economic value as well. The present review discusses the background, proof-of-concept, and validation studies associated with high-content screening (HCS) by two major pharmaceutical companies (Pfizer Inc and Jansen Pharmaceutical Companies of Johnson & Johnson) for detecting compounds with the potential to cause human DILI. These HCS assays use fluorescent-based markers of cell injury in either human hepatocytes or HepG2 cells. In collaboration with Evotec, an independent contract lab, these two companies also independently evaluated larval zebrafish as an early-stage in vivo screen for hepatotoxicity in independently conducted, blinded assessments. Details about this model species, the need for bioanalysis, and, specifically, the outcome of the phenotypic-based zebrafish screens are presented. Comparing outcomes in zebrafish against both HCS assays suggests an enhanced detection for hepatotoxicants of most DILI concern when used in combination with each other, based on the U.S. Food and Drug Administration DILI classification list.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Hígado/efectos de los fármacos , Modelos Animales , Pez Cebra , Animales , Diseño de Fármacos , Humanos , Hígado/patología , Reproducibilidad de los Resultados , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo
2.
Crit Rev Toxicol ; 42(5): 443-64, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22512667

RESUMEN

Testing for embryotoxicity in vitro is an attractive alternative to animal experimentation. The embryonic stem cell test (EST) is such a method, and it has been formally validated by the European Centre for the Validation of Alternative Methods. A number of recent studies have underscored the potential of this method. However, the EST performed well below the 78% accuracy expected from the validation study using a new set of chemicals and pharmaceutical compounds, and also of toxicity criteria, tested to enlarge the database of the validated EST as part of the Work Package III of the ReProTect Project funded within the 6th Framework Programme of the European Union. To assess the performance and applicability domain of the EST we present a detailed review of the substances and their effects in the EST being nitrofen, ochratoxin A, D-penicillamine, methylazoxymethanol, lovastatin, papaverine, warfarin, ß-aminopropionitrile, dinoseb, furosemide, doxylamine, pravastatin, and metoclopramide. By delineation of the molecular mechanisms of the substances we identify six categories of reasons for misclassifications. Some of these limitations might also affect other in vitro methods assessing embryotoxicity. Substances that fall into these categories need to be included in future validation sets and in validation guidelines for embryotoxicity testing. Most importantly, we suggest conceivable improvements and additions to the EST which will resolve most of the limitations.


Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Embrión de Mamíferos/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Teratógenos/toxicidad , Xenobióticos/toxicidad , Animales , Europa (Continente) , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Teratógenos/clasificación , Xenobióticos/clasificación
3.
J Proteome Res ; 9(11): 5727-38, 2010 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-20919680

RESUMEN

There are new challenges for hazard and risk assessment in the chemical industry with regard to REACH legislation in Europe and related activities in the U.S. and Japan, which require the development of novel in vitro models for the molecular characterization of drug- or chemical-related effects replacing conventional animal testing. In the frame of a European FP6 project on reproductive toxicology ( www.reprotect.eu ), we prepared protein samples from mouse embryonic stem cells differentiated into contracting cardiomyocytes according to the validated embryonic stem cell test (EST) protocol, which had been exposed to toxic substances selected by an expert committee from different in vivo categories of embryotoxicity. Lysates were used to carry out the following investigations: (i) identify optimal dose range conditions in the EST that are suitable for (ii) performing a differential quantitative proteomic study of underlying molecular pathways, (iii) define classes of substances with similar proteomic response patterns, (iv) relate these classes to the traditional in vivo categories of embryotoxicity with (v) the final goal to identify novel surrogate protein biomarker candidates for embryo toxicity. We found two distinct classes of toxic substances (Dinoseb, Ochratoxin-A, and Nitrofen vs ß-aminoproprionitril, Metoclopramide, Doxylamine succinate, and d-penicillamine) with clear pathway-related differences in their proteomic patterns. Most notably, different responses to cluster 1 and cluster 2 substances were observed for Heat shock protein ß-1, Ras-GTPase-activating protein SH3-domain binding protein, Ran binding protein 5, and Calreticulin, Dihydropyrimidinase-like 2 (Ulip2 protein). On the other hand, Heat shock protein 8 and Fscn1 protein were down-regulated by all compounds from both clusters.


Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Células Madre Embrionarias/efectos de los fármacos , Proteínas/efectos de los fármacos , Pruebas de Toxicidad , Animales , Biomarcadores , Diferenciación Celular , Células Cultivadas , Ratones , Miocitos Cardíacos
4.
Sci Rep ; 10(1): 13262, 2020 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-32764586

RESUMEN

Phenomic profiles are high-dimensional sets of readouts that can comprehensively capture the biological impact of chemical and genetic perturbations in cellular assay systems. Phenomic profiling of compound libraries can be used for compound target identification or mechanism of action (MoA) prediction and other applications in drug discovery. To devise an economical set of phenomic profiling assays, we assembled a library of 1,008 approved drugs and well-characterized tool compounds manually annotated to 218 unique MoAs, and we profiled each compound at four concentrations in live-cell, high-content imaging screens against a panel of 15 reporter cell lines, which expressed a diverse set of fluorescent organelle and pathway markers in three distinct cell lineages. For 41 of 83 testable MoAs, phenomic profiles accurately ranked the reference compounds (AUC-ROC ≥ 0.9). MoAs could be better resolved by screening compounds at multiple concentrations than by including replicates at a single concentration. Screening additional cell lineages and fluorescent markers increased the number of distinguishable MoAs but this effect quickly plateaued. There remains a substantial number of MoAs that were hard to distinguish from others under the current study's conditions. We discuss ways to close this gap, which will inform the design of future phenomic profiling efforts.


Asunto(s)
Productos Biológicos/farmacología , Proteínas Luminiscentes/genética , Fenómica/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Células A549 , Línea Celular , Descubrimiento de Drogas , Regulación de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Proteínas Luminiscentes/metabolismo
5.
Toxicol In Vitro ; 22(7): 1789-96, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18672049

RESUMEN

In order to support drug research in the selection process for non-embryotoxic pharmaceutical compounds, a screening method for embryotoxicity is needed. The murine embryonic stem cell test (EST) is a validated in vitro test based on two permanent mouse cell lines and delivering results in 10-days. Implementation of this test within our laboratory, revealed variability in the differentiation potential of the embryonic stem cells and, as a consequence, a lot of assays needed to be rejected due the fact the acceptance criteria were not reached. In order to gain a better yield of contracting myocardial cells, we used (1) a stringent control of the cell growth during subcultivation and a standardised hanging drop culture method and (2) a non-enzymatic cell harvest instead of a trypsin/EDTA cell harvest. Implementing of these cell culture modifications resulted in a decreased variability in the size of embryonic bodies, an increase of the number of acceptable tests and a significant increase of the differentiation potential of embryonic cells into strong beating myocardium, which made scoring less time consuming. Testing of 6 reference compounds in the optimized EST showed that the cell culture modifications did not changed the in vitro classification.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Pruebas de Toxicidad/métodos , Animales , Células 3T3 BALB , Ácido Edético/metabolismo , Células Madre Embrionarias/metabolismo , Ratones , Miocardio/citología , Tripsina/metabolismo
6.
Environ Mol Mutagen ; 46(1): 30-42, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15880423

RESUMEN

According to the current Organization of Economic Cooperation and Development (OECD) and International Committee on Harmonization (ICH) guidelines for the mammalian erythrocyte micronucleus (MN) test, analysis of peripheral blood reticulocytes (RETs) for the presence of micronuclei can be performed using flow cytometry. The MicroFlow PLUS method (Litron Laboratories, Rochester, NY) for MN analysis by flow cytometry is based on the binding of FITC-labeled antibodies to the CD71 transferrin receptor of immature RETs, on parallel RNA degradation, and on propidium iodide staining of DNA present as micronuclei. The objective of this study was to assess the sensitivity of this flow cytometry method to detect time- and dose-dependent induction of micronuclei in mouse peripheral blood RETs after treatment with nine chemical agents. Five known clastogens, two known aneugens, and two compounds previously reported to be inactive in the mouse bone marrow MN test were evaluated at three dose levels. Multiple blood sampling of the same animal before and at two time points after treatment was conducted. All known mutagens produced a dose-dependent increase in micronucleated reticulocytes (MN-RETs); the compounds previously shown to be inactive in the in vivo MN test were also negative using the present methodology. The highest frequency of MN-RETs was observed at 48 hr after treatment, except for 5-fluorouracil, which had its peak response at 72 hr. The results indicate that micronuclei can be measured by multiple blood sampling of the same animal before and after treatment without altering the sensitivity of the assay. The results confirm that the flow cytometric assessment of MN-RETs in mouse peripheral blood using MicroFlow PLUS is a sensitive method with high analysis throughput, and robust quality control.


Asunto(s)
Recolección de Muestras de Sangre/métodos , Citometría de Flujo , Micronúcleos con Defecto Cromosómico/inducido químicamente , Mutágenos/toxicidad , Reticulocitos/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Pruebas de Micronúcleos/métodos , Reticulocitos/metabolismo , Factores de Tiempo
8.
Toxicol In Vitro ; 24(5): 1417-25, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20430096

RESUMEN

Drug-induced phospholipidosis is a side effect for which drug candidates can be screened in the drug discovery phase. The numerous in silico models that have been developed as a first line of screening are based on the characteristic physicochemical properties of phospholipidosis-inducing drugs, e.g. high logP and pK(b) values. However, applying these models on a predominantly high lipophilic, basic CNS chemistry results in a high false positive rate and consequently in a wrong classification of a large number of valuable drug candidates. Here, we tested 33 CNS-compounds (24 in vivo negative and 9 in vivo positive phospholipidosis-inducers) in our in house developed in vitro phospholipidosis screening assay (Mesens et al., 2009) and compared its predictivity with the outcome of three different, well established in silico prediction models. Our in vitro assay demonstrates an increased specificity of 79% over the in silico models (29%). Moreover, by considering the proposed plasma concentration at the efficacious dose we can show a clear correlation between the in vitro and in vivo occurrence of phospholipidosis, improving the specificity of prediction to 96%. Through its high predictive value, the in vitro low throughput assay is thus preferred above high throughput in silico assays, characterized by a high false positive rate.


Asunto(s)
Fármacos del Sistema Nervioso Central/toxicidad , Ensayos Analíticos de Alto Rendimiento , Lipidosis/inducido químicamente , Fosfolípidos/metabolismo , Línea Celular , Fármacos del Sistema Nervioso Central/administración & dosificación , Simulación por Computador , Evaluación Preclínica de Medicamentos/métodos , Humanos , Lipidosis/diagnóstico
9.
Reprod Toxicol ; 30(1): 121-30, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20493250

RESUMEN

Novel molecular content for fast in vitro strategies in the context of safety tests concerning developmental toxicity has a potential to substantially reduce animal experiments according to the "3R" concept (Reduce/Refine/Replace). Here we present and discuss data from a differential proteomic profiling of samples generated using embryonic stem cell derived in vitro models treated with a set of model substances. Among substance-dependent proteomic changes, potential surrogate markers were some isoforms of heat shock proteins and a component of the Ras pathway, present in several redundant isoforms due to posttranslational modifications. Both proteins are implicated in cell migration, cell survival, growth and embryonic development. Using the examples of warfarin and lovastatin, two substances with entirely different primary targets, the surrogate marker signature nevertheless indicates a common embryotoxic mode of action. We discuss these findings observed in in vitro toxicity tests, in a context of clinical validation and evidence-based toxicology.


Asunto(s)
Alternativas a las Pruebas en Animales , Células Madre Embrionarias/efectos de los fármacos , Lovastatina/toxicidad , Teratógenos/toxicidad , Pruebas de Toxicidad/métodos , Warfarina/toxicidad , Células 3T3 , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Determinación de Punto Final , Proteínas de Choque Térmico/biosíntesis , Concentración 50 Inhibidora , Ratones , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Pruebas de Toxicidad/normas , Proteínas ras/biosíntesis
10.
Toxicol Sci ; 105(2): 342-50, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18593728

RESUMEN

The ability of murine-derived embryonic stem cells (D3) to differentiate into cardiomyocytes is the basis of the embryonic stem cell test (EST). With the EST, chemicals and pharmaceuticals can be assessed for their embryotoxic potency early on in the development process. In order to come to a higher throughput EST, a 96-well based method was developed based on low attachment well plates that allow for the formation of embryonic bodies from which the stem cells can differentiate. Twelve test compounds were selected based on their reported in vitro and in vivo embryotoxic potency. In the 96-well based EST, reportedly strong embryotoxic compounds 5-fluorouracil, 6-aminonicotinamide (6AN), methylmercury chloride, and hydroxyurea were correctly ranked with corresponding Relative Embryotoxic Potency values (REP, based on the EC(50) (microM) value of 6AN) of 2.6 +/- 2.9, 1, 2.0 +/- 3.1, and 0.07 +/- 0.05, respectively. Moderately embryotoxic compounds valproic acid, boric acid, methoxyacetic acid, and lithium chloride resulted in a correct ranking with REP values of 0.01 +/- 0.003, 0.001 +/- 0.001, 0.0007 +/- 0.001, and 0.0006 +/- 0.0004, respectively. The included nonembryotoxic compounds Penicillin G, acrylamide, and saccharin did not result in an inhibition of D3 cells to differentiate into cardiomyocytes, other than related to cytotoxicity (REP value of 0.00001). However, diphenhydramine resulted in an inhibitory effect similarly to the strong embryotoxic compound hydroxyurea, with a REP value of 0.40 +/- 0.36. However, further evaluation suggested this was due to direct inhibition of the contractile capacity of the D3 cardiomyocytes, rather than an embryotoxic mechanism. The 96-well based EST is a promising addition to the screening process of newly developed chemicals and pharmaceuticals.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Teratógenos/toxicidad , Pruebas de Toxicidad , Alternativas a las Pruebas en Animales , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células Madre Embrionarias/patología , Ratones , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/patología , Reproducibilidad de los Resultados , Teratógenos/clasificación
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