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1.
Toxicol Sci ; 163(2): 409-419, 2018 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-28329870

RESUMEN

A number of drugs can cause precipitates within renal tubules leading to crystal nephropathy. Crystal nephropathy is usually an exposure-related finding and is not uncommon in preclinical studies, where high doses are tested. An understanding of the nature of precipitates is important for human risk assessment and further development. Our aim was to investigate the ability of various imaging techniques to detect the presence of drugs or metabolites in renal crystals. We applied matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) imaging, Raman and infrared microspectroscopy, scanning electron microscopy coupled with energy dispersive X-ray (SEM/EDX) spectroscopy and standard histopathology to cases of drug-induced crystal nephropathy, induced in rodents and primates by 4 compounds. MALDI-FTICR MS imaging enabled the identification of the drug-related crystal content in all 4 cases of nephropathy, without reference material and with high accuracy. Crystals were composed of unchanged parent drug and/or metabolites. Similar results were obtained using Raman and infrared microspectroscopy for 2 compounds. In the absence of reference standards of metabolites, Raman and infrared microspectroscopy showed that the crystals consisted of components similar, but not identical, to the administered drug for the other compounds, a limitation for these techniques. SEM/EDX showed which counter ions were colocalized with the identified drug-related material, complementing the MALDI-FTICR MS findings. Therefore, we recommend MALDI-FTICR MS as a first-line methodology to characterize crystal nephropathies. Raman and infrared microspectroscopy may be useful when MALDI-FTICR MS imaging cannot be applied. SEM/EDX could be considered as a complementary technology.


Asunto(s)
Lesión Renal Aguda/diagnóstico por imagen , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico por imagen , Riñón/efectos de los fármacos , Preparaciones Farmacéuticas/química , Animales , Cristalización , Evaluación Preclínica de Medicamentos , Riñón/diagnóstico por imagen , Macaca fascicularis , Ratones , Estructura Molecular , Preparaciones Farmacéuticas/análisis , Ratas , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrofotometría Infrarroja , Espectrometría Raman
2.
Toxicol Pathol ; 45(5): 676-686, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28830332

RESUMEN

Obinutuzumab (GA101, Gazyva™, Gazyvaro®, F. Hoffmann-La Roche AG, Basel, Switzerland) is a humanized, glycoengineered type II antibody targeted against CD20. The preclinical safety evaluation required to support clinical development and marketing authorization of obinutuzumab included repeat-dose toxicity studies in cynomolgus monkeys for up to 6-month dosing with a 9-month recovery period. Results from those studies showed decreases in circulating B cells and corresponding B-cell depletion in lymphoid tissues, consistent with the desired pharmacology of obinutuzumab. Hypersensitivity reactions were noted at all doses in the 6-month study and were attributed to the foreign recognition of the drug construct in cynomolgus monkeys. Findings in monkeys were classified as acute hypersensitivity reactions that were evident immediately after dosing, such as excessive salivation, erythema, pruritus, irregular respiration, or ataxia, or chronic hypersensitivity reactions characterized by glomerulonephritis, arteritis/periarteritis, and inflammation in several tissues including serosal/adventitial inflammation. Immune complex deposits were demonstrated in tissues by immunohistochemistry, immunofluorescence, and electron microscopy. Some of, but not all, the animals that developed these reactions had detectable antidrug antibodies or circulating immune complexes accompanied by loss of drug exposure and pharmacodynamic effect. On the basis of clinical evidence to date, hypersensitivity reactions following obinutuzumab are rare, further supporting the general view that incidence and manifestation of immunogenicity in nonclinical species are generally not predictive for humans.


Asunto(s)
Anticuerpos Monoclonales Humanizados , Hipersensibilidad a las Drogas , Macaca fascicularis , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/toxicidad , Antígenos CD20/análisis , Antígenos CD20/metabolismo , Linfocitos B/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Epidídimo/efectos de los fármacos , Femenino , Glomerulonefritis/inducido químicamente , Glomerulonefritis/patología , Humanos , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/patología , Masculino , Músculos/efectos de los fármacos , Pruebas de Toxicidad Crónica
3.
Cell Chem Biol ; 24(5): 624-634.e3, 2017 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-28434878

RESUMEN

Today, novel therapeutics are identified in an environment which is intrinsically different from the clinical context in which they are ultimately evaluated. Using molecular phenotyping and an in vitro model of diabetic cardiomyopathy, we show that by quantifying pathway reporter gene expression, molecular phenotyping can cluster compounds based on pathway profiles and dissect associations between pathway activities and disease phenotypes simultaneously. Molecular phenotyping was applicable to compounds with a range of binding specificities and triaged false positives derived from high-content screening assays. The technique identified a class of calcium-signaling modulators that can reverse disease-regulated pathways and phenotypes, which was validated by structurally distinct compounds of relevant classes. Our results advocate for application of molecular phenotyping in early drug discovery, promoting biological relevance as a key selection criterion early in the drug development cascade.


Asunto(s)
Biología Computacional/métodos , Descubrimiento de Drogas/métodos , Fenotipo , Minería de Datos , Evaluación Preclínica de Medicamentos , Humanos
4.
J Immunother ; 39(7): 279-89, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27404941

RESUMEN

CEA TCB is a novel T-cell-bispecific (TCB) antibody targeting the carcinoembryonic antigen (CEA) expressed on tumor cells and the CD3 epsilon chain (CD3e) present on T cells, which is currently in Phase 1 clinical trials (NCT02324257) for the treatment of CEA-positive solid tumors. Because the human CEA (hCEA) binder of CEA TCB does not cross-react with cynomolgus monkey and CEA is absent in rodents, alternative nonclinical safety evaluation approaches were considered. These included the development of a cynomolgus monkey cross-reactive homologous (surrogate) antibody (cyCEA TCB) for its evaluation in cynomolgus monkey and the development of double-transgenic mice, expressing hCEA and human CD3e (hCEA/hCD3e Tg), as a potential alternative species for nonclinical safety studies. However, a battery of nonclinical in vitro/ex vivo experiments demonstrated that neither of the previous approaches provided a suitable and pharmacologically relevant model to assess the safety of CEA TCB. Therefore, an alternative approach, a minimum anticipated biological effect level (MABEL), based on an in vitro tumor lysis assay was used to determine the starting dose for the first-in-human study. Using the most conservative approach to the MABEL assessment, a dose of 52 µg was selected as a safe starting dose for clinical study.


Asunto(s)
Anticuerpos Biespecíficos/metabolismo , Complejo CD3/inmunología , Antígeno Carcinoembrionario/inmunología , Inmunoterapia/métodos , Neoplasias/terapia , Animales , Apoptosis , Células Cultivadas , Ensayos Clínicos Fase I como Asunto , Reacciones Cruzadas , Cálculo de Dosificación de Drogas , Evaluación Preclínica de Medicamentos , Humanos , Macaca fascicularis , Ratones , Ratones Transgénicos , Neoplasias/inmunología , Ratas , Homología Estructural de Proteína
5.
Toxicol Appl Pharmacol ; 279(3): 419-427, 2014 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-24997324

RESUMEN

The incidence of micronucleated-cells is considered to be a marker of a genotoxic event and can be caused by direct- or indirect-DNA reactive mechanisms. In particular, small increases in the incidence of micronuclei, which are not associated with toxicity in the target tissue or any structurally altering properties of the compound, trigger the suspicion that an indirect mechanism could be at play. In a bone marrow micronucleus test of a synthetic peptide (a dual agonist of the GLP-1 and GIP receptors) that had been integrated into a regulatory 13-week repeat-dose toxicity study in the rat, small increases in the incidence of micronuclei had been observed, together with pronounced reductions in food intake and body weight gain. Because it is well established that folate plays a crucial role in maintaining genomic integrity and pronounced reductions in food intake and body weight gain were observed, folate levels were determined from plasma samples initially collected for toxicokinetic analytics. A dose-dependent decrease in plasma folate levels was evident after 4 weeks of treatment at the mid and high dose levels, persisted until the end of the treatment duration of 13-weeks and returned to baseline levels during the recovery period of 4 weeks. Based on these properties, and the fact that the compound tested (peptide) per se is not expected to reach the nucleus and cause DNA damage, the rationale is supported that the elevated incidence of micronucleated polychromatic erythrocytes is directly linked to the exaggerated pharmacology of the compound resulting in a decreased folate level.


Asunto(s)
Deficiencia de Ácido Fólico/inducido químicamente , Pruebas de Mutagenicidad/métodos , Mutágenos , Péptidos/toxicidad , Animales , Temperatura Corporal/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/ultraestructura , Línea Celular Tumoral , Aberraciones Cromosómicas/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Deficiencia de Ácido Fólico/genética , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/ultraestructura , Desnutrición/inducido químicamente , Ratones , Ratones Noqueados , Pruebas de Micronúcleos , Ratas , Receptores de la Hormona Gastrointestinal/metabolismo , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética
6.
Drug Discov Today ; 18(23-24): 1138-43, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23942260

RESUMEN

Nonclinical safety testing of new biotherapeutic entities represents its own challenges and opportunities in drug development. Hot topics in this field have been discussed recently at the 2nd Annual BioSafe European General Membership Meeting. In this feature article, discussions on the challenges surrounding the use of PEGylated therapeutic proteins, selection of cynomolgus monkey as preclinical species, unexpected pharmacokinetics of biologics and the safety implications thereof are summarized. In addition, new developments in immunosafety testing of biologics, the use of transgenic mouse models and PK and safety implications of multispecific targeting approaches are discussed. Overall, the increasing complexity of new biologic modalities and formats warrants tailor-made nonclinical development strategies and experimental testing.


Asunto(s)
Productos Biológicos/toxicidad , Terapia Biológica/efectos adversos , Diseño de Fármacos , Animales , Productos Biológicos/administración & dosificación , Productos Biológicos/farmacocinética , Terapia Biológica/métodos , Humanos , Macaca fascicularis , Ratones , Ratones Transgénicos , Modelos Animales , Polietilenglicoles/química
7.
Toxicol Appl Pharmacol ; 268(1): 1-16, 2013 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-23352505

RESUMEN

Drug-induced liver injury (DILI) is the major cause for liver failure and post-marketing drug withdrawals. Due to species-specific differences in hepatocellular function, animal experiments to assess potential liabilities of drug candidates can predict hepatotoxicity in humans only to a certain extent. In addition to animal experimentation, primary hepatocytes from rat or human are widely used for pre-clinical safety assessment. However, as many toxic responses in vivo are mediated by a complex interplay among different cell types and often require chronic drug exposures, the predictive performance of hepatocytes is very limited. Here, we established and characterized human and rat in vitro three-dimensional (3D) liver co-culture systems containing primary parenchymal and non-parenchymal hepatic cells. Our data demonstrate that cells cultured on a 3D scaffold have a preserved composition of hepatocytes, stellate, Kupffer and endothelial cells and maintain liver function for up to 3months, as measured by the production of albumin, fibrinogen, transferrin and urea. Additionally, 3D liver co-cultures maintain cytochrome P450 inducibility, form bile canaliculi-like structures and respond to inflammatory stimuli. Upon incubation with selected hepatotoxicants including drugs which have been shown to induce idiosyncratic toxicity, we demonstrated that this model better detected in vivo drug-induced toxicity, including species-specific drug effects, when compared to monolayer hepatocyte cultures. In conclusion, our results underline the importance of more complex and long lasting in vitro cell culture models that contain all liver cell types and allow repeated drug-treatments for detection of in vivo-relevant adverse drug effects.


Asunto(s)
Técnicas de Cocultivo/métodos , Evaluación Preclínica de Medicamentos/métodos , Células Endoteliales/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hígado/citología , Hígado/efectos de los fármacos , Adulto , Albúminas/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Células Endoteliales/citología , Inhibidores Enzimáticos/farmacología , Fibrinógeno/metabolismo , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Ratas , Ratas Wistar , Transferrina/metabolismo , Urea/metabolismo
8.
J Clin Pharmacol ; 52(8): 1222-39, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21659628

RESUMEN

The authors have previously demonstrated rate-independent QT variability in the dog and cynomolgus monkey, where the QT associated with any RR was a normally distributed value that was accurately evaluated as the distribution mean. The present study investigated the rate-independent characteristics of the human QT. Digital electrocardiographs (1000 Hz) were collected for 24 hours in 51 patients (thorough QT study) and analyzed by computer. Distribution-based analysis was applied to the placebo and moxifloxacin (400 mg) arms to characterize the nature of the QT interval and to assess the efficacy of distribution-based analysis for QTc determination. Novel statistics using continuous means and bootstrapped 95% confidence intervals were developed to facilitate QT analysis. Machine-read QT values were compared with core laboratory semiautomated values for verification. RR intervals demonstrated repetitive protocol-dependent variations (50-250 milliseconds); QT intervals were normally distributed, spanning 60 to 100 milliseconds for each RR interval. Distribution-based analysis detected a moxifloxacin response identical to semiautomated analysis, but with reduced variability and improved statistical power, where n = 12 satisfied the ICH E14 criteria for a positive control. Distribution-based analysis has the potential to provide a universal method for clinical QT heart rate correction, enabling accurate detection of QT changes when limited numbers of volunteers are exposed to drug.


Asunto(s)
Compuestos Aza/uso terapéutico , Electrocardiografía/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Síndrome de QT Prolongado/tratamiento farmacológico , Síndrome de QT Prolongado/fisiopatología , Quinolinas/uso terapéutico , Adulto , Femenino , Fluoroquinolonas , Frecuencia Cardíaca/fisiología , Humanos , Masculino , Persona de Mediana Edad , Moxifloxacino , Adulto Joven
9.
Genome Res ; 21(10): 1746-56, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21862625

RESUMEN

The long-tailed macaque, also referred to as cynomolgus monkey (Macaca fascicularis), is one of the most important nonhuman primate animal models in basic and applied biomedical research. To improve the predictive power of primate experiments for humans, we determined the genome sequence of a Macaca fascicularis female of Mauritian origin using a whole-genome shotgun sequencing approach. We applied a template switch strategy that uses either the rhesus or the human genome to assemble sequence reads. The sixfold sequence coverage of the draft genome sequence enabled discovery of about 2.1 million potential single-nucleotide polymorphisms based on occurrence of a dimorphic nucleotide at a given position in the genome sequence. Homology-based annotation allowed us to identify 17,387 orthologs of human protein-coding genes in the M. fascicularis draft genome, and the predicted transcripts enabled the design of a M. fascicularis-specific gene expression microarray. Using liver samples from 36 individuals of different geographic origin we identified 718 genes with highly variable expression in liver, whereas the majority of the transcriptome shows relatively stable and comparable expression. Knowledge of the M. fascicularis draft genome is an important contribution to both the use of this animal in disease models and the safety assessment of drugs and their metabolites. In particular, this information allows high-resolution genotyping and microarray-based gene-expression profiling for animal stratification, thereby allowing the use of well-characterized animals for safety testing. Finally, the genome sequence presented here is a significant contribution to the global "3R" animal welfare initiative, which has the goal to reduce, refine, and replace animal experiments.


Asunto(s)
Evaluación Preclínica de Medicamentos , Macaca fascicularis/genética , Modelos Animales , Animales , Sistema Enzimático del Citocromo P-450/genética , Citocinas/genética , ADN/genética , ADN/aislamiento & purificación , Femenino , Perfilación de la Expresión Génica/métodos , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hígado/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Transportadores de Anión Orgánico/genética , Filogenia , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Transcripción Genética
10.
Nat Rev Drug Discov ; 9(4): 325-38, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20305665

RESUMEN

Monoclonal antibodies (mAbs) are now established as targeted therapies for malignancies, transplant rejection, autoimmune and infectious diseases, as well as a range of new indications. However, administration of mAbs carries the risk of immune reactions such as acute anaphylaxis, serum sickness and the generation of antibodies. In addition, there are numerous adverse effects of mAbs that are related to their specific targets, including infections and cancer, autoimmune disease, and organ-specific adverse events such as cardiotoxicity. In March 2006, a life-threatening cytokine release syndrome occurred during a first-in-human study with TGN1412 (a CD28-specific superagonist mAb), resulting in a range of recommendations to improve the safety of initial human clinical studies with mAbs. Here, we review some of the adverse effects encountered with mAb therapies, and discuss advances in preclinical testing and antibody technology aimed at minimizing the risk of these events.


Asunto(s)
Anticuerpos Monoclonales/efectos adversos , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Ensayos Clínicos como Asunto , Citocinas/inmunología , Evaluación Preclínica de Medicamentos , Humanos
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