RESUMEN
Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes in diabetes, oncology, and neurology. N-(3-(1H-1,2,4-triazol-1-yl)propyl)-5-(3-chloro-4-methoxyphenyl)oxazole-4-carboxamide (PF-04802367 or PF-367) has been identified as a highly potent inhibitor, which is among the most selective antagonists of GSK-3 to date. Its efficacy was demonstrated in modulation of tau phosphorylation inâ vitro and inâ vivo. Whereas the kinetics of PF-367 binding in brain tissues are too fast for an effective therapeutic agent, the pharmacokinetic profile of PF-367 is ideal for discovery of radiopharmaceuticals for GSK-3 in the central nervous system. A (11) C-isotopologue of PF-367 was synthesized and preliminary PET imaging studies in non-human primates confirmed that we have overcome the two major obstacles for imaging GSK-3, namely, reasonable brain permeability and displaceable binding.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/diagnóstico por imagen , Neuroimagen , Oxazoles/farmacología , Tomografía de Emisión de Positrones , Inhibidores de Proteínas Quinasas/farmacología , Triazoles/farmacología , Proteínas tau/antagonistas & inhibidores , Encéfalo/metabolismo , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Oxazoles/síntesis química , Oxazoles/química , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Triazoles/síntesis química , Triazoles/química , Proteínas tau/metabolismoRESUMEN
The toxicity of hydroxyurea, a treatment for specific neoplasms, sickle-cell disease, polycythemia, and thrombocytosis that kills cells in mitosis, was assessed in repeat-dose, oral gavage studies in rats and dogs and a cardiovascular study in telemetered dogs. Hydroxyurea produced hematopoietic, lymphoid, cardiovascular, and gastrointestinal toxicity with steep dose response curves. In rats dosed for 10 days, 50 mg/kg/day was tolerated; 500 mg/kg/day produced decreased body weight gain; decreased circulating leukocytes, erythrocytes, and platelets; decreased cellularity of thymus, lymph nodes, and bone marrow; and epithelial degeneration and/or dysplasia of the stomach and small intestine; 1,500 mg/kg/day resulted in deaths on day 5. In dogs, a single dose at ≥ 250 mg/kg caused prostration leading to unscheduled euthanasia. Dogs administered 50 mg/kg/day for 1 month had decreased circulating leukocytes, erythrocytes, and platelets; increased bone marrow cellularity with decreased maturing granulocytes; increased creatinine kinase activity; and increased iron pigment in bone marrow and hepatic sinusoidal cells. In telemetered dogs, doses ≥ 15 mg/kg decreased systolic blood pressure (BP); 50 mg/kg increased diastolic BP, heart rate, and change in blood pressure over time (+dP/dt), and decreased QT and PR intervals and maximum left ventricular systolic and end diastolic pressures with measures returning to control levels within 24 hr.
Asunto(s)
Hidroxiurea/toxicidad , Animales , Presión Sanguínea/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Perros , Femenino , Corazón/efectos de los fármacos , Hidroxiurea/administración & dosificación , Masculino , Miocardio/patología , Ratas , Ratas Sprague-Dawley , Pruebas de ToxicidadRESUMEN
Glucokinase activators (GKAs) are being developed for the treatment of type 2 diabetes. The toxicity of 4 GKAs (PF-04279405, PF-04651887, piragliatin, and PF-04937319) was assessed in mice, rats, dogs, and/or monkeys. GKAs were administered for 2 to 8 weeks. Standard endpoints, glucose, and insulin were assessed. All compounds produced varying degrees of hypoglycemia in all species. Brain neuronal necrosis and/or peripheral neuropathy were observed with most compounds. These findings are consistent with literature reports linking hypoglycemia with nervous system effects. Arteriopathy, mainly of cardiac vessels, was observed at a low frequency in monkey and/or dog. Arteriopathy occurred only at doses that produced severe and prolonged periods of repeated hypoglycemia. Since this lesion occurred in multiple studies with structurally distinct GKAs, these results suggested arteriopathy was related to GKA pharmacology. The morphological characteristics of the arteriopathy were consistent with that produced by experimental catecholamine administration. We hypothesize that the prolonged periods of hypoglycemia resulted in increased local and/or systemic concentrations of catecholamines via a counterregulatory and/or stress-related mechanism. Alternatively, prolonged hypoglycemia may have resulted in endothelial dysfunction leading to arteriopathy. This risk can be managed in human patients in clinical studies by careful glucose monitoring and intervention to avoid prolonged episodes of hypoglycemia.
Asunto(s)
Azetidinas/efectos adversos , Bencenoacetamidas/efectos adversos , Benzofuranos/efectos adversos , Hipoglucemia/patología , Necrosis/patología , Enfermedades del Sistema Nervioso Periférico/patología , Pirimidinas/efectos adversos , Animales , Azetidinas/sangre , Bencenoacetamidas/sangre , Benzofuranos/sangre , Cromatografía Líquida de Alta Presión , Perros , Evaluación Preclínica de Medicamentos , Femenino , Hipoglucemia/inducido químicamente , Hipoglucemiantes/efectos adversos , Insulina/sangre , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos ICR , Necrosis/inducido químicamente , Neuronas/efectos de los fármacos , Neuronas/patología , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Pirimidinas/sangre , Ratas , Ratas Sprague-DawleyRESUMEN
Several classes of drugs have been shown to cause drug-induced vascular injury (DIVI) in preclinical toxicity studies. Measurement of blood flow and vessel diameter in numerous vessels and across various tissues by ultrasound imaging has the potential to be a noninvasive translatable biomarker of DIVI. Our objective was to demonstrate the utility of high-frequency ultrasound imaging for measuring changes in vascular function by evaluating blood flow and vessel diameter in the superior mesenteric arteries (SMA) of rats treated with compounds that are known to cause DIVI and are known vasodilators in rat: fenoldopam, CI-1044, and SK&F 95654. Blood flow, vessel diameter, and other parameters were measured in the SMA at 4, 8, and 24 hr after dosing. Mild to moderate perivascular accumulations of mononuclear cells, neutrophils in tunica adventitia, and superficial tunica media as well as multifocal hemorrhage and necrosis in the tunica media were found in animals 24 hr after treatment with fenoldopam and SK&F 95654. Each compound caused marked increases in blood flow and shear stress as early as 4 hr after dosing. These results suggest that ultrasound imaging may constitute a functional correlate for the microscopic finding of DIVI in the rat.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Ultrasonografía/métodos , Lesiones del Sistema Vascular/inducido químicamente , Lesiones del Sistema Vascular/patología , Animales , Azepinas/efectos adversos , Fenoldopam/efectos adversos , Hemodinámica , Masculino , Arterias Mesentéricas/diagnóstico por imagen , Arterias Mesentéricas/efectos de los fármacos , Niacinamida/efectos adversos , Niacinamida/análogos & derivados , Piridazinas/efectos adversos , Piridinas/efectos adversos , Ratas , Ratas Sprague-Dawley , Lesiones del Sistema Vascular/diagnóstico por imagenRESUMEN
Previously we found that regulation of eNOS is an important part of the pathogenic process of Drug-induced vascular injury (DIVI) for PDE4i. The aims of the current study were to examine the phosphorylation of eNOS in mesentery versus aorta at known regulatory sites across DIVI-inducing drug classes and to compare changes across species. We found that phosphorylation at S615 in rats was elevated 35-fold 2 hr after the last dose of CI-1044 in mesentery versus 3-fold in aorta. Immunoprecipitation studies revealed that many of the upstream regulators of eNOS activation were associated with eNOS in 1 or more signalosome complexes. Next rats were treated with drugs from 4 other classes known to cause DIVI. Each drug was given alone and in combination with SIN-1 (NO donor) or L-NAME (eNOS inhibitor), and the level of eNOS phosphorylation in mesentery and aorta tissue was correlated with the extent of vascular injury and measured serum nitrite. Drugs or combinations produced altered serum nitrite levels as well as vascular injury score in the mesentery. The results suggested that phosphorylation of S615 may be associated with DIVI activity. Studies with the species-specific A2A adenosine agonist CI-947 in rats versus primates showed a similar pattern.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Lesiones del Sistema Vascular/inducido químicamente , Lesiones del Sistema Vascular/patología , Adenosina/administración & dosificación , Adenosina/efectos adversos , Adenosina/análogos & derivados , Animales , Aorta/metabolismo , Azepinas/administración & dosificación , Azepinas/efectos adversos , Relación Dosis-Respuesta a Droga , Masculino , Niacinamida/administración & dosificación , Niacinamida/efectos adversos , Niacinamida/análogos & derivados , Óxido Nítrico/sangre , Óxido Nítrico Sintasa de Tipo III/genética , Nitritos/sangre , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
Better biomarkers are needed to identify, characterize, and/or monitor drug-induced vascular injury (DIVI) in nonclinical species and patients. The Predictive Safety Testing Consortium (PSTC), a precompetitive collaboration of pharmaceutical companies and the U.S. Food and Drug Administration (FDA), formed the Vascular Injury Working Group (VIWG) to develop and qualify translatable biomarkers of DIVI. The VIWG focused its research on acute DIVI because early detection for clinical and nonclinical safety monitoring is desirable. The VIWG developed a strategy based on the premise that biomarkers of DIVI in rat would be translatable to humans due to the morphologic similarity of vascular injury between species regardless of mechanism. The histomorphologic lexicon for DIVI in rat defines degenerative and adaptive findings of the vascular endothelium and smooth muscles, and characterizes inflammatory components. We describe the mechanisms of these changes and their associations with candidate biomarkers for which advanced analytical method validation was completed. Further development is recommended for circulating microRNAs, endothelial microparticles, and imaging techniques. Recommendations for sample collection and processing, analytical methods, and confirmation of target localization using immunohistochemistry and in situ hybridization are described. The methods described are anticipated to aid in the identification and qualification of translational biomarkers for DIVI.
Asunto(s)
Biomarcadores/sangre , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Lesiones del Sistema Vascular/inducido químicamente , Lesiones del Sistema Vascular/patología , Animales , Evaluación Preclínica de Medicamentos/tendencias , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Humanos , Músculo Liso/efectos de los fármacos , Músculo Liso/patología , Estados Unidos , United States Food and Drug AdministrationRESUMEN
MOTIVATION: Although numerous methods have been developed to better capture biological information from microarray data, commonly used single gene-based methods neglect interactions among genes and leave room for other novel approaches. For example, most classification and regression methods for microarray data are based on the whole set of genes and have not made use of pathway information. Pathway-based analysis in microarray studies may lead to more informative and relevant knowledge for biological researchers. RESULTS: In this paper, we describe a pathway-based classification and regression method using Random Forests to analyze gene expression data. The proposed methods allow researchers to rank important pathways from externally available databases, discover important genes, find pathway-based outlying cases and make full use of a continuous outcome variable in the regression setting. We also compared Random Forests with other machine learning methods using several datasets and found that Random Forests classification error rates were either the lowest or the second-lowest. By combining pathway information and novel statistical methods, this procedure represents a promising computational strategy in dissecting pathways and can provide biological insight into the study of microarray data. AVAILABILITY: Source code written in R is available from http://bioinformatics.med.yale.edu/pathway-analysis/rf.htm.
Asunto(s)
Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reconocimiento de Normas Patrones Automatizadas , Algoritmos , Animales , Simulación por Computador , Interpretación Estadística de Datos , Humanos , Ratones , Modelos Biológicos , Modelos Estadísticos , Ratas , Análisis de Regresión , Programas InformáticosRESUMEN
Drug-induced vascular injury (DIVI) in preclinical studies can delay, if not terminate, a drug development program. Clinical detection of DIVI can be very difficult as there are no definitive biomarkers known to reliably detect this disorder in all instances. The preclinical identification of DIVI requires detailed microscopic examination of a wide range of tissues although one of the most commonly affected areas in rats is the mesenteric vasculature. The reason for this predisposition of mesenteric arteries in rats as well as the exact mechanism and cell types involved in the initial development of these lesions have not been fully elucidated. We hypothesized that by using a mixed culture of cells from rat mesenteric tissue, we would be able to identify an RNA expression signature that could predict the invivo development of DIVI. Five compounds designed to inhibit Phosphodiesterase 4 activity (PDE4i) were chosen as positive controls. PDE4i's are well known to induce DIVI in the mesenteric vasculature of rats and there is microscopic evidence that this is associated, at least in part, with a proinflammatory mechanism. We surveyed, by qRT-PCR, the expression of 96 genes known to be involved in inflammation and using a Random-Forest model, identified 12 genes predictive of invivo DIVI outcomes in rats. Using these genes, we were able to cross-validate the ability of the Random-Forest modeling to predict the concentration at which PDE4i caused DIVI invivo.
Asunto(s)
Arterias Mesentéricas/citología , Inhibidores de Fosfodiesterasa 4/toxicidad , Lesiones del Sistema Vascular/inducido químicamente , Animales , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The blood-brain barrier (BBB) is the cellular interface between the circulating blood and neural environment, and is created by apposed endothelial cells and their intercellular tight junctions. Many aspects of how the BBB functions at the molecular level remain unresolved; therefore, we report for the first time a comprehensive gene expression profile of rat brain microvessels using serial analysis of gene expression (SAGE). We assembled a full and quantitative SAGE catalog containing 101,364 tags, of which 33% of the tags matched known genes, 51% matched expressed sequence tags (ESTs) in the Unigene database, and 16% of the tags were unassigned. The transcriptome catalog contains many new and novel transcripts among known BBB genes. A large compliment of junctional proteins and an extensive assortment of facilitated carrier and ATP-dependent transporters are included. To identify microvessel-enriched transcripts, we compared the microvessel SAGE catalog to cortex and hippocampus SAGE catalogs. This resulted in identification of 864 genes, including several known for their abundant expression at the BBB, such as the transferrin receptor (TrnR). Sorting enriched genes based on function revealed groups that encode transporters (11%), receptors (5%), proteins involved in vesicle trafficking (4%), structural proteins (10%), and components of signal transduction pathways (17%). This genomic repertoire emphasizes the unique cellular phenotype existing within the brain and further implicates the BBB as a mediator between the brain and periphery. These results may provide a useful resource and reference point from which to determine the effects of different physiological, developmental, and disease processes on BBB gene expression.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/ultraestructura , Perfilación de la Expresión Génica , Transcripción Genética , Animales , Vasos Sanguíneos/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Interpretación Estadística de Datos , Biblioteca de Genes , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
The diffusion of monocarboxylates such as lactate and pyruvate across the plasma membrane of mammalian cells is facilitated by a family of integral membrane transport proteins, the monocarboxylate transporters (MCTs). Currently, at least eight unique members of the MCT family have been discovered and orthologs to each have been identified in a variety of species. Four MCTs (MCT1-MCT4) have been functionally characterized. Each isoform possesses unique biochemical properties such as kinetic constants and sensitivity to known MCT inhibitors. Several fold changes in the expression of MCTs may be evoked by altered physiological conditions, yet the molecular mechanisms underlying the regulation of MCTs are poorly understood. Post-translational regulation of MCT1 and MCT4 occurs, in part, by interaction with CD147, an accessory protein that is necessary for trafficking, localization, and functional expression of these transporters. Because of the physiological importance of monocarboxylates to the overall maintenance of metabolic homeostasis, the function of MCTs is significant to several pathologies that occur with disease, such as ischemic stroke and cancer. Finally, the expression of MCT1 in the epithelium of the small intestine and colon and in the blood-brain barrier may provide routes for the intestinal and blood to brain transfer of carboxylated pharmaceutical agents and other exogenous monocarboxylates.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Humanos , Enfermedades Metabólicas/tratamiento farmacológico , Enfermedades Metabólicas/metabolismo , Transportadores de Ácidos Monocarboxílicos/química , Transportadores de Ácidos Monocarboxílicos/genéticaRESUMEN
Drug-induced vascular injury (DIVI) is observed in rat mesenteric arterioles in response to treatment with phosphodiesterase-4 inhibitors (PDE4i). However, the mechanisms responsible for causing the characteristic vascular lesions are unclear. Nitrotyrosine (NT) adducts, markers of local nitric oxide (NO) production, have been observed in close proximity to the arterial lesions and in the inflammatory cells associated with DIVI. To determine if NO has a direct role in DIVI, rats were treated with the PDE4i CI-1044 at 10, 20, or 40 mg/kg alone or in combination with the nitric oxide synthase inhibitor L-NAME (60 mg/kg) or the nitric oxide donor SIN-1 (30 mg/kg). Mesenteries were collected and processed for microscopic evaluation. NT formation was evaluated in situ via immunohistochemical staining. Serum nitrite (SN), a marker of in vivo NO production, was measured. Compared with vehicle controls, treatment with CI-1044 alone resulted in dose-related increases in the frequency and severity of vascular injury, SN levels, and NT residues. SIN-1 coadministration caused vascular injury to occur at lower doses of CI-1044, compared with CI-1044 alone, with the overall incidence and severity of injury being greater across all CI-1044-dose groups. Following administration of 20 or 40 mg/kg CI-1044, there were also increases in NT immunoreactivity when SIN-1 was coadministered and significant increases in SN. Conversely, coadministration of L-NAME resulted in marked reduction of injury, NT, and SN when compared with CI-1044 alone. The present study suggests that NO production is closely linked to PDE4i-induced vascular injury.
Asunto(s)
Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Inhibidores de Fosfodiesterasa 4/metabolismo , Lesiones del Sistema Vascular/inducido químicamente , Animales , Azepinas/efectos adversos , Biomarcadores/análisis , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/efectos adversos , Inmunohistoquímica , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/patología , Molsidomina/análogos & derivados , Molsidomina/metabolismo , NG-Nitroarginina Metil Éster/efectos adversos , Niacinamida/efectos adversos , Niacinamida/análogos & derivados , Óxido Nítrico/análisis , Óxido Nítrico Sintasa/metabolismo , Nitritos/sangre , Ratas , Ratas Sprague-Dawley , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
Acute vascular injury that leads to vascular inflammation is a common finding in the preclinical toxicity testing of drugs in rats and dogs. However, the relevance of this finding for risk to humans is unclear. Concern about the safety of these drugs is heightened by the current lack of noninvasive clinical methods to predict the onset of vascular damage in animals or humans. Determining the relevance of this poorly understood preclinical outcome for humans requires a better understanding of the molecular mechanisms of injury in addition to the development of sensitive and specific leading biomarkers for the clinical diagnosis of acute vascular damage. Most molecular research on this toxicity has been performed in rats, but recent development of canine gene expression microarrays makes transcriptomic studies now possible in the dog. In this study, we investigated the molecular mechanisms of drug-induced vascular injury in dogs using gene arrays. After treating Beagles with toxic doses of CI-947, an adenosine receptor agonist, we profiled gene expression in the coronary arteries and correlated those changes with histopathology at 16 and 24 hours after dosing. The results demonstrated that pathobiological processes such as stimulation of the innate immune response, increased extracellular matrix turnover and oxidative stress were active at times of very early injury.