RESUMEN
Aging of hematopoietic stem cells (HSCs) is linked to various blood disorders and malignancies. SIRT1 has been implicated in healthy aging, but its role in HSC aging is poorly understood. Surprisingly, we found that Sirt1 knockout improved the maintenance of quiescence of aging HSCs and their functionality as well as mouse survival in serial bone marrow transplantation (BMT) recipients. The majority of secondary and tertiary BMT recipients of aging wild type donor cells developed B/myeloid mixed phenotype acute leukemia (MPAL), which was markedly inhibited by Sirt1 knockout. SIRT1 inhibition also reduced the growth and survival of human B/myeloid MPAL cells. Sirt1 knockout suppressed global gene activation in old HSCs, prominently the genes regulating protein synthesis and oxidative metabolism, which may involve multiple downstream transcriptional factors. Our results demonstrate an unexpected role of SIRT1 in promoting HSC aging and age-dependent MPAL and suggest SIRT1 may be a new therapeutic target for modulating functions of aging HSCs and treatment of MPAL.
Asunto(s)
Leucemia , Sirtuina 1 , Envejecimiento/genética , Animales , Células Madre Hematopoyéticas/metabolismo , Leucemia/genética , Leucemia/metabolismo , Ratones , Fenotipo , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
Chronic myeloid leukemia (CML) is an age-dependent blood malignancy. Like many other age-dependent human diseases, laboratory animal research of CML uses young mice that do not factor in the influence of aging. To understand how aging may impact animal modeling of human age-dependent diseases, we established the first aging mouse model of human CML in BALB/c mice in the advanced age defined by 75% survival. This model was developed by noncytotoxic depletion of bone marrow lineage-positive cells followed by BCR-ABL retroviral transduction and transplantation. CML developed in aging mice shared many similarities to that in young mice, but had increased incidence of anemia that is often seen in human CML. Importantly, we showed that aging of both donor hematopoietic stem cells and recipient bone marrow niche impacted BCR-ABL mediated leukemogenesis and leukemia spectrum. Optimal CML induction relied on age-matching for donors and recipients, and cross-transplantation between young and old mice produced a mixture of different leukemia. Therefore, our model provides initial evidence of the feasibility and merit of CML modeling in aging mice and offers a new tool for future studies of CML stem cell drug resistance and therapeutic intervention in which aging would be taken into consideration as an influencing factor.
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Leucemia Mielógena Crónica BCR-ABL Positiva , Animales , Células de la Médula Ósea , Proteínas de Fusión bcr-abl , Células Madre Hematopoyéticas , Ratones , Ratones Endogámicos BALB C , RetroviridaeRESUMEN
PURPOSE: Proliferating cell nuclear antigen (PCNA) plays an essential role in regulating DNA synthesis and repair and is indispensable to cancer cell growth and survival. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was ubiquitously expressed in a broad range of cancer cells and tumor tissues, but not significantly in nonmalignant cells. We found the L126-Y133 region of caPCNA is structurally altered and more accessible to protein-protein interaction. A cell-permeable peptide harboring the L126-Y133 sequence blocked PCNA interaction in cancer cells and selectively kills cancer cells and xenograft tumors. On the basis of these findings, we sought small molecules targeting this peptide region as potential broad-spectrum anticancer agents. EXPERIMENTAL DESIGN: By computer modeling and medicinal chemistry targeting a surface pocket partly delineated by the L126-Y133 region of PCNA, we identified a potent PCNA inhibitor (AOH1160) and characterized its therapeutic properties and potential toxicity. RESULTS: AOH1160 selectively kills many types of cancer cells at below micromolar concentrations without causing significant toxicity to a broad range of nonmalignant cells. Mechanistically, AOH1160 interferes with DNA replication, blocks homologous recombination-mediated DNA repair, and causes cell-cycle arrest. It induces apoptosis in cancer cells and sensitizes them to cisplatin treatment. AOH1160 is orally available to animals and suppresses tumor growth in a dosage form compatible to clinical applications. Importantly, it does not cause significant toxicity at 2.5 times of an effective dose. CONCLUSIONS: These results demonstrated the favorable therapeutic properties and the potential of AOH1160 as a broad-spectrum therapeutic agent for cancer treatment.
Asunto(s)
Antineoplásicos/farmacología , Biomarcadores de Tumor , Antígeno Nuclear de Célula en Proliferación/metabolismo , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Roturas del ADN/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Desarrollo de Medicamentos , Humanos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Antígeno Nuclear de Célula en Proliferación/química , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
microRNA-146a (miR-146a) has been previously implicated as an essential molecular brake, preventing immune overreaction and malignant transformation by attenuating NF-κB signaling, putatively via repression of the Traf6 and Irak1 genes. The exact contribution of miR-146a-mediated silencing of these genes to the control of immune activation is currently unknown. Therefore, we defined the role of the miR-146a-Traf6 signaling axis in the regulation of immune homeostasis using a genetic epistasis analysis in miR-146a-/- mice. We have uncovered a surprising separation of functions at the level of miR-146a targets. Lowering the Traf6 gene dose and consequent attenuation of NF-κB activation rescued several significant miR-146a-/- phenotypes, such as splenomegaly, aberrant myeloproliferation, and excessive inflammatory responses. In contrast, decreasing Traf6 expression had no effect on the development of the progressive bone marrow failure phenotype, as well as lymphomagenesis in miR-146a-/- mice, indicating that miR-146a controls these biological processes through different molecular mechanisms.
Asunto(s)
Autoinmunidad , Células Madre Hematopoyéticas/citología , Inflamación/inmunología , MicroARNs/inmunología , Mielopoyesis , Neoplasias/inmunología , Factor 6 Asociado a Receptor de TNF/inmunología , Animales , Femenino , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/inmunología , Homeostasis , Humanos , Inflamación/genética , Inflamación/fisiopatología , Masculino , Ratones , MicroARNs/genética , Células Mieloides/citología , Células Mieloides/inmunología , Neoplasias/genética , Neoplasias/fisiopatología , Factor 6 Asociado a Receptor de TNF/genéticaRESUMEN
The tumor suppressor gene RASSF1A is epigenetically silenced in most human cancers. As a binding partner of the kinases MST1 and MST2, the mammalian orthologs of the Drosophila Hippo kinase, RASSF1A is a potential regulator of the Hippo tumor suppressor pathway. RASSF1A shares these properties with the scaffold protein SAV1. The role of this pathway in human cancer has remained enigmatic inasmuch as Hippo pathway components are rarely mutated in tumors. Here we show that Rassf1a homozygous knockout mice develop liver tumors. However, heterozygous deletion of Sav1 or codeletion of Rassf1a and Sav1 produced liver tumors with much higher efficiency than single deletion of Rassf1a. Analysis of RASSF1A-binding partners by mass spectrometry identified the Hippo kinases MST1, MST2, and the oncogenic IκB kinase TBK1 as the most enriched RASSF1A-interacting proteins. The transcriptome of Rassf1a(-/-) livers was more deregulated than that of Sav1(+/-) livers, and the transcriptome of Rassf1a(-/-), Sav1(+/-) livers was similar to that of Rassf1a(-/-) mice. We found that the levels of TBK1 protein were substantially upregulated in livers lacking Rassf1a. Furthermore, transcripts of several ß-tubulin isoforms were increased in the Rassf1a-deficient livers presumably reflecting a role of RASSF1A as a microtubule-stabilizing protein. In human liver cancer, RASSF1A frequently undergoes methylation at the promoter but this was not observed for MST1, MST2, or SAV1. Our results suggest a multifactorial role of RASSF1A in suppression of liver carcinogenesis. Cancer Res; 76(9); 2824-35. ©2016 AACR.
Asunto(s)
Proteínas de Ciclo Celular/genética , Transformación Celular Neoplásica/genética , Neoplasias Hepáticas/genética , Proteínas Supresoras de Tumor/genética , Adenoma de Células Hepáticas/genética , Adenoma de Células Hepáticas/patología , Animales , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular/metabolismo , Modelos Animales de Enfermedad , Inmunoprecipitación , Neoplasias Hepáticas/patología , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Even when treated with aggressive current therapies, most patients with glioblastoma survive less than two years. Rapid tumor growth, an invasive nature, and the blood-brain barrier, which limits the penetration of large molecules into the brain, all contribute to the poor tumor response associated with conventional therapies. Immunotherapy has emerged as a therapeutic approach that may overcome these challenges. We recently reported that single-walled carbon nanotubes (SWCNTs) can be used to dramatically increase the immunotherapeutic efficacy of CpG oligonucleotides in a mouse model of glioma. Following implantation in the mouse brain, the tumor cell line used in these previous studies (GL261) tends to form a spherical tumor with limited invasion into healthy brain. In order to evaluate SWCNT/CpG therapy under more clinically-relevant conditions, here we report the treatment of a more invasive mouse glioma model (K-Luc) that better recapitulates human disease. In addition, a CpG sequence previously tested in humans was used to formulate the SWCNT/CpG which was combined with temozolomide, the standard of care chemotherapy for glioblastoma patients. We found that, following two intracranial administrations, SWCNT/CpG is well-tolerated and improves the survival of mice bearing invasive gliomas. Interestingly, the efficacy of SWCNT/CpG was enhanced when combined with temozolomide. This enhanced anti-tumor efficacy was correlated to an increase of tumor-specific cytotoxic activity in splenocytes. These results reinforce the emerging understanding that immunotherapy can be enhanced by combining it with chemotherapy and support the continued development of SWCNT/CpG.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Dacarbazina/análogos & derivados , Glioma/tratamiento farmacológico , Inmunoterapia , Nanotubos de Carbono/química , Oligodesoxirribonucleótidos/uso terapéutico , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Dacarbazina/uso terapéutico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Glioma/patología , Inflamación/patología , Lípidos/química , Ratones Endogámicos C57BL , Invasividad Neoplásica , Polietilenglicoles/química , Bazo/patología , Temozolomida , Resultado del TratamientoRESUMEN
Epidermal growth factor receptor variant III (EGFRvIII) is a cancer-specific deletion mutant observed in approximately 25% to 50% of glioblastoma multiforme (GBM) patients. An antibody drug conjugate, AMG 595, composed of the maytansinoid DM1 attached to a highly selective anti-EGFRvIII antibody via a noncleavable linker, was developed to treat EGFRvIII-positive GBM patients. AMG 595 binds to the cell surface and internalizes into the endo-lysosomal pathway of EGFRvIII-expressing cells. Incubation of AMG 595 with U251 cells expressing EGFRvIII led to potent growth inhibition. AMG 595 treatment induced significant tumor mitotic arrest, as measured by phospho-histone H3, in GBM subcutaneous xenografts expressing EGFRvIII. A single intravenous injection of AMG 595 at 17 mg/kg (250 µg DM1/kg) generated complete tumor regression in the U251vIII subcutaneous xenograft model. AMG 595 mediated tumor regression in the D317 subcutaneous xenograft model that endogenously expresses EGFRvIII. Finally, AMG 595 treatment inhibited the growth of D317 xenografts orthotopically implanted into the brain as determined by magnetic resonance imaging. These results demonstrate that AMG 595 is a promising candidate to evaluate in EGFRvIII-expressing GBM patients.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Receptores ErbB/inmunología , Glioblastoma/tratamiento farmacológico , Inmunoconjugados/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Glioblastoma/inmunología , Glioblastoma/metabolismo , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/inmunología , Inmunohistoquímica , Inyecciones Intravenosas , Maitansina/análogos & derivados , Maitansina/inmunología , Maitansina/farmacología , Ratones Desnudos , Ratones SCID , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Carga Tumoral/inmunologíaRESUMEN
MicroRNAs (miRNAs) are a class of powerful posttranscriptional regulators implicated in the control of diverse biological processes, including regulation of hematopoiesis and the immune response. To define the biological functions of miR-142, which is preferentially and abundantly expressed in immune cells, we created a mouse line with a targeted deletion of this gene. Our analysis of miR-142(-/-) mice revealed a critical role for this miRNA in the development and homeostasis of lymphocytes. Marginal zone B cells expand in the knockout spleen, whereas the number of T and B1 B cells in the periphery is reduced. Abnormal development of hematopoietic lineages in miR-142(-/-) animals is accompanied by a profound immunodeficiency, manifested by hypoimmunoglobulinemia and failure to mount a productive immune response to soluble antigens and virus. miR-142(-/-) B cells express elevated levels of B-cell-activating factor (BAFF) receptor (BAFF-R) and as a result proliferate more robustly in response to BAFF stimulation. Lowering the BAFF-R gene dose in miR-142(-/-) mice rescues the B-cell expansion defect, suggesting that BAFF-R is a bona fide miR-142 target through which it controls B-cell homeostasis. Collectively, our results uncover miR-142 as an essential regulator of lymphopoiesis, and suggest that lesions in this miRNA gene may lead to primary immunodeficiency.
Asunto(s)
Linfocitos B/patología , Eliminación de Gen , Síndromes de Inmunodeficiencia/genética , Trastornos Inmunoproliferativos/genética , Linfopoyesis , MicroARNs/genética , Animales , Receptor del Factor Activador de Células B/genética , Linfocitos B/inmunología , Linfocitos B/metabolismo , Femenino , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Inmunidad Celular , Inmunidad Humoral , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/patología , Trastornos Inmunoproliferativos/inmunología , Trastornos Inmunoproliferativos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/inmunologíaRESUMEN
The ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is one of the most hotly pursued targets for the treatment of Alzheimer's disease. We used a structure- and property-based drug design approach to identify 2-aminooxazoline 3-azaxanthenes as potent BACE1 inhibitors which significantly reduced CSF and brain Aß levels in a rat pharmacodynamic model. Compared to the initial lead 2, compound 28 exhibited reduced potential for QTc prolongation in a non-human primate cardiovascular safety model.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Xantenos/química , Xantenos/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Línea Celular , Células HEK293 , Humanos , Inhibidores de Proteasas/síntesis química , Ratas , Xantenos/síntesis químicaRESUMEN
Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic ß-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.
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Proteínas Portadoras/antagonistas & inhibidores , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales , Animales , Glucemia/metabolismo , Proteínas Portadoras/metabolismo , Núcleo Celular/enzimología , Cristalografía por Rayos X , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/enzimología , Modelos Animales de Enfermedad , Hepatocitos , Humanos , Hiperglucemia/sangre , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/enzimología , Hipoglucemiantes/química , Hígado/citología , Hígado/enzimología , Hígado/metabolismo , Masculino , Modelos Moleculares , Especificidad de Órganos , Fosforilación/efectos de los fármacos , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/farmacología , Piperazinas/uso terapéutico , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Sulfonamidas/uso terapéuticoRESUMEN
Sunitinib malate (SUTENT) is a multitargeted receptor tyrosine kinase (RTK) inhibitor that is approved multinationally for the treatment of imatinib-resistant/-intolerant gastrointestinal stromal tumor and advanced renal cell carcinoma. This paper characterizes the organ toxicity of sunitinib in Sprague-Dawley rats and cynomolgus monkeys, and the reversibility of any treatment-induced effects. Rats and monkeys received sunitinib (0-15 and 0-20 mg/kg/day, respectively) orally on a consecutive daily dosing schedule for thirteen weeks or on an intermittent daily dosing schedule for up to nine months. Clinical observations and laboratory parameters were recorded. Necropsy was conducted following treatment/recovery periods, and histologic examinations were performed. In rats, sunitinib was generally tolerated at 0.3 and 1.5 mg/kg/day, and findings were reversible. In monkeys, the level at which there were no observed adverse effects was 1.5 mg/kg/day, and findings were similarly reversible (except for uterine/ovarian weight changes and skin pallor). Data suggest that inhibition of multiple RTK pathways may induce pharmacologic effects on organ systems in nonclinical species. Key pharmacologic effects of sunitinib included reversible inhibition of neovascularization into the epiphyseal growth plate, and impaired corpora lutea formation and uterine development during estrus. Similar observations have been noted with this class of RTK signaling inhibitors and are consistent with pharmacologic perturbations of physiologic/angiogenic processes associated with the intended molecular targets.
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Antineoplásicos/toxicidad , Indoles/toxicidad , Pirroles/toxicidad , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Pruebas de Toxicidad Crónica/métodos , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/patología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Femenino , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/patología , Placa de Crecimiento/efectos de los fármacos , Placa de Crecimiento/patología , Incisivo/efectos de los fármacos , Incisivo/patología , Indoles/administración & dosificación , Indoles/farmacocinética , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/patología , Macaca fascicularis , Masculino , Microscopía Electrónica , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/patología , Ovario/efectos de los fármacos , Ovario/patología , Páncreas/efectos de los fármacos , Páncreas/patología , Pirroles/administración & dosificación , Pirroles/farmacocinética , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , SunitinibAsunto(s)
Lipidosis/inducido químicamente , Lipidosis/metabolismo , Fosfolípidos/metabolismo , Pruebas de Toxicidad/métodos , Animales , Biomarcadores/metabolismo , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Humanos , Lipidosis/diagnóstico , Medición de Riesgo , Pruebas de Toxicidad/tendencias , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Regulatory CD4(+)CD25(+) T cells (Tregs) suppress autoimmune and inflammatory diseases through mechanisms that are only partly understood. Previous studies suggest that Tregs can suppress bacterially triggered intestinal inflammation and respond to LPS through TLRs with enhanced suppressive activity. In this study, we have used murine cecal ligation and puncture as a model of polymicrobial sepsis to explore the effects of adoptive transfer of Tregs on septic outcome. Adoptive transfer of in vitro-stimulated Tregs in both prevention and therapeutic modes significantly improved survival of cecal ligation and puncture mice. Furthermore, the effect was dependent on both the number of Tregs adoptively transferred and the presence of host T cells. Animals that received stimulated Tregs had significantly increased peritoneal mast cells and peritoneal TNF-alpha production. More importantly, adoptive transfer of in vitro-stimulated Tregs significantly improved bacterial clearance, which resulted in improved survival. Our results suggest a novel role for Tregs in sepsis.
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Traslado Adoptivo , Activación de Linfocitos/inmunología , Receptores de Interleucina-2/biosíntesis , Sepsis/inmunología , Sepsis/terapia , Linfocitos T Reguladores/microbiología , Linfocitos T Reguladores/trasplante , Traslado Adoptivo/métodos , Animales , Movimiento Celular/inmunología , Células Cultivadas , Relación Dosis-Respuesta Inmunológica , Femenino , Inyecciones Intravenosas , Ligadura , Mastocitos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Peritoneo/citología , Peritoneo/inmunología , Peritoneo/microbiología , Punciones , Sepsis/microbiología , Sepsis/mortalidad , Análisis de Supervivencia , Linfocitos T Reguladores/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Many cationic amphiphilic (phospholipidosis-inducing) drugs (CADs) accumulate in tissues following repeated dosing in preclinical models, and this is sometimes associated with dose-limiting toxicities. Plasma drug levels cannot be used to estimate tissue accumulation of CADs since it occurs in tissues despite stabilization of plasma levels. Severe myopathy was found in skeletal muscles of rats during the initial safety evaluation of a dopamine D3 receptor antagonist, PNU-177864, and was associated with phospholipidosis in numerous tissues. The myopathy was observed only when plasma levels of PNU-177864 remained essentially constant throughout the 24-hour dosing period. A repeat dose drug distribution study using whole body autoradiography demonstrated that drug-related material did not accumulate in skeletal muscle or other tissues following repeated doses at levels considered within the therapeutic range and showing toxicokinetic profiles acceptable for further development. These observations provided support for the continued development of and longer-term toxicity studies with this candidate compound.
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Antagonistas de Dopamina/farmacocinética , Antagonistas de Dopamina/toxicidad , Músculo Esquelético/patología , Enfermedades Musculares/inducido químicamente , Animales , Autorradiografía , Antagonistas de Dopamina/sangre , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Lipidosis/inducido químicamente , Masculino , Microscopía Electrónica , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/ultraestructura , Fosfolípidos/análisis , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Repeat dose oral toxicity studies were conducted in rat and dog to assess the safety for human clinical testing of the potent dopamine D3 receptor antagonist, PNU-177864. Systemic phospholipidosis was the principal treatment-related change with epididymal epithelial cell phospholipidosis being the most prominent finding in rats and dogs. Epididymal epithelial cells had no histologic evidence of degeneration; sperm density and morphology were normal histologically in both species; and sperm concentration, morphology, and motility in the dog were comparable to dogs given vehicle. Other sites with phospholipidosis included lymphoid tissues (lymph nodes, Peyer's patches, and/or spleen), pulmonary alveolar macrophages, and peripheral blood lymphocytes in rats and dogs and adrenal cortex, liver, pituitary, hair follicles, bone marrow lymphocytes and plasma cells, and skeletal muscle in rats only. The phospholipidosis was resolved after a 6-week recovery period in all tissues but epididymis. There was no evidence of cell injury in tissues that had phospholipid accumulations except in rat skeletal muscle that had multifocal myofiber degeneration and necrosis. For clinical trials, serum AST and CK and peripheral blood lymphocyte vacuolation were considered potential safety biomarkers for skeletal muscle degeneration and phospholipidosis, respectively.
Asunto(s)
Antagonistas de Dopamina/toxicidad , Epidídimo/efectos de los fármacos , Lipidosis/inducido químicamente , Animales , Biomarcadores , Perros , Evaluación Preclínica de Medicamentos , Epidídimo/patología , Epidídimo/ultraestructura , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Células Epiteliales/ultraestructura , Linfocitos/efectos de los fármacos , Linfocitos/patología , Masculino , Microscopía Electrónica , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Fosfolípidos , Ratas , Especificidad de la Especie , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Vacuolas/efectos de los fármacos , Vacuolas/patología , Vacuolas/ultraestructuraRESUMEN
Reactive oxygen species (ROS) can directly induce or enhance tumor necrosis factor (TNF)-mediated apoptosis in a number of different cell lines. To test the relevance of intracellular ROS in modulating apoptotic signaling in vivo, we evaluated hepatocellular apoptosis mediated by the TNF or Fas receptor in wild-type and glutathione peroxidase-1 (Gpx1-/-)-deficient mice (129SV/B6 background). Apoptosis developed in livers of wild-type animals 4-6 h after intraperitoneal administration of 700 mg/kg galactosamine/100 micro g/kg endotoxin. Apoptosis was indicated by processing of procaspases-3 (assessed by western blotting), a fivefold increase in caspase-3 activity (DEVD-AMC as substrate), and a 44-fold increase in DNA fragmentation (ELISA). The time course and magnitude of apoptosis were the same in Gpx1-/- mice. In contrast, Gpx1-/- mice had higher plasma alanine aminotransferase (ALT) levels and more severe hemorrhage compared to wild-type animals at 6 h. Treatment of wild-type mice with the anti-Fas antibody Jo-2 (0.6 mg/kg i.v.) resulted in processing of procaspase-3 and a sevenfold increase in caspase-3 activity in both wild-type and Gpx1-/- mice. However, higher plasma ALT values in Gpx1-/- mice at 3 h may reflect a trend to develop more rapidly secondary necrosis. These data suggest that, under our experimental conditions, intracellular ROS did not modulate the death receptor-initiated apoptotic signaling cascade in hepatocytes. As Gpx1 is located in the cytosol and in mitochondria, which are the main cellular compartments involved in apoptotic signaling, our findings indicate that the oxidant stress in vivo was insufficient to modulate these signaling pathways. However, Gpx1 deficiency enhances the susceptibility for secondary necrosis or neutrophil-induced cell injury.
Asunto(s)
Apoptosis/fisiología , Glutatión Peroxidasa/deficiencia , Glutatión Peroxidasa/metabolismo , Hígado/fisiología , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Receptor fas/fisiología , Alanina Transaminasa/sangre , Animales , Apoptosis/genética , Caspasa 3 , Caspasas/metabolismo , Fragmentación del ADN , Modelos Animales de Enfermedad , Precursores Enzimáticos/metabolismo , Galactosamina/toxicidad , Glutatión Peroxidasa/genética , Hemorragia/etiología , Hígado/efectos de los fármacos , Hígado/patología , Ratones , Ratones Endogámicos , Ratones Noqueados , Necrosis , Glutatión Peroxidasa GPX1RESUMEN
The pace at which new drug candidates are being identified by Discovery Research demands that they be screened for preclinical attributes rapidly and efficiently. The early identification and elimination of compounds with toxic liabilities will produce safer drugs in a shorter time period, and with an increased rate of success. Most major pharmaceutical companies now recognize the strategic role of pathology support for research and have developed specific units to effect this outcome. The early interaction of these pathologists with drug discovery teams to identify compounds with toxic liabilities is critical. Approaches being used include high throughput in vitro screens to predict the relative toxicity of discovery compounds and to provide early indications of underlying mechanisms, target profiling to predict consequences of receptor-ligand interactions at other-than-indicated target sites, and acute in vivo studies to establish tolerability limits and target organs of toxicity. These approaches include the application of contemporary tools such as genomics, proteomics, metabonomics, and genetically engineered animal models. To maximize the benefit of discovery pathology, it is critical that pharmaceutical companies also actively participate in non-proprietary knowledge sharing and the education of pathologists and toxicologists to lead these efforts in the future.