RESUMO
Direct delivery of therapeutics to the central nervous system (CNS) greatly expands opportunities to treat neurological diseases but is technically challenging. This opinion outlines principal technical aspects of direct CNS delivery via intracerebroventricular (ICV) or intrathecal (IT) injection to common nonclinical test species (rodents, dogs, and nonhuman primates) and describes procedure-related clinical and histopathological effects that confound interpretation of test article-related effects. Direct dosing is by ICV injection in mice due to their small body size, while other species are dosed IT in the lumbar cistern. The most frequent procedure-related functional effects are transient absence of lower spinal reflexes after IT injection or death soon after ICV dosing. Common procedure-related microscopic findings in all species include leukocyte infiltrates in CNS meninges or perivascular (Virchow-Robin) spaces; nerve fiber degeneration in the spinal cord white matter (especially dorsal and lateral tracts compressed by dosing needles or indwelling catheters), spinal nerve roots, and sciatic nerve; meningeal fibrosis at or near IT injection sites; hemorrhage; and gliosis. Findings typically are minimal to occasionally mild. Findings tend to be more severe and/or have a higher incidence in the spinal cord segments and spinal nerve roots at or close to the site of administration.
Assuntos
Oligonucleotídeos , Roedores , Cães , Camundongos , Animais , Sistema Nervoso Central/patologia , Medula Espinal/patologia , Degeneração Neural/patologia , PrimatasRESUMO
Modulation of the cell cycle may underlie the toxicologic or pharmacologic responses of a potential therapeutic agent and contributes to decisions on its preclinical and clinical safety and efficacy. The descriptive and quantitative assessment of normal, aberrant, and degenerate mitotic figures in tissue sections is an important end point characterizing the effect of xenobiotics on the cell cycle. Historically, pathologists used manual counting and special staining visualization techniques such as immunohistochemistry for quantification of normal, aberrant, and degenerate mitotic figures. We designed an automated image analysis algorithm for measuring these mitotic figures in hematoxylin and eosin (H&E)-stained sections. Algorithm validation methods used data generated from a subcutaneous human transitional cell carcinoma xenograft model in nude rats treated with the cell cycle inhibitor Eg5. In these studies, we scanned and digitized H&E-stained xenografts and applied a complex ruleset of sequential mathematical filters and shape discriminators for classification of cell populations demonstrating normal, aberrant, or degenerate mitotic figures. The resultant classification system enabled the representations of three identifiable degrees of morphological change associated with tumor differentiation and compound effects. The numbers of mitotic figure variants and mitotic indices data generated corresponded to a manual assessment by a pathologist and supported automated algorithm verification and application for both efficacy and toxicity studies.
Assuntos
Algoritmos , Antineoplásicos/farmacologia , Processamento de Imagem Assistida por Computador/métodos , Cinesinas/antagonistas & inibidores , Mitose/efeitos dos fármacos , Animais , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Humanos , Ratos , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUNDSpinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein. New SMN-enhancing therapeutics are associated with variable clinical benefits. Limited knowledge of baseline and drug-induced SMN levels in disease-relevant tissues hinders efforts to optimize these treatments.METHODSSMN mRNA and protein levels were quantified in human tissues isolated during expedited autopsies.RESULTSSMN protein expression varied broadly among prenatal control spinal cord samples, but was restricted at relatively low levels in controls and SMA patients after 3 months of life. A 2.3-fold perinatal decrease in median SMN protein levels was not paralleled by comparable changes in SMN mRNA. In tissues isolated from nusinersen-treated SMA patients, antisense oligonucleotide (ASO) concentration and full-length (exon 7 including) SMN2 (SMN2-FL) mRNA level increases were highest in lumbar and thoracic spinal cord. An increased number of cells showed SMN immunolabeling in spinal cord of treated patients, but was not associated with an increase in whole-tissue SMN protein levels.CONCLUSIONSA normally occurring perinatal decrease in whole-tissue SMN protein levels supports efforts to initiate SMN-inducing therapies as soon after birth as possible. Limited ASO distribution to rostral spinal and brain regions in some patients likely limits clinical response of motor units in these regions for those patients. These results have important implications for optimizing treatment of SMA patients and warrant further investigations to enhance bioavailability of intrathecally administered ASOs.FUNDINGSMA Foundation, SMART, NIH (R01-NS096770, R01-NS062869), Ionis Pharmaceuticals, and PTC Therapeutics. Biogen provided support for absolute real-time RT-PCR.
Assuntos
Envelhecimento , Neurônios Motores , Atrofia Muscular Espinal , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Medula Espinal , Envelhecimento/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Autopsia , Sobrevivência Celular , Feminino , Humanos , Masculino , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Proteína 2 de Sobrevivência do Neurônio Motor/antagonistas & inibidores , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Activating mutations in KRAS underlie the pathogenesis of up to 20% of human tumors, and KRAS is one of the most frequently mutated genes in cancer. Developing therapeutics to block KRAS activity has proven difficult, and no direct inhibitor of KRAS function has entered clinical trials. We describe the preclinical evaluation of AZD4785, a high-affinity constrained ethyl-containing therapeutic antisense oligonucleotide (ASO) targeting KRAS mRNA. AZD4785 potently and selectively depleted cellular KRAS mRNA and protein, resulting in inhibition of downstream effector pathways and antiproliferative effects selectively in KRAS mutant cells. AZD4785-mediated depletion of KRAS was not associated with feedback activation of the mitogen-activated protein kinase (MAPK) pathway, which is seen with RAS-MAPK pathway inhibitors. Systemic delivery of AZD4785 to mice bearing KRAS mutant non-small cell lung cancer cell line xenografts or patient-derived xenografts resulted in inhibition of KRAS expression in tumors and antitumor activity. The safety of this approach was demonstrated in mice and monkeys with KRAS ASOs that produced robust target knockdown in a broad set of tissues without any adverse effects. Together, these data suggest that AZD4785 is an attractive therapeutic for the treatment of KRAS-driven human cancers and warrants further development.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas ras/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Modelos Animais de Doenças , Humanos , Camundongos , Mutação/genética , Oligonucleotídeos Antissenso/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/antagonistas & inibidoresRESUMO
Measuring the activity and temperature of rats is commonly required in biomedical research. Conventional approaches necessitate single housing, which affects their behavior and wellbeing. We have used a subcutaneous radiofrequency identification (RFID) transponder to measure ambulatory activity and temperature of individual rats when group-housed in conventional, rack-mounted home cages. The transponder location and temperature is detected by a matrix of antennae in a baseplate under the cage. An infrared high-definition camera acquires side-view video of the cage and also enables automated detection of vertical activity. Validation studies showed that baseplate-derived ambulatory activity correlated well with manual tracking and with side-view whole-cage video pixel movement. This technology enables individual behavioral and temperature data to be acquired continuously from group-housed rats in their familiar, home cage environment. We demonstrate its ability to reliably detect naturally occurring behavioral effects, extending beyond the capabilities of routine observational tests and conventional monitoring equipment. It has numerous potential applications including safety pharmacology, toxicology, circadian biology, disease models and drug discovery.
Assuntos
Comportamento Animal , Abrigo para Animais , Comportamento Social , Temperatura , Gravação em Vídeo/métodos , Animais , Automação , Implantes Experimentais , Masculino , Movimento , Dispositivo de Identificação por Radiofrequência , Ratos Wistar , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
This is a report on a 2-day joint meeting between the British Society of Toxicological Pathology (BSTP) and the Safety Pharmacology Society (SPS) held in the UK in November 2013. Drug induced adverse effects on the cardiovascular system are associated with the attrition of more marketed and candidate drugs than any other safety issue. The objectives of this meeting were to foster inter-disciplinary approaches to address cardiovascular risk assessment, improve understanding of the respective disciplines, and increase awareness of new technologies. These aims were achieved. This well attended meeting covered both 'purely functional' cardiovascular adverse effects of drugs (e.g., electrophysiological and haemodynamic changes) as well as adverse effects encompassing both functional and pathological changes. Most of the presentations focused on nonclinical safety data, with information on translation to human where known. To reflect the content of the presentations we have cited key references and review articles.