RESUMEN
Macrophages in the arterial intima sustain chronic inflammation during atherogenesis. Under hypercholesterolemic conditions murine Ly6C(high) monocytes surge in the blood and spleen, infiltrate nascent atherosclerotic plaques, and differentiate into macrophages that proliferate locally as disease progresses. Spleen tyrosine kinase (SYK) may participate in downstream signaling of various receptors that mediate these processes. We tested the effect of the SYK inhibitor fostamatinib on hypercholesterolemia-associated myelopoiesis and plaque formation in Apoe(-/-) mice during early and established atherosclerosis. Mice consuming a high cholesterol diet supplemented with fostamatinib for 8 weeks developed less atherosclerosis. Histologic and flow cytometric analysis of aortic tissue showed that fostamatinib reduced the content of Ly6C(high) monocytes and macrophages. SYK inhibition limited Ly6C(high) monocytosis through interference with GM-CSF/IL-3 stimulated myelopoiesis, attenuated cell adhesion to the intimal surface, and blocked M-CSF stimulated monocyte to macrophage differentiation. In Apoe(-/-) mice with established atherosclerosis, however, fostamatinib treatment did not limit macrophage accumulation or lesion progression despite a significant reduction in blood monocyte counts, as lesional macrophages continued to proliferate. Thus, inhibition of hypercholesterolemia-associated monocytosis, monocyte infiltration, and differentiation by SYK antagonism attenuates early atherogenesis but not established disease when local macrophage proliferation dominates lesion progression.
Asunto(s)
Aterosclerosis/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Monocitos/efectos de los fármacos , Mielopoyesis/efectos de los fármacos , Oxazinas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridinas/uso terapéutico , Aminopiridinas , Animales , Aterosclerosis/inmunología , Aterosclerosis/prevención & control , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Progresión de la Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Macrófagos/efectos de los fármacos , Ratones , Morfolinas , Oxazinas/farmacología , Piridinas/farmacología , Pirimidinas , Distribución Aleatoria , Quinasa SykRESUMEN
Drug combination testing in the pharmaceutical industry has typically been driven by late-stage opportunistic strategies rather than by early testing to identify drug combinations for clinical investigation that may deliver improved efficacy. A rationale for combinations exists across a number of diseases in which pathway redundancy or resistance to therapeutics are evident. However, early assays are complicated by the absence of both assay formats representative of disease biology and robust infrastructure to screen drug combinations in a medium-throughput capacity. When applying drug combination testing studies, it may be difficult to translate a study design into the required well contents for assay plates because of the number of compounds and concentrations involved. Dispensing these plates increases in difficulty as the number of compounds and concentration points increase and compounds are subsequently rolled onto additional labware. We describe the development of a software tool, in conjunction with the use of acoustic droplet technology, as part of a compound management platform, which allows the design of an assay incorporating combinations of compounds. These enhancements to infrastructure facilitate the design and ordering of assay-ready compound combination plates and the processing of combinations data from high-content organotypic assays.
Asunto(s)
Automatización de Laboratorios/métodos , Tecnología Biomédica/métodos , Evaluación Preclínica de Medicamentos/métodos , Interacciones Farmacológicas , Programas Informáticos , Acústica , Combinación de Medicamentos , SolucionesRESUMEN
The discovery and optimisation of a series of zwitterionic CCR3 antagonists is described. Optimisation of the structure led to AZ12436092, a compound with excellent selectivity over activity at hERG and outstanding pharmacokinetics in preclinical species.
Asunto(s)
Descubrimiento de Drogas , Piperidinas/química , Piperidinas/farmacocinética , Receptores CCR3/antagonistas & inhibidores , Animales , Humanos , Concentración 50 Inhibidora , Estructura Molecular , RatasRESUMEN
The ability of adherent cells such as fibroblasts to enter the cell cycle and progress to S phase is strictly dependent on the extent to which individual cells can attach to and spread on a substratum. Here we have used microengineered adhesive islands of 22 and 45 mum diameter surrounded by a nonadhesive substratum of polyhydroxyl methacrylate to accurately control the extent to which individual Swiss 3T3 fibroblasts may spread. The effect of cell shape on mitogen-evoked Ca2+ signaling events that accompany entry into the cell cycle was investigated. In unrestricted cells, the mitogens bombesin and fetal calf serum evoked a typical biphasic change in the cytoplasmic free Ca2+ concentration. However, when the spreading of individual cells was restricted, such that progression to S phase was substantially reduced, both bombesin and fetal calf serum caused a rapid transient rise in the cytoplasmic free Ca2+ concentration but failed to elicit the normal sustained influx of Ca2+ that follows Ca2+ release. As expected, restricting cell spreading led to the loss of actin stress fibers and the formation of a ring of cortical actin. Restricting cell shape did not appear to influence mitogen-receptor interactions, nor did it influence the presence of focal adhesions. Because Ca2+ signaling is an essential component of mitogen responses, these findings implicate Ca2+ influx as a necessary component of cell shape-dependent control of the cell cycle.