RESUMO
RTH258 (brolucizumab) is a humanized single chain antibody fragment, the smallest functional unit of an antibody designed to target vascular endothelial growth factor in angiogenic retinal disease. To further understand the safe use of RTH258, this study assessed the potential impact of intravitreal RTH258 on pre- and postnatal development in the offspring of cynomolgus monkeys following administration to the mother. Three groups of 16 pregnant females were included: a low dose group (RTH258 3 mg/50 µl [60 mg/ml]), a high dose group (RTH258 6 mg/50 µl [120 mg/ml]), and a control group. Maternal animals were administered a single injection of 50 µl in the right eye once every four weeks. Animals were observed daily and detailed observations were collected before and after the first dose, and then weekly thereafter. Following parturition, observations of infants included external, morphological, and ophthalmic examinations; neurobehavioral test battery; grip strength; and skeletal development. Blood samples for hematology, coagulation, and clinical chemistry were collected from non-fasted maternal and infant animals. No RTH258-related deaths occurred in maternal dams or infants. No RTH258-related clinical observations were noted in maternal animals or in surviving infants - there were no changes in gestation length; pregnancy loss; deaths; body weight/weight change; infant grip strength; infant external, morphological, or skeletal evaluations; ophthalmoscopy or neurobehavioral observations; or clinical pathology parameters. RTH258 had no impact on pregnancy or parturition; embryo-fetal development; or survival, growth, or postnatal development of offspring when administered via repeated intravitreal administration.
Assuntos
Anticorpos Monoclonais Humanizados , Fator A de Crescimento do Endotélio Vascular , Humanos , Gravidez , Animais , Feminino , Macaca fascicularis , Anticorpos Monoclonais Humanizados/farmacologia , PartoRESUMO
Small molecule pharmaceutical products are assumed to reach concentrations in semen similar to those in blood plasma. Exposure modeling for these small-molecule products in humans assumes a daily dose of 5mL of semen and 100% absorption from the vagina with distribution to the conceptus through the maternal systemic circulation. Monoclonal antibody drugs are present in semen at concentrations about 2% or less of those in blood, and the modeling used for small molecules will over-estimate the possibility of conceptus exposure to immunoglobulins. It is not known whether peptide products reach semen, but in general peptide medications are destroyed by vaginal peptidases, and conceptus exposure is predicted to be minimal. Theoretical exposure routes to pharmaceuticals that might result in exposure of the conceptus greater than that of maternal systemic exposures include direct access through the cervical canal, adsorption to sperm for carriage into the oocyte, and direct delivery from the vaginal veins or lymphatics to the uterine artery. There is some evidence for direct access to the uterus for progesterone, terbutaline, and danazol, but the evidence does not involve exposures during pregnancy in most instances. Studies in mice, rats, rabbits, and monkeys do not suggest that exposure to small molecule pharmaceuticals in semen imposes risks to the conceptus beyond those that can be predicted using modeling of systemic maternal exposure. Monoclonal antibody and peptide exposure in semen does not pose a significant risk to the conceptus.
Assuntos
Anticorpos Monoclonais/metabolismo , Embrião de Mamíferos/metabolismo , Feto/metabolismo , Peptídeos/metabolismo , Preparações Farmacêuticas/metabolismo , Sêmen/metabolismo , Vagina/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Anticorpos Monoclonais/toxicidade , Transporte Biológico , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Feto/efeitos dos fármacos , Haplorrinos , Humanos , Masculino , Exposição Materna , Camundongos , Modelos Animais , Modelos Biológicos , Exposição Paterna , Peptídeos/química , Peptídeos/toxicidade , Permeabilidade , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/química , Coelhos , Medição de Risco , Absorção VaginalRESUMO
Cartilage formation in the embryonic limb is presaged by a cellular condensation phase that is mediated by both cell-cell and cell-matrix interactions. N-Cadherin, a Ca(2+)-dependent cell-cell adhesion molecule, is expressed at higher levels in the condensing mesenchyme, followed by down-regulation upon chondrogenic differentiation, strongly suggesting a functional role in the cellular condensation process. To further examine the role of N-cadherin, we have generated expression constructs of wild type and two deletion mutants (extracellular and intracellular) of N-cadherin in the avian replication-competent, RCAS retrovirus, and transfected primary chick limb mesenchymal cell cultures with these constructs. The effects of altered, sustained expression of N-cadherin and its mutant forms on cellular condensation, on the basis of peanut agglutinin (DNA) staining, and chondrogenesis, based on expression of chondrocyte phenotypic markers, were characterized. Cellular condensation was relatively unchanged in cultures overexpressing wild type N-cadherin, compared to controls on all days in culture. However, expression of either of the deletion mutant forms of N-cadherin resulted in decreased condensation, with the extracellular deletion mutant demonstrating the most severe inhibition, suggesting a requirement for N-cadherin mediated cell-cell adhesion and signaling in cellular condensation. Subsequent chondrogenic differentiation was also affected in all cultures overexpressing the N-cadherin constructs, on the basis of metabolic sulfate incorporation, the presence of the cartilage matrix proteins collagen type II and cartilage proteoglycan link protein, and alcian blue staining of the matrix. The characteristics of the cultures suggest that the N-cadherin mutants disrupt proper cellular condensation and subsequent chondrogenesis, while the cultures overexpressing wild type N-cadherin appear to condense normally, but are unable to proceed toward differentiation, possibly due to the prolonged maintenance of increased cell-cell adhesiveness. Thus, spatiotemporally regulated N-cadherin expression and function, at the level of both homotypic binding and linkage to the cytoskeleton, is required for chondrogenesis of limb mesenchymal cells.
Assuntos
Caderinas/fisiologia , Agregação Celular/fisiologia , Condrogênese/fisiologia , Botões de Extremidades/metabolismo , Mesoderma/metabolismo , Animais , Caderinas/biossíntese , Cartilagem/metabolismo , Embrião de Galinha , Colágeno Tipo II/metabolismo , Técnicas de Cultura , Proteínas do Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Botões de Extremidades/citologia , Mesoderma/citologia , Proteoglicanas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retroviridae/genética , Fatores de Tempo , Transativadores/metabolismo , Transfecção , beta CateninaRESUMO
Wnt signaling has been implicated in the regulation of limb mesenchymal chondrogenesis. In this study, we have analyzed the molecular mechanism of Wnt-7a inhibition of chondrogenic differentiation by examining the involvement of mitogen-activated protein kinase (MAPK) pathways, i.e., Erk and p38. The combination of Wnt-7a misexpression and Erk inhibition partially recovers Wnt-7a inhibition of chondrogenic differentiation, whereas the combination of Wnt-7a misexpression and p38 inhibition acts in a synergistic chondro-inhibitory fashion. Although Wnt-7a misexpression has no direct effect on Erk signaling, it increases the activity of one of the ultimate targets of the MAPK pathway, c-jun, a major component of the activator protein-1 (AP-1) transcription factor complex. In addition, Wnt-7a misexpression enhances the activity of an AP-1 promoter-luciferase reporter construct by approximately 2.3-fold in vitro. Interestingly, misexpression of wild-type N-cadherin in these micromass cultures suppresses the activity of the same AP-1 promoter by approximately 40%, whereas misexpression of an extracellular 390-amino-acid N-terminal deletion mutant of N-cadherin has a stimulatory effect on the AP-1 promoter activity by approximately 2.6-fold. Thus, our results suggest that at least a part of the chondro-inhibitory effect of Wnt-7a misexpression may involve AP-1 transcription factor stimulation. Furthermore, a very tightly regulated level of AP-1 activity is necessary for the process of limb mesenchymal chondrogenesis, and signals from Wnt-ligands (e.g., Wnt-7a), cell adhesion molecules (e.g., N-cadherin), and MAPK pathways (e.g., Erk and p38) are interactively involved in this regulation.