RESUMEN
Monoclonal antibodies against tumor necrosis factor-alpha (TNFα) are widely used for treatment of inflammatory diseases. However, despite the inhibitory effect this class of drugs has on the immune system, anti-drug antibodies are often formed with continuous use. Particles formed during stress conditions, which can be used to simulate storage and handling conditions of commercial antibodies, have previously been associated with the formation of anti-drug antibodies. This study investigates the relationship between particles, oligomerization, folding and chemical degradation on the in vitro cytokine response toward the TNFα inhibitor adalimumab. Adalimumab aggregates generated using stir and heat stress were fractionated into distinct sub-populations, and their structure and immunogenic potential were evaluated. A chemically degraded sample of adalimumab was included to compare particle composition with the milder accelerated heat and stir stressed conditions. Particles from stressed adalimumab samples induced elevated cytokine levels and CD4+ T cell proliferation in vitro compared to non-stressed samples. Samples enriched with both submicron and subvisible particles of adalimumab induced the strongest cytokine release and the strongest CD4+ T cell proliferation despite maintaining some TNFα inhibitory functionality. Samples that were stressed and subsequently purified of subvisible and submicron particles did not elicit a significantly higher cytokine response or show increased CD4+ T cell proliferation compared to a non-stressed sample. Oxidation-induced chemical modifications in adalimumab, mainly in Met, His, Trp, and Tyr, were not found to be sufficient in absence of particle formation to induce increased CD4+ T cell proliferation or cytokine release despite less decreased TNFα inhibitory activity of adalimumab. These observations provide further evidence that particles do indeed potentiate the immunogenic potential of adalimumab.
Asunto(s)
Anticuerpos Monoclonales , Factor de Necrosis Tumoral alfa , Adalimumab/farmacología , Anticuerpos Monoclonales/química , CitocinasRESUMEN
Peptides play important roles in regulating biological processes and form the basis of a multiplicity of therapeutic drugs. To date, only about 300 peptides in human have confirmed bioactivity, although tens of thousands have been reported in the literature. The majority of these are inactive degradation products of endogenous proteins and peptides, presenting a needle-in-a-haystack problem of identifying the most promising candidate peptides from large-scale peptidomics experiments to test for bioactivity. To address this challenge, we conducted a comprehensive analysis of the mammalian peptidome across seven tissues in four different mouse strains and used the data to train a machine learning model that predicts hundreds of peptide candidates based on patterns in the mass spectrometry data. We provide in silico validation examples and experimental confirmation of bioactivity for two peptides, demonstrating the utility of this resource for discovering lead peptides for further characterization and therapeutic development.
Asunto(s)
Aprendizaje Automático , Péptidos , Humanos , Ratones , Animales , Espectrometría de Masas , Péptidos/química , MamíferosRESUMEN
In this research, the tableting properties of α-melibiose monohydrate were studied. Melibiose is a disaccharide which bears structural resemblance to lactose, because they both consist of galactose and glucose monosaccharide subunits. Compactibility and deformation behavior of two melibiose batches from different suppliers were studied and compared with α-lactose monohydrate and some other typical tableting excipients. Differences in the deformation behavior were determined comparing the shape of the Heckel plots, the yield pressure values and the strain rate sensitivity (SRS) indexes. In addition, the effect of moisture on the tabletability was studied. According to the yield pressures and SRS indexes melibiose was concluded to be fragmenting, even at higher degree than lactose monohydrate. However, the overall deformation behavior of melibiose was found to be similar to that of lactose monohydrate. Increase in moisture content resulted in higher tensile strengths of tablets for both melibiose batches, but it seemed to have more effect on compactibility of the other batch. In conclusion, melibiose has potential to be used as an excipient in tablet formulations.
Asunto(s)
Química Farmacéutica/métodos , Melibiosa/química , Fuerza Compresiva , Melibiosa/análisis , Tamaño de la Partícula , ComprimidosRESUMEN
Encapsulated human retinal pigment epithelial cell line ARPE-19 has been successfully used in experimental cell therapy of retinal degenerations and Parkinson's disease, but the long-term storage of encapsulated cells is still an unresolved question. Reconstitution of viable encapsulated cells from dry form would benefit the development of cell therapy products. We freeze dried and reconstituted microencapsulated ARPE19 and ARPE19-SEAP cells. Cross-linked alginate matrix with polycation (poly-l-lysine, cationic starch) coating was used for microencapsulation. Cell viability was assessed with fluorescence microscopy and oxygen consumption of the cells. Freeze dried and reconstituted cell microcapsules were imaged using scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM). We show partial viability of microencapsulated cells after freeze-drying. Unlike poly-l-lysine (PLL) coating, cationic starch supported microcapsule shape and cell viability during freeze-drying. Trehalose pre-treatment augmented cell viability. Likewise, some lyoprotectants (trehalose, glycerol) enabled preservation of cell viability. Upon reconstitution the freeze dried cell microcapsules rapidly regained their original spherical shape. This proof-of-concept study demonstrates that microencapsulated cells can retain their viability during freeze-drying. Therefore, this approach can be further optimized for the benefit of cell therapy product development.