RESUMO
Peptides and proteins commonly have complex structural landscapes allowing for transformation into a wide array of species including oligomers, aggregates, and fibrils. The formation of undesirable forms including aggregates and fibrils poses serious risks from the perspective of drug development and disease. Liraglutide, a GLP-1 agonist for the treatment of diabetes, is a conjugated peptide that forms oligomers that can be stabilized by pH and organic solvents. We have developed an analytical toolkit to overcome challenges inherent to Liraglutide's conjugated acyl chain and probed the impact its oligomers have on its physical stability. Our studies show that Liraglutide's oligomer states have significant and potentially detrimental impacts on its propensity to aggregate and form fibrils as well as its potency. Liraglutide delivered as a synthetic peptide is able to maintain its oligomerization state in dried lyophilized powders, acting as a memory effect from its synthetic process and purification. Through Liraglutide's oligomer memory effect, we demonstrate the importance and impact the process for synthetic peptides can have on drug development spanning from discovery to formulation development.
Assuntos
Bioensaio/métodos , Estabilidade de Medicamentos , Peptídeo 1 Semelhante ao Glucagon/agonistas , Liraglutida/farmacologia , Peptídeos/química , Animais , Disponibilidade Biológica , Células CHO , Dicroísmo Circular , Cricetulus , Composição de Medicamentos/métodos , Descoberta de Drogas/métodos , Excipientes/química , Liofilização , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Microscopia Eletrônica de Transmissão , Agregados Proteicos , Estrutura Secundária de Proteína , SolubilidadeRESUMO
A rabbit blood sugar bioidentity assay is required by the FDA to evaluate biological activity for all insulin and its analogs per USP<121> guideline. Not only are a large number of live animals used, but the rabbit blood sugar method is also highly variable and expensive. Our goal is to develop a functional cell-based assay to replace rabbit blood sugar method. An H4IIE G6P-Luc reporter assay was developed by utilizing insulin's role in regulating hepatic gluconeogenesis pathway. It is known that Glucose 6-phosphatase is a rate-limiting enzyme in the gluconeogenesis pathway, and the mRNA expression of its catalytic subunit, G6PC, is highly regulated by insulin. A G6P-Luc stable cell line in H4IIE hepatocytes was first generated by stably expressing luciferase reporter gene driven by human G6PC promoter via lentivirus technology. The cell-based assay was developed and optimized to demonstrate good dose-dependent responsiveness to insulin. We further qualified the assay with two analysts through multiple runs, and demonstrated excellent performance characteristics of linearity, accuracy, and precision. A robustness study was then conducted to define critical factors for assay performance. We compared this newly developed assay with a previously established cell-based pIR MSD assay, which measures insulin receptor phosphorylation (pIR) in HepG2 cell line using Meso-Scale Discovery (MSD) technology. The comparability study was conducted to compare the two assays using samples generated from forced degradation. This study showed high correlation between assays, and both are stability indicating. Compared with the pIR MSD assay, the G6P-Luc assay not only has a significantly lower variability in qualification studies, but also offers many other advantages, including ease of use in a quality control laboratory with fewer steps, lower cost, and does not depend on a single vendor. In conclusion, we have developed a physiologically relevant and robust functional cell-based assay that is suitable to replace rabbit blood sugar method.
Assuntos
Alternativas aos Testes com Animais , Glicemia/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Insulina Glargina/farmacologia , Animais , Glicemia/análise , Linhagem Celular , Relação Dose-Resposta a Droga , Gluconeogênese/fisiologia , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Hipoglicemiantes/administração & dosagem , Insulina Glargina/administração & dosagem , Luciferases/genética , Regiões Promotoras Genéticas , Coelhos , RatosRESUMO
Tumor necrosis factor (TNF) is a key regulator of inflammatory responses and has been implicated in many pathological conditions. We used structure-based design to engineer variant TNF proteins that rapidly form heterotrimers with native TNF to give complexes that neither bind to nor stimulate signaling through TNF receptors. Thus, TNF is inactivated by sequestration. Dominant-negative TNFs represent a possible approach to anti-inflammatory biotherapeutics, and experiments in animal models show that the strategy can attenuate TNF-mediated pathology. Similar rational design could be used to engineer inhibitors of additional TNF superfamily cytokines as well as other multimeric ligands.