Modelo in vitro para agregação amiloide em formulação de insulina regular e o uso de polifenóis e vitaminas como excipientes para estabilização

Abstract

Diabetes Mellitus (DM) is a disease characterized by the increased glucose blood concentration (hyperglycemia), which may result from defects in secretion or action of insulin hormone, produced by pancreatic islet beta cells. Insulin is responsible for metabolizing glucose for energy production, and when some dysfunction in this hormone production happens, it is necessary to control the glucose in the bloodstream, by an exogenous replacement of insulin. However, care should be taken, as amyloid aggregates can be formed, which results from molecular destructing of the protein, causing it to lose its biological activity. These amyloid aggregates in insulin are known in the literature, but are not very well characterized, besides there are no reports on amyloid tests directly in the biopharmaceutical formulation. It was developed a simple and reproducible model that mimics the transport and storage of insulin in unrefrigerated condition, to analyze the formation of amyloid aggregates within the formulation of commercial insulin. Insulin formulations were submitted to agitation conditions (175 rpm) and temperature of 37 °C, and its activity was monitored by Tioflavin T fluorescence (ThT) and Congo Red spectrophotometry (CR), and the morphology of the aggregates was verified though the use of transmission electron microscopy (TEM). It was possible to observe that there is formation of amyloid aggregates in commercial insulin formulations, which may present a direct relationship with the stability and efficacy of this drug in its daily use. Thus, the described system can be used in the study of amyloid aggregation and in the search for molecules with antiamyloidogenic potential. From the proposed aggregation model, substances were evaluated as excipients to increase the stability of this biopharmaceutical. Results showed that polyphenols (curcumin, quercetin and rutin) had no effect, while the use of vitamins (ascorbic acid, retinol and α – tocopherol) had, it and retinol and α – tocopherol demonstrated a more pronounced effect that was confirmed by the 3 techniques applied. Therefore, these two substances presented potential for use as excipients in improving the stability of pharmaceutical formulations, which can generate in increasing the efficacy and safety of treatment using this drug