Investigation of factors that cause insulin precipitation and/or amyloid formation in insulin formulations

Abstract

BackgroundMultiple daily subcutaneous injections (MDSIs) are mainly used for formulating an insulin therapy for diabetic patients; however, they also cause insulin-derived amyloidosis (IDA) and lead to poor glycemic control. In addition, for the continuous subcutaneous insulin infusion system (CSII), precipitation frequently causes catheter occlusion and, if the precipitate in the formulations is amyloid, the injection of the insoluble amyloid into the subcutaneous tissue leads to IDA. The aim of this study was to conduct in vitro experiments and present a situation where insulin formulations cause precipitation and amyloid formation.MethodsHumulin®R and NovoRapid® were used as model formulations for MDSIs and CSII, respectively. The generation of the precipitation was evaluated by measuring turbidity, and amyloid formation was evaluated by using Thioflavin T. Humulin®R was mixed with saline buffer solutions and glucose solutions to evaluate the effect of dilution. In addition, we created an experimental system to consider the effect of the time course of condition changes, and investigated the effects of insulin concentration, m-cresol existence, and pH change on the generation of the precipitate and amyloid in the formulation.ResultsIn both the original and diluted formulations, physical stimulation resulted in the formation of a precipitate, which in most cases was an amyloid. The amyloid was likely to be formed at a near neutral pH. On the contrary, although a precipitate formed when the pH was decreased to near the isoelectric point, this precipitate was not an amyloid. Further decreases in pH resulted in the formation of amyloids, suggesting that both the positive and negative charged states of insulin tended to form amyloids. The formulation additive m-cresol suppressed amyloid formation. When additives were removed from the formulation, the amyloid-containing gel was formed in the field of substance exchange.ConclusionsTo consider changes in conditions that may occur for insulin formulations, the relationship between the formation of precipitates and amyloids was demonstrated in vitro by using insulin formulations. From the in vitro study, m-cresol was shown to have an inhibitory effect on amyloid formation.

Phenolic Preservative Removal from Commercial Insulin Formulations Reduces Tissue Inflammation while Maintaining Euglycemia

“…tubing set, the maximum dwell time would be up to 4 h from insulin reservoir to delivery . Previous studies in vitro have not shown fibrillation or degradation occurring at 4 h. ,,, However, it remains to be determined how exposure to tubing and filter conditions will impact protein stability without PP over time.…”

“…tubing set, the maximum dwell time would be up to 4 h from insulin reservoir to delivery. 54 Previous studies in vitro have not shown fibrillation or degradation occurring at 4 h. 32,36,63,64 However, it remains to be determined how exposure to tubing and filter conditions will impact protein stability without PP over time. Notwithstanding these conformational changes, we tested insulin activity following Z-Y filtration by infusion in chemically induced diabetic mice.…”

Quantifying the Effects of Vibration on Medicines in Transit Caused by Fixed-Wing and Multi-Copter Drones

“…Ohno et al [34] reported a frequency dependency towards the stability of aqueous insulin solutions where, at a very low stimulation of 0.5 Hz, no loss of stability was seen, however after 24 h of constant vibration at approximately 14 Hz at 37 • C, a significant increase in the turbidity was observed. Drone delivery services are typically of a much shorter duration, but flight times to distant islands, (e.g., isolated communities in the South Pacific) that require larger drones powered by combustion engines, may generate the exposure times reported in the insulin laboratory studies described by Ohno et al [34].…”

Downstream processing of recombinant human insulin and its analogues production from E. coli inclusion bodies

“…Specific additives can be added to the formulation to prevent bacterial growth and protein aggregation, or to modify the in vivo absorption kinetics of insulin. For example, m-cresol could be added to suppress insulin amyloid formation (Ohno et al 2019). Complexation of insulin with zinc and/or protamine can generate intermediate-or long-acting formulations (Waller and Sampson 2017).…”