Continuous Subcutaneous Insulin Infusion (CSII) is superior to conventional insulin therapy as it improves glycemic control thus reducing the probability of diabetic complications. Notwithstanding CSII's benefits, insulin dependent diabetic patients rarely achieve optimal glucose control. Moreover, CSII is only FDA approved for 3 days and often fails prematurely for reasons that have not been fully elucidated. We hypothesize that phenolic compounds, such as m‐cresol and phenol, which are present in all commercial insulin formulations are responsible for the tissue reaction occurring at the insulin infusion site. This hypothesis was examined with in vitro cell cultures and a mouse air‐pouch model to determine cellular and tissue reactions following infusions with saline, phenolic compounds, (i.e., commercial diluent), and insulin. We demonstrated that diluent and insulin were cytotoxic to cells in culture at sub‐clinical concentrations (e.g., >1:10 of commercial insulin). Air pouch studies demonstrated that infusion of either diluted insulin or diluent itself induced three to five‐fold level of recruited leukocytes as compared to saline. At both 3‐ and 7‐days post infusion, these were predominantly neutrophils and macrophages. We conclude that phenolic compounds in commercial insulin preparations are cell and tissue toxic, which contributes to the failure of effective insulin infusion therapy.

Background: Exogenous insulin therapy requires stabilization of the insulin molecule, which is achieved through the use of excipients (e.g., phenolic preservatives (PP)) that provide protein stability, sterility and prolong insulin shelf life. However, our laboratory recently reported that PP, (e.g., m-creosol and phenol) are also cytotoxic, inducing inflammation and fibrosis. Optimizing PP levels through filtration would balance the need for insulin preservation with PP-induced inflammation. Method: Zeolite Y (Z-Y), a size-exclusion-based resin, was employed to remove PP from commercial insulin formulations (Humalog) before infusion. Results: PP removal significantly decreased cell toxicity in vitro and inflammation in vivo. Infusion site histological analysis after a 3 day study demonstrated that leukocyte accumulation increased with nonfiltered preparations but decreased after filtration. Additional studies demonstrated that a Z-Y fabricated filter effectively removed excess PP such that the filtered insulin solution achieved equivalent glycemic control in diabetic mice when compared to nonfiltered insulin. Conclusion: This approach represents the proof of concept that using Z-Y for in-line PP removal assists in lowering inflammation at the site of insulin infusion and thus could lead to extending the functional lifespan of insulin infusion sets in vivo.

Background: Effective exogenous insulin delivery is the cornerstone of insulin dependent diabetes mellitus management. Recent literature indicates that commercial insulin-induced tissue reaction and cellular cytotoxicity may contribute to variability in blood glucose as well as permanent loss of injection or infusion site architecture and function. It is well accepted that insulin formulations are susceptible to mechanical and chemical stresses that lead to insulin fibril formation. This study aims to characterize in vitro and in vivo toxicity, as well as pro-inflammatory activity of insulin fibrils. Method: In vitro cell culture evaluated cytotoxicity and fibril uptake by macrophages and our modified murine air-pouch model quantified inflammatory activity. The latter employed FLOW cytometry and histopathology to characterize fibril-induced inflammation in vivo, which included fibril uptake by inflammatory phagocytes. Results: These studies demonstrated that insulin derived fibrils are cytotoxic to cells in vitro. Furthermore, inflammation is induced in the murine air-pouch model in vivo and in response, macrophages uptake fibrils both in vitro and in vivo. Conclusions: Administration of insulin fibrils can lead to cytotoxicity in macrophages. In vivo data demonstrate insulin fibrils to be pro-inflammatory which over time can lead to cumulative cell/tissue toxicity, inflammation, and destructive wound healing. Long term, these tissue reactions could contribute to loss of insulin injection site architecture and function.

The approximation of euglycemia is the most effective means of preventing diabetic complications, which is achieved through effective insulin delivery. Recent reports indicate that insulin phenolic preservatives, which are found in all commercial insulin formulations, are cytotoxic, pro-inflammatory and induce secondary fibrosis. Therefore, we hypothesize that these preservatives induce an inflammatory response at the site of insulin infusion leading to diminished glycemic control and adverse pharmacokinetic outcomes. Insulin degradation by inflammatory cell proteases was quantitated following protease treatment in vitro. A modified murine air pouch model was utilized to evaluate the relative inflammatory responses following infusions of saline, insulin preservatives, and insulin, utilizing the adjuvant irritant thioglycolate. Blood glucose levels were monitored in diabetic mice with and without air pouch irritation. A pharmacokinetic analysis evaluated insulin effectiveness for diabetic mice between these two conditions. Inflammatory cells are significantly present in insulin preservative-induced inflammation, which effects diminished blood glucose control by both insulin uptake and degradation. Insulin containing these preservatives resulted in similar degrees of inflammation as observed with the irritant thioglycolate. These studies imply that the preservative agents found in commercial insulin formulations induce an intense localized inflammatory reaction. This inflammatory reaction may be responsible for the premature failure of insulin infusion devices. Future studies directed at reducing this inflammatory reaction may prove to be an important step in extending the lifespan of insulin infusion devices.

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