Amyloid Fibrillation of Insulin: Amelioration Strategies and Implications for Translation

Abstract

Insulin is a therapeutically relevant molecule with use in treating diabetes patients. Unfortunately, it undergoes a range of untoward and often unpredictable physical transformations due to alterations in its biochemical environment, including pH, ionic strength, temperature, agitation, and exposure to hydrophobic surfaces. The transformations are prevalent in its physiologically active monomeric form, while the zinc cationcoordinated hexamer, although physiologically inactive, is stable and less susceptible to fibrillation. The resultant molecular reconfiguration, including unfolding, misfolding, and hydrophobic interactions, often results in agglomeration, amyloid fibrillogenesis, and precipitation. As a result, a part of the dose is lost, causing a compromised therapeutic efficacy. Besides, the amyloid fibrils form insoluble deposits, trigger immunologic reactions, and harbor cytotoxic potential. The physical transformations also hold back a successful translation of non-parenteral insulin formulations, in addition to challenges related to encapsulation, chemical modification, purification, storage, and dosing. This review revisits the mechanisms and challenges that drive such physical transformations in insulin, with an emphasis on the observed amyloid fibrillation, and presents a critique of the current amelioration strategies before prioritizing some future research objectives.

Peptide Inhibitors of Insulin Fibrillation: Current and Future Challenges

“…Other factors that accelerate insulin fibrillation include heating, mechanical agitation, and high ionic strength. All these conditions can promote the exposure of the hydrophobic surfaces, while ions can screen the charges and enable hydrophobic interactions [ 42 ]. Shifting the equilibrium to the hexameric form could be an idea to avoid insulin fibrillation, but this approach would also bear disadvantages, considering that insulin is active in the monomeric form.…”

“…Subsequent additions of monomers to the nuclei proceed to form the fibrils [ 44 ]. However, the experimentally observed kinetics are more complex and suggest the formation of secondary nuclei, as well as fibrils’ fragmentation and branching [ 42 ]. Interestingly, the disulfide bridges do not undergo rearrangement during fibrillation and remain intact [ 45 ].…”

“…At 37 °C and acidic pH, there is a decrease in the lag phase, and an increase in the rate with which fibrils grow, relative to what happens at physiological pH values; although, in the pH range from 1.6 to 3.0, there is actually an increase in the lag time and a decrease in the rate of fibril growth [ 44 , 54 ]. Certain preservatives, such as methylparaben, can undergo hydrolysis and generate acidic species, such as 4-hydroxybenzoic acid [ 55 ], which can lower the pH of insulin formulations and promote fibrillation [ 42 ]. In the case of infusion pumps, the pH may become acidic as a result of carbon dioxide mixing or leaching of tubing components [ 56 ].…”

Semisynthesis of A6–A11 lactam insulin

“…The ZR-A1-A5 selenoester peptide 4 was then introduced (8.9 mg, 8.37 μmol), and the solution was shaken for 4 h, followed by aspiration with nitrogen gas for 1 h to remove the DMF. A-chain is combined with recombinant B-chain (7) via thiolysis to form ZR-A6-A11 lactam insulin (8). For this study, the A1-A5 and A12-A21 fragments were conveniently assembled via chemical synthesis, although it should be noted that both these peptide precursors can also be prepared via scalable recombinant techniques.…”

An Investigation into the Acidity-Induced Insulin Agglomeration: Implications for Drug Delivery and Translation

“…However, an acidic environment remains the key diver behind insulin agglomeration. A similar agglomeration of insulin can be induced in a suspension by administering heat, a fluctuating ionic ambiance, exposure to hydrophobic surfaces and to additives . Unfortunately, such unwarranted and often unwelcome agglomeration risks deactivating a considerable part of an administered dose.…”