Abstract
PurposeComparison of the dissociation kinetics of rapid-acting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions.MethodsDissociation kinetics after dilution were monitored directly in terms of the average molecular mass using combined static and dynamic light scattering. Changes in tertiary structure were detected by near-UV circular dichroism.ResultsGlulisine forms compact hexamers in formulation even in the absence of Zn2+. Upon severe dilution, these rapidly dissociate into monomers in less than 10 s. In contrast, in formulations of lispro and aspart, the presence of Zn2+ and phenolic compounds is essential for formation of compact R6 hexamers. These slowly dissociate in times ranging from seconds to one hour depending on the concentration of phenolic additives. The disadvantage of the long dissociation times of lispro and aspart can be diminished by a rapid depletion of the concentration of phenolic additives independent of the insulin dilution. This is especially important in conditions similar to those after subcutaneous injection, where only minor dilution of the insulins occurs.ConclusionKnowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins.
Highlights
One of the great challenges for biotechnological research is the optimum substitution of the natural regulation of the active insulin concentration in blood lost in diabetic patients by appropriate insulin analogs and advanced injection techniques
The association states at insulin concentrations of 0.17 mg/ml marked by a dashed vertical line correspond to the final states that are approached during kinetic experiments using 1:20 dilution jumps
The results show the influence of specific modifications of the formulation conditions of lispro, aspart, glulisine and human insulin (HI) on the association states and the dissociation kinetics
Summary
One of the great challenges for biotechnological research is the optimum substitution of the natural regulation of the active insulin concentration in blood lost in diabetic patients by appropriate insulin analogs and advanced injection techniques. This comprises both the establishment of the basal level of insulin by long-acting insulins and the fast adjustment of the instantaneous insulin concentration in accordance to food uptake by rapid-acting insulins (RAI) [1]. Two opposing requirements, high stability of insulin in formulation and fast dissociation after subcutaneous injection play an important role for the development of RAI
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