REVERSIBLE ADSORPTION OF SOLUBLE HEXAMERIC INSULIN ONTO THE SURFACE OF INSULIN CRYSTALS COCRYSTALLIZED WITH PROTAMINE - AN ELECTROSTATIC INTERACTION

Mixing pharmaceutical preparations of soluble neutral regular insulin solution (NRI) and neutral protamine Hagedorn (NPH) crystalline insuli n suspension leads to a reduction in the measurable amount of soluble insulin in the formulation supernatant. However in spite of the loss i n soluble insulin, the time-actions of these components have been show n, in clinical trials, to be unaffected. The interaction between these different physical forms of insulin has been studied using reversed-p hase HPLC, isothermal titrating calorimetry, and Doppler electrophoret ic light scattering analysis. Sorbent surface and solution perturbatio n studies revealed that the NRI adsorbs to the surface of the NPH crys tal with an equilibrium constant ranging from 10(4) M(-1) to 10(7) M(- 1), depending on the protamine concentration, pH, ionic strength, and temperature. This adsorption behavior suggests that the binding is med iated by electrostatic interactions arising between the positively-cha rged NPH crystal and the negatively-charged NRI hexamer. Doppler elect rophoretic light scattering results, used to probe the pH-dependent su rface charge of NPH and soluble insulin hexamer, support the conclusio n that electrostatic interactions mediate the adsorption process. Adso rption studies under physiological conditions indicate that the elevat ed temperature and ionic strength, in a subcutaneous depot, are suffic ient to lead to the dissociation of the NRI/NPH complex that exists in these NPH mixture formulations.