1 Pharmaceutical Design and Drug Delivery, Department of Pharmacy, Faculty of Health and Medical Sciences, Københavns Universitet2 Analytical Biosciences, Department of Pharmacy, Faculty of Health and Medical Sciences, Københavns Universitet3 Department of Pharmacy, Faculty of Pharmaceutical Sciences, Københavns Universitet4 Novo Nordisk A/S5 Department of Pharmacy, Faculty of Pharmaceutical Sciences, Københavns Universitet6 Pharmaceutical Design and Drug Delivery, Department of Pharmacy, Faculty of Health and Medical Sciences, Københavns Universitet7 Analytical Biosciences, Department of Pharmacy, Faculty of Health and Medical Sciences, Københavns Universitet
Assessment of release kinetics of subcutaneously administered protein therapeutics remains a complex challenge. In vitro methods capable of visualizing and characterizing drug transport properties, in the formulation as well as surrounding subcutaneous tissue environment, are desirable in drug development. Diffusion is a key process in drug release and transport. Thus, our objective was to develop a UV imaging in vitro method for direct visualization and characterization of insulin diffusivity and self-association behavior. Agarose hydrogels were used for mimicking subcutaneous tissue. Diffusivity, self-association, and apparent size of insulin were further characterized by Taylor dispersion analysis, size exclusion chromatography, and dynamic light scattering. At low insulin concentrations and pH 3.0, the hydrodynamic radius of insulin was determined by Taylor dispersion analysis to 1.5±0.1nm, corresponding to the size of insulin monomer. Increasing concentration and pH to 1mM and pH 7.4, respectively, favoring insulin hexamers, increased the insulin hydrodynamic radius to 3.0±0.1nm. The UV imaging method developed was adequately sensitive to identify and characterize, in terms of diffusion coefficients, the changes in insulin transport in hydrogel due to pH and concentration changes. In conclusion, UV imaging allowed insulin diffusion in hydrogel matrixes to be studied in real-time, and showed that insulin self-association properties were reflected in the diffusion behavior. UV imaging is a useful tool for characterization of the influence of environmental conditions on protein mass transport. Hydrogels combined with UV imaging may be of utility for in vitro testing of protein therapeutics.
Journal of Pharmaceutical and Biomedical Analysis, 2014, Vol 92, p. 203-10