Jensen Sabrine S, Jensen Henrik, Cornett Claus, Møller Eva H, Østergaard Jesper
Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
J Pharm Biomed Anal. 2014 Apr;92:203-10. doi: 10.1016/j.jpba.2014.01.022. Epub 2014 Jan 26.
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.
皮下注射蛋白质治疗药物释放动力学的评估仍然是一项复杂的挑战。在药物研发过程中，需要能够可视化并表征制剂以及皮下组织周围环境中药物转运特性的体外方法。扩散是药物释放和转运的关键过程。因此，我们的目标是开发一种紫外成像体外方法，用于直接可视化并表征胰岛素的扩散率和自缔合行为。使用琼脂糖水凝胶模拟皮下组织。通过泰勒分散分析、尺寸排阻色谱法和动态光散射进一步表征胰岛素的扩散率、自缔合和表观尺寸。在低胰岛素浓度和pH 3.0条件下，通过泰勒分散分析确定胰岛素的流体动力学半径为1.5±0.1nm，对应于胰岛素单体的尺寸。将浓度和pH分别提高到1mM和pH 7.4，有利于胰岛素六聚体的形成，使胰岛素流体动力学半径增加到3.0±0.1nm。所开发的紫外成像方法具有足够的灵敏度，能够根据扩散系数识别和表征水凝胶中胰岛素转运因pH和浓度变化而发生的改变。总之，紫外成像能够实时研究胰岛素在水凝胶基质中的扩散，并表明胰岛素的自缔合特性反映在扩散行为中。紫外成像对于表征环境条件对蛋白质质量转运的影响是一种有用的工具。水凝胶与紫外成像相结合可能对蛋白质治疗药物的体外测试有用。
