Strickley R G, Anderson B D
Department of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, Salt Lake City 84112, USA.
J Pharm Sci. 1997 Jun;86(6):645-53. doi: 10.1021/js9700311.
Previous studies have established that at low pH human insulin decomposition proceeds through a two-step mechanism involving rate-limiting intramolecular formation of a cyclic anhydride intermediate at the C-terminal AsnA21 followed by intermediate partitioning to various products, most notably desamido insulin and covalent dimers, in both aqueous solution and in the amorphous (lyophilized) solid state. This study examines the product distribution resulting from insulin degradation in lyophilized powders as a function of water content and the phase behavior of the solid (glassy versus rubbery) between pH 3 and 5. In amorphous solids at low water content (glassy state), the cyclic anhydride intermediate of insulin reacts predominantly with water to form deamidated insulin, whereas the intermolecular reaction with another insulin molecule to form a covalent dimer accounts for < or = 15% of the total degradation. Increasing water content reduces the glass transition temperature of insulin to < 35 degrees C, and covalent dimer formation becomes increasingly favored relative to deamidation. An increase in solid-state pH also favors dimerization as deprotonation of the terminal amino groups of insulin renders them more nucleophilic. Covalent dimerization was almost totally suppressed by incorporation into a glassy matrix of trehalose, which both minimizes molecular mobility and physically separates the insulin molecules. The kinetics and product distribution of human insulin in lyophilized powders between pH 3 and 5 illustrate the differential sensitivities of various solid-state reaction types to the effects of water activity and solid-phase behavior. The intramolecular cyclization at the AsnA21 position requires only short-range conformational flexibility and thus is only modestly restricted even in the glassy state. On the other hand, the competing bimolecular reactions involving either water or another molecule of insulin combining with the intermediate anhydride are dependent on molecular mobility of the reactants, in accord with predictions of free volume theory. In the glassy state, deamidation (reaction with water) is favored because of the restricted molecular mobility of proteins in rigid matrices. Increasing plasticization with increasing water content favors covalent aggregate formation because of the higher dependence of protein mobility on free volume within the solid matrix.
先前的研究已证实，在低pH值条件下，人胰岛素的分解通过两步机制进行，该机制包括在C端天冬酰胺A21处限速的分子内环酐中间体形成，随后中间体分配为各种产物，最显著的是脱酰胺胰岛素和共价二聚体，这一过程在水溶液和无定形（冻干）固态中均会发生。本研究考察了冻干粉末中胰岛素降解产生的产物分布与含水量以及pH值在3至5之间时固体（玻璃态与橡胶态）相行为的函数关系。在低含水量的无定形固体（玻璃态）中，胰岛素的环酐中间体主要与水反应形成脱酰胺胰岛素，而与另一个胰岛素分子发生分子间反应形成共价二聚体的反应占总降解量的比例≤15%。含水量增加会使胰岛素的玻璃化转变温度降至<35℃，相对于脱酰胺反应，共价二聚体的形成变得越来越有利。固态pH值的升高也有利于二聚化，因为胰岛素末端氨基的去质子化使其亲核性增强。通过将胰岛素掺入海藻糖的玻璃态基质中，共价二聚化几乎被完全抑制，这既使分子流动性最小化，又使胰岛素分子在物理上相互分离。人胰岛素在pH值3至5之间的冻干粉末中的动力学和产物分布说明了各种固态反应类型对水活性和固相结合行为影响的不同敏感性。天冬酰胺A21位置的分子内环化仅需要短程构象灵活性，因此即使在玻璃态下也仅受到适度限制。另一方面，涉及水或另一个胰岛素分子与中间酸酐结合的竞争性双分子反应取决于反应物的分子流动性，这与自由体积理论的预测一致。在玻璃态下，脱酰胺反应（与水反应）占优，因为蛋白质在刚性基质中的分子流动性受限。随着含水量增加而导致的增塑作用有利于共价聚集体的形成，因为蛋白质流动性对固体基质内自由体积的依赖性更高。
