Sadhale Y, Shah J C
Department of Pharmaceutical Sciences, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA.
Int J Pharm. 1999 Nov 25;191(1):51-64. doi: 10.1016/s0378-5173(99)00288-4.
The main objective of the study was to evaluate if the liquid crystalline cubic phase gel of glyceryl monooleate (GMO) protects insulin from agitation induced aggregation. The aggregation of Humulin(R), Regular Iletin I(R) and Regular Iletin II(R), in cubic phase GMO gels at 30 U/g of gel was compared with that in PBS at 100 oscillations/min at 37 degrees C using optical density at 600 nm. The effect of agitation on the secondary structure of insulin in solution and in the gels was determined with circular dichroism (CD) spectroscopy, and the time course of aggregation was also followed by HPLC. A sigmoidal increase in optical density of solution with time indicated formation of increasing amounts of insoluble insulin aggregates. However, in the gels, optical density values stayed at, or around, the initial optical density value, comparable with that of a blank gel suggesting that insulin had not aggregated in the gel. CD spectroscopy of the soluble insulin showed a total loss of native conformation upon aggregation of insulin in solution. In contrast, CD spectra of insulin in the gel were unaltered suggesting protection from aggregation during agitation. Furthermore, agitation of insulin in gels for a duration as long as 2 months at 37 degrees C, had very little adverse effect on the native conformation of insulin, as indicated by the lack of a significant change in its CD spectrum. Therefore, the cubic phase gel was indeed able to protect insulin from agitation-induced aggregation and subsequent precipitation. Although the majority of insulin in solution appeared to have aggregated and precipitated after 8 days by UV and CD spectroscopy, RP-HPLC results indicated the presence of some soluble aggregates of insulin. In summary, the liquid crystalline cubic phase gel of GMO protects peptides, like insulin, from agitation-induced aggregation.
该研究的主要目的是评估单油酸甘油酯（GMO）的液晶立方相凝胶是否能保护胰岛素免受搅拌诱导的聚集。将含30 U/g凝胶的立方相GMO凝胶中优泌林（R）、正规胰岛素I（R）和正规胰岛素II（R）的聚集情况，与在37℃下以100次振荡/分钟在PBS中的聚集情况进行比较，通过600 nm处的光密度来测定。用圆二色性（CD）光谱法测定搅拌对溶液和凝胶中胰岛素二级结构的影响，并用高效液相色谱法跟踪聚集的时间进程。溶液光密度随时间呈S形增加，表明形成了越来越多的不溶性胰岛素聚集体。然而，在凝胶中，光密度值保持在初始光密度值或其附近，与空白凝胶相当，这表明胰岛素在凝胶中未发生聚集。可溶性胰岛素的CD光谱显示，胰岛素在溶液中聚集时天然构象完全丧失。相比之下，凝胶中胰岛素的CD光谱未发生改变，表明在搅拌过程中受到了防止聚集的保护。此外，在37℃下将凝胶中的胰岛素搅拌长达2个月，对胰岛素的天然构象几乎没有不利影响，这从其CD光谱没有显著变化可以看出。因此，立方相凝胶确实能够保护胰岛素免受搅拌诱导的聚集和随后的沉淀。尽管通过紫外和CD光谱法显示，溶液中的大多数胰岛素在8天后似乎已经聚集和沉淀，但反相高效液相色谱结果表明存在一些可溶性胰岛素聚集体。总之-GMO的液晶立方相凝胶可保护胰岛素等肽免受搅拌诱导的聚集。
