Gietz U, Arvinte T, Häner M, Aebi U, Merkle H P
Department of Applied BioSciences, Drug Formulation and Delivery, Swiss Federal Institute of Technology Zurich (ETH), Switzerland.
Eur J Pharm Sci. 2000 Jul;11(1):33-41. doi: 10.1016/s0928-0987(00)00072-5.
Sustained release formulations for recombinant hirudin (rHir), an anticoagulant thrombin-specific inhibitor, were developed. Zn-rHir suspensions were formed by precipitation with zinc salts at neutral pH. Characterization of protein precipitation was by UV analysis, capillary electrophoresis (CE), zinc analysis, light and electron microscopy, and particle size analysis. The precipitation of aqueous rHir solution with ZnCl(2) solution at neutral pH resulted in Zn-rHir suspensions. Optimum yields of pelletized Zn-rHir were obtained between pH 7.0 and 7.4. For complete precipitation ( approximately 100%) a molar ratio of zinc to rHir of >28 was necessary. As shown by electron microscopy, the smallest resolvable unit of Zn-rHir suspensions was 20 nm. Agglomerates of up to 200 microm were observed by light microscopy. Zinc salt-induced precipitation phenomena were also investigated using ZnBr(2), ZnI(2), Zn(NO(3))(2) and ZnSO(4) instead of ZnCl(2). ZnSO(4) showed the lowest precipitation efficiency. All other salts behaved similar to ZnCl(2). Upon storage the pelletized protein content of the ZnCl(2) based precipitates was stable ( approximately 95% rHir after 1 year at room temperature), whereas the pelletized protein content of ZnSO(4) based precipitates dropped sharply after precipitation (2% remaining after 13 days at room temperature). This indicates a transition of the ZnSO(4) based precipitates to hexagonal basic zinc sulfate plates and free rHir. The driving force is the lower aqueous solubility of basic zinc sulfate as compared to the higher solubility of basic zinc chloride.
研发了用于重组水蛭素（rHir，一种抗凝性凝血酶特异性抑制剂）的缓释制剂。通过在中性pH值下用锌盐沉淀形成Zn-rHir悬浮液。通过紫外分析、毛细管电泳（CE）、锌分析、光学和电子显微镜以及粒度分析对蛋白质沉淀进行表征。在中性pH值下，rHir水溶液与ZnCl₂溶液沉淀生成Zn-rHir悬浮液。在pH 7.0至7.4之间可获得最佳产率的造粒Zn-rHir。为实现完全沉淀（约100%），锌与rHir的摩尔比必须大于28。电子显微镜显示，Zn-rHir悬浮液的最小可分辨单元为20 nm。光学显微镜观察到尺寸达200 µm的团聚体。还使用ZnBr₂、ZnI₂、Zn(NO₃)₂和ZnSO₄替代ZnCl₂研究了锌盐诱导的沉淀现象。ZnSO₄的沉淀效率最低。所有其他盐的表现与ZnCl₂相似。储存时，基于ZnCl₂的沉淀物的造粒蛋白质含量稳定（室温下放置1年后约95%为rHir），而基于ZnSO₄的沉淀物的造粒蛋白质含量在沉淀后急剧下降（室温下放置13天后仅剩余2%）。这表明基于ZnSO₄的沉淀物转变为六方碱式硫酸锌板和游离rHir。驱动力是碱式硫酸锌的水溶性低于碱式氯化锌。
