Gietz U, Arvinte T, Mader E, Oroszlan P, Merkle H P
Swiss Federal Institute of Technology Zürich (ETH), Switzerland.
Eur J Pharm Biopharm. 1998 May;45(3):259-64. doi: 10.1016/s0939-6411(98)00008-3.
In humans, recombinant hirudin (rHir), an anticoagulant protein, has a relatively short half-life (about 1 h). Therefore, a rHir formulation with sustained biological activity was previously proposed to result from complexing zinc salts and rHir (Zn-rHir). The purpose of this paper is to introduce and validate an in vitro release model for subcutaneous Zn-rHir formulations. In glass vials the formulations were suspended in agarose gel (2%) and coated with an extra layer of protein-free agarose. The agarose layers were covered with receiver solution, either buffered solutions (HEPES or PBS, pH 7.4) or human serum. To validate the release model and to demonstrate its potential to discriminate between different formulations, several commercial insulin and Zn-insulin formulations were also tested. The release profiles were evaluated by statistical moment analysis (mean times). Only in HEPES buffer was good discrimination between the investigated insulin formulations observed. The mean times of in vitro release of the insulin formulations and the proposed duration of their biological activities were in correlation. Low discrimination was found in PBS. For rHir, clear discrimination between the investigated rHir formulations was achieved in HEPES buffer, whereas low discrimination was found in PBS or in serum. The developed release model may be a sensitive in vitro test to assure the quality of subcutaneous insulin and rHir formulations, and may also be applicable to assess other slow-release protein and low molecular weight drug injectables.
在人类中，重组水蛭素（rHir）作为一种抗凝蛋白，半衰期相对较短（约1小时）。因此，之前有人提出通过锌盐与rHir复合（Zn-rHir）来制备具有持续生物活性的rHir制剂。本文的目的是介绍并验证一种用于皮下注射Zn-rHir制剂的体外释放模型。在玻璃瓶中，将制剂悬浮于琼脂糖凝胶（2%）中，并用一层无蛋白琼脂糖覆盖。琼脂糖层覆盖有接收液，接收液可以是缓冲溶液（HEPES或PBS，pH 7.4）或人血清。为了验证该释放模型并证明其区分不同制剂的潜力，还测试了几种市售胰岛素和Zn-胰岛素制剂。通过统计矩分析（平均时间）评估释放曲线。仅在HEPES缓冲液中观察到所研究的胰岛素制剂之间有良好的区分度。胰岛素制剂的体外释放平均时间与其预期的生物活性持续时间相关。在PBS中发现区分度较低。对于rHir，在HEPES缓冲液中实现了所研究的rHir制剂之间的清晰区分，而在PBS或血清中发现区分度较低。所开发的释放模型可能是一种敏感的体外试验，可确保皮下胰岛素和rHir制剂的质量，也可能适用于评估其他缓释蛋白和低分子量药物注射剂。
