Formulation of Synthetic Vectors as Lyophilized Products

描述（由申请人提供）：合成多核苷酸递送系统的开发受到这些胶体悬浮液作为液体制剂的极端不稳定性的阻碍。这种不稳定性也影响了基础研究，迫使科学家对载体制剂进行实验，而这些载体制剂在批次之间容易出现显著的变异性。因此，人们有兴趣开发将允许合成载体配制成稳定的脱水制剂的方法，所述制剂可以大批量制备，并在环境温度下运输和储存。本文提出的工作重点是开发脱水合成载体的方法，使其物理特性和生物活性在急性冻干应激和长期储存期间得以保持。先前关于这些问题的工作已经证明，通过采用大量的糖，可以在急性冻干应激期间实现稳定。不幸的是，稳定化所需的糖的量与肌内或皮下注射在药理学上不相容;对于许多应用，例如，DNA疫苗我们提出了机制研究，以确定在急性冻干应激过程中的载体聚集（损伤的主要机制）的因果关系，并追求在等渗渗透压下实现稳定性的策略。这些初步研究的结果将应用于研究两种方法以提高储存稳定性的实验。在第一种方法中，将直接评估制剂在储存期间减少活性氧物质积累的能力。这些实验利用了一种新的荧光技术，首次允许监测干燥制剂中氧自由基的产生。第二种方法与最近的报告一致，并质疑固态稳定性领域中“越干燥越好”的传统教条。最终，这些方法的结果将在完全优化制剂的2年储存稳定性研究中合并。认识到不同类型的合成载体正在被优化用于基因/多核苷酸递送，这些实验采用不同的模型载体（例如，脂质复合物和聚合复合物）以开发通常适用于大分子复合物稳定化的合理制剂指南（例如，疫苗、病毒、纳米颗粒）。

DESCRIPTION (provided by applicant): The development of synthetic polynucleotide delivery systems is hampered by the extreme instability of these colloidal suspensions as liquid formulations. This instability also affects basic research by forcing scientists to conduct experiments with vector preparations that are prone to significant batch-to-batch variability. As a result, there is interest in developing methods that would allow synthetic vectors to be formulated as stable, dehydrated preparations that could be prepared in large batches, and shipped and stored at ambient temperatures. The work proposed here focuses on developing methods of dehydrating synthetic vectors such that their physical characteristics and biological activity are maintained during both acute lyophilization stress and prolonged storage. Previous work on these problems has demonstrated that stabilization can be achieved during acute lyophilization stress by employing high amounts of sugars. Unfortunately, the amount of sugar needed for stabilization is not osmotically compatible with intramuscular or subcutaneous injection; the preferred method of administration for many applications, e.g., DNA vaccines. We propose mechanistic studies to determine the causative effects of vector aggregation (the major mechanism of damage) during acute lyophilization stress, and pursue strategies that achieve stability at isotonic osmolalities. The findings from these initial studies will be applied to experiments investigating two approaches to enhance storage stability. In the first approach, formulations will be directly assessed for their ability to reduce the accumulation of reactive oxygen species during storage. These experiments utilize a novel fluorescence technique that, for the first time, allows the generation of oxygen radicals in dried preparations to be monitored. The second approach is consistent with recent reports and questions the conventional dogma in the field of solid-state stability that "drier is better". Ultimately, the results from these approaches will be combined in a 2-year storage stability study on fully-optimized formulations. Recognizing that different types of synthetic vectors are being optimized for gene/polynucleotide delivery, these experiments employ different model vectors (e.g., lipoplexes and polyplexes) in order to develop rational formulation guidelines that are generally applicable to the stabilization of macromolecular complexes (e.g., vaccines, viruses, nanoparticles) during freeze-drying and storage.