Human aminopeptidase B (APB) is a labile enzyme that is being investigated as a biocatalyst for intranasal delivery of prodrug/enzyme combinations. Therefore, the stability of APB is a major concern to ensure a viable drug product. Lyophilization is one technique commonly used to extend shelf life of enzymes. However, the lyophilization process itself can cause conformational changes and aggregation, leading to inactivation of enzymes. In this study, we demonstrate the use of the substrate avizafone (AVF), a prodrug for diazepam, as a stabilizer to minimize inactivation of APB during lyophilization. Permutations of APB samples combined with AVF, trehalose, and/or mannitol were snap-frozen and lyophilized, and subsequently reconstituted to measure the activity of APB. Of the formulation permutations, an APB + AVF + trehalose combination resulted in minimum degradation with 71% retention of activity. This was followed by APB + AVF and APB + trehalose with 60 and 56% retention of activity, respectively. In comparison, APB + mannitol and APB alone retained only 16 and 6.4% activity, respectively. Lyophilizates of the APB + AVF + trehalose formulation were subjected to a 6 month accelerated stability study, at the end of which negligible reduction in activity was observed. These results suggest that colyophilization of an enzyme with its substrate can impart stability on par with the commonly used lyoprotectant, trehalose, but the combination of substrate and trehalose provides a greater stabilizing effect than either additive alone.

中文翻译：

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地西p前药可在冻干过程中稳定人氨肽酶B。

人氨基肽酶B（APB）是一种不稳定的酶，正在研究作为鼻内递送前药/酶组合的生物催化剂。因此，APB的稳定性是确保可行的药物产品的主要关注点。冻干是通常用于延长酶的保存期限的一种技术。但是，冻干过程本身会导致构象变化和聚集，从而导致酶失活。在这项研究中，我们证明了使用底物阿维扎酮（AVF）（地西m的前药）作为稳定剂，可最大程度地减少冻干过程中APB的失活。将速冻和冻干的结合有AVF，海藻糖和/或甘露醇的APB样品的排列，然后冻干，以测量APB的活性。在公式排列中，APB + AVF +海藻糖的组合可将降解降到最低，并保留71％的活性。其次是APB + AVF和APB +海藻糖，分别保留了60％和56％的活性。相比之下，单独的APB +甘露醇和APB仅保留16％和6.4％的活性。对APB + AVF +海藻糖制剂的冻干物进行了为期6个月的加速稳定性研究，最后观察到活性可忽略不计。这些结果表明，酶及其底物的共冻干可以赋予与常用的冻干保护剂海藻糖同等的稳定性，但是底物和海藻糖的组合比单独使用任何一种添加剂都具有更好的稳定作用。其次是APB + AVF和APB +海藻糖，分别保留了60％和56％的活性。相比之下，单独的APB +甘露醇和APB仅保留16％和6.4％的活性。对APB + AVF +海藻糖制剂的冻干物进行了为期6个月的加速稳定性研究，最后观察到活性可忽略不计。这些结果表明，酶及其底物的共冻干可以赋予与常用的冻干保护剂海藻糖同等的稳定性，但是底物和海藻糖的组合比单独使用任何一种添加剂都具有更好的稳定作用。其次是APB + AVF和APB +海藻糖，分别保留了60％和56％的活性。相比之下，单独的APB +甘露醇和APB仅保留16％和6.4％的活性。对APB + AVF +海藻糖制剂的冻干物进行了为期6个月的加速稳定性研究，最后观察到活性可忽略不计。这些结果表明，酶及其底物的共冻干可以赋予与常用的冻干保护剂海藻糖同等的稳定性，但是底物和海藻糖的组合比单独使用任何一种添加剂都具有更好的稳定作用。对APB + AVF +海藻糖制剂的冻干物进行了为期6个月的加速稳定性研究，最后观察到活性可忽略不计。这些结果表明，酶及其底物的共冻干可以赋予与常用的冻干保护剂海藻糖同等的稳定性，但是底物和海藻糖的组合比单独使用任何一种添加剂都具有更好的稳定作用。对APB + AVF +海藻糖制剂的冻干物进行了为期6个月的加速稳定性研究，最后观察到活性可忽略不计。这些结果表明，酶及其底物的共冻干可以赋予与常用的冻干保护剂海藻糖同等的稳定性，但是底物和海藻糖的组合比单独使用任一种添加剂都具有更好的稳定作用。