Diazepam Prodrug Stabilizes Human Aminopeptidase B during Lyophilization.
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.
['Rautiola|Davin|D|0000-0002-1472-2739', 'Updyke|Joel L|JL|', 'Nelson|Kathryn M|KM|0000-0001-8274-2064', 'Siegel|Ronald A|RA|0000-0002-9591-1282']
D000626:Aminopeptidases D055162:Biocatalysis D003975:Diazepam D004151:Dipeptides D004355:Drug Stability D004795:Enzyme Stability D005612:Freeze Drying D005615:Freezing D006801:Humans D008353:Mannitol D011355:Prodrugs D059748:Proteolysis D014199:Trehalose
prodrug stability aminopeptidase B lyophilization avizafone enzyme stabilization freeze-drying temperature dependence