Development and Evaluation of Botulinum Neurotoxin Therapeutics

Botulinum neurotoxins (BoNTs) are highly potent neuroparalytic agents that act by inhibiting release of acetylcholine from motor neurons and neuroeffector junctions. Exposure to BoNT results in generalized muscle weakness, culminating in respiratory collapse and death in the absence of critical and supportive care. In previous studies, the potassium channel blocker 3,4-diaminopyridne (3,4-DAP) was shown to counteract BoNT/A-induced muscle paralysis in ex vivo nerve muscle preparations, but protection in systemically intoxicated animals was transient due to the short in vivo half-life of 3,4-DAP. It was hypothesized that the brief half-life of 3,4-DAP may be mitigated by continuous infusion of the drug. Accordingly, we studied the actions of 3,4-DAP infusion in rats intoxicated with sub- and supra-lethal doses of BoNT using survival as the primary endpoint and prolongation of survival time and symptomatic relief as secondary endpoints. The results indicate that a 14-day infusion of 3,4-DAP could protect against BoNT-mediated morbidity and mortality, especially in rats intoxicated by BoNT serotype A (BoNT/A). We have also performed a preliminary study to evaluate the suitability of using the rabbit as a large animal model for carrying out advanced studies on BoNT medical countermeasures. For the current effort, we have determined the LD50 of BoNT/A in New Zealand white rabbits using the animal conserving up-and-down method. The data on rabbits are presented as Part 2 of this report.