Novel Antidotal Treatment of Botulism Using an FDA‐Approved Small Molecule

Botulinum neurotoxins (BoNTs) are exceedingly potent neuroparalytic toxins which block release of neurotransmitter from motor nerve terminals, causing respiratory failure at lethal doses. The only treatment for botulism is post‐exposure prophylaxis with antitoxin. However, antitoxin is most effective when given prior to symptomatic emergence, and consequently 70% of symptomatic patients require artificial ventilation for a median of two weeks. The voltage‐gated potassium channel antagonist 3,4‐diaminopyridine (DAP) has been proposed as a symptomatic treatment to mitigate muscle weakness in botulism patients. However, experimental support for this hypothesis is lacking. To test the efficacy of DAP in treating toxic signs of botulism, we first evaluated concentration‐dependent effects of DAP on synaptic transmission and muscle function in mouse hemidiaphragm preparations following intoxication by BoNT serotypes A, B or E. For all serotypes, DAP increased quantal content and restored muscle contraction, with effect sizes that were inversely proportional to the degree of paralysis. Based on these findings, we evaluated the effects of human‐equivalent doses of DAP in mice intoxicated with the same BoNT serotypes. In a sublethal intoxication model, DAP transiently increased respiratory function at multiple time points for all serotypes. In lethal challenges with BoNT/A, repeated administration of DAP sustained respiratory function and prolonged survival throughout the treatment period. These findings suggested that DAP could have antidotal efficacy if continuously delivered. To test for antidotal capability, DAP was continuously infused at nontoxic levels in rats intoxicated with 2 LD50 of BoNT/A, starting after signs of botulism emerged. DAP enabled 100% survival, whereas saline‐treated rats died in 2.8 days. Infusion was stopped at two weeks once respiratory signs of botulism resolved, and rats remained healthy and active through study end at three weeks, without symptomatic rebound. Following euthanasia, electrophysiological characterization revealed a large fraction of phrenic endplates that continued to exhibit impaired neuromuscular transmission, despite the apparent absence of physiological signs of botulism. These data suggest that treatment with DAP allowed for the emergence of compensatory changes in neuromuscular function that promoted physiological recovery. Not only do the underlying mechanisms represent fertile ground for therapeutic exploitation, but they are also important for understanding regenerative responses following transient chemical denervation of the respiratory system. These studies indicate that DAP has unique potential to improve respiratory function during botulism. Furthermore, they demonstrate the first antidotal treatment of type A botulism in vivo. We are currently preparing to evaluate the effects of DAP on respiratory function and intensity and duration of supportive care in patients suffering natural and iatrogenic botulism. Support or Funding Information This project was funded by the Defense Threat Reduction Agency and NIH (R01A193504); and supported by appointments to the Postgraduate Research Participation Program at USAMRICD administered by the Oak Ridge Institute for Science and Education.