Combined treatment with antitoxin and 3,4-diaminopyridine improves survival outcomes after lethal botulinum neurotoxin challenge.

Abstract

Botulinum neurotoxins (BoNTs) are the most potent toxins known, causing life-threatening flaccid paralysis by blocking acetylcholine release at neuromuscular junctions. Clinical botulism results in respiratory failure, requiring prolonged artificial ventilation for survival. The only specific therapy is antitoxin, which neutralizes circulating toxin but cannot affect toxin within neurons, resulting in a narrow therapeutic window. Due to its high potency and lack of treatment options, large-scale BoNT exposures present profound risks to human life. Thus, there is an urgent need for rapidly acting symptomatic therapies that can reverse respiratory paralysis and sustain survival until resolution of toxin effects. We previously identified the FDA-approved, voltage-gated potassium channel blocker 3,4-diaminopyridine (3,4-DAP) as a fast-acting symptomatic treatment in preclinical botulism models. Here, we expand upon those findings by evaluating continuous infusion of 3,4-DAP in rats exposed to a typical range of BoNT/A doses. Infusion of clinically relevant doses of 3,4-DAP improved survival, reversed respiratory paralysis, and rapidly alleviated clinical signs at toxin doses substantially higher than previously studied. Therapeutic efficacy of 3,4-DAP correlated inversely with toxin exposure, consistent with its proposed mechanism of enhancing acetylcholine release from residual functional synaptic vesicle pools. 3,4-DAP treatment remained effective even when initiated at advanced stages of botulism, when antitoxin monotherapy provided no benefit, significantly extending the clinical treatment window. Combining 3,4-DAP infusion with antitoxin had robust effects on clinical outcomes, reversing clinical symptoms and improving survival compared to either treatment alone. Monitoring of body temperature further revealed that significant hypothermia precedes overt clinical symptoms, providing a novel biomarker of intoxication and treatment efficacy, as toxin-induced hypothermia resolved within hours after 3,4-DAP administration. Collectively, these findings provide robust preclinical evidence supporting clinical translation of 3,4-DAP as a symptomatic reversal agent for botulism, potentially transforming clinical management strategies for this lethal neurotoxin-induced disease.