Antidotal treatment of botulism in rats by continuous infusion with 3,4-diaminopyridine

Abstract

Botulinum neurotoxins (BoNTs) are highly potent, select agent toxins that inhibit neurotransmitter release at motor nerve terminals, causing muscle paralysis and death by asphyxiation. Other than post-exposure prophylaxis with antitoxin, the only treatment option for symptomatic botulism is intubation and supportive care until recovery, which can require weeks or longer. In previous studies, we reported the FDA-approved drug 3,4-diaminopyridine (3,4-DAP) reverses early botulism symptoms and prolongs survival in lethally intoxicated mice. However, the symptomatic benefits of 3,4-DAP are limited by its rapid clearance. Here we investigated whether 3,4-DAP could sustain symptomatic benefits throughout the full course of respiratory paralysis in lethally intoxicated rats. First, we confirmed serial injections of 3,4-DAP stabilized toxic signs and prolonged survival in rats challenged with 2.5 LD50 BoNT/A. Rebound of toxic signs and death occurred within hours after the final 3,4-DAP treatment, consistent with the short half-life of 3,4-DAP in rats. Based on these data, we next investigated whether the therapeutic benefits of 3,4-DAP could be sustained throughout the course of botulism by continuous infusion. To ensure administration of 3,4-DAP at clinically relevant doses, three infusion dose rates (0.5, 1.0 and 1.5 mg/kg∙h) were identified that produced steady-state serum levels of 3,4-DAP consistent with clinical dosing. We then compared dose-dependent effects of 3,4-DAP on toxic signs and survival in rats intoxicated with 2.5 LD50 BoNT/A. In contrast to saline vehicle, which resulted in 100% mortality, infusion of 3,4-DAP at ≥ 1.0 mg/kg∙h from 1 to 14 d after intoxication produced 94.4% survival and full resolution of toxic signs, without rebound of toxic signs after infusion was stopped. In contrast, withdrawal of 3,4-DAP infusion at 5 d resulted in re-emergence of toxic sign and death within 12 h, confirming antidotal outcomes require sustained 3,4-DAP treatment for longer than 5 d after intoxication. We exploited this novel survival model of lethal botulism to explore neurophysiological parameters of diaphragm paralysis and recovery. While neurotransmission was nearly eliminated at 5 d, neurotransmission was significantly improved at 21 d in 3,4-DAP-infused survivors, although still depressed compared to naïve rats. 3,4-DAP is the first small molecule to reverse systemic paralysis and promote survival in animal models of botulism, thereby meeting a critical treatment need that is not addressed by post-exposure prophylaxis with conventional antitoxin. These data contribute to a growing body of evidence supporting the use of 3,4-DAP to treat clinical botulism.

Aminopyridines Restore Ventilation and Reverse Respiratory Acidosis at Late Stages of Botulism in Mice

“…1, A and B ). These initial studies used 2 mg/kg 3,4-DAP to allow direct comparison with previous reports showing improved toxic signs and activity in intoxicated mice and rats at the same dose ( Vazquez-Cintron et al, 2020 ; Machamer et al, 2022 ).…”

“…Consequently, 3,4-DAP is typically administered to LEMS patients from 3 to 6 times a day at relatively low doses to avoid adverse effects ( Thakkar et al, 2017 ). Previous studies conducted in animal models of botulism lead us to hypothesize that reversal of botulism symptoms requires approximately three- to fourfold higher exposures than are needed for typical patients with LEMS (125 ng/mL versus 32 ng/mL 3,4-DAP) ( Thakkar et al, 2017 ; Vazquez-Cintron et al, 2020 ; Machamer et al, 2022 ). Consequently, there is significant interest in identifying small molecules with more suitable physicochemical properties for the treatment of botulism and other neuromuscular indications.…”

“…Indeed, 3,4-DAP restores phrenic neurotransmission and nerve-evoked muscle contractions in isolated mouse diaphragms poisoned by multiple BoNT serotypes, with improved efficacy for BoNT/A ( Bradford et al, 2018 ). Administration of human-equivalent doses of 3,4-DAP rapidly reverses toxic signs and prolongs survival in mice at terminal stages of botulism ( Vazquez-Cintron et al, 2020 ), whereas continuous infusion of 3,4-DAP has antidotal efficacy in rats challenged with lethal doses of BoNT/A ( Machamer et al, 2022 ).…”

The Necessity of a Locally Active Antidote in the Clinical Practice of Botulinum Neurotoxin Therapy: Short Communication

“…The attempt to influence the duration of the action of BoNT/A by the additional application of BoNT/E, which also cleaves SNAP25 but in a different place than BoNT/A, failed to reduce BoNT/A-induced paralysis [ 33 ]. However, continuous infusion with 3,4-DP showed a symptomatic reversal of BoNT-induced paralysis and antidotal efficacy after a lethal systemic injection with BoNT/A [ 18 ]. Therefore, the systemic application of 3,4-DP may have a beneficial influence on the second type of BoNT/A-induced side effects.…”

“…Ouabain blocked the BoNT action in vitro but was found to enhance BoNT-induced paralysis in vivo [ 12 ]. The reduction in the BoNT action by the application of 3,4-diaminopyridine (DP) in vitro and in vivo has been well known for decades [ 13 , 14 , 15 , 16 , 17 , 18 ], and its clinical efficacy in foodborne botulism is promising [ 19 ]. Furthermore, so far, the clinical efficacy of DP application on BoNT-induced paralysis in humans has been studied systematically.…”