Evaluation of the preventive and therapeutic administration of an inhibitor of the Thioredoxin - Thioredoxin Reductase (Trx-TrxR) enzyme complex (Ebselen) in human botulism

Background: Botulism is a severe neuroparalytic disease caused by botulinum neurotoxins (BoNTs), which act in the cytosol of nerve cells by specifically cleaving proteins of the SNARE complex, thereby blocking the release of acetylcholine contained in synaptic vesicles. This results in flaccid neuroparalysis that can lead to death due to respiratory failure. BoNTs have been classified by the Centers for Disease Control and Prevention (CDC) in Atlanta as category A agents with potential bioterrorism use. To date, no adequate therapies are available to prevent the effects of BoNTs, and the only available treatment is post-exposure, based on the administration of serotype-specific antisera. However, these antisera can induce individual hypersensitivity reactions and require long preparation times. The study of the molecular mechanism of action of botulinum toxins has enabled the identification of new strategies for the development of effective pharmacological approaches. Preclinical studies indicate that the reduction of the disulfide bond is mediated by the Thioredoxin–Thioredoxin Reductase (Trx–TrxR) redox system located on the cytosolic surface of synaptic vesicles. The identification of this mechanism has led to the discovery of inhibitors of Trx and TrxR, particularly Ebselen, which has been shown to be highly effective in preventing paralysis caused by BoNTs. For this reason, the Trx–TrxR system represents a novel potential therapeutic target for future drug development. Methods: The project included preclinical evidence with translational and clinical development activities. In particular, preclinical studies demonstrating the efficacy of Ebselen in preventing BoNT-induced neuroparalysis were used to support clinical translation. Based on this evidence, a protocol was designed for a randomized, double blind, placebo-controlled phase I/II clinical trial in healthy volunteers, using a human challenge model based on the injection of botulinum neurotoxin type A (BoNT/A) into the extensor digitorum brevis (EDB) muscle. Pharmacodynamic effects will be assessed through electrophysiological measurements of the compound muscle action 4 potential (CMAP). To enable the initiation of the clinical trial, the project included evaluation of the active pharmaceutical ingredient (API) and production of the investigational medicinal product (IMP) in compliance with Good Manufacturing Practice (GMP). In addition, regulatory and ethical aspects were addressed in accordance with Good Clinical Practice (GCP), the European Clinical Trials Regulation (EU No. 536/2014), and CTIS requirements, including preparation for submission to the Ethics Committee. To ensure proper study conduct, a Contract Research Organization (CRO) and a clinical center authorized to conduct Phase I clinical trials in humans were involved. Results: The efficacy of Ebselen in inhibiting BoNT-induced neuroparalysis across different serotypes through blockade of the Trx–TrxR system is supported by preclinical studies. Furthermore, previous clinical studies have demonstrated a favorable safety and tolerability profile in humans. These data represent a key element, as they reduce translational uncertainty and support the feasibility of a clinical trial in this new indication. The clinical trial protocol was successfully developed, integrating safety, pharmacokinetic, and exploratory pharmacodynamic endpoints within an adaptive design. The EDB muscle is confirmed to be reproducible and sensitive for detecting pharmacodynamic effects in early-phase studies; moreover, CMAP is suitable for the electrophysiological assessment of neuromuscular transmission in the EDB. Based on these premises, the regulatory submission process to the competent authorities has been initiated to obtain authorization to start the phase I/II clinical trial in healthy volunteers. Conclusions: This study demonstrates the feasibility of translating mechanistic knowledge on BoNT activation into early clinical development of a novel “host directed” therapeutic strategy. Ebselen represents a promising candidate for the treatment of botulism, with potential advantages over current antitoxin-based therapies, including broader applicability across different BoNT serotypes. Furthermore, this project provides a comprehensive translational framework for the development of medical countermeasures against high-impact neurotoxins.