Ex Vivo and In Vivo Evaluation of a Novel Broad-Spectrum Nerve Agent Antidote Formulation for Central Nervous System Delivery

Abstract

JDS364.HCl, an oxime-based reactivator developed for the treatment of organophosphorus nerve agent intoxication, has recently emerged as a promising uncharged broad-spectrum hybrid antidote. Building on previously reported in vitro stability data of JDS364.HCl formulated with cyclodextrins, the present study provides an ex vivo characterization and in vivo evaluation of the toxicity, biodistribution, metabolism, and therapeutic efficacy of the formulated reactivator in a murine model. Toxicological assessment identified injection route- and formulation-dependent NOAEL values, highlighting the influence of both the route of administration and cyclodextrin complexation on systemic toxicity. A dose-dependent inhibition of acetylcholinesterase activity was observed in specific tissues, particularly the brain and respiratory organs, providing mechanistic insights into the physiological alterations associated with higher doses of JDS364.HCl reactivator. Pharmacokinetic and biodistribution analyses revealed that formulated JDS364.HCl was rapidly distributed systemically, crossed the blood-brain barrier, and was predominantly eliminated via renal clearance. Metabolic profiling further demonstrated extensive and rapid metabolization, primarily through glucuronidation and dehydration pathways. Finally, the in vivo efficacy study indicated that, although formulation with HP-β-CD improved the stability of JDS364.HCl in solution, it did not significantly increase the protective efficacy index against VX intoxication compared with the unformulated antidote.

Introduction

Despite the prohibition of chemical weapons since 1997 (Chemical weapons conven on, 2020), nerve agents such as Novichok agents and VX remain a signi cant threat to global security, as demonstrated by their use in several targeted poisoning incidents (Wiaderek et al., 2025). These compounds, characterized by potent neurotoxicity, belong to the organophosphorus (OP) family, which also includes several widely used pes cides (Mali et al., 2023). The toxicity of OP compounds results from the irreversible inhibition of acetylcholinesterase (AChE), leading to the accumula on of acetylcholine at cholinergic synapses and sustained cholinergic oversmula on. This pathological hypersmula on can cause severe neurological impairments, respiratory failure accompanied by hypoxia, and ul mately death (Mercey et al., 2012). Although FDA-approved an dotes, such as oxime-based AChE reac vators including pralidoxime (2-PAM), are currently available, their therapeuticti efficacy remains limited. These limitations are primarily a ributed to poor blood-brain barrier (BBB) penetra on and insufficient reactivation eticacy across the full spectrum of OP compounds. Consequently, the development of more e ec ve therapeu c alterna ves is essen al. Recent research has focused on the design of novel an dotes exhibi ng enhanced reactivati on potency toward OP compound-inhibited AChE, improved BBB permeability, andbroad-spectrum ac vity (Voros et al., 2024). Among the most promising strategies is the development of uncharged hybrid reac vators (Kliachyna et al., 2014; Renou et al., 2013; Mercey et al., 2011), which combine a conven onal oxime moiety with a peripheral site ligand (PSL) connected via an alipha c linker (De Koning et al., 2011). Within this class of compounds, the patented molecule JDS364.HCl, a 3-hydroxy-2-pyridinaldoxime deriva ve bearing a 4 aminoquinoline PSL (Fig. 1), has demonstrated strong in vitro AChE reac va on ac vity (De Sousa et al., 2026). Moreover, JDS364.HCl demonstrated superior in vitro BBB permeability compared with marketed an dotes such as 2-PAM (De Sousa et al., 2026; Thiberville et al., 2025). However, further clinical development has been limited by insu cient stability in aqueous solu ons.

To overcome the instability of JDS364.HCl, several formula on strategies were considered (Thiberville et al., 2025). Among these, complexa on with cyclodextrins (CDs) emerged as the most e ec ve approach. Two CDs approved by the European Medicines Agency (EMA) for intravenous (IV) administra on, namely, hydroxypropyl-β cyclodextrin (HP-β-CD) and sulfobutyl ether-β-cyclodextrin (SBE-β-CD) (Fig. 1), were shown to complex JDS364.HCl with a 1:1 stoichiometry (Thiberville et al., 2025). Complexa on markedly enhanced the apparent aqueous stability of JDS364.HCl for more than two months at room temperature, as well as in plasma, while preserving in vitro BBB permeability. However, the higher binding a nity of the JDS364.HCl:SBE-β-CD complex limited drug dissocia on, thereby reducing BBB permeability rela ve to unformulated JDS364.HCl, as observed in a human in vitro BBB model (Thiberville et al., 2025).

In this study, the ex vivo and in vivo proper es of JDS364.HCl:HP-β-CD complexes were inves gated in a murine model to support the development of a next-genera on an dote for OP compound poisoning. First, the interac ons of JDS364.HCl with biological matrices were inves gated and characterized by assessing plasma protein binding and red blood cell (RBC) partitoning. Subsequently, an in vivo toxicity study was conducted in mice following IV administra on of JDS364.HCl:HP-β-CD complexes. The no-observed-adverse-e ect-level (NOAEL), dened as the highest dose that does not induce any detectable adverseti effects, was determined, and AChE affevity was measured in non-intoxicated mice. Pharmacokine c (PK) and biodistribution studies were then performed to evaluate brain penetration, ssue distribution, and metaboliza on. In addi on, the in uence of the route of administration was inves gated by comparing IV, intraperitoneal (IP), and intramuscular (IM) administra on. Finally, the therapeu cieticacy of JDS364.HCl:HP-β-CD complexes administered via the IP route was assessed in VX intoxicated mice, by determining the protec ve index (PI).

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Materials

JDS364.HCl (MW = 372.86 g/mol) and JDS207.HCl (MW = 461.39 g/mol) were synthesized by Prestwick Chemical and supplied by the ICPEES UMR CNRS 7515 laboratory (purity > 95 %). The same batches were used for all experiments. HP-β-CD (Kleptose® HPB; average degree of substitution = 4.3; MW = 1387 g/mol) and SBE-β-CD (Captisol®; average degree of substitution = 6.6; MW = 2163 g/mol) were kindly provided by Roquette (Lestrem, France). Ultrapure Milli-Q® (Merck Millipore, Burlington, MA, USA) water was used throughout the study. All solvents, including acetonitrile containing 0.1 % formic acid, water containing 0.1 % formic acid, ethanol, and methanol, were obtained from VWR Chemicals (France).

Léa Thiberville, Anne-Sophie Hanak, Catherine Cailleau et al. Ex Vivo and In Vivo Evaluation of a Novel Broad-Spectrum Nerve Agent Antidote Formulation for Central Nervous System Delivery, 22 September 2026, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-10974318/v1]

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