# Cyclodextrin-based formulations for delivering broad-spectrum nerve agent antidote to the central nervous system: stability, physicochemical characterization and application in a human blood-brain barrier model

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Léa Thiberville<sup>a,b</sup>, Vincent Faivre<sup>a,*</sup>, Christina Sizun<sup>c</sup>, Marie-Pierre Dehouck<sup>d</sup>, Christophe Landry<sup>d</sup>, Rachid Baati<sup>b,*</sup>, Nicolas Tsapis<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Cyclodextrins

Acetylcholinesterase reactivator

Nerve agent antidote

Human blood-brain barrier model

## ABSTRACT

Nerve agents, such as VX and sarin, represent a significant threat to global security due to their devastating neurotoxic effects and potential for misuse. The therapeutic inefficacy of current countermeasures underscores the urgent need for more effective alternatives. In this context, recent advances have identified JDS364.HCl, an uncharged hybrid antidote, as a promising candidate. However, its instability in aqueous solution remains a significant challenge. To address this, cyclodextrin-based formulations were developed using two EMA-approved cyclodextrins: HP-β-CD and SBE-β-CD. These formulations significantly improved JDS364.HCl stability for over two months at room temperature. Interaction studies revealed a 1:1 stoichiometry for both cyclodextrin complexes, with JDS364.HCl: SBE-β-CD exhibiting a 100-fold higher affinity constant, attributed to additional electrostatic interactions with SBE-β-CD sidechains. While SBE-β-CD provided superior plasma stability compared to HP-β-CD, the high binding affinity of JDS364.HCl: SBE-β-CD complexes hindered the molecule's release and reduced its ability to cross the BBB, as observed in a human BBB model. Nonetheless, the results for both cyclodextrins are encouraging, as they enhance JDS364.HCl's stability in plasma while allowing its passage across the BBB. Notably, JDS364.HCl demonstrated superior BBB permeability compared to marketed antidotes such as 2-PAM. These findings highlight the potential of cyclodextrins to improve the efficacy of JDS364.HCl as a nerve agent antidote.

## 1. Introduction

Organophosphorus compounds (OPs) are widely used in agriculture as pesticides such as paraoxon, glyphosate and chlorpyrifos (Mali et al., 2023; Guyton et al., 2015). However, their neurotoxic properties also led to their notorious use as chemical weapons, including agents like Novichok, VX, sarin and soman (Gupta, 2020; John et al., 2018; Seto et al., 1998). These compounds are highly toxic molecules with the potential to cause mass casualties. Mechanistically, they act by phosphorylating the serine in the active site of acetylcholinesterase (AChE),

thereby inhibiting this enzyme, which is crucial for neuronal transmission (Mercey et al., 2012). This inhibition prevents the hydrolysis of acetylcholine, resulting in the accumulation of the neurotransmitter at nerve synapses and leading to continuous stimulation of central nervous system (CNS) muscarinic and cholinergic receptors. Symptoms frequently observed following OP poisoning range from myosis and excessive salivation to convulsions, paralysis, and death (Sidell and Borak, 1992) due to cholinergic crisis. The rapid and severe onset of these symptoms make OPs particularly dangerous, both to military and civilian populations.

**Abbreviations:** 2-PAM, pralidoxime; AChE, acetylcholinesterase; BBB, blood-brain barrier; CD, cyclodextrin; CNS, central nervous system; EMA, European Medicines Agency; HP-β-CD, hydroxylpropyl-β-cyclodextrin; NOAEL, No Observable Adverse Effect Level; OPNA, organophosphorus-nerve agents; RT, room temperature; SBE-β-CD, sulfobutylether-β-cyclodextrin.

* Corresponding authors.

E-mail addresses: vincent.faivre@universite-paris-saclay.fr (V. Faivre), rachid.baati@unistra.fr (R. Baati), nicolas.tsapis@universite-paris-saclay.fr (N. Tsapis).

<footnote><sup>a</sup> Université Paris-Saclay, CNRS, Institut Galien Paris-Saclay, 91400 Orsay, France</footnote>

<footnote><sup>b</sup> Ecole de Chimie Polymères et Matériaux, Université de Strasbourg, Institut de Chimie et Procédés pour l'Energie, l'Environnement et la Santé UMR CNRS 7515, 67087 Strasbourg, France</footnote>

<footnote><sup>c</sup> CNRS, Université Paris-Saclay, Institut de Chimie des Substances Naturelles, 91900 Gif-sur-Yvette, France</footnote>

<footnote><sup>d</sup> Laboratoire de la Barrière Hémato-Encéphalique, Université d'Artois, Lens, France</footnote>
To reactivate the inhibited AChE, the phosphorus group bound to the enzyme must be displaced. This requires an antidote, known as an AChE reactivator, with strong nucleophilic properties to attack the electrophilic phosphorus atom, thus releasing and regenerating the native enzyme (Eyer, 2003). Currently available reactivators such as pralidoxime (2-PAM), obidoxime, and asoxime (HI-6) are based on the use of an oxime moiety (Wilson and Ginsburg, 1955; Luettringhaus and Hagedorn, 1964; Kušić et al., 1985). These compounds are typically administered intramuscularly via an autoinjector in combination with a muscarinic-receptor antagonist like atropine and an anti-convulsant drug of the benzodiazepine family (Timperley et al., 2019; Rousseau et al., 2009). Unfortunately, these reactivators are limited in treating OP poisoning due to their inability to reactivate aged AChE, their poor penetration of the blood-brain barrier (BBB) and their poor efficacy against all nerve agents (Worek et al., 2016; Kuca et al., 2007).

Given the limitations of current FDA-approved AChE reactivators, discovering more effective alternatives is essential. Recent research has focused on developing antidotes with higher reactivation potential, improved BBB penetration, enhanced affinity for AChE, and broad-spectrum activity (Voros et al., 2024). A promising new approach involves the use of uncharged hybrid reactivators (Kliachyna et al., 2014; Renou et al., 2013; Mercey et al., 2011), which combine a typical oxime structure with a peripheral site ligand (PSL) via a linker (De Koning et al., 2011). The PSL interacts with aromatic residues at the top of the enzyme's gorge, enhancing the reactivator's affinity for the inhibited AChE. The aim is to achieve an optimal balance between affinity and reactivity, characterized by the lowest dissociation constant ($k_D$) and the highest reactivation constant ($k_R$) (Worek et al., 2011). Building on this strategy, Baati and al. patented a new 3-hydroxy-2-pyridinaldoxime hybrid reactivators family (Baati et al., 2017) (Fig. 1). Among this family, JDS364.HCl, bearing the 4-aminooquinoline group as the PSL, was selected as a model molecule as it showed the most interesting in vitro properties. In particular, it showed in *in vitro* assays the best reactivation efficacy for VX and tabun-inhibited AChE (Sousa, 2015). Moreover, preliminary *in vivo* tests in Swiss mice model, including determination of the No-Observable-Adverse-Effect-Level (NOAEL) by intraperitoneal route at 35 µmol/kg, have been reported for JDS364.HCl (Trancart et al., 2024).

Despite promising *in vitro* results, no broad-spectrum uncharged hybrid reactivator has yet been approved for treatment due to challenges such as solubility, stability, bioavailability, and toxicity. Specifically, instability issues in aqueous solutions have prevented molecules like JDS364.HCl from being used for further *in vivo* efficacy studies. Indeed, its inability to be concentrated limits its suitability for intramuscular administration, which requires a small injection volume. A key strategy to overcome these limitations is the development of optimized drug delivery systems that enhance aqueous solubility. Although no formulation has yet been published for the reactivators patented by Baati and al. (2017), various AChE reactivators have been formulated to improve their penetration into the brain, including 2-PAM, which has been incorporated in zirconium-based organic polyhedral (Delgado et al., 2022), liposomes (Kuznetsova et al., 2022; Vasilieva et al., 2022; Pashirova et al., 2018) and solid lipid nanoparticles (Pashirova et al., 2017). However, formulation approaches carry the risk of delaying

blood-brain barrier (BBB) penetration (Pashirova et al., 2017) which is undesirable for emergency post-exposure treatments. Therefore, developing a formulation with a rapid-release mechanism is essential. Additionally, the ideal formulation should be simple, cost-effective, and use excipients approved by the European Medicines Agency (EMA) to facilitate industrialization.

Considering these factors, the use of co-solvents, surfactants, nanoemulsions and cyclodextrins (CDs) were considered. Briefly, co-solvent systems disrupt the self-association of water molecules, thereby reducing the interfacial tension between the aqueous solution and the hydrophobic drug. Surfactants, characterized by their amphiphilic nature, can solubilize molecules either through a direct co-solvent effect or by encapsulation into micelles (He et al., 2022). Nanoemulsions solubilize lipophilic drugs by incorporating them into oil nanodroplets, increasing their dispersibility. Finally, CDs are cyclic oligosaccharides composed of D-glucopyranose units, containing a hydrophobic central cavity and a hydrophilic outer surface (Brewster and Loftsson, 2007). Due to their unique structure, these molecules are known to form inclusion complexes with some hydrophobic active substances, significantly increasing their water-solubility (Saokham et al., 2018). The two most common water-soluble β-CD derivatives, hydroxypropyl-β-cyclodextrin (HP-β-CD) and sulfobutylether-β-cyclodextrin (SBE-β-CD) (Ferreira et al., 2022; Challa et al., 2005) are EMA-approved for intravenous administration. HP-β-CD is neutral and substituted with $-CH_2CH(OH)CH_3$ with an average degree of substitution of 2.8–10.5. SBE-β-CD, on the other hand, is polyanionic and substituted with $-(CH_2)_4SO_3Na^+$ with an average degree of substitution of 6.5.

This study aims to select the optimal formulation for stabilizing JDS364.HCl, characterize its physicochemical properties, and assess the BBB permeability of the formulated JDS364. The target concentration was set at 20 mg/ml, to enable dosing mice with an amount corresponding to the NOAEL (Trancart et al., 2024) via any parenteral route. These investigations are crucial for advancing the therapeutic potential of JDS364.HCl and paving the way for the future clinical development of a new broad-spectrum OP nerve agent reactivator.

## 2. Material & methods

### 2.1. Material

HP-β-cyclodextrin (Kleptose® HPB; average degree of substitution = 4.3; MW = 1387 Da) and SBE-β-CD (Captisol®; average degree of substitution = 6.6; MW = 2163 Da) were kindly provided by Roquette (Lestrem, France). JDS364.HCl (MW = 372.86 Da) and JDS207.HCl (MW = 461.39 Da) were synthesized by Prestwick Chemical and provided by ICPEES UMR CNRS 7515 laboratory with a purity > 95 %. The same batch was used for all experiments. JDS364 (free form) was synthesized following an optimized protocol inspired by the patent of Baati and al. (2017) (Supplementary Material A).

In all experiments, Ultrapure Milli-Q® water was used. Miglyol® 812 N was kindly provided by IOI Oleochemical (Malaysia), and refined olive oil, refined sesame oil, refined sesame oil by ADM-SIO (France). Polyethylene glycol 400 was provided by VWR and poloxamer 188 by BASF. Propylene glycol, polysorbate 80, polysorbate 20, polyvinylpyrrolidone and glycerol were provided by Sigma. For solvents, acetonitrile with 0.1 % formic acid, water with 0.1 % formic acid, ethanol and methanol were obtained from VWR Chemicals.

### 2.2. HPLC method development

A High-performance liquid chromatography (HPLC) method for quantification of JDS364.HCl was developed using an Ultimate 3000 UHPLC system (Thermo Fisher Scientific). For analyte separation, a Macherey-Nagel™ column (Nucleodur™ C8 EC, 100 x 4.6 mm, 3 µm, 100 Å) was used with gradient elution at a total flow rate of 1 mL/min. The injection volume was 5 µL and the column was kept at 30 °C using

<figure><img src="image_1.png" /><figcaption>**Fig. 1.** Chemical structure of JDS364.HCl and JDS207.HCl (Baati et al., 2017; Santoni et al., 2018).</figcaption></figure>
an oven. Samples were eluted with an aqueous phase (A) consisting of 0.1 % formic acid in water and an organic phase (B) consisting of 0.1 % formic acid in acetonitrile. The gradient started at 5 % B, was increased to 50 % B over 8 min and restored to 5 % B in 2 min; 12 min of additional equilibration time at the initial gradient conditions were allowed before the next injection. The total run time was 22 min. The automated injection needle was washed with a mixture of 9:1 water: methanol prior to and following sample introduction into the HPLC. Detection was performed at 329 nm using a Diode Array Detector. Data analysis was conducted with Chromeleon™ 7.2 software (Thermo Fisher Scientific). With this method, JDS364.HCl had a retention time of 5.4 min (chromatogram in supplementary material B, Fig. B). The quantification method of JDS364.HCl was developed using a linearity range from 10 µg/ml to 100 µg/ml in a H₂O: DMSO mixture (1:1 ratio). The linear equation was calculated to be 227.38x + 0.0351 with a correlation coefficient R² of 0.9998. The limit of detection (LOD) is 8.5 ng and the limit of quantification (LOQ) is 28 ng.

## 2.3. X-ray Diffraction (XRD)

XRD was conducted using a microfocused X-ray tube (IµS, Incoatec) that emits Cu Kα radiation (λ = 1.542 Å). The X-ray generator ran at a voltage of 50 kV for an intensity of 1000 µA. The incident beam was focused on the detector using multilayer mirrors (Montel type optics) and a 2D Kratky collimator. Wide-angle X-ray scattering (WAXS) measurements were performed with a Vantec-1 detector (Bruker), positioned perpendicular to the incident beam with an angle range from 19° to 28°. A tungsten filter was used to block the direct beam. The diffracted intensity was measured as a function of the scattering vector q, defined by the relation $q = 4πsinθ/λ$, where θ is half the scattering angle and λ is the wavelength of the radiation. The characteristic repetition distance d, indicative of structural arrangements, was determined using the relation $q(Å⁻¹) = 2π/d$ (Å). Calibration of the WAXS detector was performed using Tristearin (β form). Samples were loaded into Lindemann quartz capillaries (GLAS Muller, Berlin, Germany) with an external diameter of 1.5 mm using an appropriate needle. The capillaries were then placed in a temperature-controlled sample holder (Microcalix, Setaram) and kept at room temperature during 30-minute acquisition periods. The patterns were recorded and analyzed using ASA 3 software (Bruker, RX).

## 2.4. Solubilization in co-solvents, surfactants and oils

### 2.4.1. In co-solvents and surfactants

JDS364.HCl was added as a solid at a concentration of 20 mg/mL to the following aqueous solutions: polyethylene glycol 400 20 % (v/v); propylene glycol 10 % (v/v); propylene glycol 50 % (v/v); polysorbate 80 10 % (v/v); polysorbate 20 10 % (v/v); poloxamer 188 20 % (v/v); polyvinylpyrrolidone 10 % (v/v); ethanol 10 % (v/v); glycerol 10 % (v/v). The solutions were then mixed under magnetic stirring for 5 days. The appearance of a precipitate visible to the naked eye was used to reject the stability of the molecule in solution.

### 2.4.2. In oils for nanoemulsions

JDS364 (free form) was added as a solid at a concentration of 1 mg/mL to the following oils (pure excipient): miglyol® 812 N; refined olive oil – IV; refined soybean oil – IV; refined sesame oil – IV. The solutions were then mixed under magnetic stirring for 5 days. The insolubility of JDS364 was observed with the naked eye.

## 2.5. Preparation of JDS364.HCl: CD complexes in aqueous solution

JDS364.HCl was added as a solid to an aqueous solution of HP-β-CD or SBE-β-CD at the desired molar ratio. The solution was then vortexed for approximately one minute.

## 2.6. Stoichiometry determination by Job's plot analysis

The stoichiometry of the host–guest complex was determined by the continuous variation method proposed by Job (Job, 1928). Equimolar solutions of JDS364.HCl and CD were mixed at different ratios by varying molar ratio from 0.1 to 0.9 whereas total concentrations were kept constant at 6.7 µM. The parameter observed experimentally is a variation in guest absorbance, which is sensitive to complex formation. Absorbance values were measured by UV–Vis spectrometry at λ = 325 nm for each solution at 298.15 K. Job diagrams were plotted with ordinate ΔA × R as a function of R, where ΔA is the difference in absorbance of JDS364.HCl with and without cyclodextrin and R = [JDS364]/[JDS364]+[CD]. The Job's plot showed a maximum at a specific molar ratio (R), indicating the stoichiometry of the complex (Cristina Tablet et al., 2012).

## 2.7. Binding constant determination

The binding constant (K) of the JDS364.HCl-CD complex was determined by UV–Vis spectrometry, processing the data using three comparative methods: the Scott method (Scott, 2010), the Benesi-Hildebrand method (Benesi and Hildebrand, 1949) and an algorithmic method developed by David Landy (Landy et al., 2000). All experiments were performed at least three times, and the means and standard deviations for the binding constants are reported. To reduce the spectral variations due to experimental errors, the host guest stock solution was prepared by dissolution of CD powder directly from guest solution. Variation of the CD concentration is then obtained by mixing these two stock solutions, in which guest concentrations are strictly equal.

The equation (1) for the Benesi-Hildebrand method is as follows:

$$ \frac{1}{\Delta A} = \frac{1}{\Delta \varepsilon[G]_0 K[CD]} + \frac{1}{\Delta \varepsilon[G]_0} \quad (1) $$

where ΔA is the change in absorbance due to the addition of CD, [G]₀ is the concentration (mM) of the guest substrate (i.e. in JDS364.HCl), K (M⁻¹) is the binding constant, Δε is the extinction coefficient, and [CD] is the concentration (mM) of the CD ligand. If the plots of 1/ΔA versus 1/CD concentration are linear, the binding constants are calculated, based on the Benesi-Hildebrand equation, as the y-intercept of the linear regression divided by the slope.

The second method is the Scott equation (2), a modification of the Benesi-Hildebrand equation:

$$ \frac{[CD]}{\Delta A} = \frac{[CD]}{\Delta A_{max}} + \frac{1}{K \Delta A_{max}} \quad (2) $$

where ΔA and K are as previously defined. ΔAmax is the absorbance change between a pure sample of complex and the free component at saturation. A plot of CD concentration divided by absorbance change versus CD concentration was used with Scott's equation to determine binding constants as slope divided by y-intercept.

Finally, the algorithmic method is intended to be more reliable, since it does not use the assumption previously made in the Scott and Benesi-Hildebrand methods, which is $[CD]_T \gg [CD - JDS364]$. It also avoids linearizing the data, which can introduce errors and reduce the precision of the binding constant determination, especially if the data deviates from ideal behaviour (Landy, 2018). The algorithmic method is based on the following equations:

$$ I + CD \leftrightarrow CD - I \quad \text{hence } K = \frac{[CD - I]}{[CD] * [I]} \quad (3) $$

$$ [I]_T = [I] + [CD - I] \quad (4) $$

$$ [CD]_T = [CD] + [CD - I] \quad (5) $$
The concentration of the complex can then be written as in the equation (6):

$$[CD-I] = -\frac{1}{2}\sqrt{\left(\left(\frac{1}{K} + [CD]_T + [I]_T\right)^2 - 4[CD]_T[I]_T\right) + \frac{1}{2}\left(\frac{1}{K} + [CD]_T + [I]_T\right)} \quad (6)$$

$[CD-I]$ is therefore only a function of the total cyclodextrin and guest molecule concentrations (known quantities) and the formation constant (unknown value). To solve this equation with two unknowns, it is possible to use experimental data and an algorithmic procedure. The quality of the experimental data can be assessed by a low standard deviation of the molar absorptivity.

## 2.8. Nuclear magnetic resonance (NMR)

Pure JDS364.HCl and CD solutions (5 mM) were prepared in D₂O in 5 mm NMR tubes. JDS364.HCl and CD solutions were mixed in a 1:1 M ratio (5 mM: 5 mM) in the NMR tubes. An additional sample was prepared from a mixture of JDS364.HCl and HP-β-CD with a 1:5 M ratio (5 mM:25 mM). One-dimensional (1D) ¹H NMR and two-dimensional (2D) off-resonance ROESY (Rotating-Frame Overhauser Enhancement Spectroscopy) spectra were recorded on a Bruker Avance III spectrometer at a magnetic field of 16.4 T ($ν$ (¹H) = 698.7 MHz) with a TXO cryoprobe equipped with Z gradients, at a temperature of 25 °C. ROESY experiments were performed with 250 ms mixing time and 4.2 kHz cw spin-lock.

Spectra were processed with MNova (version 14.2.0–26256) or Bruker TopSpin 4.0.9 software. Baseline correction was applied to each 1D spectrum after Fourier transformation, either with or without a 0.3 Hz exponential apodization, and phase correction. ROESY spectra were processed with a squared sine bell apodization and baseline corrected in the F2 dimension. For ROESY spectra, each FID contained 1 k points with a spectral window of 9 ppm. The relaxation delay D1 was set to 3 s, the number of scans NS was 32, and the number of points in the indirect dimension was 400 (spectral window 9 ppm), resulting in a total acquisition time of 12 h. Assignment of chemical shifts was done by acquiring and analyzing 2D COSY spectra and completed by analysis of ROESY spectra.

## 2.9. Stability study in rat plasma

Fresh rat blood was collected in Li-Heparin tubes and then quickly centrifuged to obtain plasma. The plasma was then frozen in liquid nitrogen and stored in a freezer at -20 °C. Prior to use, the plasma was thawed. 20 µL of JDS364.HCl alone or JDS364.HCl: CD formulations ([JDS364.HCl] = 2.6 mg/ml; JDS364.HCl: HP-β-CD with a ratio 1:4; JDS364.HCl: SBE-β-CD with a ratio 1:2) were injected to 200 µL of rat plasma. The solutions were then incubated at 37 °C for various times (0; 15; 180 min). The final JDS364.HCl plasma concentration was 0.26 mg/ml, which corresponds to the maximum concentration expected in plasma for a NOAEL of 35 µmol/kg. This concentration was chosen to take into account the dilution factor upon injection. For the extraction of JDS364.HCl, 50 µL of the molecule JDS207.HCl (used here as an internal standard) was added to the mixture, followed by vortexing for 30 s. Then, 1 mL of methanol was added to precipitate the proteins. The solutions were centrifuged at 25000 rpm for 30 min at 4 °C. The supernatant was collected, evaporated under nitrogen flow and redissolved in a H₂O: DMSO (1:1) mixture. The solutions were then filtered through a 0.2 µm PTFE filter and JDS364.HCl was quantified using HPLC as described above.

## 2.10. In vitro blood-brain barrier (BBB) crossing studies

### 2.10.1. Design of the in vitro human BBB model

In vitro human BBB models were produced using human brain-like endothelial cells (BLECs) (Cecchelli et al., 2014). Initially, CD34+ hematopoietic stem cells isolated from human umbilical cord blood were differentiated in endothelial cells (Pedroso et al., 2011). BLECs were then produced through a non-contact co-culture with brain pericytes (PCs) using Transwell (TW) systems. The model was developed in 12 TW plates (12-well format, 1.12 cm² culture area, polycarbonate membrane, 0.4 µm pore size, Corning, New York, USA) as a co-culture with human pericytes provided by Professor Takashi Kanda's group (Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Ube, Japan) (Shimizu et al., 2011). The permeability of each tested compound across the Transwell inserts, both with and without BLECs, was evaluated.

To produce the human coculture BBB model, brain PCs were seeded onto the bottom wells (simulating the abluminal compartment) of plates coated with rat tail type I collagen (10 µg/cm²; Corning, New York, USA), at a density of 50,000 PCs per well, in Endothelial Cells Growth Medium MV2 (ECGMV2 Promocell™) supplemented with 50 µg/ml gentamicin (Biowest VWR). The cells were incubated for 3 h at 37 °C. Next, CD34+ -derived ECs were seeded into Transwell inserts (simulating the luminal compartment) coated with Matrigel™ diluted by 1/48 (v/v) (BD Biosciences, San Jose, CA, USA), at a density of 80,000 ECs per insert and immediately cocultured with brain PCs previously seeded on the bottom wells of the plates. The TW system was stored at 37 °C in an incubator chamber (95 % humidity with 5 % CO₂) with the medium being renewed every 2 days. After 6 days of co-culture with brain pericytes, the endothelial cells acquired the BBB phenotype and were subsequently used for the following experiments (as illustrated in Supplementary Material C. Fig D. (A)).

### 2.10.2. Toxicity and transport experiments using the in vitro BBB model

To evaluate toxicity and transport of the reactivators through the endothelium of the in vitro BBB model, inserts containing BLECs were inserted into wells containing Ringer-HEPES buffer (50 mM NaCl, 5.2 mM KCl, 2.2 mM CaCl₂, 0.2 mM MgCl₂·6H₂O, 6 mM NaHCO₃, 5 mM HEPES, 2.8 mM glucose; pH 7.4) at 37 °C. At T0, the different formulations diluted in Ringer-HEPES buffer were added to the luminal compartment ([2-PAM] = 50 µM; [JDS364] = 50 µM; for JDS364.HCl: HP-β-CD: molar ratio 1:6; for JDS364.HCl: SBE-β-CD: molar ratio 1:2). Lucifer Yellow was added to each formulation ([Lucifer Yellow] = 25 µM) and used as a paracellular integrity marker to monitor product toxicity. Under physiological conditions, this small hydrophilic molecule shows low BBB penetration, and its endothelial permeability reveals the integrity of the endothelial cell monolayer.

At each time point (t = 20; 40; 60; 120 min), the insert was changed from one well to another to renew the Ringer-HEPES solution contained in the abluminal part in order to maintain the diffusion gradient. Each time, samples were taken from the abluminal sections to quantify Lucifer Yellow and JDS364.HCl or 2-PAM. At the end, the luminal part was also sampled to perform a mass balance.

Co-incubation of the molecule with Lucifer Yellow was performed in triplicate. This co-incubation made it possible to simultaneously demonstrate JDS364.HCl's toxicity (by assessing the endothelial permeability of Lucifer Yellow) and its transport through the BBB (by assessing the endothelial permeability of JDS364.HCl) (Supplementary Material C, Fig D. (B)). For toxicity assay, BLEC monolayers incubated with only Lucifer Yellow acted as negative control and DMSO 3 % was used as a BBB breakdown control. For transport assay, Lucifer Yellow is used as a negative control as it poorly crosses the BBB.

Endothelial permeability coefficients (P<sub>e</sub>, cm/min) for JDS364.HCl, 2-PAM and Lucifer Yellow were calculated as follows (Dehouck et al., 1992). For each replicate (control inserts without cells and inserts with cells), the clearance was calculated according to the equation (7):
$$ \text{Clearance (µL)} = \frac{[C]_a * V_a}{[C]_t} \qquad (7) $$

Where $[C]_t$ was the initial molecule concentration in the luminal compartment, $[C]_a$ was the molecule concentration in the abluminal compartment, $V_a$ was the volume in the abluminal compartment.

Lucifer Yellow was quantified by fluorescence (excitation/emission wavelength 432/538 nm) using a fluorimeter (Synergy H1; BioTek, Winooski, VT, USA) in a 12-well plate. JDS364.HCl and 2-PAM were quantified with a LC-MS method (see below).

The mean cleared volume was plotted as a function of time, and the slope was estimated by linear regression analysis. Permeability-surface area products (PS, cm³/min) and $P_e$ values were subsequently calculated according to the equation (8):

$$ \frac{1}{PSe} = \frac{1}{PSt} - \frac{1}{PSf} \qquad (8) $$

The PSe value was then divided by the endothelium surface area (1.12 cm²) to obtain the endothelial permeability coefficient (Pe in cm/min).

Compound loss caused, for example by adsorption on TW plates, metabolism in the cells or insolubility of the compound was assessed by calculating the mass balance (MB) according to the equation (9):

$$ MB(\%) = 100 * \frac{[C]_a * V_a + [C]_t * V_t}{[C]_0 * V_t} \qquad (9) $$

where $[C]_a$ was the molecule concentration in the abluminal compartment; $[C]_t$ was the molecule concentration in the luminal compartment; $[C]_0$ was the initial concentration in the luminal compartment; $V_a$, $V_t$ were the volumes in the abluminal and luminal compartments, respectively.

### 2.10.3. LC-MS method development

For *in vitro* testing on the human BBB model, a Liquid Chromatography Mass Spectrometry (LC-MS/MS) method for quantification of JDS364.HCl was developed. Analyses were performed using a quadrupole time-of-flight Triple TOF 5600+ mass spectrometer coupled with an Eksigent Ekspert nanoLC 425 system (Sciex, Redwood City, USA). Samples were diluted in acetonitrile/water/formic acid (50:50:0.1). For analyte separation, 5 µL of standards or samples were injected into a Eksigent Halo C18 column (50 x 0.5 mm; 2.7 µm, 90 A). Samples were eluted using a gradient elution at a total flow rate of 10 µL/min with an aqueous phase (A) consisting of 0.1 % formic acid in water and an organic phase (B) consisting of 0.1 % formic acid in acetonitrile. The gradient started at 40 % B, was increased to 80 % B over 3 min, maintained at 80 % B until 6 min and restored to 40 % B in 0.1 min; 3 more min of equilibration time at the initial gradient conditions were allowed before the next injection. Mass spectrometer parameters were: precursor ion *m/z* 337.2 [M + H]⁺, quantifier ion *m/z* 319.2 and qualifier ion *m/z* 175.1 with ion spray voltage set to 5500 V and collision energy to 40 V. In these conditions, JDS364.HCl calibration curves shown linearity from 1 to 500 nM and a LOD of 0.5 nM with a retention time at 4.5 min (chromatogram and product ion spectrum in Supplementary Material B, Fig. C). Quantification of 2-PAM was performed as previously described by Calas et al., 2020 (Calas et al., 2020).

### 2.11. Data presentation and statistical analysis

One-way ANOVA were performed to evaluate the statistical significance using GraphPad Prism 8 (GraphPad, Inc., San Diego, CA, USA). Dunnett's multiple comparison was performed after ANOVA testing. All the results are presented as mean ± SD unless otherwise stated. The value with $P \le 0.05$ was considered significant. The P values correspond to the following legend: * $P \le 0.05$; ** = $P \le 0.01$; *** $P \le 0.001$; **** $P \le 0.0001$.

## 3. Results and discussion

### 3.1. Stability study

#### 3.1.1. Solubility and stability of JDS364.HCl

A solubility and stability study of JDS364.HCl at 20 mg/ml was conducted at room temperature in water, physiological serum and HEPES buffer (10 mM HEPES, 145 mM NaCl, pH 7.4). JDS364.HCl had a solubility limit of 2.03 ± 0.02 mg/ml in HEPES, so a stability study was not performed in this medium. However, as shown in Table 1, JDS364. HCl was soluble at 20 mg/mL in both water and physiological serum, after which it precipitated to a concentration of 6.20 ± 0.05 mg/mL and 3.17 ± 0.01 mg/mL, respectively, after 7 days.

This observation raised two issues: what is the cause of the molecule's precipitation, and how to avoid it?

#### 3.1.2. Characterization of JDS364.HCl precipitate

To understand JDS364.HCl precipitation, the precipitate obtained in aqueous solution was further characterized. A HRMS study showed that the mass-to-charge ratio of JDS364.HCl and JDS364.HCl precipitate was 337.1644 [M + H]⁺ in both cases. Moreover, an NMR study showed that the proton spectrum of the precipitate contained all the peaks corresponding to JDS364.HCl. These two studies indicated that the chemical structure of JDS364.HCl remains unchanged in the precipitate.

Then, to investigate a possible difference in crystalline structure for both solids, a wide-angle X-Ray scattering (WAXS) study was carried out. The well-defined peaks on the precipitate diffractogram are characteristic of a crystalline form, while the native JDS364.HCl profile is characteristic of an amorphous state (Fig. 2). Therefore, the WAXS analysis reveals that precipitation is linked to the crystallization of JDS364.HCl in aqueous solution, likely due to the formation of a hydrate.

#### 3.1.3. Selection of the optimal formulation

Given the constraints involved in formulating an AChE reactivator, several approaches were considered including the use of water-miscible co-solvents and surfactants, nanoemulsions and cyclodextrins.

##### 3.1.3.1. Water-miscible co-solvents and surfactants.
The concentrations of co-solvents and surfactants used here correspond to those already used in commercially available injectable formulations (Strickley, 2004). A 20 mg/ml solution of JDS364.HCl was solubilized by several co-solvents and surfactants. The stability of the solutions was observed over 5 days at room temperature (Table 2). None of them, except propylene glycol, prevented precipitation (Table 2, entry 3). Although propylene glycol 50 % was the only co-solvent that prevented JDS364. HCl precipitation, its use in injectable products comes with significant limitations. These include the risk of precipitation, pain, inflammation, and hemolysis upon injection. More importantly, propylene glycol is known to cause thrombophlebitis when used in intravenous formulations at high concentrations (Doenicke et al., 1992). Other less restrictive approaches were therefore explored.

##### 3.1.3.2. Nanoemulsions.
An alternative approach involves using JDS364 in its more hydrophobic free form rather than as a hydrochloride salt which is more hydrophilic. The solubility of the free form was explored in various EMA-approved oils with the aim to formulate JDS364 as oil-in-water nanoemulsions (Strickley, 2004) (Table 3). The

<table><caption>**Table 1**
JDS364.HCl concentration (mg/ml) after incubation in different solvents at room temperature (n = 3).</caption><thead><tr><th>Solvent</th><th>T0 (theoretical)</th><th>T0</th><th>T0 + 7 days</th></tr></thead><tbody><tr><td>H<sub>2</sub>O</td><td>20</td><td>20.29 ± 0.33</td><td>6.20 ± 0.05</td></tr><tr><td>NaCl 0.9 %</td><td>20</td><td>19.84 ± 0.37</td><td>3.17 ± 0.01</td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>**Fig. 2.** Wide angle X-Ray scattering of the native JDS364.HCl and its precipitate from aqueous solution.</figcaption></figure>

**Table 2**

Solubilization and stabilization of a 20 mg/ml JDS364.HCl solution by several co-solvents and surfactants at room temperature.

<table><thead><tr><th>Entry</th><th>Excipient</th><th>Solubility</th><th>Stability<br>(5 days at RT)</th></tr></thead><tbody><tr><td>1</td><td>Polyethylene glycol 400 20 % (v/v)</td><td>Yes</td><td>No</td></tr><tr><td>2</td><td>Propylene glycol 10 % (v/v)</td><td>Yes</td><td>No</td></tr><tr><td>3</td><td>Propylene glycol 50 % (v/v)</td><td>Yes</td><td>Yes</td></tr><tr><td>4</td><td>Polysorbate 80 10 % (v/v)</td><td>Yes</td><td>No</td></tr><tr><td>5</td><td>Polysorbate 20 10 % (v/v)</td><td>Yes</td><td>No</td></tr><tr><td>6</td><td>Poloxamer 188 20 % (v/v)</td><td>Yes</td><td>No</td></tr><tr><td>7</td><td>Polyvinylpyrrolidone 10 % (v/v)</td><td>Yes</td><td>No</td></tr><tr><td>8</td><td>Ethanol 10 % (v/v)</td><td>Yes</td><td>No</td></tr><tr><td>9</td><td>Glycerol 10 % (v/v)</td><td>Yes</td><td>No</td></tr></tbody></table>

**Table 3**

Solubilization of 1 mg/ml JDS364 (free form) in several EMA-approved oils at room temperature.

<table><thead><tr><th>Excipient</th><th>Solubility</th></tr></thead><tbody><tr><td>Miglyol® 812 N</td><td>No</td></tr><tr><td>Refined olive oil – IV</td><td>No</td></tr><tr><td>Refined soybean oil – IV</td><td>No</td></tr><tr><td>Refined sesame oil – IV</td><td>No</td></tr></tbody></table>

selected oils represent common choices from different families, including medium-chain triglycerides (Miglyol®) and long-chain triglycerides (refined olive oil, refined soybean oil, and refined sesame oil).

It was found that no oil could effectively solubilize JDS364 in its free form. Consequently, the use of oil-in-water emulsions was discarded.

### 3.1.3.3. CD-based formulation.
Cyclodextrins were then considered to enhance the stability of JDS364.HCl solutions. Practically, to observe the potential increase in solubility in the presence of CDs, different amounts of HP-β-CD and SBE-β-CD were added to a 20 mg/ml JDS364. HCl solution to obtain different JDS364.HCl: CD molar ratios. After 5 days of agitation, the final concentration of JDS364.HCl in the supernatant was compared with the initial concentration (after filtration through a 0.22 µm filter). Gratifyingly, as shown in Table 4, a 1:3 JDS364.HCl: HP-β-CD molar ratio was enough to obtain a stable formulation. Similarly, a JDS364.HCl: SBE-β-CD molar ratio of 1:2 was sufficient to stabilize the formulation.

A stability study was then carried out up to 57 days. To promote equilibrium towards complex formation, a higher molar ratio of 1:6 was chosen for the JDS364.HCl: HP-β-CD complex. The presence of ionizable

**Table 4**

% JDS364.HCl in solution after 5 days incubation at room temperature at different JDS364.HCl: CD ratios for [JDS364.HCl]₀ = 20 mg/ml.

<table><thead><tr><th>Molar ratio JDS364.HCl: HP-β-CD</th><th>% JDS364.HCl in solution</th></tr></thead><tbody><tr><td>1:0</td><td>35 %</td></tr><tr><td>1:1</td><td>43 %</td></tr><tr><td>1:2</td><td>98 %</td></tr><tr><td>1:3</td><td>101 %</td></tr><tr><td>1:4</td><td>101 %</td></tr></tbody></table><table><thead><tr><th>Molar ratio JDS364.HCl: SBE-β-CD</th><th>% JDS364.HCl in solution</th></tr></thead><tbody><tr><td>1:0</td><td>39 %</td></tr><tr><td>1:0.5</td><td>43 %</td></tr><tr><td>1:1</td><td>85 %</td></tr><tr><td>1:2</td><td>100 %</td></tr><tr><td>1:3</td><td>100 %</td></tr></tbody></table>

sulfate groups in SBE-β-CD increases the osmolarity of a solution more than HP-β-CD at equivalent concentrations. Therefore, in order to remain within an injectable osmolarity range, the minimum molar ratio (1:2) to stabilize the formulation was used in the stability study for the JDS364.HCl: SBE-β-CD complex. As shown in Fig. 3, JDS364.HCl complexed with both CDs remained stable at 20 mg/mL, with no significant variations observed after two months at room temperature. Comparison of these results with Table 1 showed that HP-β-CD and SBE-β-CD, at molar ratios of 1:6 and 1:2, respectively, increased the stability of JDS364.HCl at least sixfold compared to physiological serum and threefold compared to water.

Therefore, as CD-based formulations successfully achieved the target concentration of 20 mg/mL by providing enhanced stability, they were identified as the optimal formulations for JDS364.HCl and the JDS364. HCl:CD inclusion complexes were further characterized.

## 3.2. Characterization of cyclodextrin-based formulations

### 3.2.1. Stoichiometry determination by Job's plot analysis

The stoichiometry of the complexes was first determined by using the continuous variation method (Job, 1928) as detailed in the material and methods section. For both types of cyclodextrins, the Job plots showed a maximum at R = 0.5 and symmetrical shapes, indicating that the JDS364.HCl: HP-β-CD and JDS364.HCl: SBE-β-CD complexes have a 1:1

<figure><img src="image_3.png" /><figcaption>**Fig. 3.** Stability of JDS364.HCl: HP-β-CD (ratio 1:6, in aqueous medium) and JDS364.HCl: SBE-β-CD (ratio 1:2, in aqueous medium) complexes at room temperature for [JDS364.HCl] = 20 mg/ml (2-way ANOVA analysis, n = 3).</figcaption></figure>
stoichiometry (Fig. 4). It should be noted that despite this stoichiometry, JDS364.HCl was formulated with an excess of cyclodextrins to promote complex formation rather than a strict 1:1 M ratio. This approach helps to shift the equilibrium toward complexation, ensuring a higher proportion of JDS364.HCl is complexed, as shown in the literature (Saokham et al., 2018).

### 3.2.2. Binding constant determination

The binding constant (K) of the JDS364.HCl: CD complex was evaluated using UV-Vis spectroscopy. Data analysis was performed through three distinct methods: Benesi-Hildebrand method (Benesi and Hildebrand, 1949), Scott method (Scott, 2010), and an algorithm developed by David Landy (Landy et al., 2000) as detailed above. Isothermal Titration Calorimetry (ITC) was also considered, but as the complex is athermal, it was not possible to draw conclusions using this method. For the Benesi-Hildebrand and Scott methods, binding constants were obtained from the slope and y-intercept (Supplementary Material D, Fig. E). It may be observed that the standard deviations are high, especially for JDS364.HCl: HP-β-CD complexes, since the binding constant was low. It was therefore appropriate to use an algorithmic method to obtain a reliable constant with greater accuracy. With this method, the mean standard deviation of the molar absorptivity for both cyclodextrins was very low, attesting to the quality of the experimental data (Supplementary Material D, Table C). Table 5 summarizes the binding constants obtained with the different methods. The binding constants were consistent with those of other drugs reported in the literature (Kurkov et al., 2012). The binding constant for JDS634.HCl: HP-β-CD complexes were 100 M⁻¹ and 100 times higher ~10000 M⁻¹ for JDS364. HCl: SBE-β-CD complexes. This stronger interaction may allow greater affinity of JDS364.HCl with SBE-β-CD, but it may also reduce the bioavailability of JDS364.HCl. Further tests are required to assess the impact of such a difference in binding constant. As SBE-β-CD is a polyanionic molecule, the stronger interaction observed could arise from additional electrostatic forces in addition to inclusion. An NMR study was conducted to confirm this assumption.

### 3.2.3. Molecular interaction analysis

The interactions between CDs and guest molecules often involve shifts in the chemical signals of specific protons (Várnai et al., 2022; Deng et al., 2016). Therefore, to characterize the molecular interactions, ¹H NMR spectra were recorded for CDs alone, JDS364.HCl alone and for JDS364.HCl complexed with CDs in D₂O (Supplementary Material E).

On the one hand, chemical shifts were compared between CDs alone and CDs in complex form (Table 6.). Upfield shifts of H5 and H3 protons (Δ (δ) = -0.02 ppm) were observed for both complexes. As shown in Fig. 5 (B), the H3 and H5 protons are located inside the internal cavity of the CDs. Therefore, these shifts can be associated to the formation of inclusion complexes between JDS364.HCl and CDs. In the case of SBE-β-CD, large chemical shift differences were also observed for (CH₂)₄SO₃

<table><caption>**Table 5**
Binding constants (K) at 25 °C.</caption><thead><tr><th></th><th>K (JDS364.HCl: HP-β-CD)<br>(M<sup>-1</sup>)</th><th>K (JDS364.HCl: SBE-β-CD)<br>(M<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Benesi-Hildebrand<br>method</td><td>131 ± 57</td><td>7622 ± 355</td></tr><tr><td>Scott method</td><td>227 ± 95</td><td>7396 ± 1837</td></tr><tr><td>Algorithmic method</td><td>111 ± 40</td><td>9809 ± 754</td></tr></tbody></table>

<table><caption>**Table 6**
¹H chemical shift differences of HP-β-CD or SBE-β-CD protons in 1:1 JDS364.HCl:CD mixtures and CDs alone. Δ (δ) = δ(JDS364.HCl:CD) – δ(CD)).</caption><thead><tr><th>Proton<br>HP-β-CD</th><th>Δ (δ) (ppm)</th></tr></thead><tbody><tr><td>H1, H1'</td><td>&lt;-0.01</td></tr><tr><td>H3</td><td>-0.01</td></tr><tr><td>H6</td><td>-0.00</td></tr><tr><td>H5</td><td>-0.02</td></tr><tr><td>H2</td><td>0.00</td></tr><tr><td>H4</td><td>0.00</td></tr><tr><td>CH₃</td><td>&lt;-0.01</td></tr></tbody><thead><tr><th>Proton<br>SBE-β-CD</th><th>Δ (δ) (ppm)</th></tr></thead><tbody><tr><td>H1, H1'</td><td>-0.03</td></tr><tr><td>H3</td><td>-0.02</td></tr><tr><td>H5</td><td>-0.02</td></tr><tr><td>H6</td><td>-0.02</td></tr><tr><td>H2</td><td>-0.04</td></tr><tr><td>H4</td><td>-0.01</td></tr><tr><td>(CH₂)₄SO₃</td><td>-0.05</td></tr><tr><td>(CH₂)₄SO₃</td><td>-0.04</td></tr></tbody></table>

signals (Δ (δ) = -0.04 and -0.05 ppm). This suggests that SBE-β-CD is also involved in the complexation through its negatively charged sidechains, both on the narrower and the wider side of the CD.

On the other hand, chemical shifts were compared between JDS364. HCl alone and JDS364.HCl in the complex form (Table 7.). For the JDS364.HCl: HP-β-CD complex, the largest chemical shift differences were observed for protons 20 and 21, indicating the inclusion of the pyridine moiety inside the HP-β-CD cavity.

In the case of the JDS364.HCl: SBE-β-CD complex, very large chemical shift changes of 0.10 ppm were detected for proton 20, as well as proton 22, which is consistent with the encapsulation of the pyridine ring with the CD cavity. This is corroborated by the chemical shift change observed for aliphatic chain proton 15. Strikingly, with SBE-β-CD, large chemical shift differences were also observed for protons 4, 5 and 7. These differences likely result from interactions with the negatively charged sidechains of SBE-β-CD, due to the proximity of the protonated N-heteroatom on the quinoline ring. These findings confirm

<figure><img src="image_4.png" /><figcaption>**Fig. 4.** Job's plot analysis (A) for the JDS364.HCl: HP-β-CD complex; (B) for the JDS364.HCl: SBE-β-CD complex. ΔA is the difference in absorbance of JDS364.HCl with and without cyclodextrin and R = [JDS364]/[JDS364]+[CD] the molar ratio.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig. 5. General structure of (A) JDS364.HCl (B) CD.</figcaption></figure>

<caption>**Table 7**</caption>

¹H chemical shifts differences of JDS364.HCl protons between JDS364.HCl alone and the JDS364.HCl: CD complexes. Δ (δ) = δ(JDS364.HCl: CD) – δ(JDS364.HCl)).

<table><thead><tr><th>Proton<br>JDS364.HCl</th><th>Δδ (JDS364.HCl – HP-β-CD)</th><th>Δδ (JDS364.HCl – SBE-β-CD)</th></tr></thead><tbody><tr><td>4</td><td>0.01</td><td>0.08</td></tr><tr><td>7</td><td>0.01</td><td>-0.09</td></tr><tr><td>9</td><td>0.00</td><td>-0.03</td></tr><tr><td>10</td><td>0.00</td><td>-0.02</td></tr><tr><td>8</td><td>0.00</td><td>-0.02</td></tr><tr><td>20</td><td>-0.04</td><td>0.10</td></tr><tr><td>21</td><td>-0.04</td><td>0.02</td></tr><tr><td>5</td><td>0.01</td><td>0.09</td></tr><tr><td>12</td><td>0.01</td><td>0.04</td></tr><tr><td>15</td><td>-0.01</td><td>0.05</td></tr><tr><td>14</td><td>0.00</td><td>0.01</td></tr><tr><td>13</td><td>0.00</td><td>0.02</td></tr><tr><td>22</td><td>0.01</td><td>0.25</td></tr></tbody></table>

that SBE-β-CD interacts with JDS364.HCl through both its internal cavity and its sidechains via electrostatic interactions (Várnai et al., 2022). Moreover, this is consistent with the previous hypothesis that the binding constant of SBE-β-CD is higher than that of HP-β-CD, due to the contribution of electrostatic interactions.

To go further, a 2D ROESY NMR study was conducted. As shown in Fig. 6. (A), spatial proximity was observed between JDS364.HCl protons

and the protons H3 and H5 from the internal cavity of HP-β-CD. More precisely, they interacted with the protons 20, 21, and 22 of JDS364.HCl (i.e. with the pyridine). A cross-peak was also detected between HP-β-CD and the aliphatic side chain proton 15 of JDS364.HCl, when using a higher concentration of HP-β-CD, 25 mM instead of 5 mM. Regarding SBE-β-CD, the JDS364.HCl protons 20, 21 and 22 were also spatially close to SBE-β-CD protons H3, H5 and/or H6 (Fig. 6. (B)). One cross-peak could be unambiguously assigned to an interaction between SBE-β-CD protons H3 and/or H5 and JDS364.HCl proton 14. Signal overlay between SBE-β-CD and JDS364.HCl side chain protons precluded observation of other contacts. Moreover, cross-peaks were observed between the quinoline protons 4 and 7 and the sidechain of SBE-β-CD.

For both cyclodextrins, the 2D ROESY results were consistent with the findings from 1D ¹H NMR: the pyridine moiety and the aliphatic chain of JDS364.HCl interact with the internal cavity of both types of CDs. Weak cross-peaks were also observed between protons 8, 9, and 10 of JDS364.HCl and H3 and H5 protons of SBE-β-CD. This indicates that the quinoline moiety of JDS364 could enter the SBE-β-CD cavity. However, these cross-peaks are much less intense than those observed with pyridine protons, so that this can be considered as a minor complexation mode. Since no intramolecular ROE signals were observed between pyridine and quinolone protons of JDS364.HCl, we exclude that the two moieties are simultaneously present in the cavity.

These results are promising because the 4-carbon aliphatic chain and the reactivator moiety, which included pyridine and oxime groups, are common to a large family of AChE reactivators (Baati et al., 2017). Thus,

<figure><img src="image_6.png" /><figcaption>Fig. 6. 2D ROESY NMR spectra (698.7 MHz, 25 °C, 250 ms spin-lock time) of (A) JDS364.HCl: HP-β-CD, and (B) JDS364.HCl: SBE-β-CD. The mixtures contained 5 mM JDS364.HCl and 5 mM CD. The strip in panel A at 2.8 ppm was extracted from a ROESY spectrum acquired with a mixture prepared with 25 mM HP-β-CD. Positive contours are in grey, negative contours in black. The corresponding 1D ¹H NMR spectra are represented on each side of the 2D spectra. Boxes indicate intermolecular cross-peaks between JDS364.HCl and CDs.</figcaption></figure>
inclusion complexes with cyclodextrins may be applicable to other
molecules similar to JDS364.HCl. This hypothesis was confirmed by a
study with the reactivator JDS207.HCl (Supplementary material F).

3.3. *Stability study in rat plasma*

Regarding *ex vivo* tests, preliminary studies first confirmed that JDS364.HCl and its CD-based formulations did not induce a hemolytic effect on red blood cells (Supplementary material G). Then, stability tests in rat plasma were conducted to evaluate the stability of JDS364. HCl at a concentration that accounts for the dilution factor upon injection. The results demonstrated that JDS364.HCl complexed with the two CDs was significantly more stable than unformulated JDS364.HCl for up to 15 min after incubation (Fig. 7). Moreover, in the case of JDS364.HCl: SBE-β-CD complexes, the molecule was significantly more stable than the unformulated JDS364.HCl up to 3 h after incubation, with a JDS364. HCl loss of 17.4 ± 4.3 % (compared to a loss of 24.2 ± 2.6 %). These results suggest that cyclodextrins enhance the stability of JDS364.HCl, with SBE-β-CD providing greater stability likely due to its higher binding constant with JDS364.HCl.

3.4. Toxicity and transport experiments on a human in vitro BBB model

An *in vitro* human BBB model was used to assess the toxicity and BBB permeability of JDS364.HCl: CD complexes.

3.4.1. Toxicity

Toxicity was evaluated by measuring the permeability coefficient of
Lucifer Yellow, a marker for BBB integrity. The reliability of the ex-
periments was confirmed by conducting a mass balance between the
initial and final quantities of Lucifer Yellow in the luminal and abl-
minal compartments. All experiments showed a mass balance of Lucifer
Yellow between 80 % and 120 %. BLECs incubated without excipients
acted as negative control and DMSO 3 % was used as a BBB breakdown
control.

First, the BBB integrity was assessed after incubation with HP-β-CD
and SBE-β-CD from 100 to 4000 µM and 50 to 2500 µM, respectively

(Fig. 8 (A) and (B)). Results showed no significant differences (p > 0.05) in the endothelial permeability of the integrity marker (Pe<sub>LY</sub>) between the control and the cyclodextrin solutions over different concentrations. The BBB tightness integrity was not impacted; hence cyclodextrins did not show toxicity up to 4000 µM for HP-β-CD and 2500 µM for SBE-β-CD. Subsequently, BBB integrity was tested in the presence of JDS364.HCl and JDS364.HCl: CD complexes (Fig. 8 (C)). Once again, results showed no significant differences (p > 0.05) in the endothelial permeability to the integrity marker Lucifer Yellow (Pe<sub>LY</sub>) proving that neither JDS364. HCl nor JDS364.HCl complexed with CDs are toxic in this BBB model.

3.4.2. Transport experiments

Then, a transport study of JDS364.HCl across the BBB was conducted to assess its ability to reach AChE in the CNS. JDS364.HCl without formulation was tested to demonstrate its inherent ability to cross the BBB. Lucifer Yellow was used as a negative control and 2-PAM was used as a comparative molecule since it is a marketed AChE reactivator.

The reliability of the experiments was ensured by performing a mass
balance between the initial and final quantities of JDS364.HCl in the
upper and lower compartments of the *in vitro* BBB model. All experi-
ments consistently showed a mass balance between 80 % and 120 %. These high recoveries of JDS364.HCl in the BBB transport assays indicate the absence of excessive non-specific binding to cell culture materials or metabolism in the cells.

As shown in Fig. 9, the endothelial permeability of 2-PAM (Pe = 0.88 ± 0.04. 10⁻³ cm/min) was not significantly higher than the one of the negative control (Pe = 0.79 ± 0.04. 10⁻³ cm/min), confirming its limited ability to cross the BBB. In contrast, the permeability values of JDS364. HCl, both in its free form (Pe = 3.00 ± 0.49. 10⁻³ cm/min) and when complexed with HP-β-CD (Pe = 2.1 ± 0.29. 10⁻³ cm/min) and SBE-β-CD (Pe = 1.54 ± 0.12. 10⁻³ cm/min), were significantly higher than the one of 2-PAM. This demonstrates a key finding: JDS364.HCl and its CD- formulation exhibits a greater ability to cross the BBB compared to 2- PAM, a marketed antidote.

It can also be observed that the presence of CD reduced the endo-
thelial permeability value. This result can be attributed to the fact that
CDs do not cross the BBB efficiently (Calias, 2017; Pontikis et al., 2013;
Camargo et al., 2001). Consequently, the JDS364.HCl:CD complex
exhibited limited BBB penetration, with JDS364.HCl crossing only after
dissociation from the complex. Moreover, the reduction in BBB perme-
ability was more pronounced for JDS364.HCl: SBE-β-CD complexes. As
shown in previous sections, SBE-β-CD has a higher binding constant for
JDS364.HCl compared to HP-β-CD. Dissociation of JDS364.HCl: SBE-
β-CD complexes in the BBB model is therefore lower, resulting in
reduced passage of JDS364.HCl and hence low endothelial permeability
in the transport experiment.

Overall, this study demonstrated that JDS364.HCl can cross the BBB more efficiently than 2-PAM even when formulated with CDs. Here, the CDs play a crucial role in stabilizing and transporting JDS364.HCl to the BBB and a rapid-release mechanism is expected from CD complexes upon injection. It would allow JDS364.HCl to cross the barrier independently. The *in vitro* BBB model advocates for the use of HP-β-CD to facilitate the release of JDS364.HCl from the complex and cross the BBB. *In vivo* experiments are needed to confirm this observation.

4. Conclusion

Uncharged hybrid reactivators like JDS364.HCl have emerged as
promising candidates for more effective AChE reactivation. However,
JDS364.HCl's instability in aqueous solution is a significant limitation,
particularly for intramuscular injections, which require concentrated
solutions. Here, to overcome this limitation, several formulations were
screened, including co-solvents, surfactants, oils, and cyclodextrins. The
most promising results were obtained with two EMA-approved cyclo-
dextrins: HP-β-CD and SBE-β-CD. Complexation with these CDs signifi-
cantly increased the stability of JDS364.HCl at 20 mg/ml for over two

<figure><img src="image_7.png" /><figcaption>Fig. 7. Percentage of JDS364.HCl in plasma as a function of time (experimental conditions: [JDS364.HCl]: 0.26 mg/ml; formulation: JDS364.HCl: HP-β-CD (ratio 1:4); JDS364.HCl: SBE-β-CD (ratio 1:2); incubated at 37 °C) (mixed-effects analysis (* = p < 0.05)).</figcaption></figure>
A

<figure><img src="image_8.png" /></figure>

B

<figure><img src="image_9.png" /></figure>

C

<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Evaluation of BBB integrity after addition of (A) HP-β-CD at different concentrations; (B) SBE-β-CD at different concentrations; (C) JDS364.HCl, JDS364.HCl: HP-β-CD (ratio 1:6), JDS364.HCl: SBE-β-CD (ratio 1:2) (integrity marker: lucifer yellow (LY); [JDS364.HCl] = 50 µM; POS CTRL: 3 % DMSO; NEG CTRL (LY alone); one way ANOVA with CTRL NEG as reference, n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 9.** BBB permeability of Lucifer Yellow (NEG CTRL), 2-PAM, JDS364.HCl, JDS364.HCl: HP-β-CD (ratio 1:6), JDS364.HCl: SBE-β-CD (ratio 1:2) ([LY] = 25 µM; [2-PAM] = 50 µM; [JDS364.HCl] = 50 µM; one way ANOVA analysis with negative control as reference, n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

months at room temperature. Physicochemical characterizations showed a 1:1 stoichiometry for both complexes, with the JDS364.HCl: SBE-β-CD complex exhibiting a 100-fold higher affinity constant due to additional electrostatic interactions with SBE-β-CD sidechains.

Interestingly, both cyclodextrin complexes significantly improved

plasma stability compared to JDS364.HCl alone, with the JDS364.HCl: SBE-β-CD complex demonstrating even superior plasma stability due to its higher binding constant. As observed in an *in vitro* model of the human BBB, JDS364.HCl crossed the BBB model even better than marketed antidotes such as 2-PAM. Although the high binding affinity of the JDS364.HCl: SBE-β-CD complex limited drug release and reduced BBB permeability, JDS364.HCl formulated with either CD still showed better BBB penetration than 2-PAM.

These findings are promising, as they do not only demonstrate the advantage of cyclodextrins as stabilizers and reactivator carriers for JDS364.HCl but also suggest a broader applicability of cyclodextrins to other broad-spectrum reactivators developed by Baati and al. Future work will focus on a biodistribution study to assess whether JDS364.HCl formulated with cyclodextrins can achieve effective concentrations in the brain, with the ultimate goal of improving its therapeutic potential for OP poisoning.

### CRediT authorship contribution statement

**Léa Thiberville:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis. **Vincent Faivre:** Writing – review & editing, Supervision, Conceptualization. **Christina Sizun:** Writing – review & editing, Investigation, Formal analysis. **Marie-Pierre Dehouck:** Writing – review & editing, Methodology, Formal analysis. **Christophe Landry:** Investigation. **Rachid Baati:** Writing – review & editing, Supervision, Funding acquisition, Conceptualization. **Nicolas Tsapis:** Writing – review & editing, Supervision, Conceptualization.

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lea Thiberville reports financial support was provided by French Government Defense Innovation Agency. Reports a relationship with that includes: Has patent pending to. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in
this paper.

## Acknowledgements

The authors gratefully acknowledge the Agence de l'Innovation de la Défense (AID) for funding LT's PhD fellowship (Allocation de Thèse Classique AID n° 2022024). They also thank Dr. François-Xavier Legrand (IGPS) for his advice on CD-based formulations, Lynda Benrabah and Martin Souce for providing access to the HPLC platform (IGPS). The authors acknowledge Bastien Prost and Dr Audrey Solgadi, SAMM core facility from IPSIT, for providing HRMS analysis. The mass spectrometer of the Spectrométrie de Masse de l'Artois (SMART) core facilities used in this study was funded by the European Regional Development Fund (ERDF), the Conseil Régional Hauts de France and University of Artois.

## Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpharm.2025.125505.

## Data availability

Data will be made available on request.

## References

Baati, R., Brown, R.C.D., Dias, J., Nachon, F., Sousa, J., 2017. Broad spectrum reactivators of OPNA-inhibition of human cholinesterases. WO 2017 (021319), A1.

Benesi, H.A., Hildebrand, J.H., 1949. A Spectrophotometric Investigation of the Interaction of Iodine with Aromatic Hydrocarbons. J. Am. Chem. Soc. 71, 2703-2707. https://doi.org/10.1021/ja01176a030.

Brewster, M.E., Loftsson, T., 2007. Cyclodextrins as pharmaceutical solubilizers. Drug Solubility Meas. It Improve It 59, 645-666. https://doi.org/10.1016/j.addr.2007.05.012.

Calas, A.-G., Hanak, A.-S., Jaffré, N., Nervo, A., Dias, J., Rousseau, C., Courageux, C., Brazzolotto, X., Villa, P., Obrecht, A., Goossens, J.-F., Landry, C., Hachani, J., Gosselet, F., Dehouck, M.-P., Yerri, J., Kliachyna, M., Baati, R., Nachon, F., 2020. Efficacy Assessment of an Uncharged Reactivator of NOP-Inhibited Acetylcholinesterase Based on Tetrahydroacridine Pyridine-Aldoxime Hybrid in Mouse Compared to Pralidoxime. Biomolecules 10, 858. https://doi.org/10.3390/biom10060858.

Calias, P., 2017. 2-Hydroxypropyl-β-cyclodextrins and the Blood-Brain Barrier: Considerations for Niemann-Pick Disease Type C1. Curr. Pharm. Des. 23, 6231-6238. https://doi.org/10.2174/1381612823666171019164220.

Camargo, F., Erickson, R.P., Garver, W.S., Hossain, G.S., Carbone, P.N., Heidenreich, R. A., Blanchard, J., 2001. Cyclodextrins in the treatment of a mouse model of Niemann-Pick C disease. Life Sci. 70, 131-142. https://doi.org/10.1016/S0024-3205(01)01318-4.

Cecchelli, R., Aday, S., Sevin, E., Almeida, C., Culot, M., Dehouck, L., Coisne, C., Engelhardt, B., Dehouck, M.-P., Ferreira, L., 2014. A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells. PLoS One 9, e99733. https://doi.org/10.1371/journal.pone.0099733.

Challa, R., Ahuja, A., Ali, J., Khar, R.K., 2005. Cyclodextrins in drug delivery: an updated review. AAPS PharmSciTech 6, E329-E357. https://doi.org/10.1208/pt060243.

Cristina Tablet, Iulia Matei, Mihaela Hillebrand, 2012. The Determination of the Stoichiometry of Cyclodextrin Inclusion Complexes by Spectral Methods: Possibilities and Limitations, in: Alessio Innocenti (Ed.), Stoichiometry and Research. IntechOpen, Rijeka, p. Ch. 3. DOI: 10.5772/34287.

De Koning, M.C., Joosen, M.J.A., Noort, D., Van Zuylen, A., Tromp, M.C., 2011. Peripheral site ligand-oxime conjugates: A novel concept towards reactivation of nerve agent-inhibited human acetylcholinesterase. Bioorg. Med. Chem. 19, 588-594. https://doi.org/10.1016/j.bmc.2010.10.059.

Dehouck, M.P., Jolliet-Riant, P., Brée, F., Fruchart, J.C., Cecchelli, R., Tillement, J.P., 1992. Drug transfer across the blood-brain barrier: correlation between in vitro and in vivo models. J. Neurochem. 58, 1790-1797. https://doi.org/10.1111/j.1471-4159.1992.tb10055.x.

Delgado, P., Martin-Romera, J.D., Perona, C., Vismara, R., Galli, S., Maldonado, C.R., Carmona, F.J., Padial, N.M., Navarro, J.A.R., 2022. Zirconium Metal-Organic Polyhedra with Dual Behavior for Organophosphate Poisoning Treatment. ACS Appl. Mater. Interfaces 14, 26501-26506. https://doi.org/10.1021/acsami.2c06025.

Deng, Y., Pang, Y., Guo, Y., Ren, Y., Wang, F., Liao, X., Yang, B., 2016. Host-guest inclusion systems of daidzein with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and sulfobutyl ether-β-cyclodextrin (SBE-β-CD): Preparation, binding behaviors and water solubility. J. Mol. Struct. 1118, 307-315. https://doi.org/10.1016/j.molstruc.2016.04.040.

Doenicke, A., Nebauer, A.E., Hoernecke, R., Mayer, M., Roizen, M.F., 1992. Osmolalities of propylene glycol-containing drug formulations for parenteral use. Should

propylene glycol be used as a solvent? Anesth. Analg. 75, 431-435. https://doi.org/10.1213/00000539-199209000-00020.

Eyer, P., 2003. The role of oximes in the management of organophosphorus pesticide poisoning. Toxicol. Rev. 22, 165-190. https://doi.org/10.2165/00139709-200322030-00004.

Ferreira, L., Campos, J., Veiga, F., Cardoso, C., Paiva-Santos, A.C., 2022. Cyclodextrin-based delivery systems in parenteral formulations: A critical update review. Eur. J. Pharm. Biopharm. 178, 35-52. https://doi.org/10.1016/j.ejpb.2022.07.007.

Gupta, R.C., 2020. Handbook of toxicology of chemical warfare agents. An Imprint of Elsevier, Third edition. ed. Academic Press, London, United Kingdom; San Diego, CA.

Guyton, K.Z., Loomis, D., Grosse, Y., El Ghissassi, F., Benbrahim-Tallaa, L., Guha, N., Scoccianti, C., Mattock, H., Straif, K., 2015. Carcinogenicity of tetrachlorvinphos, parathion, malathion, diazinon, and glyphosate. Lancet Oncol. 16, 490-491. https://doi.org/10.1016/S1470-2045(15)70134-8.

He, M., Zheng, W., Wang, N., Gao, H., Ouyang, D., Huang, Z., 2022. Molecular Dynamics Simulation of Drug Solubilization Behavior in Surfactant and Solventless Injections. Pharmaceutics 14, 2366. https://doi.org/10.3390/pharmaceutics14112366.

Job, P., 1928. Formation and stability of inorganic complexes in solution. Ann. Chim. 113-203.

John, H., van der Schans, M.J., Koller, M., Spruit, H.E.T., Worek, F., Thiermann, H., Noort, D., 2018. Fatal sarin poisoning in Syria 2013: forensic verification within an international laboratory network. Forensic Toxicol. 36, 61-71. https://doi.org/10.1007/s11419-017-0376-7.

Kliachyna, M., Santoni, G., Nussbaum, V., Renou, J., Sanson, B., Colletier, J.-P., Arboléas, M., Loiodice, M., Weik, M., Jean, L., Renard, P.-Y., Nachon, F., Baati, R., 2014. Design, synthesis and biological evaluation of novel tetrahydroquinoline pyridine- aldoxime and -amidoxime hybrids as efficient uncharged reactivators of nerve agent-inhibited human acetylcholinesterase. Eur. J. Med. Chem. 78, 455-467. https://doi.org/10.1016/j.ejmech.2014.03.044.

Kuca, K., Jun, D., Bajgar, J., 2007. Currently Used Cholinesterase Reactivators Against Nerve Agent Intoxication: Comparison of Their Effectiveness in Vitro. Drug Chem. Toxicol. 30, 31-40. https://doi.org/10.1080/01480540601017637.

Kurkov, S.V., Madden, D.E., Carr, D., Loftsson, T., 2012. The Effect of Parenterally Administered Cyclodextrins on the Pharmacokinetics of Coadministered Drugs. J. Pharm. Sci. 101, 4402-4408. https://doi.org/10.1002/jps.23329.

Kusić, R., Bošković, B., Vojvodić, V., Jovanović, D., 1985. HI-6 in Man: Blood Levels, Urinary Excretion, and Tolerance after Intramuscular Administration of the Oxime to Healthy Volunteers. Toxicol. Sci. 5, 89-97. https://doi.org/10.1093/toxsci/5.6part2.89.

Kuznetsova, D.A., Gaynanova, G.A., Vasilieva, E.A., Pavlov, R.V., Zueva, I.V., Babaev, V. M., Kuznetsov, D.M., Voloshina, A.D., Petrov, K.A., Zakharova, L.Y., Sinyashin, O.G., 2022. Oxime Therapy for Brain AChE Reactivation and Neuroprotection after Organophosphate Poisoning. Pharmaceutics 14, 1950. https://doi.org/10.3390/pharmaceutics14091950.

Landy, D., 2018. Measuring Binding Constants of Cyclodextrin Inclusion Compounds. In: Fourmentin, S., Crini, G., Lichtfouse, E. (Eds.), Cyclodextrin Fundamentals, Reactivity and Analysis. Springer International Publishing, Cham, pp. 223-255. https://doi.org/10.1007/978-3-319-76159-6-5.

Landy, D., Fourmentin, S., Salome, M., Surpateanu, G., 2000. Analytical Improvement in Measuring Formation Constants of Inclusion Complexes between β-Cyclodextrin and Phenolic Compounds. J. Incl. Phenom. Macrocycl. Chem. 38, 187-198. https://doi.org/10.1023/A:1008156110999.

Luettringhaus, A., Hagedorn, I., 1964. Quaternary hydroxyiminomethylpyridinium salts. the dischloride of bis-(4-hydroxyiminomethyl-1-pyridinium-methyl)-ether (lueh6), a new reactivator of acetylcholinesterase inhibited by organic phosphoric acid esters. Arzneimittelforschung. 14, 1-5.

Mali, H., Shah, C., Raghunandan, B.H., Prajapati, A.S., Patel, D.H., Trivedi, U., Subramanian, R.B., 2023. Organophosphate pesticides an emerging environmental contaminant: Pollution, toxicity, bioremediation progress, and remaining challenges. J. Environ. Sci. 127, 234-250. https://doi.org/10.1016/j.jes.2022.04.023.

Mercey, G., Verdelet, T., Renou, J., Kliachyna, M., Baati, R., Nachon, F., Jean, L., Renard, P.-Y., 2012. Reactivators of Acetylcholinesterase Inhibited by Organophosphorus Nerve Agents. Acc. Chem. Res. 45, 756-766. https://doi.org/10.1021/ar2002864.

Mercey, G., Verdelet, T., Saint-André, G., Gillon, E., Wagner, A., Baati, R., Jean, L., Nachon, F., Renard, P.-Y., 2011. First efficient uncharged reactivators for the dephosphylation of poisoned human acetylcholinesterase. Chem. Commun. 47, 5295-5297. https://doi.org/10.1039/C1CC10787A.

Pashirova, T.N., Zueva, I.V., Petrov, K.A., Babaev, V.M., Lukashenko, S.S., Rizvanov, I.K., Souto, E.B., Nikolsky, E.E., Zakharova, L.Y., Masson, P., Sinyashin, O.G., 2017. Nanoparticle-Delivered 2-PAM for Rat Brain Protection against Paraoxon Central Toxicity. ACS Appl. Mater. Interfaces 9, 16922-16932. https://doi.org/10.1021/acsami.7b04163.

Pashirova, T.N., Zueva, I.V., Petrov, K.A., Lukashenko, S.S., Nizameev, I.R., Kulik, N.V., Voloshina, A.D., Almasy, L., Kadirov, M.K., Masson, P., Souto, E.B., Zakharova, L.Y., Sinyashin, O.G., 2018. Mixed cationic liposomes for brain delivery of drugs by the intranasal route: The acetylcholinesterase reactivator 2-PAM as encapsulated drug model. Colloids Surf. B Biointerfaces 171, 358-367. https://doi.org/10.1016/j.colsurfb.2018.07.049.

Pedroso, D.C.S., Tellechea, A., Moura, L., Fidalgo-Carvalho, I., Duarte, J., Carvalho, E., Ferreira, L., 2011. Improved Survival, Vascular Differentiation and Wound Healing Potential of Stem Cells Co-Cultured with Endothelial Cells. PLoS ONE 6, e16114. https://doi.org/10.1371/journal.pone.0016114.

Pontikis, C.C., Davidson, C.D., Walkley, S.U., Platt, F.M., Begley, D.J., 2013. Cyclodextrin alleviates neuronal storage of cholesterol in Niemann-Pick C disease without
evidence of detectable blood-brain barrier permeability. J. Inherit. Metab. Dis. 36, 491-498. https://doi.org/10.1007/s10545-012-9583-x.

Renou, J., Mercey, G., Verdelet, T., Páunescu, E., Gillon, E., Arboléas, M., Loiodice, M., Kliachyna, M., Baati, R., Nachon, F., Jean, L., Renard, P.-Y., 2013. Syntheses and in vitro evaluations of uncharged reactivators for human acetylcholinesterase inhibited by organophosphorus nerve agents. Chem. Biol. Interact. 203, 81–84. https://doi.org/10.1016/j.cbi.2012.09.023.

Rousseau, J.-M., Besse Bardot, I., Franck, L., Libert, N., Lallement, G., Clair, P., 2009. Interest of In Europe syringe for nerve agent intoxication. Ann. Fr. Anesth. Reanim. 28, 482–488. https://doi.org/10.1016/j.annfar.2009.02.039.

Santoni, G., De Sousa, J., De La Mora, E., Dias, J., Jean, L., Sussman, J.L., Silman, I., Renard, P.-Y., Brown, R.C.D., Weik, M., Baati, R., Nachon, F., 2018. Structure-Based Optimization of Nonquaternary Reactivators of Acetylcholinesterase Inhibited by Organophosphorus Nerve Agents. J. Med. Chem. 61, 7630–7639. https://doi.org/10.1021/acs.jmedchem.8b00592.

Saokham, P., Muankaew, C., Jansook, P., Loftsson, T., 2018. Solubility of Cyclodextrins and Drug/Cyclodextrin Complexes. Mol. Basel Switz. 23, 1161. https://doi.org/10.3390/molecules23051161.

Scott, R.L., 2010. Some comments on the Benesi-Hildebrand equation. Recl. Trav. Chim. Pays-Bas 75, 787–789.

Seto, Y., Tsunoda, N., Kataoka, M., Tsuge, K., Nagano, T., 1998. Blood Cholinesterase Activity Levels of Victims Intoxicated with Sarin in Matsumoto and Tokyo Subway System. In: Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. (Eds.), Structure and Function of Cholinesterases and Related Proteins. Springer, US, Boston, MA, p. 302. https://doi.org/10.1007/978-1-4899-1540-5_86.

Shimizu, F., Sano, Y., Abe, M., Maeda, T., Ohtsuki, S., Terasaki, T., Kanda, T., 2011. Peripheral nerve pericytes modify the blood–nerve barrier function and tight junctional molecules through the secretion of various soluble factors. J. Cell. Physiol. 226, 255–266. https://doi.org/10.1002/jcp.22337.

Sidell, F.R., Borak, J., 1992. Chemical warfare agents: II. nerve agents. Ann. Emerg. Med. 21, 865–871. https://doi.org/10.1016/S0196-0644(05)81036-4.

Sousa, J. de, 2015. Design, synthesis and biological evaluation of novel uncharged bifunctional hybrid reactivators for organophosphorus nerve agent-inhibited human acetylcholinesterare (These de doctorat). Strasbourg.

Strickley, R.G., 2004. Solubilizing excipients in oral and injectable formulations. Pharm. Res. 21, 201–230. https://doi.org/10.1023/b:pham.0000016235.32639.23.

Timperley, C.M., Forman, J.E., Abdollahi, M., Al-Amri, A.S., Baulig, A., Benachour, D., Borrett, V., Cariño, F.A., Geist, M., Gonzalez, D., Kane, W., Kovarik, Z., Martínez-Alvarez, R., Mourão, N.M.F., Neffe, S., Raza, S.K., Rubaylo, V., Suárez, A.G., Takeuchi, K., Tang, C., Trifirò, F., Van Straten, F.M., Vanninen, P.S., Vučinić, S., Zaitsev, V., Zafar-Uz-Zaman, M., Zina, M.S., Holen, S., 2019. Advice on assistance and protection provided by the Scientific Advisory Board of the Organisation for the Prohibition of Chemical Weapons: Part 1. On medical care and treatment of injuries from nerve agents. Toxicology 415, 56–69. https://doi.org/10.1016/j.tox.2019.01.004.

Trancart, M., Hanak, A.-S., Dambrune, C., Madi, M., Voros, C., Baati, R., Calas, A.-G., 2024. No-observed-adverse-effect-level (NOAEL) assessment as an optimized dose of cholinesterase reactivators for the treatment of exposure to warfare nerve agents in mice. Chem. Biol. Interact. 392, 110929. https://doi.org/10.1016/j.cbi.2024.110929.

Várnai, B., Zsila, F., Szakács, Z., Garádi, Z., Malanga, M., Béni, S., 2022. Sulfobutylation of Beta-Cyclodextrin Enhances the Complex Formation with Mitragynine: An NMR and Chiroptical Study. Int. J. Mol. Sci. 23. https://doi.org/10.3390/ijms23073844.

Vasilieva, E.A., Kuznetsova, D.A., Valeeva, F.G., Kuznetsov, D.M., Zakharov, A.V., Amerhanova, S.K., Voloshina, A.D., Zueva, I.V., Petrov, K.A., Zakharova, L.Y., 2022. Therapy of Organophosphate Poisoning via Intranasal Administration of 2-PAM-Loaded Chitosomes. Pharmaceutics 14, 2846. https://doi.org/10.3390/pharmaceutics14122846.

Voros, C., Dias, J., Timperley, C.M., Nachon, F., Brown, R.C.D., Baati, R., 2024. The risk associated with organophosphorus nerve agents: from their discovery to their unavoidable threat, current medical countermeasures and perspectives. Chem. Biol. Interact. 395, 110973. https://doi.org/10.1016/j.cbi.2024.110973.

Wilson, I.B., Ginsburg, S., 1955. A powerful reactivator of alkylphosphate-inhibited acetylcholinesterase. Biochim. Biophys. Acta 18, 168–170. https://doi.org/10.1016/0006-3002(55)90040-8.

Worek, F., Aurbek, N., Wille, T., Eyer, P., Thiermann, H., 2011. Kinetic prerequisites of oximes as effective reactivators of organophosphate-inhibited acetylcholinesterase: a theoretical approach. J. Enzyme Inhib. Med. Chem. 26, 303–308. https://doi.org/10.3109/14756366.2010.504673.

Worek, F., Thiermann, H., Wille, T., 2016. Oximes in organophosphate poisoning: 60 years of hope and despair. 12th Int. Meet. Cholinesterases 6th Conf. Paraoxonase 259, 93–98. https://doi.org/10.1016/j.cbi.2016.04.032.