Recent events have shown that organophosphorus nerve agents (OPNAs) are a serious threat. Cholinesterase inhibition by OPNAs results in acetylcholine accumulation, a cholinergic crisis leading to death if untreated. Efficacy assessment of new medical countermeasures against OPNAs relies on translational animal models. We developed a swine model of percutaneous VX intoxication and a simple plate reader-based enzymatic method to quantify plasmatic VX over time. Juvenile pigs anesthetized with sevoflurane were poisoned with a single supralethal (n = 5; 1200 μg/kg) or sublethal (n = 6; 320 μg/kg) percutaneous dose of VX. These intoxicated animals were compared to 7 control animals. Repeated blood sampling was performed up to 6 h post-intoxication. Blood cholinesterase activities were measured using the Ellman assay. Nanomolar plasma concentrations of VX were measured by exogenous butyrylcholinesterase added to an aliquot of plasma. As expected, we observed a steady increase in plasma concentration of VX over time concomitant to a decrease in blood cholinesterase activities for all intoxicated pigs. Despite the simplicity of the enzymatic method, the results obtained are in good agreement with those of the liquid chromatography-mass spectrometry method. This method is also applicable to other OPNAs such as novichoks with minor adaptations.

Organophosphorus (OP) compounds inhibit central and peripheral acetylcholinesterase (AChE) activity, overstimulating cholinergic receptors and causing autonomic dysfunction (e.g., bronchoconstriction, excess secretions), respiratory impairment, seizure and death at high doses. Current treatment for OP poisoning in the United States includes reactivation of OP-inhibited AChE by the pyridinium oxime 2-pyridine aldoxime (2-PAM). However, 2-PAM has a narrow therapeutic index and its efficacy is confined to a limited number of OP agents. The bis-pyridinium oxime MMB4, which is a more potent reactivator than 2-PAM with improved pharmaceutical properties and therapeutic range, is under consideration as a potential replacement for 2-PAM. Similar to other pyridinium oximes, high doses of MMB4 lead to off-target effects culminating in respiratory depression and death. To understand the toxic mechanisms contributing to respiratory depression, we evaluated the effects of MMB4 (0.25-16 mM) on functional and neurophysiological parameters of diaphragm and limb muscle function in rabbits and rats. In both species, MMB4 depressed nerve-elicited muscle contraction by blocking muscle endplate nicotinic receptor currents while simultaneously prolonging endplate potentials by inhibiting AChE. MMB4 increased quantal content, endplate potential rundown and tetanic fade during high frequency stimulation in rat but not rabbit muscles, suggesting species-specific effects on feedback mechanisms involved in sustaining neurotransmission. These data reveal multifactorial effects of MMB4 on cholinergic neurotransmission, with the primary toxic modality being reduced muscle nicotinic endplate currents. Evidence of species-specific effects on neuromuscular function illustrates the importance of comparative toxicology when studying pyridinium oximes and, by inference, other quaternary ammonium compounds.

(1) Background: Human exposure to organophosphorus compounds employed as pesticides or as chemical warfare agents induces deleterious effects due to cholinesterase inhibition. One therapeutic approach is the reactivation of inhibited acetylcholinesterase by oximes. While currently available oximes are unable to reach the central nervous system to reactivate cholinesterases or to display a wide spectrum of action against the variety of organophosphorus compounds, we aim to identify new reactivators without such drawbacks. (2) Methods: This study gathers an exhaustive work to assess in vitro and in vivo efficacy, and toxicity of a hybrid tetrahydroacridine pyridinaldoxime reactivator, KM297, compared to pralidoxime. (3) Results: Blood-brain barrier crossing assay carried out on a human in vitro model established that KM297 has an endothelial permeability coefficient twice that of pralidoxime. It also presents higher cytotoxicity, particularly on bone marrow-derived cells. Its strong cholinesterase inhibition potency seems to be correlated to its low protective efficacy in mice exposed to paraoxon. Ventilatory monitoring of KM297-treated mice by double-chamber plethysmography shows toxic effects at the selected therapeutic dose. This breathing assessment could help define the No Observed Adverse Effect Level (NOAEL) dose of new oximes which would have a maximum therapeutic effect without any toxic side effects.

Obviously, delivery of the medications to the brain is more difficult than other tissues due to the existence of a strong obstacle, which is called blood-brain barrier (BBB). Because of the lipophilic nature of this barrier, it would be a complex (and in many cases impossible) process to cross the medications with hydrophilic behavior from BBB and deliver them to the brain. Thus, novel intricate drug-carriers in nano scales have been recently developed and suitably applied for this purpose. One of the most important categories of these hydrophilic medications, are reactivators for acetyl cholinesterase (AChE) enzyme that facilitates the breakdown of acetylcholine (as a neurotransmitter). The AChE function is inhibited by organophosphorus (OP) nerve agents that are extremely used in military conflicts. In this review, the abilities of the nanosized drug delivery systems to perform as suitable vehicles for AChE reactivators are comprehensively discussed.

The acute toxicity of organophosphorus-based compounds is primarily a result of acetylcholinesterase inhibition in the central and peripheral nervous systems. The resulting cholinergic crisis manifests as seizure, paralysis, respiratory failure and neurotoxicity. Though overstimulation of muscarinic receptors is the mechanistic basis of central organophosphorus (OP) toxicities, short-term changes in synapse physiology that precede OP-induced seizures have not been investigated in detail. To study acute effects of OP exposure on synaptic function, field excitatory postsynaptic potentials (fEPSPs) were recorded from Schaffer collateral synapses in the mouse hippocampus CA1 stratum radiatum during perfusion with various OP compounds. Administration of the OPs paraoxon, soman or VX rapidly and stably depressed fEPSPs via a presynaptic mechanism, while the non-OP proconvulsant tetramethylenedisulfotetramine had no effect on fEPSP amplitudes. OP-induced presynaptic long-term depression manifested prior to interictal spiking, occurred independent of recurrent firing, and did not require NMDA receptor currents, suggesting that it was not mediated by activity-dependent calcium uptake. Pharmacological dissection revealed that the presynaptic endocannabinoid type 1 receptor (CB1R) as well as postsynaptic M1 and M3 muscarinic acetylcholine receptors were necessary for OP-LTD. Administration of CB1R antagonists significantly reduced survival in mice after a soman challenge, revealing an acute protective role for endogenous CB1R signaling during OP exposure. Collectively these data demonstrate that the endocannabinoid system alters glutamatergic synaptic function during the acute response to OP acetylcholinesterase inhibitors.

Organophosphorus compounds (OPs) continue to represent a significant chemical threat to humans due to exposures from their use as weapons, their potential storage hazards, and from their continued use agriculturally. Existing methods for detection include ELISA and mass spectrometry. The new approach presented here provides an innovative first step toward a portable OP quantification method that surmounts conventional limitations involving sensitivity, selectivity, complexity, and portability. DNA affinity probes, or aptamers, represent an emerging technology that, when combined with a mix-and-read, free-solution assay (FSA) and a compensated interferometer (CI) can provide a novel alternative to existing OP nerve agent (OPNA) quantification methods. Here it is shown that FSA can be used to rapidly screen prospective aptamers in the biological matrix of interest, allowing the identification of a \'best-in-class\' probe. It is also shown that combining aptamers with FSA-CI enables quantification of the OPNA metabolites, Sarin (NATO designation \"G-series, B\", or GB) and Venomous Agent X (VX) acids, rapidly with high selectivity at detection limits of sub-10 pg/mL in 25% serum (by volume in PBS). These results suggest there is potential to directly impact diagnostic specificity and sensitivity of emergency response testing methods by both simplifying sample preparation procedures and making a benchtop reader available for OPNA metabolite quantification.

This work describes a novel and sensitive non-isotope dilution method for simultaneous quantification of organophosphorus nerve agents (OPNAs) soman (GD) and VX adducts to butyrylcholinesterase (BChE), their aged methylphosphonic acid (MeP) adduct and unadducted BChE in plasma exposed to OPNA. OPNA-BChE adducts were isolated with an off-column procainamide-gel separation (PGS) from plasma, and then digested with pepsin into specific adducted FGES*AGAAS nonapeptide (NP) biomarkers. The resulting NPs were detected by UHPLC-MS/MS MRM. The off-column PGS method can capture over 90% of BChE, MeP-BChE, VX-BChE and GD-BChE from their respective plasma materials. One newly designed and easily synthesized phosphorylated BChE nonapeptide with one Gly-to-Ala mutation was successfully reported to serve as internal standard instead of traditional isotopically labeled BChE nonapeptide. The linear range of calibration curves were from 1.00-200ngmL-1 for VX-NP, 2.00-200ngmL-1 for GD-NP and MeP-NP (R2≥0.995), and 3.00-200ngmL-1 for BChE NP (R2≥0.990). The inter-day precision had relative standard deviation (%RSD) of <8.89%, and the accuracy ranged between 88.9-120%. The limit of detection was calculated to be 0.411, 0.750, 0.800 and 1.43ngmL-1 for VX-NP, GD-NP, MeP-NP and BChE NP, respectively. OPNA-exposed quality control plasma samples were characterized as part of method validation. Investigation of plasma samples unexposed to OPNA revealed no baseline values or interferences. Using the off-column PGS method combined with UHPLC-MS/MS, VX-NP and GD-NP adducts can be unambiguously detected with high confidence in 0.10ngmL-1 and 0.50ngmL-1 of exposed human plasma respectively, only requiring 0.1mL of plasma sample and taking about four hours without special sample preparation equipment. These improvements make it a simple, sensitive and robust PGS-UHPLC-MS/MS method, and this method will become an attractive alternative to immunomagnetic separation (IMS) method and a useful diagnostic tool for retrospective detection of OPNA exposure with high confidence. Furthermore, using the developed method, the adducted BChE levels from VX and GD-exposed (0.10-100ngmL-1) plasma samples were completely characterized, and the fact that VX being more active and specific to BChE than GD was re-confirmed.

Organophosphorus nerve agents (OPNAs) are toxic compounds that are classified as prohibited Schedule 1 chemical weapons. In the body, OPNAs bind to butyrylcholinesterase (BChE) to form nerve agent adducts (OPNA-BChE). OPNA-BChE adducts can provide a reliable, long-term protein biomarker for assessing human exposure. A major challenge facing OPNA-BChE detection is hydrolysis (aging), which can continue to occur after a clinical specimen has been collected. During aging, the o-alkyl phosphoester bond hydrolyzes, and the specific identity of the nerve agent is lost. To better identify OPNA exposure events, a high-throughput method for the detection of five aged OPNA-BChE adducts was developed. This is the first diagnostic panel to allow for the simultaneous quantification of any Chemical Weapons Convention Schedule 1 OPNA by measuring the aged adducts methyl phosphonate, ethyl phosphonate, propyl phosphonate, ethyl phosphoryl, phosphoryl and unadducted BChE. The calibration range for all analytes is 2.00-250. ng/mL, which is consistent with similar methodologies used to detect unaged OPNA-BChE adducts. Each analytical run is 3 min, making the time to first unknown results, including calibration curve and quality controls, less than 1 h. Analysis of commercially purchased individual serum samples demonstrated no potential interferences with detection of aged OPNA-BChE adducts, and quantitative measurements of endogenous levels of BChE were similar to those previously reported in other OPNA-BChE adduct assays.

A series of new uncharged functional acetylcholinesterase (AChE) reactivators including heterodimers of tetrahydroacridine with 3-hydroxy-2-pyridine aldoximes and amidoximes has been synthesized. These novel molecules display in vitro reactivation potencies towards VX-, tabun- and paraoxon-inhibited human AChE that are superior to those of the mono- and bis-pyridinium aldoximes currently used against nerve agent and pesticide poisoning. Furthermore, these uncharged compounds exhibit a broader reactivity spectrum compared to currently approved remediation drugs.