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Organophosphorus (OP) pesticides and insecticides are used in agriculture and other industries can also cause adverse effects through environmental exposures in the people working in agricultural and pesticide industries. OP nerve agent exposures have been associated with delayed neurotoxic effects including sleep disorders, cognitive malfunctions, and brain damage in Gulf War victims, and Japanese victims of terrorist attacks with nerve agents. However, the mechanisms behind such prolonged adverse effects after chronic OP nerve agent's exposures in survivors are not well understood. In the present study, male Wistar rats were subcutaneously exposed to nerve agent soman (0.25XLD 50 ) for 21 consecutive days to evaluate the neurobehavioral, neuropathological and biochemical alterations (oxidative stress and antioxidants levels). Neurobehavioral studies using Elevated Plus Maze (EPM), T-Maze, and rotarod tests revealed that chronic soman exposure produced alterations in behavioral functions including increased anxiety and reduction in working memory and neuromuscular strength. Biochemical studies showed that antioxidants enzyme (glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) levels were reduced and oxidative stress (reduced glutathione (GSH) and lipid peroxidation levels (malondialdehyde (MDA)) were significantly increased in brain at 30 days in soman exposed rats as compared to control rats. Neuroselective fluorojade-c stain was used to examine the brain damage after chronic soman exposure. Results demonstrated that chronic soman exposure induced neurodegeneration as brain damage was detected at 30- and 90-days post exposure. The present study results suggest that chronic nerve agent exposures even at low doses may produce long-term adverse effects like neurobehavioral deficits in rats., Competing Interests: Declaration of Competing Interest All authors declare that there are none in this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Organophosphates (OPs) and nerve agents are potent neurotoxic compounds that cause seizures, status epilepticus (SE), brain injury, or death. There are persistent long-term neurologic and neurodegenerative effects that manifest months to years after the initial exposure. Current antidotes are ineffective in preventing these long-term neurobehavioral and neuropathological changes. Additionally, there are few effective neuroprotectants for mitigating the long-term effects of acute OP intoxication. We have pioneered neurosteroids as novel anticonvulsants and neuroprotectants for OP intoxication and seizures. In this study, we evaluated the efficacy of two novel synthetic, water-soluble neurosteroids, valaxanolone (VX) and lysaxanolone (LX), in combating the long-term behavioral and neuropathological impairments caused by acute OP intoxication and SE. Animals were exposed to the OP nerve agent surrogate diisopropylfluorophosphate (DFP) and were treated with VX or LX in addition to midazolam at 40 minutes postexposure. The extent of neurodegeneration, along with various behavioral and memory deficits, were assessed at 3 months postexposure. VX significantly reduced deficits of aggressive behavior, anxiety, memory, and depressive-like traits in control (DFP-exposed, midazolam-treated) animals; VX also significantly prevented the DFP-induced chronic loss of NeuN(+) principal neurons and PV(+) inhibitory neurons in the hippocampus and other regions. Additionally, VX-treated animals exhibited a reduced inflammatory response with decreased GFAP(+) astrogliosis and IBA1(+) microgliosis in the hippocampus, amygdala, and other regions. Similarly, LX showed significant improvement in behavioral and memory deficits, and reduced neurodegeneration and cellular neuroinflammation. Together, these results demonstrate the neuroprotectant effects of the novel synthetic neurosteroids in mitigating the long-term neurologic dysfunction and neurodegeneration associated with OP exposure. SIGNIFICANCE STATEMENT: Survivors of nerve agents and organophosphate (OP) exposures suffer from long-term neurological deficits. Currently, there is no specific drug therapy for mitigating the impact of OP exposure. However, novel synthetic neurosteroids that activate tonic inhibition provide a viable option for treating OP intoxication. The data from this study indicates the neuroprotective effects of synthetic, water-soluble neurosteroids for attenuation of long-term neurological deficits after OP intoxication. These findings establish valaxanolone and lysaxanolone as potent and efficacious neuroprotectants suitable for injectable dosing., (Copyright © 2024 by The Author(s).)

Organophosphates cause hyperstimulation of the central nervous system, leading to extended seizures, convulsions, and brain damage. Sarin is a highly toxic organophosphate nerve agent that has been employed in several terrorist attacks. The prolonged toxicity of sarin may be enhanced by the neuroinflammatory response initiated by the inflammasome, caspase involvement, and generation/release of proinflammatory cytokines. Since neurodegeneration and neuroinflammation are prevalent in sarin-exposed animals, we were interested in evaluating the capacity of quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh), a pan caspase inhibitor to attenuate neuroinflammation following sarin exposure. To test this hypothesis, sarin-exposed C57BL/6 mice were treated with Q-VD-OPh or negative control quinolyl-valyl-O-methylglutamyl-[-2,6-difluorophenoxy]-methyl ketone, sacrificed at 2- and 14-day time points, followed by removal of the amygdala and hippocampus. A Bio-Rad 23-Plex cytokine analysis was completed on each tissue. The results suggest that exposure to sarin induced a dramatic increase in interleukin-1 β and 6 other cytokines and a decrease in 2 of the 23 cytokines at 2 days in the amygdala compared with controls. Q-VD-OPh attenuated these changes at the 2-day time point. At 14 days, six of these cytokines were still significantly different from controls. Hippocampus was less affected at both time points. Diazepam, a neuroprotective drug against nerve agents, caused an increase in several cytokines but did not have a synergistic effect with Q-VD-OPh. Treatment of sarin exposure with apoptosis inhibitors appears to be a worthwhile approach for further testing as a comprehensive counteragent against organophosphate exposure. SIGNIFICANCE STATEMENT: A pan inhibitor of caspases (Q-VD-OPh) was proposed as a potential antidote for sarin-induced neuroinflammation by reducing the level of inflammation via inflammasome caspase inhibition. Q-VD-OPh added at 30 minutes post-sarin exposure attenuated the inflammatory response of a number of cytokines and chemokines in the amygdala and hippocampus, two brain regions sensitive to organophosphate exposure. Apoptotic marker reduction at 2 and 14 days further supports further testing of inhibitors of apoptosis as a means to lessen extended organophosphate toxicity in the brain., (Copyright © 2024 by The Author(s).)

Exposure to highly toxic organophosphorus compounds causes inhibition of the enzyme acetylcholinesterase resulting in a cholinergic toxidrome and innervation of receptors in the neuromuscular junction may cause life-threatening respiratory effects. The involvement of several receptor systems was therefore examined for their impact on bronchoconstriction using an ex vivo rat precision-cut lung slice (PCLS) model. The ability to recover airways with therapeutics following nerve agent exposure was determined by quantitative analyses of muscle contraction. PCLS exposed to nicotine resulted in a dose-dependent bronchoconstriction. The neuromuscular nicotinic antagonist tubocurarine counteracted the nicotine-induced bronchoconstriction but not the ganglion blocker mecamylamine or the common muscarinic antagonist atropine. Correspondingly, atropine demonstrated a significant airway relaxation following ACh-exposure while tubocurarine did not. Atropine, the M3 muscarinic receptor antagonist 4-DAMP, tubocurarine, the β 2 -adrenergic receptor agonist formoterol, the Na + -channel blocker tetrodotoxin and the K + ATP -channel opener cromakalim all significantly decreased airway contractions induced by electric field stimulation. Following VX-exposure, treatment with atropine and the Ca 2+ -channel blocker magnesium sulfate resulted in significant airway relaxation. Formoterol, cromakalim and magnesium sulfate administered in combinations with atropine demonstrated an additive effect. In conclusion, the present study demonstrated improved airway function following nerve agent exposure by adjunct treatment to the standard therapy of atropine., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The fluorinated bis-pyridinium oximes were designed and synthesized with the aim of increasing their nucleophilicity and potential to reactivate phosphorylated human recombinant acetylcholinesterase (AChE) and human purified plasmatic butyrylcholinesterase (BChE) in relation to chlorinated and non-halogenated oxime analogues. Compared to non-halogenated oximes, halogenated oximes showed lower pK a of the oxime group (fluorinated < chlorinated < non-halogenated) along with higher level of oximate anion formation at the physiological pH, and had a higher binding affinity of both AChE and BChE. The stability tests showed that the fluorinated oximes were stable in water, while in buffered environment di-fluorinated oximes were prone to rapid degradation, which was reflected in their lower reactivation ability. Mono-fluorinated oximes showed comparable reactivation to non-halogenated (except asoxime) and mono-chlorinated oximes in case of AChE inhibited by sarin, cyclosarin, VX, and tabun, but were less efficient than di-chlorinated ones. The same trend was observed in the reactivation of inhibited BChE. The advantage of halogen substituents in the stabilization of oxime in a position optimal for in-line nucleophilic attack were confirmed by extensive molecular modelling of pre-reactivation complexes between the analogue oximes and phosphorylated AChE and BChE. Halogen substitution was shown to provide oximes with additional beneficial properties, e.g., fluorinated oximes gained antioxidative capacity, and moreover, halogens themselves did not increase cytotoxicity of oximes. Finally, the in vivo administration of highly efficient reactivator and the most promising analogue, 3,5-di-chloro-bispyridinium oxime with trimethylene linker, provided significant protection of mice exposed to sarin and cyclosarin., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Maintaining a long-term continuous and stable reactivator blood concentration to treat organophosphorus nerve agent poisoning using acetylcholinesterase (AChE) reactivator pralidoxime chloride (2-PAM) is very important yet difficult. Because the flexible framework of MIL-88B(Fe) nanoparticles (NPs) can swell in polar solvents, pralidoxime chloride (2-PAM) was loaded in MIL-88B(Fe) NPs (size: ca. 500 nm) by stirring and incubation in deionized water to obtain 2-PAM@MIL-88B(Fe), which had a maximum drug loading capacity of 12.6 wt %. The as-prepared composite was characterized by IR, powder X-ray diffraction (P-XRD), scanning electron microscopy (SEM), ζ-potential, Brunauer-Emmett-Teller (BET), and thermogravimetry/differential thermal analysis (TG/DTA). The results showed that under constant conditions, the maximum drug release rates of 2-PAM@MIL-88B(Fe) in absolute ethanol, phosphate-buffered saline (PBS) solution (pH = 7.4), and PBS solution (pH = 4) at 150 h were 51.7, 80.6, and 67.1%, respectively. This was because the composite showed different swelling behaviors in different solvents. In PBS solution with pH = 2, the 2-PAM@MIL-88B(Fe) framework collapsed after 53 h and released 100% of 2-PAM. For mice after intragastric poisoning with sarin (a neurotoxic agent), an atropine-assisted 2-PAM@MIL-88B(Fe) treatment experiment revealed that 2-PAM@MIL-88B(Fe) continuously released 2-PAM for more than 72 h so that poisoned AChE was continuously and steadily reactivated. The reactivation rate of AChE was 56.7% after 72 h. This composite is expected to provide a prolonged, stable therapeutic drug for the mid- and late-stage treatment of neurotoxic agent poisoning.

Residual effects of the 1990-1991 Gulf War (GW) still plague veterans 30 years later as Gulf War Illness (GWI). Thought to stem mostly from deployment-related chemical overexposures, GWI is a disease with multiple neurological symptoms with likely immunological underpinnings. Currently, GWI remains untreatable, and the long-term neurological disease manifestation is not characterized fully. The present study sought to expand and evaluate the long-term implications of prior GW chemicals exposure on neurological function 6-8 months post GWI-like symptomatology induction. Additionally, the beneficial effects of delayed treatment with the glycan immunotherapeutic lacto-N-fucopentaose III (LNFPIII) were evaluated. Male C57BL/6J mice underwent a 10-day combinational exposure (i.p.) to GW chemicals, the nerve agent prophylactic pyridostigmine bromide (PB) and the insecticide permethrin (PM; 0.7 and 200 mg/kg, respectively). Beginning 4 months after PB/PM exposure, a subset of the mice were treated twice a week until study completion with LNFPIII. Evaluation of cognition/memory, motor function, and mood was performed beginning 1 month after LNFPIII treatment initiation. Prior exposure to PB/PM produced multiple locomotor, neuromuscular, and sensorimotor deficits across several motor tests. Subtle anxiety-like behavior was also present in PB/PM mice in mood tests. Further, PB/PM-exposed mice learned at a slower rate, mostly during early phases of the learning and memory tests employed. LNFPIII treatment restored or improved many of these behaviors, particularly in motor and cognition/memory domains. Electrophysiology data collected from hippocampal slices 8 months post PB/PM exposure revealed modest aberrations in basal synaptic transmission and long-term potentiation in the dorsal or ventral hippocampus that were improved by LNFPIII treatment. Immunohistochemical analysis of tyrosine hydroxylase (TH), a dopaminergic marker, did not detect major PB/PM effects along the nigrostriatal pathway, but LNFPIII increased striatal TH. Additionally, neuroinflammatory cells were increased in PB/PM mice, an effect reduced by LNFPIII. Collectively, long-term neurobehavioral and neurobiological dysfunction associated with prior PB/PM exposure was characterized; delayed LNFPIII treatment provided multiple behavioral and biological beneficial effects in the context of GWI, highlighting its potential as a GWI therapeutic., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Since several decades oximes have been used as part of treatment of nerve agent intoxication with the aim to restore the biological function of the enzyme acetylcholinesterase after its covalent inhibition by organophosphorus compounds such as pesticides and nerve agents. Recent findings have illustrated that, besides oximes, certain Mannich phenols can reactivate the inhibited enzyme very effectively, and may therefore represent an attractive complementary class of reactivators. In this paper we further probe the effect of structural variation on the in vitro efficacy of Mannich phenol based reactivators. Thus, we present the synthesis of 14 compounds that are close variants of the previously reported 4-amino-2-(1-pyrrolidinylmethyl)-phenol, a very effective non-oxime reactivator, and 3 dimeric Mannich phenols. All compounds were assessed for their ability to reactivate human acetylcholinesterase inhibited by the nerve agents VX, tabun, sarin, cyclosarin and paraoxon in vitro. It was confirmed that the potency of the compounds is highly sensitive to small structural changes, leading to diminished reactivation potency in many cases. However, the presence of 4-substituted alkylamine substituents (as exemplified with the 4-benzylamine-variant) was tolerated. More surprisingly, the dimeric compounds demonstrated non-typical behavior and displayed some reactivation potency as well. Both findings may open up new avenues for designing more effective non-oxime reactivators., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The implementation of the Chemical Weapons Convention (CWC) in 1997 was a milestone in the prohibition of chemical warfare agents (CWA). Yet, the repeated use of CWA underlines the ongoing threat to the population. Organophosphorus (OP) nerve agents still represent the most toxic CWA subgroup. Defensive research on nerve agents is mainly focused on the "classical five", namely tabun, sarin, soman, cyclosarin and VX, although Schedule 1 of the CWC covers an unforeseeable number of homologues. Likewise, an uncounted number of OP pesticides have been produced in previous decades. Our aim was to determine the in vitro inhibition kinetics of selected organophosphono- and organophosphorothioates with human AChE, as well as hydrolysis of the agents in human plasma and reactivation of inhibited AChE, in order to derive potential structure-activity relationships. The investigation of the interactions of selected OP compounds belonging to schedule 1 (V-agents) and schedule 2 (amiton) of the CWC with human AChE revealed distinct structural effects of the P -alkyl, P - O -alkyl and N , N -dialkyl residues on the inhibitory potency of the agents. Irrespective of structural modifications, all tested V-agents presented as highly potent AChE inhibitors. The high stability of the tested agents in human plasma will most likely result in long-lasting poisoning in vivo, having relevant consequences for the treatment regimen. In conclusion, the results of this study emphasize the need to investigate the biological effects of nerve agent analogues in order to assess the efficacy of available medical countermeasures.

Nerve agents, the deadliest chemical warfare agents, are potent inhibitors of acetylcholinesterase (AChE) and cause rapid cholinergic crisis with serious symptoms of poisoning. Oxime reactivators of AChE are used in medical practice in the treatment of nerve agent poisoning, but the search for novel improved reactivators with central activity is an ongoing pursuit. For numerous oximes synthesized, in vitro reactivation is a standard approach in biological evaluation with little attention given to the pharmacokinetic properties of the compounds. This study reports a comprehensive physicochemical, pharmacokinetic, and safety profiling of five lipophilic 3-hydroxy-2-pyridine aldoximes, which were recently shown to be potent AChE reactivators with a potential to be centrally active. The oxime JR595 was singled out as highly metabolically stable in human liver microsomes, noncytotoxic oxime for SH-SY5Y neuroblastoma and 1321N1 astrocytoma cell lines, and its pharmacokinetic profile was determined after intramuscular administration in mice. JR595 was rapidly absorbed into blood after 15 min with simultaneous distribution to the brain at up to about 40% of its blood concentration; however, it was eliminated from both the brain and blood within an hour. In addition, the MDCKII-MDR1 cell line assay showed that oxime JR595 was not a P-glycoprotein efflux pump substrate. Finally, the preliminary antidotal study against multiple LD 50 doses of VX and sarin in mice showed the potential of JR595 to provide desirable therapeutic outcomes with future improvements in its circulation time.

Although there has been an increasing appreciation for functional differences between the dorsal (dH) and ventral (vH) hippocampal sectors, there is a lack of information characterizing the cholinergic and noncholinergic mechanisms of acetylcholinesterase inhibitors on synaptic transmission along the hippocampal dorsoventral axis. Diisopropylfluorophosphate (DFP) is an organophosphate (OP) that is commonly employed as a nerve agent surrogate in vitro as well as in rodent models of disease states, such as Gulf War Illness. The present study investigated the cholinergic and noncholinergic mechanisms responsible for the effects of acute DFP exposure on dH and vH synaptic transmission in a hippocampal slice preparation. A paired-pulse extracellular recording protocol was used to monitor the population spike (PS) amplitude as well as the PS paired-pulse ratio (PS-PPR) in the CA1 subfield of the dH and the vH. We observed that DFP-induced PS1 inhibition was produced by a cholinergic mechanism in the dH, whereas a noncholinergic mechanism was indispensable in mediating the inhibitory effect of DFP on the PS1 in the vH. PS-PPR in both dH and vH sectors was increased by acute DFP exposure, an effect that was blocked by an N-methyl-D-aspartate receptor antagonist but not by cholinergic antagonists. Clinical reports have indicated dorsoventral-specific hippocampal abnormalities in cases of OP intoxications. Therefore, the observed dorsoventral-specific noncholinergic mechanisms underlying the effects of DFP on hippocampal synaptic transmission may have important implications for the treatment of OP overexposures. SIGNIFICANCE STATEMENT: It is unknown if acetylcholinesterase inhibitors differentially impact dorsal and ventral hippocampal synaptic transmission. The data in the present study show that an organophosphate, diisopropylfluorophosphate, impacts glutamatergic transmission along the dorsoventral axis in a hippocampal slice preparation via distinct cholinergic and noncholinergic mechanisms. These findings may provide insight into investigations of therapeutic agents that target noncholinergic mechanisms in cases of organophosphate overexposures., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Introduction: First responders and those who work with organophosphate (OP) compounds can experience ocular symptoms similar to those caused by exposure to low levels of nerve agents. This study was designed to examine the efficacy of a safe, clinically available, simulant that reproduces ocular symptoms associated with low-level OP exposure. Among these ocular symptoms are a constriction of the pupils (miosis), decreased visual acuity, and changes in accommodation., Materials and Methods: Volunteers aged 18-40 were assigned to groups receiving either a two-drop or three-drop dose of FDA approved 2% pilocarpine ophthalmic solution. Baseline visual performance measurements were taken before eye drop instillation and a timer was started following the first drop of pilocarpine. Once eye drops were administered, visual performance including distant and near vision, pupil size, and accommodation were measured every 5 minutes for 2 hours., Results: Both groups experienced significant miosis in excess of 90 minutes. Visual acuity was significantly reduced because of accommodative changes. The three-drop group experienced longer lasting combined effects when compared to the two-drop group., Conclusions: 2% pilocarpine ophthalmic solution can safely simulate major ocular symptoms of OP exposure for behavioral research studies for at least 60 minutes., (© Association of Military Surgeons of the United States 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In March 2018 the term Novichok (Hoвичoκ) became publically known following an attempted murder of a former Russian spy in Salisbury, UK. Novichok is the name of a group of nerve agents secretly produced by Russia in the later stages of the Cold War. These compounds were never declared under the Chemical Weapons Convention and very little is known about the actual identity and characteristics of these compounds. Structures of some of the Novichoks have been reported by a former Russian chemist, Vil Mirzayanov, previously working at the Russian State Scientific Research Institute of Organic Chemistry and Technology (GOSNIIOKhT). It was in this context claimed that at least two compounds of the Novichok family, known as Novichok-5 and Novichok-7 were 5-8 times more potent than the hitherto most toxic nerve agent, VX. The present study elucidates, applying a series of QSAR models toxicity, skin permeation, pharmacokinetic aspects as well as the environmental fate of a series of Novichoks. Virtually the results from the different studies related to human health point in the same direction, i. e., the Novichoks are significantly less toxic than VX and the skin permeation much lower and less efficient than observed for VX. Hence, the claim by Mirzayanov could not be substantiated., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Organophosphorus nerve agents (OPNAs) irreversibly block acetylcholinesterase activity, resulting in accumulation of excess acetylcholine at neural synapses, which can lead to a state of prolonged seizures known as status epilepticus (SE). Benzodiazepines, the current standard of care for SE, become less effective as latency to treatment increases. In a mass civilian OPNA exposure, concurrent trauma and limited resources would likely cause a delay in first response time. To address this issue, we have developed a rat model to test novel anticonvulsant/ neuroprotectant adjuncts at delayed time points., Methods: For model development, adult male rats with cortical electroencephalographic (EEG) electrodes were exposed to soman and administered saline along with atropine, 2-PAM, and midazolam 5, 20, or 40 min after SE onset. We validated our model using three drugs: scopolamine, memantine, and phenobarbital. Using the same procedure outlined above, rats were given atropine, 2-PAM, midazolam and test treatment 20 min after SE onset., Results: Using gamma power, delta power, and spike rate to quantify EEG activity, we found that scopolamine was effective, memantine was minimally effective, and phenobarbital had a delayed effect on terminating SE. Fluoro-Jade B staining was used to assess neuroprotection in five brain regions. Each treatment provided significant protection compared to saline + midazolam in at least two brain regions., Discussion: Because our data agree with previously published studies on the efficacy of these compounds, we conclude that this model is a valid way to test novel anticonvulsants/ neuroprotectants for controlling benzodiazepine-resistant OPNA-induced SE and subsequent neuropathology., (Published by Elsevier Inc.)

Objectives: Children and adults are likely to be among the casualties in a civilian nerve agent exposure. This study evaluated the efficacy of valnoctamide (racemic-VCD), sec-butylpropylacetamide (racemic-SPD), and phenobarbital for stopping nerve agent seizures in both immature and adult rats., Methods: Female and male postnatal day (PND) 21, 28, and 70 (adult) rats, previously implanted with electroencephalography (EEG) electrodes were exposed to seizure-inducing doses of the nerve agents sarin or VX and EEG was recorded continuously. Five minutes after seizure onset, animals were treated with SPD, VCD, or phenobarbital. The up-down method was used over successive animals to determine the anticonvulsant median effective dose (ED 50 ) of the drugs., Results: SPD-ED 50 values in the VX model were the following: PND21, 53 mg/kg (male) and 48 mg/kg (female); PND28, 108 mg/kg (male) and 43 mg/kg (female); and PND70, 101 mg/kg (male) and 40 mg/kg (female). SPD-ED 50 values in the sarin model were the following: PND21, 44 mg/kg (male) and 28 mg/kg (female); PND28, 79 mg/kg (male) and 34 mg/kg (female); and PND70, 53 mg/kg (male) and 53 mg/kg (female). VCD-ED 50 values in the VX model were the following: PND21, 34 mg/kg (male) and 43 mg/kg (female); PND28, 165 mg/kg (male) and 59 mg/kg (female); and PND70, 87 mg/kg (male) and 91 mg/kg (female). VCD-ED 50 values in the sarin model were the following: PND21, 45 mg/kg (male), 48 mg/kg (female); PND28, 152 mg/kg (male) 79 mg/kg (female); and PND70, 97 mg/kg (male) 79 mg/kg (female). Phenobarbital-ED 50 values in the VX model were the following: PND21, 43 mg/kg (male) and 18 mg/kg (female); PND28, 48 mg/kg (male) and 97 mg/kg (female). Phenobarbital-ED 50 values in the sarin model were the following: PND21, 32 mg/kg (male) and 32 mg/kg (female); PND28, 58 mg/kg (male) and 97 mg/kg (female); and PND70, 65 mg/kg (female)., Significance: SPD and VCD demonstrated anticonvulsant activity in both immature and adult rats in the sarin- and VX-induced status epilepticus models. Phenobarbital was effective in immature rats, whereas in adult rats, higher doses were required that were accompanied by toxicity. Overall, significantly less drug was required to stop seizures in PND21 animals than in the older animals, and overall, males required higher amounts of drug than females., (Wiley Periodicals, Inc. © 2019 International League Against Epilepsy.)

Nerve agents belong to the most dangerous chemical warfare agents and can be/were misused by terrorists. Effective prophylaxis and treatment is necessary to diminish their effect. General principles of prophylaxis are summarized (protection against acetylcholinesterase inhibition, detoxification, treatment "in advance" and use of different drugs). They are based on the knowledge of mechanism of action of nerve agents. Among different examinations, it is necessary to test prophylactic effectivity in vivo and compare the results with protection in vitro. Chemical and biological approaches to the development of new prophylactics would be applied simultaneously during this research. Though the number of possible prophylactics is relatively high, the only four drugs were introduced into military medical practice. At present, pyridostigmine seems to be common prophylactic antidote; prophylactics panpal (tablets with pyridostigmine, trihexyphenidyl and benactyzine), transant (transdermal patch containing HI-6) are other means introduced into different armies as prophylactics. Scavenger commercionally available is Protexia®. Future development will be focused on scavengers, and on other drugs either reversible cholinesterase inhibitors (e.g., huperzine A, gallantamine, physostigmine, acridine derivatives) or other compounds., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Six chlorinated bispyridinium mono-oximes, analogous to potent charged reactivators K027, K048, and K203, were synthesized with the aim of improving lipophilicity and reducing the p K a value of the oxime group, thus resulting in a higher oximate concentration at pH 7.4 compared to nonchlorinated analogues. The nucleophilicity was examined and the p K a was found to be lower than that of analogous nonchlorinated oximes. All the new compounds efficiently reactivated human AChE inhibited by nerve agents cyclosarin, sarin, and VX. The most potent was the dichlorinated analogue of oxime K027 with significantly improved ability to reactivate the conjugated enzyme due to improved binding affinity and molecular recognition. Its overall reactivation of sarin-, VX-, and cyclosarin-inhibited AChE was, respectively, 3-, 7-, and 8-fold higher than by K027. Its universality, PAMPA permeability, favorable acid dissociation constant coupled with its negligible cytotoxic effect, and successful ex vivo scavenging of nerve agents in whole human blood warrant further analysis of this compound as an antidote for organophosphorus poisoning.

Organophosphates (OP) such as the pesticide diisopropylfluorophosphate (DFP) and the nerve agent sarin are lethal chemicals that induce seizures, status epilepticus (SE), and brain damage. Midazolam, a benzodiazepine modulator of synaptic GABA-A receptors, is currently considered as a new anticonvulsant for nerve agents. Here, we characterized the time course of protective efficacy of midazolam (0.2-5 mg/kg, i.m.) in rats exposed to DFP, a chemical threat agent and surrogate for nerve agents. Behavioral and electroencephalogram (EEG) seizures were monitored for 24 hours after DFP exposure. The extent of brain injury was determined 3 days after DFP exposure by unbiased stereologic analyses of valid markers of neurodegeneration and neuroinflammation. Seizures were elicited within ∼8 minutes after DFP exposure that progressively developed into persistent SE lasting for hours. DFP exposure resulted in massive neuronal injury or necrosis, neurodegeneration of principal cells and interneurons, and neuroinflammation as evident by extensive activation of microglia and astrocytes in the hippocampus, amygdala, and other brain regions. Midazolam controlled seizures, neurodegeneration, and neuroinflammation when given early (10 minutes) after DFP exposure, but it was less effective when given at 40 minutes or later. Delayed therapy (≥40 minutes), a simulation of the practical therapeutic window for first responders or hospital admission, was associated with reduced seizure protection and neuroprotection. These results strongly reaffirm that the DFP-induced seizures and brain damage are progressively resistant to delayed treatment with midazolam, confirming the benzodiazepine refractory SE after OP intoxication. Thus, novel anticonvulsants superior to midazolam or adjunct therapies that enhance its efficacy are needed for effective treatment of refractory SE., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Organophosphorous (OP) compounds (such as nerve agents) inhibit the enzyme acetylcholinesterase (AChE) by covalent phosphylation of a key serine residue in the active site of the enzyme resulting in severe symptoms and ultimately death. OP intoxications are currently treated by administration of certain oxime compounds. The presently fielded oximes reactivate OP-inhibited AChE by liberating the phosphylated serine. Recent research towards new reactivators was predominantly devoted to design, synthesis and evaluation of new oxime-based compounds dedicated to overcoming some of the major limitations such as their intrinsic toxicity, their permanent charge which thwarts penetration of brain tissues and their inability to effectively reactivate all types of nerve agent inhibited AChEs. However, in over six decades of research only limited success has been achieved, indicating that there is a need for alternative classes of compounds that could reactivate OP-inhibited AChE. Recently, a number of non-oxime compounds was discovered in which the 4-amino-2-((diethylamino)methyl)phenol (ADOC) motif proved to be able to reactivate OP-inhibited AChE to some extent. In this paper several structural derivatives of ADOC were synthesized and screened for their ability to reactivate human AChE (hAChE) inhibited by the nerve agents VX, sarin, tabun, cyclosarin and paraoxon. We here disclose that one of those compounds showed a remarkable ability to reactivate OP-inhibited hAChE in vitro and that it is the most potent non-oxime reported to date., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

After the inhibition of acetylcholinesterase (AChE) by organophosphorus (OP) nerve agents, a dealkylation reaction of the phosphylated serine, referred to as aging, can occur. When aged, known reactivators of OP-inhibited AChE are no longer effective. Realkylation of aged AChE may provide a route to reversing aging. We designed and synthesized a library of quinone methide precursors (QMPs) as proposed realkylators of aged AChE. Our lead compound (C8) from an in vitro screen successfully resurrected 32.7 and 20.4% of the activity of methylphosphonate-aged and isopropyl phosphate-aged electric-eel AChE, respectively, after 4 days. C8 displays properties of both resurrection (recovery from the aged to the native state) and reactivation (recovery from the inhibited to the native state). Resurrection of methylphosphonate-aged AChE by C8 was significantly pH-dependent, recovering 21% of activity at 4 mM and pH 9 after only 1 day. C8 is also effective against isopropyl phosphate-aged human AChE.

Organophosphorus agents such as sarin and soman that phosphylate the active site serine of the enzyme acetylcholinesterase are notorious and pernicious, not only because they have been used by tyrants to effect mass murder of their own populations but also because they are sought by terrorists to inflict mass casualties on civilian populations. These threats underscore the need to develop effective antidotes against such agents. Phosphylation of acetylcholinesterase produces two adducts, an initial neutral adduct that can be reactivated with oxime nucleophiles, and a subsequent monoanionic adduct (called aged acetylcholinesterase) which has proven over two generations to be impervious to reactivation. This Viewpoint discusses a recent article in the journal that describes the first successful efforts to resurrect the activity of aged acetylcholinesterase.

Organophosphorus nerve agents (NAs) irreversibly inhibit acetylcholinesterase (AChE), the enzyme responsible for breaking down the neurotransmitter acetylcholine (ACh). The over accumulation of ACh after NA exposure leads to cholinergic toxicity, seizure, and death. Current medical countermeasures effectively mitigate peripheral symptoms, however; the brain is often unprotected. Alternative acute treatment with the adenosine A 1 receptor agonist N 6 -cyclopentyladensosine (CPA) has previously been demonstrated to prevent AChE inhibition as well as to suppress neuronal activity. The mechanism of AChE protection is unknown. To elucidate the feasibility of potential CPA-AChE interaction mechanisms, we applied a truncated molecular model approach and density functional theory. The candidate mechanisms studied are reversible enzyme inhibition, enzyme reactivation, and NA blocking prior to enzyme conjugation. Our thermodynamic data suggest that CPA can compete with the NAs sarin and soman for the active site of AChE, but may, in contrast to NAs, undergo back-reaction. We found a strong interaction between CPA and NA conjugated AChE, making enzyme reactivation unlikely but possibly allowing for CPA protection through the prevention of NA aging. The data also indicates that there is an affinity between CPA and unbound NAs. The results from this study support the hypothesis that CPA counters NA toxicity via multiple mechanisms and is a promising therapeutic strategy that warrants further development., (Published by Elsevier Ltd.)

Nerve agents are organophosphorus chemical warfare agents that exert their action through the irreversible inhibition of acetylcholinesterase, with a consequent overstimulation of cholinergic transmission followed by its shutdown. Beyond warfare, they have notoriously been employed in acts of terrorism as well as high profile assassinations. After a brief historical introduction on the development and deployment of nerve agents, this review provides a survey of their chemistry, the way they affect cholinergic transmission, the available treatment options, and the current directions for their improvement. As the review illustrates, despite their merits, the currently available treatment options present several shortcomings. Current research directions involve the search for improved antidotes, antagonists of the nicotinic receptors, small-molecule pretreatment options, as well as bioscavengers as macromolecular pretreatment options. These efforts are making good progress in many different directions and, hopefully, will lead to a lower target susceptibility, thus reducing the appeal of nerve agents as chemical weapons.

Neuroprotection from nerve agent such as soman-induced neural damage is a major challenge for existing drugs. Nerve agent exposure can cause many neural effects in survivors arising mainly due to acetylcholinesterase (AChE) inhibition or death within minutes. Unraveling the mechanisms underlying the nerve agent-induced multiple neurological effects is useful to develop better and safe drugs. The present study aimed to understand the molecular response during soman exposure and to evaluate the neuroprotective efficacy of galantamine on nerve agent-induced neurotoxic changes. mRNA expression studies using quantitative real-time PCR revealed significant changes in S-100β, Gfap, c-fos, and Bdnf in the hippocampus and piriform cortex after soman (90 μg/kg, s.c) exposure. Immunoblot analysis showed acute soman exposure significantly increased the protein levels of neuroglial markers (S100-β and GFAP); c-Fos and protein oxidation in discrete rat brain areas indicate their role in nerve agent-induced neurotoxicity. Induction of BDNF levels during soman exposure may indicate the recovery mechanisms activation. AChE was inhibited in the blood and brain up to 82% after soman exposure. Antidotal treatment with galantamine alone (3 mg/kg) and galantamine plus atropine (10 mg/kg) has protected animals from nerve agent-induced intoxication, death, and soman-inhibited AChE up to 45% in the blood and brain. Animal received galantamine displayed increased levels of neuroprotective genes (nAChRα-7, Bcl-2, and Bdnf) in the brain suggest the neuroprotective value of galantamine. Neuroglial changes, c-Fos, and protein oxidation levels significantly reduced after galantamine and galantamine plus atropine treatment indicate their potential antidotal value in nerve agent treatment.

Human butyrylcholinesterase is a performant stoichiometric bioscavenger of organophosphorous nerve agents. It is either isolated from outdated plasma or functionally expressed in eukaryotic systems. Here, we report the production of active human butyrylcholinesterase in a prokaryotic system after optimization of the primary sequence through the Protein Repair One Stop Shop process, a structure- and sequence-based algorithm for soluble bacterial expression of difficult eukaryotic proteins. The mutant enzyme was purified to homogeneity. Its kinetic parameters with substrate are similar to the endogenous human butyrylcholinesterase or recombinants produced in eukaryotic systems. The isolated protein was prone to crystallize and its 2.5-Å X-ray structure revealed an active site gorge region identical to that of previously solved structures. The advantages of this alternate expression system, particularly for the generation of butyrylcholinesterase variants with nerve agent hydrolysis activity, are discussed., Competing Interests: The authors declare no conflict of interest.

The threat of chemical warfare agents like nerve agents requires life saving measures of medical pretreatment combined with treatment after exposure. Pretreatment (pyridostigmine) may cause some side effects in a small number of individuals. A comprehensive research on animals has been performed to clarify effects on behavior. The results from these studies are far from unambiguous, since pyridostigmine may produce adverse effects on behavior in animals in relatively high doses, but not in a consistent way. Other animal studies have examined the potential of drugs like physostigmine, galantamine, benactyzine, trihexyphenidyl, and procyclidine, but they all produce marked behavioral impairment at doses sufficient to contribute to protection against a convulsant dose of soman. Attempts have also been made to develop a combination of drugs capable of assuring full protection (prophylaxis) against nerve agents. However, common to all combinations is that they at anticonvulsant doses cause behavioral deficits. Therefore, the use of limited pretreatment doses may be performed without marked side effects followed by post-exposure therapy with a combination of drugs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The terroristic availability of highly toxic nerve agents (NAs) highlights the necessity for a deep understanding of their toxicities and effective medical treatments. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for a characterization of the NAs poisoning and an evaluation on the efficacy of reactivators in in vitro was developed for the first time. After exposure to sarin or VX and pepsin digestion, the specific peptides of acetylcholinesterase (AChE) in a purified status, i.e. undecapeptide "GESAGAASVGM" in free, unaged, or aged status was identified and quantified. A key termination procedure is focused to make the reaction system "frozen" and precisely "capture" the poisoning, aging and spontaneous reactivation status of AChE, and the abundance of such specific peptides can thus be simultaneously measured. In our established method, as low as 0.72% and 0.84% inhibition level of AChE induced by 0.5nM sarin and VX can be detected from the measurement of peptide adducts, which benefits a confirmation of NAs exposure, especially at extremely low levels. Comparing with conventional colorimetric Ellman assays, our method provides not only enzyme activity and inhibition rate, but also the precise poisoning status of NAs exposed AChE. Based on the full information provided by this method, the efficacy of reactivators, such as HI-6, obidoxime and pralidoxime, in the typical treatment of NAs poisoned AChE in in vitro was further evaluated. Our results showed that this method is a promising tool for the characterization of NAs poisoning and the evaluation of reactivator efficacy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Changes in electrodermal activity (EDA) correlate with arousal and stress during stimulating experiences. We hypothesized that associations exist between short-term performance gains and changes in EDA. A total of 187 combat medics were randomly assigned to simulation (S), live tissue (L), or video (V) based training in the recognition and treatment of nerve agent casualties. Change in EDA from baseline to training was quantified for tonic and phasic responses and was categorized as positive (>+10%), no change (±10%), or negative (<-10%). Cognitive and psychomotor skills assessments were applied before and after the baseline/training period to quantify short-term performance changes. Statistically significant differences in both EDA arousal measures between training modalities (p < 0.001 with L > S ∼ V) were observed. Notably, larger proportions of trainees experienced negative changes in tonic (67%) and phasic (21%) EDA measures in the V group when compared to the L and S groups. Regardless of training modality, negative tonic and phasic EDA responses were associated with lower psychomotor performance gains and this finding approached statistical significance (tonic: p = 0.056, phasic: p = 0.08). No significant differences were noted in pre- to post-training cognitive performance between EDA response categories. As quantified by EDA response to training, reduced arousal was associated with lower short-term psychomotor, but not cognitive, performance gains., (Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.)

Nerve agents are organophosphates acting as potent inhibitors of acetylcholinesterase (AChE), the enzyme responsible for the hydrolysis of acetylcholine and, consequently, the termination of the transmission of nerve impulses. The inhibition of AChE by an organophosphate can be reversed by a nucleophilic agent able to dephosphorylate a serine residue in the active site of AChE. In this sense, the oximes are compounds capable of removing the nerve agent and reactivate the enzyme. Here, we have applied a methodology involving theoretical docking and Quantum Mechanics/Molecular Mechanics, using the softwares Molegro(®) and Spartan(®), to evaluate the kinetic constants of reactivation and the interactions of the oxime BI-6 with AChE inhibited by different organophosphorus compounds in comparison to in vitro data. Results confirm that this method is suitable for the prediction of kinetic and thermodynamic parameters of oximes, which may be useful in the design and selection of new and more effective oximes.

Oxime K203 seems to be the most promising oxime in case of reactivation of tabun-inhibited acetylcholinesterase (AChE). Although it was originally developed for treatment of tabun intoxications, it is able to reactivate cholinesterases inhibited by other nerve agents. This study is aimed at the evaluation of its potency in vitro against other nerve agents. For this purpose, sarin, tabun, cyclosarin, soman, VX, Russian VX and DFP were selected as members of the nerve agent family to check its universality. At high concentrations (10(-3) M), oxime K203 reached promising reactivation activity. At low concentrations, relevant for human use (10(-5) M), promising reactivation potency was obtained only with tabun. In conclusion, oxime K203 reactivates other nerve agents-inhibited cholinesterases, however its broad-spectrum reactivation is limited at high, for human not attainable, concentrations only.

Elucidation of noncholinesterase protein targets of organophosphates, and nerve agents in particular, may reveal additional mechanisms for their high toxicity as well as clues for novel therapeutic approaches toward intoxications with these agents. Within this framework, we here describe the synthesis of the activity-based probe 3, which contains a phosphonofluoridate moiety, a P-Me moiety, and a biotinylated O-alkyl group, and its use in activity-based protein profiling with two relevant biological samples, that is, rhesus monkey liver and cultured human A549 lung cells. In this way, we have unearthed eight serine hydrolases (fatty acid synthase, acylpeptide hydrolase, dipeptidyl peptidase 9, prolyl oligopeptidase, carboxylesterase, long-chain acyl coenzyme A thioesterase, PAF acetylhydrolase 1b, and esterase D/S-formyl glutathione hydrolase) as targets that are modified by the nerve agent sarin. It is also shown that the newly developed probe 3 might find its way into the development of alternative, less laborious purification protocols for human butyrylcholinesterase, a potent bioscavenger currently under clinical investigation as a prophylactic/therapeutic for nerve agent intoxications.