Learning and memory function preserved by delayed A1 adenosine receptor agonist treatment following soman intoxication in rats and a humanized esterase mouse model.

Exposure to organophosphorus compounds, such as soman (GD), cause widespread toxic effects, sustained status epilepticus, neuropathology, and death. The A1 adenosine receptor agonist N-bicyclo-(2.2.1)-hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA), when given 1 min after GD exposure, provides neuroprotection and prevents behavioral impairments. Here, we tested the ability of ENBA at delayed treatment times to improve behavioral outcomes via a two-way active avoidance task in two male animal models, each consisting of saline and GD exposure groups. In a rat model, animals received medical treatments (atropine sulfate [A], 2-PAM [P], and midazolam [MDZ]) or AP + MDZ + ENBA at 15 or 30 min after seizure onset and were subjected to behavioral testing for up to 14 days. In a human acetylcholinesterase knock-in serum carboxylesterase knock-out mouse model, animals received AP, AP + MDZ, AP + ENBA, or AP + MDZ + ENBA at 15 min post seizure onset and were subjected to the behavioral task on days 7 and 14. In rats, the GD/AP + MDZ + ENBA group recovered to saline-exposed avoidance levels while the GD/AP + MDZ group did not. In mice, in comparison with GD/AP + MDZ group, the GD/AP + MDZ + ENBA showed decreases in escape latency, response latency, and pre-session crossings, as well as increases in avoidances. In both models, only ENBA-treated groups showed control level inter-trial interval crossings by day 14. Our findings suggest that ENBA, alone and as an adjunct to medical treatments, can improve behavioral and cognitive outcomes when given at delayed time points after GD intoxication.

Seizure suppression and neuroprotection in soman-exposed rats following delayed intramuscular treatment of adenosine A1 receptor agonist as an adjunct to standard medical treatment.

Soman produces excitotoxic effects by inhibiting acetylcholinesterase in the cholinergic synapses and neuromuscular junctions, resulting in soman-induced sustained status epilepticus (SSE). Our previous work showed delayed intramuscular (i.m.) treatment with A1 adenosine receptor agonist N-bicyclo-[2.2.1]-hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA) alone suppressed soman-induced SSE and prevented neuropathology. Using this same rat soman seizure model, we tested if delayed therapy with ENBA (60 mg/kg, i.m.) would terminate seizure, protect neuropathology, and aid in survival when given in conjunction with current standard medical countermeasures (MCMs): atropine sulfate, 2-PAM, and midazolam (MDZ). Either 15- or 30-min following soman-induced SSE onset, male rats received atropine and 2-PAM plus either MDZ or MDZ + ENBA. Electroencephalographic (EEG) activity, physiologic parameters, and motor function were recorded. Either 2- or 14-days following exposure surviving rats were euthanized and perfused for histology. All animals treated with MDZ + ENBA at both time points had 100% EEG seizure termination and reduced total neuropathology compared to animals treated with MDZ (2-day, p = 0.015 for 15-min, p = 0.002 for 30-min; 14-day, p < 0.001 for 15-min, p = 0.006 for 30-min), showing ENBA enhanced MDZ's anticonvulsant and neuroprotectant efficacy. However, combined MDZ + ENBA treatment, when compared to MDZ treatment groups, had a reduction in the 14-day survival rate regardless of treatment time, indicating possible enhancement of MDZ's neuronal inhibitory effects by ENBA. Based on our findings, ENBA shows promise as an anticonvulsant and neuroprotectant in a combined treatment regimen following soman exposure; when given as an adjunct to standard MCMs, the dose of ENBA needs to be adjusted.

Treatment of Organophosphorus Poisoning with 6-Alkoxypyridin-3-ol Quinone Methide Precursors: Resurrection of Methylphosphonate-Aged Acetylcholinesterase.

Organophosphorus (OP) nerve agents inhibit acetylcholinesterase (AChE), creating a cholinergic crisis in which death can occur. The phosphylated serine residue spontaneously dealkylates to the OP-aged form, which current therapeutics cannot reverse. Soman's aging half-life is 4.2 min, so immediate recovery (resurrection) of OP-aged AChE is needed. In 2018, we showed pyridin-3-ol-based quinone methide precursors (QMPs) can resurrect OP-aged electric eel AChE in vitro, achieving 2% resurrection after 24 h of incubation (pH 7, 4 mM). We prepared 50 unique 6-alkoxypyridin-3-ol QMPs with 10 alkoxy groups and five amine leaving groups to improve AChE resurrection. These compounds are predicted in silico to cross the blood-brain barrier and treat AChE in the central nervous system. This library resurrected 7.9% activity of OP-aged recombinant human AChE after 24 h at 250 µM, a 4-fold increase from our 2018 report. The best QMP (1b), with a 6-methoxypyridin-3-ol core and a diethylamine leaving group, recovered 20.8% (1 mM), 34% (4 mM), and 42.5% (predicted maximum) of methylphosphonate-aged AChE activity over 24 h. Seven QMPs recovered activity from AChE aged with Soman and a VX degradation product (EA-2192). We hypothesize that QMPs form the quinone methide (QM) to realkylate the phosphylated serine residue as the first step of resurrection. We calculated thermodynamic energetics for QM formation, but there was no trend with the experimental biochemical data. Molecular docking studies revealed that QMP binding to OP-aged AChE is not the determining factor for the observed biochemical trends; thus, QM formation may be enzyme-mediated.

Hypothermia as potential therapeutic approach to attenuating soman-induced seizure, neuropathology, and mortality with an adenosine A1 receptor agonist and body cooling.

Organophosphorus nerve agents, such as soman (GD), produce excitotoxic effects resulting in sustained status epilepticus (SSE) and brain damage. Previous work shows that neuronal inhibitory effects of A1 adenosine receptor (A1AR) agonists, such as N6- Bicyclo (2.2.1)-hept-2-yl-5'-chloro-5'-deoxyadenosine (Cl-ENBA), suppresses GD-induced SSE and improves neuropathology. Some other physiologic effects of these agonists are hypothermia, hypotension, and sedation. Hypothermia may also shield the brain from injury by slowing down chemical insults, lessening inflammation, and contributing to improved neurological outcomes. Therefore, we attempted to isolate the hypothermic effect from ENBA by assessing the neuroprotective efficacy of direct surface body cooling in a rat GD-induced SSE model, and comparing the effects on seizure termination, neuropathology, and survival. Male rats implanted with a body temperature (Tb) transponder and electroencephalographic (EEG) electrodes were primed with asoxime (HI-6), exposed to GD 30 min later, and then treated with Cl-ENBA or had Tb lowered directly via body cooling at 30 min after the onset of seizure activity. Afterwards, they were either allowed to develop hypothermia as expected, or received thermal support to maintain normothermic Tb for a period of 6-h. Neuropathology was assessed at 24 h. Regardless of Cl-ENBA or surface cooling, all hypothermic GD-exposed groups had significantly improved 24-h survival compared to rats with normothermic Tb (81% vs. 39%, p < 0.001). Cl-ENBA offered neuroprotection independently of hypothermic Tb. While hypothermia enhanced the overall efficacy of Cl-ENBA by improving survival outcomes, body cooling didn't reduce seizure activity or neuropathology following GD-induced SSE.

Use of carbonyldiimidazole as a derivatization agent for the detection of pinacolyl alcohol, a forensic marker for Soman, by EI-GC-MS and LC-HRMS in official OPCW proficiency test matrices.

Pinacolyl alcohol (PA), a key forensic marker for the nerve agent Soman (GD), is a particularly difficult analyte to detect by various analytical methods. In this work, we have explored the reaction between PA and 1,1'-carbonyldiimidazole (CDI) to yield pinacolyl 1H-imidazole-1-carboxylate (PIC), a product that can be conveniently detected by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Regarding its GC-MS profile, this new carbamate derivative of PA possesses favorable chromatographic features such as a sharp peak and a longer retention time (RT = 16.62 min) relative to PA (broad peak and short retention time, RT = 4.1 min). The derivative can also be detected by LC-HRMS, providing an avenue for the analysis of this chemical using this technique where PA is virtually undetectable unless present in large concentrations. From a forensic science standpoint, detection of this low molecular weight alcohol signals the past or latent presence of the nerve agent Soman (GD) in a given matrix (i.e., environmental or biological). The efficiency of the protocol was tested separately in the analysis and detection of PA by EI-GC-MS and LC-HRMS when present at a 10 µg/mL in a soil matrix featured in the 44th PT and in a glycerol-rich liquid matrix featured in the 48th Official Organization for the Prohibition of Chemical Weapons (OPCW) Proficiency Test when present at a 5 µg/mL concentration. In both scenarios, PA was successfully transformed into PIC, establishing the protocol as an additional tool for the analysis of this unnatural and unique nerve agent marker by GC-MS and LC-HRMS.

The estimation of acute oral toxicity (LD50) of G-series organophosphorus-based chemical warfare agents using quantitative and qualitative toxicology in silico methods.

The idea of this study was the estimation of the theoretical acute toxicity (t-LD50, rat, oral dose) of organophosphorus-based chemical warfare agents from the G-series (n = 12) using different in silico methods. Initially identified in Germany, the G-type nerve agents include potent compounds such as tabun, sarin, and soman. Despite their historical significance, there is a noticeable gap in acute toxicity data for these agents. This study employs qualitative (STopTox and AdmetSAR) and quantitative (TEST; CATMoS; ProTox-II and QSAR Toolbox) in silico methods to predict LD50 values, offering an ethical alternative to animal testing. Additionally, we conducted quantitative extrapolation from animals, and the results of qualitative tests confirmed the acute toxicity potential of these substances and enabled the identification of toxicophoric groups. According to our estimations, the most lethal agents within this category were GV, soman (GD), sarin (GB), thiosarin (GBS), and chlorosarin (GC), with t-LD50 values (oral administration, extrapolated from rat to human) of 0.05 mg/kg bw, 0.08 mg/kg bw, 0.12 mg/kg bw, 0.15 mg/kg bw, and 0.17 mg/kg bw, respectively. On the contrary, compounds with a cycloalkane attached to the phospho-oxygen linkage, specifically methyl cyclosarin and cyclosarin, were found to be the least toxic, with values of 2.28 mg/kg bw and 3.03 mg/kg bw. The findings aim to fill the knowledge gap regarding the acute toxicity of these agents, highlighting the need for modern toxicological methods that align with ethical considerations, next-generation risk assessment (NGRA) and the 3Rs (replacement, reduction and refinement) principles.

Chronic sub lethal nerve agent (Soman) exposure induced long-term neurobehavioral, histological, and biochemical alterations in rats.

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 (0.25XLD50) 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.

Efficacy of Lacosamide and Rufinamide as Adjuncts to Midazolam-Ketamine Treatment Against Cholinergic-Induced Status Epilepticus in Rats.

Benzodiazepine pharmacoresistance develops when treatment of status epilepticus (SE) is delayed. This response may result from gamma-aminobutyric acid A receptors (GABAAR) internalization that follows prolonged SE; this receptor trafficking results in fewer GABAAR in the synapse to restore inhibition. Increase in synaptic N-methyl-D-aspartate receptors (NMDAR) also occurs in rodent models of SE. Lacosamide, a third-generation antiseizure medication (ASM), acts on the slow inactivation of voltage-gated sodium channels. Another ASM, rufinamide, similarly acts on sodium channels by extending the duration of time spent in the inactivation stage. Combination therapy of the benzodiazepine midazolam, NMDAR antagonist ketamine, and ASMs lacosamide (or rufinamide) was investigated for efficacy against soman (GD)-induced SE and neuropathology. Adult male rats implanted with telemetry transmitters for monitoring electroencephalographic (EEG) activity were exposed to a seizure-inducing dose of GD and treated with an admix of atropine sulfate and HI-6 1 minute later and with midazolam monotherapy or combination therapy 40 minutes after EEG seizure onset. Rats were monitored continuously for seizure activity for two weeks, after which brains were processed for assessment of neurodegeneration, neuronal loss, and neuroinflammatory responses. Simultaneous administration of midazolam, ketamine, and lacosamide (or rufinamide) was more protective against GD-induced SE compared with midazolam monotherapy. In general, lacosamide triple therapy had more positive outcomes on measures of epileptogenesis, EEG power integral, and the number of brain regions protected from neuropathology compared with rats treated with rufinamide triple therapy. Overall, both drugs were well tolerated in these combination models. SIGNIFICANCE STATEMENT: We currently report on improved efficacy of antiseizure medications lacosamide and rufinamide, each administered in combination with ketamine (NMDAR antagonist) and midazolam (benzodiazepine), in combatting soman (GD)-induced seizure, epileptogenesis, and brain pathology over that provided by midazolam monotherapy, or dual therapy of midazolam and lacosamide (or rufinamide) in rats. Administration of lacosamide as adjunct to midazolam and ketamine was particularly effective against GD-induced toxicity. However, protection was incomplete, suggesting the need for further study.

Preventing Long-Term Brain Damage by Nerve Agent-Induced Status Epilepticus in Rat Models Applicable to Infants: Significant Neuroprotection by Tezampanel Combined with Caramiphen but Not by Midazolam Treatment.

Acute exposure to nerve agents induces a peripheral cholinergic crisis and prolonged status epilepticus (SE), causing death or long-term brain damage. To provide preclinical data pertinent to the protection of infants and newborns, we compared the antiseizure and neuroprotective effects of treating soman-induced SE with midazolam (MDZ) versus tezampanel (LY293558) in combination with caramiphen (CRM) in 12- and 7-day-old rats. The anticonvulsants were administered 1 hour after soman exposure; neuropathology data were collected up to 6 months postexposure. In both ages, the total duration of SE within 24 hours after soman exposure was significantly shorter in the LY293558 plus CRM groups compared with the MDZ groups. Neuronal degeneration was substantial in the MDZ-treated groups but absent or minimal in the groups treated with LY293558 plus CRM. Loss of neurons and interneurons in the basolateral amygdala and CA1 hippocampal area was significant in the MDZ-treated groups but virtually absent in the LY293558 plus CRM groups. Atrophy of the amygdala and hippocampus occurred only in MDZ-treated groups. Neuronal/interneuronal loss and atrophy of the amygdala and hippocampus deteriorated over time. Reduction of inhibitory activity in the basolateral amygdala and increased anxiety were found only in MDZ groups. Spontaneous recurrent seizures developed in the MDZ groups, deteriorating over time; a small percentage of rats from the LY293558 plus CRM groups also developed seizures. These results suggest that brain damage can be long lasting or permanent if nerve agent-induced SE in infant victims is treated with midazolam at a delayed timepoint after SE onset, whereas antiglutamatergic treatment with tezampanel and caramiphen provides significant neuroprotection. SIGNIFICANCE STATEMENT: To protect the brain and the lives of infants in a mass exposure to nerve agents, an anticonvulsant treatment must be administered that will effectively stop seizures and prevent neuropathology, even if offered with a relative delay after seizure onset. The present study shows that midazolam, which was recently approved by the Food and Drug Administration for the treatment of nerve agent-induced status epilepticus, is not an effective neuroprotectant, whereas brain damage can be prevented by targeting glutamate receptors.

Evaluation of Midazolam-Ketamine-Allopregnanolone Combination Therapy against Cholinergic-Induced Status Epilepticus in Rats.

Status epilepticus (SE) is a life-threatening development of self-sustaining seizures that becomes resistant to benzodiazepines when treatment is delayed. Benzodiazepine pharmacoresistance is thought in part to result from internalization of synaptic GABAA receptors, which are the main target of the drug. The naturally occurring neurosteroid allopregnanolone is a therapy of interest against SE for its ability to modulate all isoforms of GABAA receptors. Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been partially effective in combination with benzodiazepines in mitigating SE-associated neurotoxicity. In this study, allopregnanolone as an adjunct to midazolam or midazolam-ketamine combination therapy was evaluated for efficacy against cholinergic-induced SE. Adult male rats implanted with electroencephalographic (EEG) telemetry devices were exposed to the organophosphorus chemical (OP) soman (GD) and treated with an admix of atropine sulfate and HI-6 at 1 minute after exposure followed by midazolam, midazolam-allopregnanolone, or midazolam-ketamine-allopregnanolone 40 minutes after seizure onset. Neurodegeneration, neuronal loss, and neuroinflammation were assessed 2 weeks after GD exposure. Seizure activity, EEG power integral, and epileptogenesis were also compared among groups. Overall, midazolam-ketamine-allopregnanolone combination therapy was effective in reducing cholinergic-induced toxic signs and neuropathology, particularly in the thalamus and hippocampus. Higher dosage of allopregnanolone administered in combination with midazolam and ketamine was also effective in reducing EEG power integral and epileptogenesis. The current study reports that there is a promising potential of neurosteroids in combination with benzodiazepine and ketamine treatments in a GD model of SE. SIGNIFICANCE STATEMENT: Allopregnanolone, a naturally occurring neurosteroid, reduced pathologies associated with soman (GD) exposure such as epileptogenesis, neurodegeneration, and neuroinflammation, and suppressed GD-induced toxic signs when used as an adjunct to midazolam and ketamine in a delayed treatment model of soman-induced status epilepticus (SE) in rats. However, protection was incomplete, suggesting that further studies are needed to identify optimal combinations of antiseizure medications and routes of administration for maximal efficacy against cholinergic-induced SE.

Discriminative detection of soman or VX exposure using europium chelated microparticle-based immunofluorescence microfluidic chip.

The retrospective detection of organophosphorus nerve agents (OPNAs) exposure has been achieved by the off-site analysis of OPNA-human serum albumin (HSA) adducts using mass spectrometry-based detection approaches. However, few specific methods are accessible for on-site detection. To address this, a novel immunofluorescence microfluidic chip (IFMC) testing system combining europium chelated microparticle (EuCM) with self-driven microfluidic chip assay has been established to unambiguously determine soman (GD) and VX exposure within 20 min, respectively. The detection system was based on the principle of indirect competitive enzyme-linked immunosorbent assay. The specific monoclonal antibodies that respectively recognized the phosphonylated tyrosine 411 of GD-HSA and VX-HSA adducts were labeled by EuCM to capture corresponding adducts in the exposed samples. The phosphonylated peptides in the test line and goat-anti-rabbit antibody in the control line were utilized to bind the EuCM-labeled antibodies for signal exhibition. The developed IFMC chip could discriminatively detect exposed HSA adducts with high specificity, demonstrating a low limit of detection at exposure concentrations of 0.5 × 10-6 mol/L VX and 1.0 × 10-6 mol/L GD. The exposed serum samples can be qualitatively detected following an additional pretreatment procedure. This is a novel rapid detection system capable of discriminating GD and VX exposure, providing an alternative method for rapidly identifying OPNA exposure.

Inhibiting Inducible Nitric Oxide Synthase with 1400W Reduces Soman (GD)-Induced Ferroptosis in Long-Term Epilepsy-Associated Neuropathology: Structural and Functional Magnetic Resonance Imaging Correlations with Neurobehavior and Brain Pathology.

Organophosphate (OP) nerve agent (OPNA) intoxication leads to long-term brain dysfunctions. The ineffectiveness of current treatments for OPNA intoxication prompts a quest for the investigation of the mechanism and an alternative effective therapeutic approach. Our previous studies on 1400W, a highly selective inducible nitric oxide synthase (iNOS) inhibitor, showed improvement in epilepsy and seizure-induced brain pathology in rat models of kainate and OP intoxication. In this study, magnetic resonance imaging (MRI) modalities, behavioral outcomes, and biomarkers were comprehensively investigated for brain abnormalities following soman (GD) intoxication in a rat model. T1 and T2 MRI robustly identified pathologic microchanges in brain structures associated with GD toxicity, and 1400W suppressed those aberrant alterations. Moreover, functional network reduction was evident in the cortex, hippocampus, and thalamus after GD exposure, and 1400W rescued the losses except in the thalamus. Behavioral tests showed protection by 1400W against GD-induced memory dysfunction, which also correlated with the extent of brain pathology observed in structural and functional MRIs. GD exposure upregulated iron-laden glial cells and ferritin levels in the brain and serum, 1400W decreased ferritin levels in the epileptic foci in the brain but not in the serum. The levels of brain ferritin also correlated with MRI parameters. Further, 1400W mitigated the overproduction of nitroxidative markers after GD exposure. Overall, this study provides direct evidence for the relationships of structural and functional MRI modalities with behavioral and molecular abnormalities following GD exposure and the neuroprotective effect of an iNOS inhibitor, 1400W. SIGNIFICANT STATEMENT: Our studies demonstrate the MRI microchanges in the brain following GD toxicity, which strongly correlate with neurobehavioral performances and iron homeostasis. The inhibition of iNOS with 1400W mitigates GD-induced cognitive decline, iron dysregulation, and aberrant brain MRI findings.

Phosphoproteome reveals long-term potentiation deficit following treatment of ultra-low dose soman exposure in mice.

Soman, a warfare nerve agent, poses a significant threat by inducing severe brain damage that often results in death. Nonetheless, our understanding of the biological changes underlying persistent neurocognitive dysfunction caused by low dosage of soman remains limited. This study used mice to examine the effects of different doses of soman over time. Phosphoproteomic analysis of the mouse brain is the first time to be used to detect toxic effects of soman at such low or ultra-low doses, which were undetectable based on measuring the activity of acetylcholinesterase at the whole-animal level. We also found that phosphoproteome alterations could accurately track the soman dose, irrespective of the sampling time. Moreover, phosphoproteome revealed a rapid and adaptive cellular response to soman exposure, with the points of departure 8-38 times lower than that of acetylcholinesterase activity. Impaired long-term potentiation was identified in phosphoproteomic studies, which was further validated by targeted quantitative proteomics, immunohistochemistry, and immunofluorescence analyses, with significantly increased levels of phosphorylation of protein phosphatase 1 in the hippocampus following soman exposure. This increase in phosphorylation inhibits long-term potentiation, ultimately leading to long-term memory dysfunction in mice.

Study of Huperzine A derivatives with extended protection against soman intoxication.

Pre-administration of huperzine A (Hup A) was validated to prevent poisoning from exposure to nerve agents (NAs) by reversibly inhibiting acetylcholinesterase (AChE). However, like the currently commonly used reversible inhibitors, Hup A has a short half-life and is unable to produce a long-term preventative effect. To extend the protective time of Hup A against NAs, 42 derivatives with a CN bond were designed based on the structure of Hup A in this study. All designed derivatives showed good binding capability with AChE via molecular docking. Six compounds (H3, H4, H11, H14, H16, and H25) with representative structures were selected for synthesis by Schiff base reaction, and their structures were stable. The modified Ellman's method showed the six compounds concentration-dependently inhibited AChE, and the half maximal inhibitory concentration (IC50) were higher than that of Hup A. Pretreatment of AChE with the derivatives significantly increased the IC50 of soman. In vivo experiments demonstrated H3, H4, H14, H16, and H25 had longer protective capacities against 1 × LD95 soman-induced death in mice than Hup A. The 12 h protective index showed that the protective ratios of H3, H4, H14 and H16 were 2.31, 1.85, 2.23 and 1.99 respectively, better than that of Hup A. The extended protection of the derivatives against soman may be explained by their transformation to Hup A in vivo. Furthermore, all six compounds showed lower acute oral toxicity than Hup A. Overall, our study provided an optional strategy to acquire pretreatment agents for NAs with extended action and low toxicity.

Disease-modifying effects of a glial-targeted inducible nitric oxide synthase inhibitor (1400W) in mixed-sex cohorts of a rat soman (GD) model of epilepsy.

BACKGROUND: Acute exposure to seizurogenic organophosphate (OP) nerve agents (OPNA) such as diisopropylfluorophosphate (DFP) or soman (GD), at high concentrations, induce immediate status epilepticus (SE), reactive gliosis, neurodegeneration, and epileptogenesis as a consequence. Medical countermeasures (MCMs-atropine, oximes, benzodiazepines), if administered in < 20 min of OPNA exposure, can control acute symptoms and mortality. However, MCMs alone are inadequate to prevent OPNA-induced brain injury and behavioral dysfunction in survivors. We have previously shown that OPNA exposure-induced SE increases the production of inducible nitric oxide synthase (iNOS) in glial cells in both short- and long- terms. Treating with a water soluble and highly selective iNOS inhibitor, 1400W, for 3 days significantly reduced OPNA-induced brain changes in those animals that had mild-moderate SE in the rat DFP model. However, such mitigating effects and the mechanisms of 1400W are unknown in a highly volatile nerve agent GD exposure. METHODS: Mixed-sex cohort of adult Sprague Dawley rats were exposed to GD (132 µg/kg, s.c.) and immediately treated with atropine (2 mg/kg, i.m) and HI-6 (125 mg/kg, i.m.). Severity of seizures were quantified for an hour and treated with midazolam (3 mg/kg, i.m.). An hour post-midazolam, 1400W (20 mg/kg, i.m.) or vehicle was administered daily for 2 weeks. After behavioral testing and EEG acquisition, animals were euthanized at 3.5 months post-GD. Brains were processed for neuroinflammatory and neurodegeneration markers. Serum and CSF were used for nitrooxidative and proinflammatory cytokines assays. RESULTS: We demonstrate a significant long-term (3.5 months post-soman) disease-modifying effect of 1400W in animals that had severe SE for > 20 min of continuous convulsive seizures. 1400W significantly reduced GD-induced motor and cognitive dysfunction; nitrooxidative stress (nitrite, ROS; increased GSH: GSSG); proinflammatory cytokines in the serum and some in the cerebrospinal fluid (CSF); epileptiform spikes and spontaneously recurring seizures (SRS) in males; reactive gliosis (GFAP + C3 and IBA1 + CD68-positive glia) as a measure of neuroinflammation, and neurodegeneration (especially parvalbumin-positive neurons) in some brain regions. CONCLUSION: These findings demonstrate the long-term disease-modifying effects of a glial-targeted iNOS inhibitor, 1400W, in a rat GD model by modulating reactive gliosis, neurodegeneration (parvalbumin-positive neurons), and neuronal hyperexcitability.

Delayed tezampanel and caramiphen treatment but not midazolam protects against long-term neuropathology after soman exposure.

Prolonged status epilepticus (SE) can cause brain damage; therefore, treatment must be administered promptly after seizure onset to limit SE duration and prevent neuropathology. Timely treatment of SE is not always feasible; this would be particularly true in a mass exposure to an SE-inducing agent such as a nerve agent. Therefore, the availability of anticonvulsant treatments that have neuroprotective efficacy even if administered with a delay after SE onset is an imperative. Here, we compared the long-term neuropathology resulting from acutely exposing 21-day-old male and female rats to the nerve agent soman, and treating them with midazolam (3 mg/kg) or co-administration of tezampanel (10 mg/kg) and caramiphen (50 mg/kg), at 1 h postexposure (~50 min after SE onset). Midazolam-treated rats had significant neuronal degeneration in limbic structures, mainly at one month postexposure, followed by neuronal loss in the basolateral amygdala and the CA1 hippocampal area. Neuronal loss resulted in significant amygdala and hippocampal atrophy, deteriorating from one to six months postexposure. Rats treated with tezampanel-caramiphen had no evidence of neuropathology, except for neuronal loss in the basolateral amygdala at the six-month timepoint. Anxiety was increased only in the midazolam-treated rats, at one, three, and six months postexposure. Spontaneous recurrent seizures appeared only in midazolam-treated rats, at three and six months postexposure in males and only at six months in females. These findings suggest that delayed treatment of nerve agent-induced SE with midazolam may result in long-lasting or permanent brain damage, while antiglutamatergic anticonvulsant treatment consisting of tezampanel and caramiphen may provide full neuroprotection.

Combination of acetylcholinesterase inhibitors and NMDA receptor antagonists increases survival rate in soman-poisoned mice.

Organophosphorus nerve agents pose a global threat to both military personnel and civilian population, because of their high acute toxicity and insufficient medical countermeasures. Commonly used drugs could ameliorate the intoxication and overall medical outcomes. In this study, we tested the drugs able to alleviate the symptoms of Alzheimer's disease (donepezil, huperzine A, memantine) or Parkinson's disease (procyclidine). They were administered to mice before soman intoxication in terms of their: i) protection potential against soman toxicity and ii) influence on post-exposure therapy consisting of atropine and asoxime (also known as oxime HI-6). Their pretreatment effect was not significant, when administered alone, but in combination (acetylcholinesterase inhibitor such as denepezil or huperzine A with NMDA antagonist such as memantine or procyclidine) they lowered the soman toxicity more than twice. These combinations also positively influenced the efficacy of post-exposure treatment in a similar fashion; the combinations increased the therapeutic effectiveness of antidotal treatment. In conclusion, the most effective combination - huperzine A and procyclidine - lowered the toxicity three times and improved the post-exposure therapy efficacy more than six times. These results are unprecedented in the published literature.

PGC 1α-Mediates Mitochondrial Damage in the Liver by Inhibiting the Mitochondrial Respiratory Chain as a Non-cholinergic Mechanism of Repeated Low-Level Soman Exposure.

This work aimed to assess whether mitochondrial damage in the liver induced by subacute soman exposure is caused by peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) and whether PGC-1α regulates mitochondrial respiratory chain damage. Toxicity mechanism research may provide theoretical support for developing anti-toxic drugs in the future. First, a soman animal model was established in male Sprague-Dawley (SD) rats by subcutaneous soman injection. Then, liver damage was biochemically evaluated, and acetylcholinesterase (AChE) activity was also determined. Transmission electron microscopy (TEM) was performed to examine liver mitochondrial damage, and high-resolution respirometry was carried out for assessing mitochondrial respiration function. In addition, complex I-IV levels were quantitatively evaluated in isolated liver mitochondria by enzyme-linked immunosorbent assay (ELISA). PGC-1α levels were detected with a Jess capillary-based immunoassay device. Finally, oxidative stress was analyzed by quantifying superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG), and reactive oxygen species (ROS) levels. Repeated low-level soman exposure did not alter AChE activity, while increasing morphological damage of liver mitochondria and liver enzyme levels in rat homogenates. Complex I, II and I + II activities were 2.33, 4.95, and 5.22 times lower after treatment compared with the control group, respectively. Among complexes I-IV, I-III decreased significantly (p < 0.05), and PGC-1α levels were 1.82 times lower after soman exposure than in the control group. Subacute soman exposure significantly increased mitochondrial ROS production, which may cause oxidate stress. These findings indicated dysregulated mitochondrial energy metabolism involves PGC-1α protein expression imbalance, revealing non-cholinergic mechanisms for soman toxicity.

Evaluation of 6-OxP-CD, an Oxime-based cyclodextrin as a viable medical countermeasure against nerve agent poisoning: Experimental and molecular dynamic simulation studies on its inclusion complexes with cyclosarin, soman and VX.

The ability of the cyclodextrin-oxime construct 6-OxP-CD to bind and degrade the nerve agents Cyclosarin (GF), Soman (GD) and S-[2-[Di(propan-2-yl)amino]ethyl] O-ethyl methylphosphonothioate (VX) has been studied using 31P-nuclear magnetic resonance (NMR) under physiological conditions. While 6-OxP-CD was found to degrade GF instantaneously under these conditions, it was found to form an inclusion complex with GD and significantly improve its degradation (t1/2 ~ 2 hrs) relative over background (t1/2 ~ 22 hrs). Consequently, effective formation of the 6-OxP-CD:GD inclusion complex results in the immediate neutralization of GD and thus preventing it from inhibiting its biological target. In contrast, NMR experiments did not find evidence for an inclusion complex between 6-OxP-CD and VX, and the agent's degradation profile was identical to that of background degradation (t1/2 ~ 24 hrs). As a complement to this experimental work, molecular dynamics (MD) simulations coupled with Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) calculations have been applied to the study of inclusion complexes between 6-OxP-CD and the three nerve agents. These studies provide data that informs the understanding of the different degradative interactions exhibited by 6-OxP-CD with each nerve agent as it is introduced in the CD cavity in two different orientations (up and down). For its complex with GF, it was found that the oxime in 6-OxP-CD lies in very close proximity (PGF⋯OOxime ~ 4-5 Å) to the phosphorus center of GF in the 'downGF' orientation for most of the simulation accurately describing the ability of 6-OxP-CD to degrade this nerve agent rapidly and efficiently. Further computational studies involving the center of masses (COMs) for both components (GF and 6-OxP-CD) also provided some insight on the nature of this inclusion complex. Distances between the COMs (ΔCOM) lie closer in space in the 'downGF' orientation than in the 'upGF' orientation; a correlation that seems to hold true not only for GF but also for its congener, GD. In the case of GD, calculations for the 'downGD' orientation showed that the oxime functional group in 6-OxP-CD although lying in close proximity (PGD⋯OOxime ~ 4-5 Å) to the phosphorus center of the nerve agent for most of the simulation, adopts another stable conformation that increase this distance to ~ 12-14 Å, thus explaining the ability of 6-OxP-CD to bind and degrade GD but with less efficiency as observed experimentally (t1/2 ~ 4 hr. vs. immediate). Lastly, studies on the VX:6-OxP-CD system demonstrated that VX does not form a stable inclusion complex with the oxime-bearing cyclodextrin and as such does not interact in a way that is conducive to an accelerated degradation scenario. Collectively, these studies serve as a basic platform from which the development of new cyclodextrin scaffolds based on 6-OxP-CD can be designed in the development of medical countermeasures against these highly toxic chemical warfare agents.

A novel genetically modified mouse seizure model for evaluating anticonvulsive and neuroprotective efficacy of an A1 adenosine receptor agonist following soman intoxication.

Recently a novel humanized mouse strain has been successfully generated, in which serum carboxylesterase (CES) knock out (KO) mice (Es1-/-) were further genetically modified by knocking in (KI), or adding, the gene that encodes the human form of acetylcholinesterase (AChE). The resulting human AChE KI and serum CES KO (or KIKO) mouse strain should not only exhibit organophosphorus nerve agent (NA) intoxication in a manner more similar to humans, but also display AChE-specific treatment responses more closely mimicking those of humans to facilitate data translation to pre-clinic trials. In this study, we utilized the KIKO mouse to develop a seizure model for NA medical countermeasure investigation, and then applied it to evaluate the anticonvulsant and neuroprotectant (A/N) efficacy of a specific A1 adenosine receptor (A1AR) agonist, N-bicyclo-(2.2.1)hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA), which has been shown in a rat seizure model to be a potent A/N compound. Male mice surgically implanted with cortical electroencephalographic (EEG) electrodes a week earlier were pretreated with HI-6 and challenged with various doses (26 to 47 µg/kg, SC) of soman (GD) to determine a minimum effective dose (MED) that induced sustained status epilepticus (SSE) activity in 100% of animals while causing minimum lethality at 24 h. The GD dose selected was then used to investigate the MED doses of ENBA when given either immediately following SSE initiation (similar to wartime military first aid application) or at 15 min after ongoing SSE seizure activity (applicable to civilian chemical attack emergency triage). The selected GD dose of 33 µg/kg (1.4 x LD50) generated SSE in 100% of KIKO mice and produced only 30% mortality. ENBA at a dose as little as 10 mg/kg, IP, caused isoelectric EEG activity within minutes after administration in naïve un-exposed KIKO mice. The MED doses of ENBA to terminate GD-induced SSE activity were determined to be 10 and 15 mg/kg when treatment was given at the time of SSE onset and when seizure activity was ongoing for 15 min, respectively. These doses were much lower than in the non-genetically modified rat model, which required an ENBA dose of 60 mg/kg to terminate SSE in 100% GD-exposed rats. At MED doses, all mice survived for 24 h, and no neuropathology was observed when the SSE was stopped. The findings confirmed that ENBA is a potent A/N for both immediate and delayed (i.e., dual purposed) therapy to victims of NA exposure and serves as a promising neuroprotective antidotal and adjunctive medical countermeasure candidate for pre-clinical research and development for human application.