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.

Rational polytherapy in the treatment of cholinergic seizures.

The initiation and maintenance phases of cholinergic status epilepticus (SE) are associated with maladaptive trafficking of synaptic GABAA and glutamate receptors. The resulting pharmacoresistance reflects a decrease in synaptic GABAA receptors and increase in NMDA and AMPA receptors, which tilt the balance between inhibition and excitation in favor of the latter. If these changes are important to the pathophysiology of SE, both should be treated, and blocking their consequences should have therapeutic potential. We used a model of benzodiazepine-refractory SE (RSE) (Tetz et al., 2006) and a model of soman-induced SE to test this hypothesis. Treatment of RSE with combinations of the GABAAR agonists midazolam or diazepam and the NMDAR antagonists MK-801 or ketamine terminated RSE unresponsive to high-dose monotherapy with benzodiazepines, ketamine or other antiepileptic drugs (AEDs). It also reduced RSE-associated neuronal injury, spatial memory deficits and the occurrence of spontaneous recurrent seizures (SRS), tested several weeks after SE. Treatment of sc soman-induced SE similarly showed much greater reduction of EEG power by a combination of midazolam with ketamine, compared to midazolam monotherapy. When treating late (40 min after seizure onset), there may not be enough synaptic GABAAR left to be able to restore inhibition with maximal GABAAR stimulation, and further benefit is derived from the addition of an AED which increases inhibition or reduces excitation by a non-GABAergic mechanism. The midazolam-ketamine-valproate combination is effective in terminating RSE. 3-D isobolograms demonstrate positive cooperativity between midazolam, ketamine and valproate, without any interaction between the toxicity of these drugs, so that the therapeutic index is increased by combination therapy between GABAAR agonist, NMDAR antagonist and selective AEDs. We compared this drug combination based on the receptor trafficking hypothesis to treatments based on clinical practice. The midazolam-ketamine-valproate combination is far more effective in stopping RSE than the midazolam-fosphenytoin-valproate combination inspired from clinical guidelines. Furthermore, sequential administration of midazolam, ketamine and valproate is far less effective than simultaneous treatment with the same drugs at the same dose. These data suggest that we should re-evaluate our traditional treatment of RSE, and that treatment should be based on pathophysiology. The search for a better drug has to deal with the fact that most monotherapy leaves half the problem untreated. The search for a better benzodiazepine should acknowledge the main cause of pharmacoresistance, which is loss of synaptic GABAAR. Future clinical trials should consider treating both the failure of inhibition and the runaway excitation which characterize RSE, and should include an early polytherapy arm.

Evaluation of the Influence of Three Newly Developed Bispyridinium Anti-nicotinic Compounds (MB408, MB442, MB444) on the Efficacy of Antidotal Treatment of Nerve Agent Poisoning in Mice.

The influence of three newly developed bispyridinium antinicotinic compounds (the non-oximes MB408, MB442 and MB444) on the therapeutic efficacy of a standard antidotal treatment (atropine in combination with an oxime) of acute poisoning by the organophosphorus nerve agents tabun and soman was studied in mice. The therapeutic efficacy of atropine in combination with an oxime with or without one of the bispyridinium non-oximes was evaluated by determination of the LD50 values of the nerve agents and measurement of the survival time after supralethal poisoning. Addition of all the tested non-oximes increased significantly the therapeutic efficacy of atropine in combination with an oxime against tabun poisoning. They also positively influenced the number of surviving mice 6 hr after supralethal poisoning with tabun. However, they were only slightly effective for the treatment of soman poisoning. The benefit of the tested bispyridinium non-oximes was dose-dependent. To conclude, the addition of bispyridinium non-oximes to the standard antidotal treatment of acute poisoning with tabun was beneficial regardless of the chosen non-oxime, but only slightly beneficial in the case of soman poisoning.

Time Course, Behavioral Safety, and Protective Efficacy of Centrally Active Reversible Acetylcholinesterase Inhibitors in Cynomolgus Macaques.

Galantamine hydrobromide and (-)huperzine A, centrally active reversible acetylcholinesterase inhibitors, are potentially superior to the current standard, pyridostigmine bromide, as a pretreatment for organophosphorus chemical warfare nerve agent intoxication. Galantamine, huperzine, and pyridostigmine were compared for time course of acetylcholinesterase inhibition in 12 cynomolgus macaques. Although both galantamine and huperzine shared a similar time course profile for acetylcholinesterase inhibition, huperzine was 88 times more potent than galantamine. The dose for 50% acetylcholinesterase inhibition (ID50) was 4.1 ug/kg for huperzine, 362 ug/kg for galantamine, and 30.9 ug/kg for pyridostigmine. In a safety assessment, galantamine, huperzine, and pyridostigmine were examined using an operant time-estimation task. Huperzine and pyridostigmine were devoid of behavioral toxicity, whereas galantamine was behaviorally toxic at doses producing peak acetylcholinesterase inhibition of about 50% and higher. Following pretreatment with galantamine, huperzine or pyridostigmine, monkeys were challenged with the median lethal dose of soman at the time of peak acetylcholinesterase inhibition and evaluated for overt signs of soman toxicity (cholinergic crisis, convulsions). Both huperzine and galantamine were equally effective at preventing overt signs of soman toxicity, but neither drug was capable of preventing soman-induced neurobehavioral disruption. In contrast, three of four pyridostigmine-pretreated animals exposed to soman exhibited convulsions and required therapy. Full functional recovery required 3-16 days. The degree of acetylcholinesterase inhibition was lower for pyridostigmine, but rates of recovery of acetylcholinesterase activity following soman challenge were comparable for all drug pretreatments. Huperzine may be the more promising centrally active reversible acetylcholinesterase inhibitor due to its greater potency and superior safety profile.

Repeated low-dose exposures to sarin, soman, or VX affect acoustic startle in guinea pigs.

Chemical warfare nerve agents (CWNAs) are known to cause behavioral abnormalities in cases of human exposures and in animal models. The behavioral consequences of single exposures to CWNAs that cause observable toxic signs are particularly well characterized in animals; however, less is known regarding repeated smaller exposures that may or may not cause observable toxic signs. In the current study, guinea pigs were exposed to fractions (0.1, 0.2, or 0.4) of a medial lethal dose (LD50) of sarin, soman, or VX for two weeks. On each exposure day, and for a post-exposure period, acoustic startle response (ASR) was measured in each animal. Although relatively few studies use guinea pigs to measure behavior, this species is ideal for CWNA-related experiments because their levels of carboxylesterases closely mimic those of humans, unlike rats or mice. Results showed that the 0.4 LD50 doses of soman and VX transiently increased peak startle amplitude by the second week of injections, with amplitude returning to baseline by the second week post-exposure. Sarin also increased peak startle amplitude independent of week. Latencies to peak startle and PPI were affected by agent exposure but not consistently among the three agents. Most of the changes in startle responses returned to baseline following the cessation of exposures. These data suggest that doses of CWNAs not known to produce observable toxic signs in guinea pigs can affect behavior in the ASR paradigm. Further, these deficits are transient and usually return to baseline shortly after the end of a two-week exposure period.

Repeated systemic administration of the nutraceutical alpha-linolenic acid exerts neuroprotective efficacy, an antidepressant effect and improves cognitive performance when given after soman exposure.

Exposure to nerve agents results in severe seizures or status epilepticus caused by the inhibition of acetylcholinesterase, a critical enzyme that breaks down acetylcholine to terminate neurotransmission. Prolonged seizures cause brain damage and can lead to long-term consequences. Current countermeasures are only modestly effective against the brain damage supporting interest in the evaluation of new and efficacious therapies. The nutraceutical alpha-linolenic acid (LIN) is an essential omega-3 polyunsaturated fatty acid that has a wide safety margin. Previous work showed that a single intravenous injection of alpha-linolenic acid (500 nmol/kg) administered before or after soman significantly protected against soman-induced brain damage when analyzed 24h after exposure. Here, we show that administration of three intravenous injections of alpha-linolenic acid over a 7 day period after soman significantly improved motor performance on the rotarod, enhanced memory retention, exerted an anti-depressant-like activity and increased animal survival. This dosing schedule significantly reduced soman-induced neuronal degeneration in four major vulnerable brain regions up to 21 days. Taken together, alpha-linolenic acid reduces the profound behavioral deficits induced by soman possibly by decreasing neuronal cell death, and increases animal survival.

Catalytic Soman Scavenging by the Y337A/F338A Acetylcholinesterase Mutant Assisted with Novel Site-Directed Aldoximes.

Exposure to the nerve agent soman is difficult to treat due to the rapid dealkylation of the soman-acetylcholinesterase (AChE) conjugate known as aging. Oxime antidotes commonly used to reactivate organophosphate inhibited AChE are ineffective against soman, while the efficacy of the recommended nerve agent bioscavenger butyrylcholinesterase is limited by strictly stoichiometric scavenging. To overcome this limitation, we tested ex vivo, in human blood, and in vivo, in soman exposed mice, the capacity of aging-resistant human AChE mutant Y337A/F338A in combination with oxime HI-6 to act as a catalytic bioscavenger of soman. HI-6 was previously shown in vitro to efficiently reactivate this mutant upon soman, as well as VX, cyclosarin, sarin, and paraoxon, inhibition. We here demonstrate that ex vivo, in whole human blood, 1 µM soman was detoxified within 30 min when supplemented with 0.5 µM Y337A/F338A AChE and 100 µM HI-6. This combination was further tested in vivo. Catalytic scavenging of soman in mice improved the therapeutic outcome and resulted in the delayed onset of toxicity symptoms. Furthermore, in a preliminary in vitro screen we identified an even more efficacious oxime than HI-6, in a series of 42 pyridinium aldoximes, and 5 imidazole 2-aldoxime N-propylpyridinium derivatives. One of the later imidazole aldoximes, RS-170B, was a 2-3-fold more effective reactivator of Y337A/F338A AChE than HI-6 due to the smaller imidazole ring, as indicated by computational molecular models, that affords a more productive angle of nucleophilic attack.

Organophosphorus pesticide and ester analysis by using comprehensive two-dimensional gas chromatography with flame photometric detection.

Thirty-seven phosphorus (P)-containing compounds comprising organophosphorus pesticides and organophosphate esters were analyzed by using comprehensive two-dimensional gas chromatography with flame photometric detection in P mode (GC × GC-FPD(P)), with a non-polar/moderately polar column set. A suitable modulation temperature and period was chosen based on experimental observation. A number of co-eluting peak pairs on the (1)D column were well separated in 2D space. Excellent FPD(P) detection selectivity, responding to compounds containing the P atom, produces clear 2D GC × GC plots with little interference from complex hydrocarbon matrices. Limits of detection (LOD) were within the range of 0.0021-0.048 µmol L(-1), and linear calibration correlation coefficients (R(2)) for all 37 P-compounds were at least 0.998. The P-compounds were spiked in 2% diesel and good reproducibility for their response areas and retention times was obtained. Spiked recoveries were 88%-157% for 5 µg L(-1) and 80%-138% for 10 µg L(-1) spiked levels. Both (1)tR and (2)tR shifts were noted when the content of diesel was in excess of 5% in the matrix. Soil samples were analyzed by using the developed method; some P-compounds were positively detected. In general, this study shows that GC × GC-FPD(P) is an accurate, sensitive and simple method for P-compound analysis in complicated environmental samples.

Capacities of metabotropic glutamate modulators in counteracting soman-induced seizures in rats.

Current treatment of nerve agent poisoning with ionotropic drugs proves inadequate, and alternative treatment strategies are searched for. Based on positive findings with metabotropic glutamate modulators in microinfusion studies, the present study was initiated to examine anticonvulsant effects of MPEP (2-Methyl-6-(phenylethynyl)pyridine hydrochloride), a metabotropic glutamate receptor 5 antagonist, and DCG-IV ((2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine), a metabotropic glutamate receptor 2/3 agonist, when administered systemically in combinations with HI-6 (1-[([4-(aminocarbonyl)pyridino]methoxy)methyl]-2-[(hydroxyimino)methyl]pyridinium) and procyclidine or HI-6 and levetiracetam relative to the combination of HI-6, procyclidine, and levetiracetam. The results showed that MPEP or DCG-IV combined with HI-6 and procyclidine resulted in substantial antidotal efficacy when administered 20 min after onset of seizures elicited by soman. MPEP or DCG-IV combined with HI-6 and levetiracetam did not terminate seizures and preserve lives. When given 20 min before challenge with soman, DCG-IV in combination with HI-6 and procyclidine provided protection, whereas MPEP combined with HI-6 and procyclidine did not. Combinations with metabotropic glutamate receptor modulators did not achieve the same high level of antidotal efficacy as the combination of HI-6, procyclidine, and levetiracetam. MPEP alone inhibited pseudocholinesterase activity in the brain markedly. A positive correlation was found between latency to seizure onset or full protection and level of pseudocholinesterase activity in brain. MPEP and DCG-IV can serve as effective anticonvulsants against nerve agent poisoning when combined with HI-6 and procyclidine. Metabotropic glutamate receptor modulators may represent an alternative or supplement to treatment with ionotropic drugs.

Identification and characterization of novel catalytic bioscavengers of organophosphorus nerve agents.

In an effort to discover novel catalytic bioscavengers of organophosphorus (OP) nerve agents, cell lysates from a diverse set of bacterial strains were screened for their capacity to hydrolyze the OP nerve agents VX, VR, and soman (GD). The library of bacterial strains was identified using both random and rational approaches. Specifically, two representative strains from eight categories of extremophiles were chosen at random. For the rational approach, the protein sequence of organophosphorus hydrolase (OPH) from Brevundimonas diminuta was searched against a non-redundant protein database using the Basic Local Alignment Search Tool to find regions of local similarity between sequences. Over 15 protein sequences with significant sequence similarity to OPH were identified from a variety of bacterial strains. Some of these matches were based on predicted protein structures derived from bacterial genome sequences rather than from bona fide proteins isolated from bacteria. Of the 25 strains selected for nerve agent testing, three bacterial strains had measurable levels of OP hydrolase activity. These strains are Ammoniphilus oxalaticus, Haloarcula sp., and Micromonospora aurantiaca. Lysates from A. oxalaticus had detectable hydrolysis of VR; Haloarcula sp. had appreciable hydrolysis of VX and VR, whereas lysates from M. aurantiaca had detectable hydrolysis of VR and GD.

[+]-Huperzine A protects against soman toxicity in guinea pigs.

The chemical warfare nerve agent (CWNA) soman irreversibly inhibits acetylcholinesterase (AChE) causing seizure, neuropathology and neurobehavioral deficits. Pyridostigmine bromide (PB), the currently approved pretreatment for soman, is a reversible AChE inhibitor that does not cross the blood-brain barrier (BBB) to protect against central nervous system damage. [-]-Huperzine A, a natural reversible AChE inhibitor, rapidly passes through the BBB and has numerous neuroprotective properties that are beneficial for protection against soman. However, [-]-Huperzine A is toxic at higher doses due to potent AChE inhibition which limits the utilization of its neuroprotective properties. [+]-Huperzine A, a synthetic stereoisomer of [-]-Huperzine A and a weak inhibitor of AChE, is non-toxic. In this study, we evaluated the efficacy of [+]-Huperzine A for protection against soman toxicity in guinea pigs. Pretreatments with [+]-Huperzine A, i.m., significantly increased the survival rate in a dose-dependent manner against 1.2× LD(50) soman exposures. Behavioral signs of soman toxicity were significantly reduced in 20 and 40 mg/kg [+]-Huperzine A treated animals at 4 and 24 h compared to vehicle and PB controls. Electroencephalogram (EEG) power spectral analysis showed that [+]-Huperzine A significantly reduces soman-induced seizure compared to PB. [+]-Huperzine A (40 mg/kg) preserved higher blood and brain AChE activity compared to PB in soman exposed animals. These data suggest that [+]-Huperzine A protects against soman toxicity stronger than PB and warrant further development as a potent medical countermeasure against CWNA poisoning.

In vitro kinetic interactions of pyridostigmine, physostigmine and soman with erythrocyte and muscle acetylcholinesterase from different species.

The low effectiveness of atropine and oxime treatment in soman poisoning may be enhanced by carbamates pre-treatment. For ethical reasons medical countermeasures can only be tested in animal models despite the fact of substantial species differences. With this kinetic in vitro study the interactions between pyridostigmine, physostigmine and soman with human, Rhesus monkey, swine and guinea pig erythrocyte AChE were investigated. In addition, the effect of the carbamates on the residual activity and enzyme recovery after soman inhibition was examined with erythrocyte and intercostal muscle AChE from these species with a dynamic in vitro model with real-time determination of AChE activity. Only small to moderate species differences of the inhibition and decarbamylation kinetics were recorded. It was possible to show that with erythrocyte and muscle AChE a similar level of protection by carbamates and reactivation after discontinuation of the carbamates and soman could be observed. Thus, these data indicate that carbamate pre-treatment is expected to protect a critical level of muscle AChE and confirm the presumption that erythrocyte AChE may serve as a surrogate for synaptic AChE. Hence, these and previous data fortify the notion that erythrocyte AChE is a proper tool for in vitro kinetic studies as well as for therapeutic monitoring in experimental and clinical studies.

Impaired auditory and contextual fear conditioning in soman-exposed rats.

Exposure to soman (GD) can result in prolonged seizures and subsequent neuropathology in a variety of brain regions including the amygdala and hippocampus. Both regions are believed to play important roles in the development and expression of fear conditioning. The purpose of this experiment was to test these conditioning tasks as a possible behavioral correlate of the observed neuropathology. Male rats were exposed to GD (1.0 or 1.2×LD50) or saline followed with injections of atropine sulfate, the oxime HI-6 and diazepam. Fear conditioning was conducted on post-exposure day (PED) 8 followed by measuring freezing to contextual and auditory conditioned stimuli on PED 9 and 10 respectively. Contextual and auditory fear conditioning was severely impaired in both the 1.0×LD50 and 1.2×LD50 GD groups. Both GD groups spent less time freezing than controls when returned to the context in which conditioning occurred. The 1.0×LD50 and 1.2×LD50 groups had very low levels of freezing following presentation of the auditory conditioned stimulus. Neuronal fiber degeneration was present in the piriform cortex, thalamus, and amygdala in GD-exposed animals regardless of dose. The present study suggests that contextual and auditory fear conditioning is impaired in GD-exposed rats possibly due to neuropathology observed in the hippocampus, amygdala and thalamus.

Modulators of metabotropic glutamate receptors microinfused into perirhinal cortex: anticonvulsant effects in rats challenged with soman.

Examination of critical subreceptors in the seizure controlling perirhinal cortex has revealed that microinfusion of ionotropic glutamatergic antagonists can exert anticonvulsant efficacy against soman-induced seizures. The purpose of the present study was to investigate whether modulators of metabotropic glutamate (mGlu) receptors may ensure anticonvulsant effects when microinfused into the perirhinal cortex. The results showed that the mGlu5 receptor antagonist MPEP hydrochloride (2-Methyl-6-(phenylethynyl)pyridine hydrochloride) and the mGlu2/3 receptor agonist DCG-IV ((2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine) caused full protection against seizures or increased latency to onset of seizures, whereas the mGlu1 receptor antagonist LY367385 ((S)-(+)-alpha-Amino-4-carboxy-2-methylbenzeneacetic acid) did not produce anticonvulsant efficacy in response to systemically administered soman (1.3 x LD(50)). Low doses of the above modulators had no anticonvulsant effects, whereas too high dose of MPEP resulted in proconvulsant effects. The results suggest that the perirhinal cortex is a likely site of cholinergic recruitment of glutamatergic hyperactivity after exposure to a convulsant dose of soman. Modulators of mGlu receptors may represent an alternative or supplement to ionotropic glutamate antagonists as anticonvulsants against nerve agent-evoked seizures.

Protection by pyridostigmine bromide of marmoset hemi-diaphragm acetylcholinesterase activity after soman exposure.

Pyridostigmine bromide (PB) was approved by the U.S. Food and Drug Administration (FDA) in 2003 as a pretreatment in humans against the lethal effects of the irreversible nerve agent soman (GD). Organophosphate (OP) chemical warfare agents such as GD exert their toxic effects by inhibiting acetylcholinesterase (AChE) from terminating the action of acetylcholine at postsynaptic sites in cholinergic nerve terminals (including crucial peripheral muscle such as diaphragm). As part of the post-marketing approval of PB, the FDA required (under 21CFR314, the "two animal rule") the study of a non-human primate model (the common marmoset Callithrix jacchus jacchus) to demonstrate increased survival against lethal GD poisoning, and protection of physiological hemi-diaphragm function after PB pretreatment and subsequent GD exposure. Marmosets (male and female) were placed in the following experimental groups: (i) control (saline pretreatment only), (ii) low dose PB (12.5 microg/kg), or (iii) high dose (39.5 microg/kg) PB. Thirty minutes after the PB dose, animals were challenged with either saline (control) or soman (GD, 45 microg/kg), followed 1 min later by atropine (2mg/kg) and 2-PAM (25mg/kg). After a further 16 min, animals were euthanized and the complete diaphragm removed; the right hemi-diaphragm was frozen immediately at -80 degrees C, and the left hemi-diaphragm was placed in a tissue bath for 4h (to allow for decarbamylation to occur), then frozen. AChE activities were determined using the automated WRAIR cholinesterase assay. Blood samples were collected for AChE activities prior to PB, before GD challenge, and after sacrifice. RBC-AChE was inhibited by approximately 18% and 50% at the low and high doses of PB, respectively, compared to control (baseline) activity. In the absence of PB pretreatment, the inhibition of RBC-AChE by GD was 98%. The recovery of hemi-diaphragm AChE activity after the 4h wash period (decarbamylation) was approximately 8% and 17%, at the low and high PB doses, respectively, compared with the baseline (control) AChE activity prior to PB pretreatment or soman exposure. The results suggest that PB pretreatment protects a critical fraction of AChE activity in the marmoset diaphragm, which is sufficient to allow the animal to breathe despite exposure to a dose of soman that is lethal in unprotected animals.

Spontaneous recurrent seizures after status epilepticus induced by soman in Sprague-Dawley rats.

PURPOSE: Exposure to toxic levels of organophosphorus (OP) nerve agents can lead to seizures, respiratory failure, and, if untreated, death. The cholinesterase inhibitor soman belongs to the class of OP nerve agents and can cause status epilepticus (SE) and brain damage due to neuroexcitotoxicity. In the present study, electroencephalographic seizures are characterized through telemetry implants in rats exposed to soman, followed by treatment with therapeutics similar to those administered after nerve agent exposure. METHODS: Cortical electroencephalography (EEG), motor activity and body temperature were recorded continuously for 2 days preexposure and 15 days postexposure to verify the occurrence of spontaneous recurrent seizures (SRS) after soman exposure. RESULTS: Behavioral seizures were monitored and the latency to SE was 7.8 ± 4.0 min after exposure. Among the rats that showed SE, approximately 90% had prolonged seizures within the initial 3 days after soman exposure. Five percent of the rats developed stage 1 seizures, 16% stage 2, 23% stage 3, 18% stage 4, and 38% stage 5. Seventy-nine percent of the rats presented SE and epileptiform-like discharges several days after SE, and 28.9% of those with SE experienced electrographic SRS. The latency to the appearance of SRS ranged from 5-10 days. Fiber degeneration evaluated through silver staining revealed damage in cortical and subcortical areas directly correlated with SE. DISCUSSION: The presence of SRS after seizures induced by soman highlights the importance of quantifying SRS in studies where the objective is to find new therapeutics against soman-induced seizures.

Effectiveness of donepezil, rivastigmine, and (+/-)huperzine A in counteracting the acute toxicity of organophosphorus nerve agents: comparison with galantamine.

Galantamine, a centrally acting cholinesterase (ChE) inhibitor and a nicotinic allosteric potentiating ligand used to treat Alzheimer's disease, is an effective and safe antidote against poisoning with nerve agents, including soman. Here, the effectiveness of galantamine was compared with that of the centrally active ChE inhibitors donepezil, rivastigmine, and (+/-)huperzine A as a pre- and/or post-treatment to counteract the acute toxicity of soman. In the first set of experiments, male prepubertal guinea pigs were treated intramuscularly with one of the test drugs and 30 min later challenged with 1.5 x LD(50) soman (42 microg/kg s.c.). All animals that were pretreated with galantamine (6-8 mg/kg), 3 mg/kg donepezil, 6 mg/kg rivastigmine, or 0.3 mg/kg (+/-)huperzine A survived the soman challenge, provided that they were also post-treated with atropine (10 mg/kg i.m.). However, only galantamine was well tolerated. In subsequent experiments, the effectiveness of specific treatment regimens using 8 mg/kg galantamine, 3 mg/kg donepezil, 6 mg/kg rivastigmine, or 0.3 mg/kg (+/-)huperzine A was compared in guinea pigs challenged with soman. In the absence of atropine, only galantamine worked as an effective and safe pretreatment in animals challenged with 1.0 x LD(50) soman. Galantamine was also the only drug to afford significant protection when given to guinea pigs after 1.0 x LD(50) soman. Finally, all test drugs except galantamine reduced the survival of the animals when administered 1 or 3 h after the challenge with 0.6 or 0.7 x LD(50) soman. Thus, galantamine emerges as a superior antidotal therapy against the toxicity of soman.

Distortion product otoacoustic emissions as non-invasive biomarkers and predictors of soman-induced central neurotoxicity: a preliminary study.

The organophosphorus nerve agent soman is an irreversible cholinesterase (ChE) inhibitor that can produce long-lasting seizures and brain damage in which the neurotransmitters acetylcholine and glutamate are involved. These same neurotransmitters play key-roles in the auditory function. It was then assumed that exploring the hearing function may provide markers of the central events triggered by soman intoxication. In the present study, distortion product otoacoustic emissions (DPOAEs), a non-invasive audiometric method, were used to monitor cochlear functionality in rats administered with a moderate dose of soman (45 microg/kg). DPOAEs were investigated either 4h or 24h post-challenge. In parallel, the effects of soman on whole blood and brain ChE activity and on brain histology were also studied. The first main result is that DPOAE intensities were significantly decreased 4h post-soman and returned to baseline at 24h. The amplitude changes were well related to the severity of symptoms, with the greatest change being recorded in the rats that survived long-lasting convulsions. The second main result is that baseline DPOAEs recorded 8 days before soman appear to predict the severity of symptoms produced by the intoxication. Indeed, the lowest baseline DPOAEs corresponded to the occurrence of long-lasting convulsions and brain damage and to the greatest inhibition in central ChE. These results thus suggest that DPOAEs represent a promising non-invasive tool to assess and predict the central consequences of nerve agent poisoning. Further investigations will be carried out to assess the potential applications and the limits of this non-invasive method.

Changes of acetylcholinesterase activity in different rat brain areas following intoxication with nerve agents: biochemical and histochemical study.

Acetylcholinesterase activity in defined brain regions was determined using biochemical and histochemical methods 30 min after treating rats with sarin, soman or VX (0.5 x LD(50)). Enzyme inhibition was high in the pontomedullar area and frontal cortex, but was low in the basal ganglia. Histochemical and biochemical results correlated well. Determination of the activity in defined brain structures was a more sensitive parameter than determination in whole brain homogenate where the activity was a "mean" of the activities in different structures. The pontomedullar area controls respiration, so that the special sensitivity of acetylcholinesterase to inhibition by nerve agents in this area is important for understanding the mechanism of death caused by nerve agents. Thus, acetylcholinesterase activity is the main parameter investigated in studies searching for target sites following nerve agent poisoning.

Evaluation of reactivation test in anaesthetized dogs with experimental intoxication with nerve agents.

Following repeated antidotal treatment of anaesthetized dogs (1 min with atropine, 10 min with atropine and obidoxime, 60 min with atropine and obidoxime) after the intoxication with soman, sarin and VX (1 x LD50, i.m.), the blood cholinesterases (erythrocyte, whole blood, plasma) were monitored and their reactivatability (whole blood) was determined. During this treatment, the activities of erythrocyte acetylcholinesterase (AChE), plasma butyrylcholinesterase (BuChE) and whole blood cholinesterases were monitored. Atropine and obidoxime did not affect cholinesterase activities in control animals, whereas administration of obidoxime to dogs intoxicated with nerve agent caused an increase in the cholinesterase activities. The sensitivity of cholinesterases decreased in the order erythrocyte AChE > whole blood cholinesterases > plasma BuChE, respectively. Following sarin intoxication, blood cholinesterases were increased after the obidoxime administration. Intoxication with VX showed a similar picture but reactivation after the obidoxime administration was greater. In soman intoxication, the picture of cholinesterase changes was similar during the first 30 min of treatment. Then the increase in AChE activity following obidoxime administration was not as high as in the case of sarin and VX intoxication. Thus, the reactivation efficacy of obidoxime during nerve agent intoxication indicates that its repeated administration could be easily monitored using the reactivation test.