Soman Toxicity Research Articles

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.

Memantine and Its Combination with Acetylcholinesterase Inhibitors in Pharmacological Pretreatment of Soman Poisoning in Mice.

Nerve agents pose a real threat to both the military and civil populations, but the current treatment of the poisoning is unsatisfactory. Thus, we studied the efficacy of prophylactic use of memantine alone or in combination with clinically used reversible acetylcholinesterase inhibitors (pyridostigmine, donepezil, rivastigmine) against soman. In addition, we tested their influence on post-exposure therapy consisting of atropine and asoxime. Pyridostigmine alone failed to decrease the acute toxicity of soman. But all clinically used acetylcholinesterase inhibitors administered alone reduced the acute toxicity, with donepezil showing the best efficacy. The combination of memantine with reversible acetylcholinesterase inhibitors attenuated soman acute toxicity significantly. The pretreatment administered alone or in combinations influenced the efficacy of post-exposure treatment in a similar fashion: (i) pyridostigmine or memantine alone did not affect the antidotal treatment, (ii) centrally acting reversible acetylcholinesterase inhibitors alone increased the antidotal treatment slightly, (iii) combination of memantine with reversible acetylcholinesterase inhibitors increased the antidotal treatment more markedly. In conclusion, memantine alone failed to decrease the acute toxicity of soman or increase post-exposure antidotal treatment efficacy. The combination of memantine with donepezil significantly increased post-exposure effectiveness (together 5.12, pretreatment alone 1.72). Both drugs, when applied together, mitigate soman toxicity and boost post-exposure treatment.

Treatment of acetylcholinesterase inhibitor-induced seizures with polytherapy targeting GABA and glutamate receptors

The initiation and maintenance of cholinergic-induced status epilepticus (SE) are associated with decreased synaptic gamma-aminobutyric acid A receptors (GABAAR) and increased N-methyl-d-aspartate receptors (NMDAR) and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). We hypothesized that trafficking of synaptic GABAAR and glutamate receptors is maladaptive and contributes to the pharmacoresistance to antiseizure drugs; targeting these components should ameliorate the pathophysiological consequences of refractory SE (RSE). We review studies of rodent models of cholinergic-induced SE, in which we used a benzodiazepine allosteric GABAAR modulator to correct loss of inhibition, concurrent with the NMDA antagonist ketamine to reduce excitation caused by increased synaptic localization of NMDAR and AMPAR, which are NMDAR-dependent. Models included lithium/pilocarpine-induced SE in rats and soman-induced SE in rats and in Es1−/− mice, which similar to humans lack plasma carboxylesterase, and may better model soman toxicity. These model human soman toxicity and are refractory to benzodiazepines administered at 40 min after seizure onset, when enough synaptic GABAAR may not be available to restore inhibition. Ketamine-midazolam combination reduces seizure severity, epileptogenesis, performance deficits and neuropathology following cholinergic-induced SE. Supplementing that treatment with valproate, which targets a non-benzodiazepine site, effectively terminates RSE, providing further benefit against cholinergic-induced SE. The therapeutic index of drug combinations is also reviewed and we show the improved efficacy of simultaneous administration of midazolam, ketamine and valproate compared to sequential drug administration. These data suggest that future clinical trials should treat both the lack of sufficient inhibition and the excess excitation that characterize RSE, and include early combination drug therapies.This article is part of the special issue entitled ‘Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield’.

Radiolabelled soman binding to sera from Rats, Guinea Pigs and Monkeys

Soman is a highly toxic organophosphorus chemical warfare compound that binds rapidly and irreversibility to a variety of serine active enzymes, i.e., butyryl- and acetyl-cholinesterases and carboxylesterase. The in vivo toxicity of soman has been reported to vary significantly in different animal species, such as rats and guinea pigs or non-human primates. This species variation makes it difficult to identify appropriate animal models for therapeutic drug development under the US Food and Drug Administration (FDA) Animal Rule. Since species variation in soman toxicity has been correlated with species variation in serum carboxylesterase, we undertook to determine if serum from guinea pigs, rats and non-human primates bound different levels of soman in vitro in the presence of equimolar concentrations of soman.Our results demonstrated that the amount of soman bound in the serum of rats was 4 uM, but essentially null in guinea pigs or non-human primates. The results strongly correlate with the presence or absence of carboxylesterase in the serum of animals and the difference in the toxic dose of soman in various species. Our results support prior suggestions that guinea pigs and non-human primates may be better animal models for the development of antidotes under the FDA Animal Rule.

Susceptibility to Soman Toxicity and Efficacy of LY293558 Against Soman-Induced Seizures and Neuropathology in 10-Month-Old Male Rats.

Acute nerve agent exposure causes prolonged status epilepticus (SE), leading to death or long-term brain damage. We have previously demonstrated that LY293558, an AMPA/GluK1 kainate receptor antagonist, terminates SE induced by the nerve agent soman and protects from long-term brain damage, in immature rats and young-adult rats, even if administered with a relatively long latency from the time of exposure. However, susceptibility to the lethal consequences of SE increases with age, and mortality by SE induced by soman is substantially greater in older animals. Therefore, in the present study, we compared the susceptibility to soman toxicity of 10-month-old male rats with that of young-adult male rats (42 to 50days old) and examined the protective efficacy of LY293558 in the older group. A lower percentage of the 10-month-old rats developed SE after injection of 1.2×LD50 soman, compared to the young adults, the latency to seizure onset was longer in the older rats, and seizure intensity did not differ between the two age groups. However, mortality rate in the older rats who developed SE was higher than in the young adults. Acetylcholinesterase activity in the amygdala, hippocampus, and piriform cortex did not differ between the two age groups. Administration of LY293558 at 20 or 60min post-exposure suppressed SE, increased 24-h survival rate, decreased the long-term risk of death, reduced neuronal degeneration in the amygdala, hippocampus, piriform, and entorhinal cortices, and facilitated recovery from body weight loss. Thus, LY293558 is an effective countermeasure against soman toxicity also in older animals.

The influence of modulators of acetylcholinesterase on the resistance of mice against soman and on the effectiveness of antidotal treatment of soman poisoning in mice

The potency of one reversible inhibitor of acetylcholinesterase (6-chlorotacrine), one reactivator of acetycholinesterase (K027) and their combination to increase the resistance of mice against soman and the efficacy of antidotal treatment of soman-poisoned mice was evaluated. While 6-chlorotacrine was able to markedly protect mice against acute toxicity of soman and the pharmacological pretreatment with 6-chlorotacrine increased the efficacy of antidotal treatment (the oxime HI-6 in combination with atropine) of soman-poisoned mice more than two times, the bispyridinium oxime K027 did not protect mice from acute toxicity of soman, however, the pharmacological pretreatment with this compound was able to markedly increase the efficacy of antidotal treatment of soman-poisoned mice. On the other hand, the combination of both modulators of acetylcholinesterase did not increase the prophylactic efficacy of 6-chlorotacrine alone. These findings demonstrate that pharmacological pretreatment of soman-poisoned mice can be promising and useful in the case of administration of 6-chlorotacrine while the administration of the oxime K027 did not bring any additional benefit when combined with 6-chlorotacrine.

The Evaluation of Benefit of Newly Prepared Reversible Inhibitors of Acetylcholinesterase and Commonly Used Pyridostigmine as Pharmacological Pretreatment of Soman-Poisoned Mice.

The ability of four newly prepared reversible inhibitors of acetylcholinesterase (6-chlorotacrine, 7-phenoxytacrine, compounds 1 and 2) and currently used carbamate pyridostigmine to increase the resistance of mice against soman and the efficacy of antidotal treatment of soman-poisoned mice was evaluated. The evaluation of the effect of pharmacological pretreatment is based on the identification of changes of soman-induced toxicity that was evaluated by the assessment of its LD50 value and its 95% confidence limit using probitlogarithmical analysis of death occurring within 24 h after administration of soman. 6-chlorotacrine was only able to markedly protect mice against acute toxicity of soman. In addition, the pharmacological pretreatment with 6-chlorotacrine or compound 2 was able to increase the efficacy of antidotal treatment (the oxime HI-6 in combination with atropine) of soman-poisoned mice. The other newly prepared reversible inhibitors of acetylcholinesterase (7-phenoxytacrine, compound 1) as well as commonly used pyridostigmine did not influence the efficacy of antidotal treatment. These findings demonstrate that pharmacological pretreatment of somanpoisoned mice can be promising and useful in the case of administration of 6-chlorotacrine and partly compound 2.

Dose Dependent Prophylactic Efficacy of 6-Chlorotacrine in Soman-Poisoned Mice.

The influence of the dose on the ability of promising newly prepared reversible inhibitor of acetylcholinesterase (6-chlorotacrine) to increase the resistance of mice against soman and the efficacy of antidotal treatment of soman-poisoned mice was evaluated. The evaluation of the effect of pharmacological pretreatment is based on the identification of changes of soman-induced toxicity that was evaluated by the assessment of its LD50 value and its 95% confidence limit using probit-logarithmical analysis of death occurring within 24 hrs after administration of soman. The dose of 6-chlorotacrine significantly influences the prophylactic efficacy of 6-chlorotacrine. Its highest dose was only able to significantly protect mice against acute toxicity of soman and increase the efficacy of antidotal treatment (atropine in combination with the oxime HI-6) of soman-poisoned mice. In addition, the highest dose of 6-chlorotacrine was significantly more effective to protect mice from soman poisoning than its lowest dose. These findings demonstrate the important influence of the dose of 6-chlorotacine on its prophylactic efficacy in the case of pharmacological pretreatment of soman poisoning in mice.

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 4.1ug/kg for huperzine, 362ug/kg for galantamine, and 30.9ug/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 3-16days. 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.

Beneficial effects of a ketamine/atropine combination in soman-poisoned rats under a neutral thermal environment

Exposure to organophosphorus (OP) compounds, such as pesticides and the chemical warfare agents (soman and sarin), respectively represents a major health problem and a threat for civilian and military communities. OP poisoning may induce seizures, status epilepticus and even brain lesions if untreated. We recently proved that a combination of atropine sulfate and ketamine, a glutamatergic antagonist, was effective as an anticonvulsant and neuroprotectant in mice and guinea-pigs exposed to soman. Since OP exposure may also occur in conditions of heat strain due to climate, wearing of protective gears or physical exercise, we previously demonstrated that ketamine/atropine association may be used in a hot environment without detrimental effects. In the present study, we assess soman toxicity and evaluate the effects of the ketamine/atropine combination on soman toxicity in a warm thermoneutral environment. Male Wistar rats, exposed to 31°C (easily reached under protective equipments), were intoxicated by soman and treated with an anesthetic dose of ketamine combined with atropine sulfate. Body core temperature and spontaneous locomotor activity were continuously monitored using telemetry. At the end of the warm exposure, blood chemistry and brain mRNA expression of some specific genes were measured. In soman-intoxicated animals, metabolic and genic modifications were related to convulsions rather than to soman intoxication by itself. In the warm environment, ketamine/atropine combination did not produce any side-effect on the assessed variables. Furthermore, the ketamine/atropine combination exhibited beneficial therapeutic effects on soman-intoxicated rats such as a limitation of convulsion-induced hyperthermia and of the increase in some blood chemistry markers.

Pretreatment with Huperzine A-Loaded Poly(lactide-co-glycolide) Nanoparticles Protects against Lethal Effects of Soman-Induced in Mice

Huperzine A (HupA), an alkaloid isolated from theChinese club moss, is a reversible inhibitor of cholinesterases which cross the blood-brain barrier and show high specificity for acetylcholinesterase (AChE). However, HupA induces unwanted side effects in an effective dose against nerve agent poisoning. In the present study, HupA–loaded poly (lactide-co-glycolide) nanoparticles (HupA-PLGA-NP) were prepared using the O/W emulsion solvent evaporation method. The results of SEM demonstrated that HupA-PLGA-NP had an spherical shape and a smooth surface without pores. It’s mean diameter and PDI were 208.5±3.6nm and 0.09±0.01 respectively. The Zeta potential was-35.3±1.8mV and the drug loading was 2.86±0.6%.In vitrodrug release studies showed that HupA-PLGA-NP had a sustained-release behavior in phosphate buffer solution, The accumulated amount of HupA was about 72.1% at 48h with a low burst release within 30min. The LD50values of HupA and HupA-PLGA-NP were 1.40 and 4.85mg/kg respectively, showing that the toxicity of HupA was reduced by 3.5 times. We evaluated the protective efficacy for different doses of HupA or HupA-PLGA-NP against 1.0×LD95(143.0μg/kg) soman toxicity. The results confirmed that HupA (0.3~0.5mg/kg) or HupA-PLGA-NP (0.5~1.5mg/kg) could ensure animals survive. However, about 10% of the animals injected with HupA (0.8mg/kg) died, while no animals died when injected with HupA-PLGA-NP (1.5mg/kg). Aim to 100% survival rate, the effective protective time (12h) of HupA-PLGA-NP (0.5mg/kg,iv) against 1.0×LD95soman toxicity in mice was significantly prolonged compared with that of HupA (4h). The study of AChE activity showed that whole-blood and supernatant of brain diluted by 80-fold and 10-fold respectively were optimum in this study. AChE inhibition after administration of HupA and HupA-PLGA-NP (0.5mg/kg,iv) was recorded and analyzed, The peak values of AChE inhibition in whole-blood and brain by HupA-PLGA-NP (17.6% and 21.8%) were lower than those by HupA (33.7% and 31.9%) and AChE inhibition time by HupA-PLGA-NP was longer than that by HupA. These data confirmed that HupA-PLGA-NP had less toxic and more longer time than HupA against 1.0×LD95soman poisoning and warrant further development as a potent medical countermeasure against chemical warfare nerve agents (CWNAs) poisoning.

Percutaneous Toxicity and Decontamination of Soman, Vx, and Paraoxon in Rats Using Detergents

Highly toxic organophosphorus compounds (OPs) were originally developed for warfare or as agricultural pesticides. Today, OPs represent a serious threat to military personnel and civilians. This study investigates the in vivo decontamination of male Wistar rats percutaneously exposed to paraoxon and two potent nerve agents--soman (GD) and VX. Four commercial detergents were tested as decontaminants--Neodekont(TM), Argos(TM), Dermogel(TM), and FloraFree(TM). Decontamination performed 2 min after exposure resulted in a higher survival rate in comparison with non-decontaminated controls. The decontamination effectiveness was expressed as protective ratio (PR, median lethal dose of agent in decontaminated animals divided by the median lethal dose of agent in untreated animals). The highest decontamination effectiveness was consistently achieved with Argos(TM) (PR=2.3 to 64.8), followed by Dermogel(TM) (PR=2.4 to 46.1). Neodekont(TM) and FloraFree(TM) provided the lowest decontamination effectiveness, equivalent to distilled water (PR=1.0 to 43.2).

Therapeutic efficacy of a novel bispyridinium oxime K203 and commonly used oximes (HI-6, obidoxime, trimedoxime, methoxime) in soman-poisoned male rats and mice

The potency of a novel oxime K203 in reactivating soman-inhibited acetylcholinesterase and reducing acute toxicity of soman was compared with commonly used oximes (HI-6, obidoxime, trimedoxime, methoxime) using in vivo methods. The study determining percentage of reactivation of soman-inhibited blood and tissue acetylcholinesterase in rats showed that the potency of the oxime K203 to reactivate soman-inhibited acetycholinesterase in the peripheral compartment is slightly higher than obidoxime and trimedoxime, especially in the diaphragm, slightly lower than methoxime and markedly lower compared to the oxime HI-6. The reactivating efficacy of the oximes studied in the peripheral compartment roughly corresponds to their potency to reduce acute toxicity of soman in mice. Based on the obtained data, we can conclude that the oxime K203 is not suitable for the replacement of the oxime HI-6 for the antidotal treatment of acute soman poisoning due to its relatively low potency to counteract acute toxicity of soman.

Polyclonal Antibody to Soman-Tyrosine

Soman forms a stable, covalent bond with tyrosine 411 of human albumin, with tyrosines 257 and 593 in human transferrin, and with tyrosine in many other proteins. The pinacolyl group of soman is retained, suggesting that pinacolyl methylphosphonate bound to tyrosine could generate specific antibodies. Tyrosine in the pentapeptide RYGRK was covalently modified with soman simply by adding soman to the peptide. The phosphonylated-peptide was linked to keyhole limpet hemocyanin, and the conjugate was injected into rabbits. The polyclonal antiserum recognized soman-labeled human albumin, soman-mouse albumin, and soman human transferrin but not nonphosphonylated control proteins. The soman-labeled tyrosines in these proteins are surrounded by different amino acid sequences, suggesting that the polyclonal recognizes soman-tyrosine independent of the amino acid sequence. Antiserum obtained after 4 antigen injections over a period of 18 weeks was tested in a competition ELISA where it had an IC50 of 10(-11) M. The limit of detection on Western blots was 0.01 μg (15 picomoles) of soman-labeled albumin. In conclusion, a high-affinity, polyclonal antibody that specifically recognizes soman adducts on tyrosine in a variety of proteins has been produced. Such an antibody could be useful for identifying secondary targets of soman toxicity.

A combination of [+] and [−]-Huperzine A improves protection against soman toxicity compared to [+]-Huperzine A in guinea pigs

The neuropathologic mechanisms after exposure to lethal doses of nerve agent are complex and involve multiple biochemical pathways. Effective treatment requires drugs that can simultaneously protect by reversible binding to the acetylcholinesterase (AChE) and blocking cascades of seizure related brain damage, inflammation, neuronal degeneration as well as promoting induction of neuroregeneration. [−]-Huperzine A ([−]-Hup A), is a naturally occurring potent reversible AChE inhibitor that penetrates the blood–brain barrier. It also has several neuroprotective effects including modification of beta-amyloid peptide, reduction of oxidative stress, anti-inflammatory, anti-apoptotic and induction and regulation of nerve growth factor. Toxicities at higher doses restrict the neuroporotective ability of [−]-Hup A for treatment. The synthetic stereoisomer, [+]-Hup A, is less toxic due to poor AChE inhibition and is suitable for both pre-/post-exposure treatments of nerve agent toxicity. [+]-Hup A block the N-methyl-d-aspartate (NMDA)-induced seizure in rats, reduce excitatory amino acid induced neurotoxicity and also prevent soman induced toxicity with minimum performance decrement. Unique combinations of two stereo-isomers of Hup A may provide an excellent pre/post-treatment drug for the nerve agent induced seizure/status epilepticus. We investigated a combination of [+]-Hup A with a small dose of [−]-Hup A ([+] and [−]-Hup A) against soman toxicity. Our data showed that pretreatment with a combination [+] and [−]-Hup A significantly increased the survival rate and reduced behavioral abnormalities after exposure to 1.2×LD50 soman compared to [+]-Hup A in guinea pigs. In addition, [+] and [−]-Hup A pretreatment inhibited the development of high power of EEG better than [+]-Hup A pretreatment alone. These data suggest that a combination of [+] and [−]-Hup A offers better protection than [+]-Hup A and serves as a potent medical countermeasure against lethal dose nerve agent toxicity in guinea pigs.

Correlation between butyrylcholinesterase variants and sensitivity to soman toxicity.

Butyrylcholinesterase (BChE) is synthesized in the liver and found in high concentrations in blood plasma, liver, heart, pancreas, vascular endothelium, skin, brain white matter, smooth muscle cells and adipocytes. BChE is a non specific enzyme that hydrolyzes different choline esters (succinylcholine, mivacurium) and many other drugs such as aspirin, cocaine and procaine. The enzyme is also considered as a bioscavenger due to its ability to neutralize the toxic effects of organophosphorus compounds (nervous system fs agents) such as soman. BChE displays several polymorphisms that influence its serum activity; therefore they could determine the individual sensitivity to chemical nerve agents. In this study, we investigated the correlation between BChE variants and the degree of enzyme inhibition and reactivation after soman application on blood samples of 726 individuals. The blood samples of individuals expressing abnormal variants, were more sensitive to soman compared to variants of homozygotes and heterozygotes for U-allele. We found significant differences in the degree of enzyme reactivation between different variants (with and without U-presence).

Protection by a transdermal patch system for the prophylaxis against soman poisoning in rhesus monkey

A transdermal patch system containing procyclidine, an N‐methyl‐D‐aspartate receptor antagonist possessing anticholinergic action, and physostigmine, a reversible cholinesterase inhibitor, was developed, and its prophylactic efficacy against soman intoxication was investigated. Male rhesus monkeys were shaved on the dorsal area, attached with a matrix‐type patch with various sizes (2 × 2 to 7 × 7 cm) for 24 or 72 hours, and challenged with 2 × LD50 doses (13 μg/kg) of soman. The smallest patch size for the protection against lethality induced by soman intoxication was 3 × 3 cm, resulting in blood procyclidine concentration of 10.8 ng/ml, blood physostigmine concentration of 0.54 ng/ml, which are much lower concentrations than maximum sign‐free doses, and blood cholinesterase inhibition of 42 %. The drug concentrations and enzyme inhibition rate corresponding to a diverging point of survivability were presumably estimated to be around 7 ng/ml for procyclidine, 0.35 ng/ml for physostigmine, and 37 % of enzyme inhibition. Separately, in combination with the patch treatment, the posttreatment consisting of atropine (0.5 mg/kg) plus 1‐[([4‐(aminocarbonyl)pyridinio]methoxy)methyl]‐2‐[(hydroxyimino)methyl]pyridinium (HI‐6, 50 mg/kg) exerted protection against 5 × LD50 challenge of soman, which means the posttreatment remarkablely augmented the efficacy of the patch. Additionally, it was found that brain injuries induced by soman toxicity were effectively prevented by the patch treatment according to histopathological examinations. These results suggest that the patch system could be an effective alternative for diazepam, an anticonvulsant, and the current pyridostigmine pretreatment, and especially in combination with atropine plus HI‐6, could be a choice for quality survival from nerve‐agent poisoning.

Neuroprotective effects of imidazenil against chemical warfare nerve agent soman toxicity in guinea pigs

The chemical warfare nerve agent, soman irreversibly inhibits acetylcholinesterase (AChE) leading to hypercholinergy and seizures which trigger glutamate toxicity and status epilepticus ultimately resulting in neuropathology and neurobehavioral deficits. The standard emergency treatment comprising of anticholinergic, AChE reactivator and anticonvulsant does not completely protect against soman toxicity. We have evaluated imidazenil, a new anticonvulsant imidazo benzodiazepine with high affinity and intrinsic efficacy at α5-, α2-, and α3- but low intrinsic efficacy at α1-containing GABAA receptors and is devoid of cardiorespiratory depression, sedative/hypnoitc and amnestic actions and does not elicit tolerance and dependence liabilities unlike diazepam, for protection against soman toxicity. Guinea pigs implanted with bipotential radiotelemetry probes for recording EEG and ECG were administered with 26μg/kg pyridostigmine bromide 30min prior to 2× LD50 soman exposure and 1min later treated with a combination of 2mg/kg atropine sulfate and 25mg/kg 2-pralidoxime and various doses of imidazenil. Intramuscular administration of imidazenil, dose-dependently protected against 2× LD50 of soman toxicity up to 1mg/kg. Further increase in the dose of imidazenil to 2.5mg/kg was less effective than 1mg/kg probably due to non-specific actions at sites other than GABAA receptors. Compared to vehicle group, 1mg/kg imidazenil treatment showed optimal increase in survival rate, reduction in behavioral manifestations and high power of EEG spectrum as well as neuronal necrosis. These data suggest that imidazenil is an effective anticonvulsant for medical countermeasure against soman-induced toxicity.

Minimum effective drug concentrations of a transdermal patch system containing procyclidine and physostigmine for prophylaxis against soman poisoning in rhesus monkeys

A transdermal patch system containing procyclidine, an N-methyl- d-aspartate receptor antagonist possessing anticholinergic action, and physostigmine, a reversible cholinesterase inhibitor, was developed, and its prophylactic efficacy against soman intoxication was investigated. Male rhesus monkeys were shaved on the dorsal area, attached with a matrix-type patch with various sizes (2 × 2 to 7 × 7 cm) for 24 or 72 h, and challenged with 2 × LD 50 doses (13 μg/kg) of soman. The smallest patch size for the protection against lethality induced by soman intoxication was 3 × 3 cm, resulting in blood procyclidine concentration of 10.8 ng/ml, blood physostigmine concentration of 0.54 ng/ml, which are much lower concentrations than maximum sign-free doses, and blood cholinesterase inhibition of 42%. The drug concentrations and enzyme inhibition rate corresponding to a diverging point of survivability were presumably estimated to be around 7 ng/ml for procyclidine, 0.35 ng/ml for physostigmine, and 37% of enzyme inhibition. Separately, in combination with the patch treatment, the post treatment consisting of atropine (0.5 mg/kg) plus 1-[([4-(aminocarbonyl)pyridinio]methoxy)methyl]-2-[(hydroxyimino)methyl]pyridinium (HI-6, 50 mg/kg) exerted protection against 5 × LD 50 challenge of soman, which means the posttreatment remarkably augmented the efficacy of the patch. Additionally, it was found that brain injuries induced by soman toxicity were effectively prevented by the patch treatment according to histopathological examinations. These results suggest that the patch system could be an effective alternative for diazepam, an anticonvulsant, and the current pyridostigmine pretreatment, and especially in combination with atropine plus HI-6, could be a choice for quality survival from nerve-agent poisoning.