Publications by authors named "Tsung-Ming Shih"

36 Publications

Intramuscularly administered A1 adenosine receptor agonists as delayed treatment for organophosphorus nerve agent-induced Status Epilepticus.

Toxicol Appl Pharmacol 2021 Mar 30;419:115515. Epub 2021 Mar 30.

Neuroscience Department, Medical Toxicology Research Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA. Electronic address:

Exposure to organophosphorus nerve agents (NAs) like sarin (GB) and soman (GD) can lead to sustained seizure activity, or status epilepticus (SE). Previous research has shown that activation of A1 adenosine receptors (A1ARs) can inhibit neuronal excitability, which could aid in SE termination. Two A1AR agonists, 2-Chloro-N6-cyclopentyladenosine (CCPA) and N-Bicyclo(2.2.1)hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA), were effective in terminating GD-induced SE in rats when administered via intraperitoneal (IP) injection. However, IP injection is not a clinically relevant route of administration. This study evaluated the efficacy of these agonists in terminating NA-induced SE when administered via intramuscular (IM) route. Adult male rats were exposed subcutaneously (SC) to either GB (150 μg/kg) or GD (90 μg/kg) and were treated with ENBA or CCPA at 15, 30, or 60 min after seizure onset or left untreated. Up to 7 days after exposure, deeply anesthetized rats were euthanized and perfused brains were removed for histologic assessment of neuropathology (i.e., neuronal damage) in six brain regions (amygdala, cerebral cortex, piriform cortex, thalamus, dorsal hippocampus, and ventral hippocampus). A total neuropathy score (0-24) was determined for each rat by adding the scores from each of the six regions. The higher the total score the more severe the neuropathology. With the GB model and 60 min treatment delay, ENBA-treated rats experienced 78.6% seizure termination (N = 14) and reduced neuropathology (11.6 ± 2.6, N = 5), CCPA-treated rats experienced 85.7% seizure termination (N = 14) and slightly reduced neuropathology (20.7 ± 1.8, N = 6), and untreated rats experienced no seizure termination (N = 13) and severe neuropathology (22.3 ± 1.0, N = 4). With the GD model and 60 min treatment delay, ENBA-treated rats experienced 92.9% seizure termination (N = 14) and reduced neuropathology (13.96 ± 1.8, N = 9), CCPA-treated rats experienced 78.6% seizure termination (N = 14) and slightly reduced neuropathology (22.0 ± 0.9, N = 10); and untreated rats experienced 16.7% seizure termination (N = 12) and severe neuropathology (22.0 ± 1.8, N = 5). While ENBA and CCPA both demonstrate a clear ability to terminate SE when administered up to 60 min after seizure onset, ENBA offers more neuroprotection, making it a promising candidate for NA-induced SE.
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http://dx.doi.org/10.1016/j.taap.2021.115515DOI Listing
March 2021

Evaluation of adenosine A1 receptor agonists as neuroprotective countermeasures against Soman intoxication in rats.

Toxicol Appl Pharmacol 2021 Apr 22;416:115466. Epub 2021 Feb 22.

U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, United States of America. Electronic address:

Soman, an organophosphorus (OP) compound, disrupts nervous system function through inactivation of acetylcholinesterase (AChE), the enzyme that breaks down acetylcholine at synapses. Left untreated, a state of prolonged seizure activity (status epilepticus, SE) is induced, causing widespread neuronal damage and associated cognitive and behavioral impairments. Previous research demonstrated that therapeutic stimulation of A1 adenosine receptors (A1ARs) can prevent or terminate soman-induced seizure. This study examined the ability of three potent A1AR agonists to provide neuroprotection and, ultimately, prevent observable cognitive and behavioral deficits following exposure to soman. Sprague Dawley rats were challenged with a seizure-inducing dose of soman (1.2 x LD) and treated 1 min later with one of the following A1AR agonists: (6)-Cyclopentyladenosine (CPA), 2-Chloro-N6-cyclopentyladenosine (CCPA) or N-bicyclo(2.2.1)hept-2-yl-5'-chloro-5'-deoxyadenosine (cdENBA). An active avoidance shuttle box task was used to evaluate locomotor responses to aversive stimuli at 3, 7 and 14 days post-exposure. Animals treated with CPA, CCPA or cdENBA demonstrated a higher number of avoidance responses and a faster reaction to the aversive stimulus than the soman/saline control group across all three sessions. Findings suggest that A1AR agonism is a promising neuroprotective countermeasure, capable of preventing the long-term deficits in learning and memory that are characteristic of soman intoxication.
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http://dx.doi.org/10.1016/j.taap.2021.115466DOI Listing
April 2021

The tertiary oxime monoisonitrosoacetone penetrates the brain, reactivates inhibited acetylcholinesterase, and reduces mortality and morbidity following lethal sarin intoxication in guinea pigs.

Toxicol Appl Pharmacol 2021 Mar 4;415:115443. Epub 2021 Feb 4.

Medical Toxicology Research Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA. Electronic address:

The brain is a critical target for the toxic action of organophosphorus (OP) inhibitors of acetylcholinesterase (AChE) such as the nerve agent sarin. However, the available oxime antidote 2-PAM only reactivates OP-inhibited AChE in peripheral tissues. Monoisonitrosoacetone (MINA), a tertiary oxime, reportedly reactivates AChE in the central nervous system (CNS). The current study investigated whether MINA would be beneficial as a supplemental oxime treatment in preventing lethality and reducing morbidity following lethal sarin exposure, MINA supplement would improve AChE recovery in the body, and MINA would be detectable in the CNS. Guinea pigs were exposed to sarin and treated with atropine sulfate and 2-PAM at one minute. Additional 2-PAM or MINA was administered at 3, 5, 15, or 30 min after sarin exposure. Survival and morbidity were assessed at 2 and 24 h. AChE activity in brain and peripheral tissues was evaluated one hour after MINA and 2-PAM treatment. An in vivo microdialysis technique was used to determine partitioning of MINA into the brain. A liquid chromatography-tandem mass spectrometry method was developed for the analysis of MINA in microdialysates. MINA-treated animals exhibited significantly higher survival and lower morbidity compared to 2-PAM-treated animals. 2-PAM was significantly more effective in reactivating AChE in peripheral tissues, but only MINA reactivated AChE in the CNS. MINA was found in guinea pig brain microdialysate samples beginning at ~10 min after administration in a dose-related manner. The data strongly suggest that a centrally penetrating oxime could provide significant benefit as an adjunct to atropine and 2-PAM therapy for OP intoxication.
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http://dx.doi.org/10.1016/j.taap.2021.115443DOI Listing
March 2021

Development of a Larval Zebrafish Model for Acute Organophosphorus Nerve Agent and Pesticide Exposure and Therapeutic Evaluation.

Toxics 2020 Nov 17;8(4). Epub 2020 Nov 17.

Medical Toxicology Research Division, Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA.

Organophosphorus compound exposure remains a present threat through agricultural accidents, warfare, or terrorist activity. The primary mechanism of organophosphorus toxicity is through inhibition of the enzyme acetylcholinesterase, with current emergency treatment including anticholinergics, benzodiazepines, and oxime reactivators. However, a need for more effective and broadly acting countermeasures remains. This study aimed to develop larval zebrafish as a high-throughput model for evaluating novel therapeutics against acute organophosphorus exposure. Larval zebrafish at six days post-fertilization were exposed to acute concentrations of seven organophosphorus compounds and treated with one of three oximes. Lethality studies indicated similar relative toxicity to that seen in the established rodent model, with chemical warfare agents proving more lethal than organophosphorus pesticides. Additionally, the organophosphorus-specific response for oxime reactivation of acetylcholinesterase was comparable to what has been previously reported. Behavioral studies measuring the visual motor response demonstrated greater efficacy for centrally acting oxime compounds than for those that are contained to the peripheral tissue. Overall, these results support the use of this larval zebrafish model as a high-throughput screening platform for evaluating novel treatments following acute organophosphorus exposure.
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http://dx.doi.org/10.3390/toxics8040106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712847PMC
November 2020

In Vivo Evaluation of A1 Adenosine Agonists as Novel Anticonvulsant Medical Countermeasures to Nerve Agent Intoxication in a Rat Soman Seizure Model.

Neurotox Res 2019 Aug 8;36(2):323-333. Epub 2019 May 8.

U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD, USA.

Organophosphorus nerve agents (NAs) irreversibly inhibit acetylcholinesterase, which results in the accumulation of acetylcholine and widespread excitotoxic seizure activity. Because current medical countermeasures (anticholinergics, AChE reactivators, and benzodiazepines) lack sufficient anti-seizure efficacy when treatment is delayed, those intoxicated are at risk for severe brain damage or death if treatment is not immediately available. Toward developing a more effective anti-seizure treatment for NA intoxication, this study evaluated the efficacy of A1 adenosine (ADO) receptor (A1AR) agonists in a rat soman seizure model. One minute after exposure to soman (1.6 × LD, subcutaneous), rats were treated intraperitoneally with one of the following agonists at increasing dose levels until anti-seizure efficacy was achieved: N6-cyclopentaladenosine (CPA), 2-chloro-N6-cyclopentyladenosine (CCPA), and (±)-5'-chloro-5'-deoxy-ENBA (ENBA). All A1AR agonists were efficacious in preventing seizure and promoting survival. The effective doses for the A1AR agonists were 60 mg/kg CPA, 36 mg/kg CCPA, and 62 mg/kg ENBA. Whereas vehicle-treated rats experienced 100% seizure and 21% survival (N = 28), ADO treatments reduced seizure occurrence and improved survival rates: 8% seizure and 83% survival with CPA (60 mg/kg, N = 12), 17% seizure and 75% survival with CCPA (36 mg/kg, N = 12), and 8% seizure, 83% survival with ENBA (62 mg/kg, N = 12). The brains of ADO-treated rats were also protected from damage as indicated by neurohistopathological analysis. While all ADO agonists provided neuroprotection, rats receiving CCPA and ENBA experienced less severe ADO-induced side effects (e.g., sedation, hypothermia, bradycardia) than with CPA. The data from this study suggest that the ADO signaling pathway is a promising mechanism for countering seizure activity induced by NAs.
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http://dx.doi.org/10.1007/s12640-019-00034-wDOI Listing
August 2019

Comparative effects of scopolamine and phencynonate on organophosphorus nerve agent-induced seizure activity, neuropathology and lethality.

Toxicol Mech Methods 2019 Jun 22;29(5):322-333. Epub 2019 Jan 22.

a Medical Toxicology Research Division , US Army Medical Research Institute of Chemical Defense , Edgewood , MD , USA.

The efficacy of anticonvulsant therapies to stop seizure activities following organophosphorus nerve agents (NAs) has been documented as being time-dependent. We utilized the guinea pig NA-seizure model to compare the effectiveness of phencynonate (PCH) and scopolamine (SCP) when given at the early (at time of seizure onset) or late (40 min after seizure onset) phase of seizure progression. PCH possesses both anticholinergic and anti-NMDA activities, while SCP is a purely anti-muscarinic compound. Animals with cortical electrodes were pretreated with pyridostigmine bromide 30 min prior to exposure to a 2.0 x LD subcutaneous dose of a NA (GA, GB, GD, GF, VR, or VX), followed one min later with atropine sulfate and 2-PAM. At either early or late phase, animals were treated with either PCH or SCP and the 24-h anticonvulsant ED doses were determined. When administered at seizure onset, PCH, and SCP were both effective at terminating seizure activity against all NAs, with ED values for SCP generally being lower. At the 40 min time, ED values were obtained following GA, GD, GF, and VR challenges for SCP, but ED value was obtained only following GD for PCH, indicating a superior efficacy of SCP. When seizure activity was controlled, a significant improvement in weight loss, neuropathology, and survival was observed, regardless of treatment or NA. Overall, these results demonstrate the differing efficacies of these two similarly structured anticholinergic compounds with delayed administration and warrant further investigation into the timing and mechanisms of the seizure maintenance phase in different animal models.
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http://dx.doi.org/10.1080/15376516.2018.1558322DOI Listing
June 2019

Assessment of mouse strain differences in baseline esterase activities and toxic response to sarin.

Toxicology 2018 12 30;410:10-15. Epub 2018 Aug 30.

U.S. Army Medical Research Institute of Chemical Defense, 8350 Ricketts Point Rd, Aberdeen Proving Ground, MD 21010, United States. Electronic address:

Genetics likely play a role in various responses to nerve agent (NA) exposure, as genetic background plays an important role in behavioral, neurological, and physiological responses. This study uses different mouse strains to identify if mouse strain differences in sarin exposure exist. In Experiment 1, basal levels of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CE) were measured in different strains of naïve mice to account for potential pharmacokinetic determinants of individual differences. In Experiment 2, median lethal dose (MLD) levels were estimated in 8 inbred mouse strains following subcutaneous (s.c.) administration of sarin. Few strain or sex differences in esterase activity levels were observed, with the exception of erythrocyte AChE activity in the C57BL/6J strain. Both sex and strain differences in toxicity were observed, with the most resistant strains being the BALB/cByJ and FVB/NJ strains and the most sensitive strain being the DBA/2J strain. These findings can be expanded to explore pathways involved in NA response, which may provide an avenue to develop therapeutics for preventing and treating the damaging effects of NA exposure.
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http://dx.doi.org/10.1016/j.tox.2018.08.016DOI Listing
December 2018

Mechanisms of acetylcholinesterase protection against sarin and soman by adenosine A receptor agonist N-cyclopentyladenosine.

Comput Biol Chem 2018 Aug 7;75:74-81. Epub 2018 May 7.

US Army Research Laboratory, Deer Creek Loop, Aberdeen Proving Ground, MD 21005-5069, USA; US Army Medical Research Institute of Chemical Defense, 2900 Ricketts Point Road, Aberdeen Proving Ground, MD 21010-5400, USA.

Organophosphorus nerve agents (NAs) irreversibly inhibit acetylcholinesterase (AChE), the enzyme responsible for breaking down the neurotransmitter acetylcholine (ACh). The over accumulation of ACh after NA exposure leads to cholinergic toxicity, seizure, and death. Current medical countermeasures effectively mitigate peripheral symptoms, however; the brain is often unprotected. Alternative acute treatment with the adenosine A receptor agonist N-cyclopentyladensosine (CPA) has previously been demonstrated to prevent AChE inhibition as well as to suppress neuronal activity. The mechanism of AChE protection is unknown. To elucidate the feasibility of potential CPA-AChE interaction mechanisms, we applied a truncated molecular model approach and density functional theory. The candidate mechanisms studied are reversible enzyme inhibition, enzyme reactivation, and NA blocking prior to enzyme conjugation. Our thermodynamic data suggest that CPA can compete with the NAs sarin and soman for the active site of AChE, but may, in contrast to NAs, undergo back-reaction. We found a strong interaction between CPA and NA conjugated AChE, making enzyme reactivation unlikely but possibly allowing for CPA protection through the prevention of NA aging. The data also indicates that there is an affinity between CPA and unbound NAs. The results from this study support the hypothesis that CPA counters NA toxicity via multiple mechanisms and is a promising therapeutic strategy that warrants further development.
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http://dx.doi.org/10.1016/j.compbiolchem.2018.04.017DOI Listing
August 2018

Evaluation of acetylcholine, seizure activity and neuropathology following high-dose nerve agent exposure and delayed neuroprotective treatment drugs in freely moving rats.

Toxicol Mech Methods 2016 Jun 22;26(5):378-88. Epub 2016 Jun 22.

a Pharmacology Branch, Research Division , US Army Medical Research Institute of Chemical Defense , MD , USA.

Organophosphorus nerve agents such as soman (GD) inhibit acetylcholinesterase, producing an excess of acetylcholine (ACh), which results in respiratory distress, convulsions and status epilepticus that leads to neuropathology. Several drugs (topiramate, clobazam, pregnanolone, allopregnanolone, UBP 302, cyclopentyladenosine [CPA], ketamine, midazolam and scopolamine) have been identified as potential neuroprotectants that may terminate seizures and reduce brain damage. To systematically evaluate their efficacy, this study employed in vivo striatal microdialysis and liquid chromatography to respectively collect and analyze extracellular ACh in freely moving rats treated with these drugs 20 min after seizure onset induced by a high dose of GD. Along with microdialysis, EEG activity was recorded and neuropathology assessed at 24 h. GD induced a marked increase of ACh, which peaked at 30 min post-exposure to 800% of control levels and then steadily decreased toward baseline levels. Approximately 40 min after treatment, only midazolam (10 mg/kg) and CPA (60 mg/kg) caused a significant reduction of ACh levels, with CPA reducing ACh levels more rapidly than midazolam. Both drugs facilitated a return to baseline levels at least 55 min after treatment. At 24 h, only animals treated with CPA (67%), midazolam (18%) and scopolamine (27%) exhibited seizure termination. While all treatments except for topiramate reduced neuropathology, CPA, midazolam and scopolamine showed the greatest reduction in pathology. Our results suggest that delayed treatment with CPA, midazolam, or scopolamine is effective at reducing GD-induced seizure activity and neuropathology, with CPA and midazolam capable of facilitating a reduction in GD-induced ACh elevation.
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http://dx.doi.org/10.1080/15376516.2016.1197992DOI Listing
June 2016

Zebrafish as a model for acetylcholinesterase-inhibiting organophosphorus agent exposure and oxime reactivation.

Ann N Y Acad Sci 2016 06 28;1374(1):68-77. Epub 2016 Apr 28.

Pharmacology Branch, Research Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland.

The current research progression efforts for investigating novel treatments for exposure to organophosphorus (OP) compounds that inhibit acetylcholinesterase (AChE), including pesticides and chemical warfare nerve agents (CWNAs), rely solely on in vitro cell assays and in vivo rodent models. The zebrafish (Danio rerio) is a popular, well-established vertebrate model in biomedical research that offers high-throughput capabilities and genetic manipulation not readily available with rodents. A number of research studies have investigated the effects of subacute developmental exposure to OP pesticides in zebrafish, observing detrimental effects on gross morphology, neuronal development, and behavior. Few studies, however, have utilized this model to evaluate treatments, such as oxime reactivators, anticholinergics, or anticonvulsants, following acute exposure. Preliminary work has investigated the effects of CWNA exposure. The results clearly demonstrated relative toxicity and oxime efficacy similar to that reported for the rodent model. This review surveys the current literature utilizing zebrafish as a model for OP exposure and highlights its potential use as a high-throughput system for evaluating AChE reactivator antidotal treatments to acute pesticide and CWNA exposure.
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http://dx.doi.org/10.1111/nyas.13051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4940279PMC
June 2016

Probing the activity of a non-oxime reactivator for acetylcholinesterase inhibited by organophosphorus nerve agents.

Chem Biol Interact 2016 Nov 7;259(Pt B):133-141. Epub 2016 Apr 7.

Research Division, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, USA.

Currently fielded treatments for nerve agent intoxication include atropine, an acetylcholine receptor antagonist, and pralidoxime (2PAM), a small molecule reactivator of acetylcholinesterase (AChE). 2PAM reactivates nerve agent-inhibited AChE via direct nucleophilic attack by the oxime moiety on the phosphorus center of the bound nerve agent. Due to a permanently charged pyridinium motif, 2PAM is not thought to cross the blood brain barrier and therefore cannot act directly in the neuronal junctions of the brain. In this study, ADOC, a non-permanently charged, non-oxime molecule initially identified using pesticide-inhibited AChE, was characterized in vitro against nerve agent-inhibited recombinant human AChE. The inhibitory and reactivation potentials of ADOC were determined with native AChE and AChE inhibited with tabun, sarin, soman, cyclosarin, VX, or VR and then compared to those of 2PAM. Several structural analogs of ADOC were used to probe the reactivation mechanism of the molecule. Finally, guinea pigs were used to examine the protective efficacy of the compound after exposure to sarin. The results of both in vitro and in vivo testing will be useful in the design of future small molecule reactivators.
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http://dx.doi.org/10.1016/j.cbi.2016.04.002DOI Listing
November 2016

Stimulation of central A1 adenosine receptors suppresses seizure and neuropathology in a soman nerve agent seizure rat model.

Toxicol Mech Methods 2014 Sep 22;24(6):385-95. Epub 2014 May 22.

US Army Medical Research Institute of Chemical Defense and.

The current regimen for treating nerve agent poisoning does not sufficiently suppress the excitotoxic activity that causes severe brain damage, especially in cases where treatment is delayed and nerve agent-induced status epilepticus develops. New therapeutic targets are required to improve survivability and minimize neuropathology after irreversible acetylcholinesterase inactivation. Earlier studies have shown that systemic delivery of adenosine agonists decreases nerve agent lethality; however, the mechanism of protection remains to be understood. The primary aim of this study was to investigate the role of central adenosine receptor (AR) stimulation in neuroprotection by directly injecting (6)-cyclopentyladenosine (CPA), an adenosine agonist specific to the A1 receptor subtype (A1R), into the brain intracerebroventricularly (ICV) in a soman seizure rat model. In addition to general A1R stimulation, we hypothesized that bilateral micro-injection of CPA into the cholinergic basal forebrain (BF) could also suppress excitotoxic activity. The results from these studies demonstrated that centrally administered adenosine agonists are anti-seizure and neuroprotective. CPA-delivered ICV prevented seizure and convulsion in 100% of the animals. Moreover, neuropathological evaluation indicated that adenosine treatments reduced brain damage from severe to minimal. Inhibition of the BF via CPA had varied results. Some animals were protected by treatment; however, others displayed similar pathology to the control. Overall, these data suggest that stimulating central ARs could be an effective target for the next generation countermeasures for nerve agent intoxication.
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http://dx.doi.org/10.3109/15376516.2014.920450DOI Listing
September 2014

Neuropharmacological specificity of brain structures involved in soman-induced seizures.

Neurotoxicology 2012 Jun 20;33(3):463-8. Epub 2012 Mar 20.

Pharmacology Branch, Research Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA.

Pharmacological control of seizure activity following nerve agent exposure is critical in reducing neuropathology and improving survival in casualties. Three classes of drugs, anticholinergics, benzodiazepines and excitatory amino acid (EAA) antagonists, have been shown to be effective at moderating nerve agent-induced seizures. However, little is known about which brain structures are involved in producing the anticonvulsant response. This study evaluated drugs from each class, injected directly into one of three specific brain structures, the perirhinal cortex, the entorhinal cortex, or the mediodorsal thalamus, for their ability to modulate seizures induced by the nerve agent soman. The drugs evaluated were the anticholinergic scopolamine, the benzodiazepine midazolam, and the EAA antagonist MK-801. For each drug treatment in each brain area, anticonvulsant ED₅₀ values were calculated using an up-down dosing procedure over successive animals. There was no statistical difference in the anticonvulsant ED₅₀ values for scopolamine and MK-801 in the perirhinal and entorhinal cortices. MK-801 pretreatment in the mediodorsal thalamus had a significantly lower anticonvulsant ED₅₀ value than any other treatment/injection site combination. Midazolam required significantly higher doses than scopolamine and MK-801 in the perirhinal and entorhinal cortices to produce an anticonvulsant response and was ineffective in the mediodorsal thalamus. These findings support the contention that specific neuroanatomical pathways are activated during nerve agentinduced seizures and that the discrete brain structures involved have unique pharmacological thresholds for producing an anticonvulsant response. This study is also the first to show the involvement of the mediodorsal thalamus in the control of nerve agent-induced seizures.
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http://dx.doi.org/10.1016/j.neuro.2012.03.006DOI Listing
June 2012

In vivo microdialysis and electroencephalographic activity in freely moving guinea pigs exposed to organophosphorus nerve agents sarin and VX: analysis of acetylcholine and glutamate.

Arch Toxicol 2011 Dec 22;85(12):1607-16. Epub 2011 Jun 22.

Pharmacology Branch, Research Division, US Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, MD 21010-5400, USA.

Organophosphorus nerve agents such as sarin (GB) and VX irreversibly inhibit acetylcholinesterase, causing a buildup of acetylcholine (ACh) in synapses and neuromuscular junctions, which leads to excess bronchial secretions, convulsions, seizures, coma, and death. Understanding the unique toxic characteristics of different nerve agents is vital in the effort to develop broad spectrum medical countermeasures. To this end, we employed a repeated measure multivariate design with striatal microdialysis collection and high-performance liquid chromatography analysis to measure changes in concentrations of several neurotransmitters (ACh, glutamate, aspartate, GABA) in the same samples during acute exposure to GB or VX in freely moving guinea pigs. Concurrent with microdialysis collection, we used cortical electrodes to monitor brain seizure activity. This robust double multivariate design provides greater fidelity when comparing data while also reducing the required number of subjects. No correlation between nerve agents' propensity for causing seizure and seizure-related lethality was observed. The GB seizure group experienced more rapid and severe cholinergic toxicity and lethality than that of the VX seizure group. Seizures generated from GB and VX exposure resulted in further elevation of ACh level and then a gradual return to baseline. Glutamate levels increased in the GB, but not in the VX, seizure group. There were no consistent changes in either aspartate or GABA as a result of either nerve agent. These observations reinforce findings with other nerve agents that seizure activity per se contributes to the elevated levels of brain ACh observed after nerve agent exposure.
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http://dx.doi.org/10.1007/s00204-011-0724-zDOI Listing
December 2011

The oxime pro-2-PAM provides minimal protection against the CNS effects of the nerve agents sarin, cyclosarin, and VX in guinea pigs.

Toxicol Mech Methods 2011 Jan 30;21(1):53-62. Epub 2010 Nov 30.

Research and Analytical Toxicology Divisions, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA.

This study examined whether pro-2-PAM, a pro-drug dihydropyridine derivative of the oxime 2-pralidoxime (2-PAM) that can penetrate the brain, could prevent or reverse the central toxic effects of three nerve agents; sarin, cyclosarin, and VX. The first experiment tested whether pro-2-PAM could reactivate guinea pig cholinesterase (ChE) in vivo in central and peripheral tissues inhibited by these nerve agents. Pro-2-PAM produced a dose-dependent reactivation of sarin- or VX-inhibited ChE in both peripheral and brain tissues, but with substantially greater reactivation in peripheral tissues compared to brain. Pro-2-PAM produced 9-25% reactivation of cyclosarin-inhibited ChE in blood, heart, and spinal cord, but no reactivation in brain or muscle tissues. In a second experiment, the ability of pro-2-PAM to block or terminate nerve agent-induced electroencephalographic seizure activity was evaluated. Pro-2-PAM was able to block sarin- or VX-induced seizures (16-33%) over a dose range of 24-32 mg/kg, but was ineffective against cyclosarin-induced seizures. Animals that were protected from seizures showed significantly less weight loss and greater behavioral function 24 h after exposure than those animals that were not protected. Additionally, brains were free from neuropathology when pro-2-PAM prevented seizures. In summary, pro-2-PAM provided modest reactivation of sarin- and VX-inhibited ChE in the brain and periphery, which was reflected by a limited ability to block or terminate seizures elicited by these agents. Pro-2-PAM was able to reactivate blood, heart, and spinal cord ChE inhibited by cyclosarin, but was not effective against cyclosarin-induced seizures.
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http://dx.doi.org/10.3109/15376516.2010.529190DOI Listing
January 2011

Comparison of extracellular striatal acetylcholine and brain seizure activity following acute exposure to the nerve agents cyclosarin and tabun in freely moving guinea pigs.

Toxicol Mech Methods 2010 Nov 4;20(9):600-8. Epub 2010 Oct 4.

Pharmacology Branch, Research Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA.

Organophosphorus nerve agents like cyclosarin and tabun are potent cholinesterase inhibitors. The inhibition of acetylcholinesterase, which is responsible for breaking down acetylcholine (ACh) at the synapse and neuromuscular junction, leads to a build-up of extracellular ACh and a series of toxic consequences including hypersecretion, tremor, convulsion/seizure, respiratory distress, coma, and death. This study employed simultaneous and continuous electroencephalographic recording and striatal microdialysis collection for quantification of ACh changes (via subsequent HPLC analysis) during acute exposure to a 1.0 × LD(50) subcutaneous dose of either cyclosarin or tabun to investigate differences in cholinergic and behavioral effects. Information about the unique mechanisms and consequences of different nerve agents is intended to aid in the development of broad-spectrum medical countermeasures for nerve agents. At the dose administered, non-seizure and sustained seizure responses were observed in both agent groups and in the tabun-exposed group some subjects experienced an unsustained seizure response. Significant extracellular ACh increases were only observed in seizure groups. Cyclosarin and tabun were found to exhibit some unique cholinergic and ictogenic characteristics. Lethality only occurred in subjects experiencing sustained seizure, and there was no difference in lethality between agent groups that progressed to sustained seizure.
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http://dx.doi.org/10.3109/15376516.2010.521208DOI Listing
November 2010

Time-dependent reduction in the anticonvulsant effectiveness of diazepam against soman-induced seizures in guinea pigs.

Drug Chem Toxicol 2010 Jul;33(3):279-83

Pharmacology Branch, Research Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5400, USA.

Near-lethal exposure to nerve agents produces prolonged epileptiform seizures requiring the administration of benzodiazepine anticonvulsant drugs, such as diazepam. Clinically, benzodiazepines are reported to lose anticonvulsant effectiveness the greater the delay between seizure onset and benzodiazepine treatment. This time-dependent diminished effectiveness of diazepam was tested in the present study. Seizures elicited by the nerve agent, soman, were produced in guinea pigs instrumented to record brain electrocorticographic (ECoG) activity. Different groups of animals were administered 10 mg/kg, intramuscularly, of diazepam at 5, 40, 60, 80, or 160 minutes after the onset of seizure activity. There was a progressive loss in the anticonvulsant efficacy of diazepam as the treatment was delayed after seizure onset, but no differences in the time for diazepam to stop seizures. The results show a diminished ability of diazepam to stop nerve-agent-induced seizures the longer treatment is delayed.
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http://dx.doi.org/10.3109/01480540903483417DOI Listing
July 2010

Reactivation of brain acetylcholinesterase by monoisonitrosoacetone increases the therapeutic efficacy against nerve agents in guinea pigs.

Chem Biol Interact 2010 Sep 15;187(1-3):318-24. Epub 2010 Mar 15.

Pharmacology Branch, Research Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA.

Current oxime therapies do not readily cross the blood-brain barrier to reactivate organophosphorus nerve agent-inhibited cholinesterase (ChE) within the CNS. We investigated the ability of monoisonitrosoacetone (MINA), a tertiary oxime, to reactivate ChE inhibited by the nerve agent sarin (GB), cyclosarin (GF), or VX, in peripheral tissues and brain of guinea pigs and determined whether reactivation in the CNS will enhance protection against the lethal effects of these three agents. In the reactivation experiment, animals were pretreated with atropine methylnitrate (1.0mg/kg, i.m.) 15 min prior to subcutaneous (s.c.) challenge with 1.0 x LD(50) of GB, GF, or VX. Fifteen minutes later animals were treated intramuscularly (i.m.) with MINA (ranging from 22.1 to 139.3mg/kg) or 2-PAM (25.0mg/kg). At 60 min after nerve agent, CNS (brainstem, cerebellum, cortex, hippocampus, midbrain, spinal cord, and striatum) and peripheral (blood, diaphragm, heart, and skeletal muscle) tissues were collected for ChE analysis. MINA reactivated nerve agent-inhibited ChE in the CNS and peripheral tissues in a dose-dependent manner in the following order of potency: GB>GF>VX. In a survival experiment, animals were injected i.m. with atropine sulfate (0.5mg/kg), 2-PAM (25.0mg/kg), or MINA (35.0, 60.0, or 100.0mg/kg) alone or in combination 1 min after challenge with varying s.c. doses of GB, GF, or VX to determine the level of protection. The rank order of MINA's efficacy in guinea pigs against nerve agent lethality was the same as for reactivation of inhibited ChE in the CNS. These data show that MINA is capable of reactivating nerve agent-inhibited ChE and that the extent of ChE reactivation within the CNS strongly relates to its therapeutic efficacy.
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http://dx.doi.org/10.1016/j.cbi.2010.03.010DOI Listing
September 2010

In vivo reactivation by oximes of inhibited blood, brain and peripheral tissue cholinesterase activity following exposure to nerve agents in guinea pigs.

Chem Biol Interact 2010 Sep 9;187(1-3):207-14. Epub 2010 Mar 9.

Pharmacology Branch, Research Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA.

This study compared the ability of nine oximes (HI-6, HLö7, MMB-4, TMB-4, carboxime, ICD585, ICD692, ICD3805, and 2-PAM) to reactivate in vivo cholinesterase (ChE) in blood, brain, and peripheral tissues in guinea pigs intoxicated by one of four organophosphorus nerve agents. Two bis-pyridinium compounds without an oxime group, SAD128 and ICD4157, served as non-oxime controls. Animals were injected subcutaneously with 1.0 x LD(50) of the nerve agents sarin, cyclosarin, VR or VX and treated intramuscularly 5 min later with one of these oximes. Toxic signs and lethality were monitored; tissue ChE activities were determined at 60 min after nerve agent. Some animals exposed to sarin or cyclosarin, with or without non-oxime treatment, died within 60 min; however, no animal treated with an oxime died. For VR or VX, all animals survived the 60 min after exposure, with or without non-oxime or oxime therapy. The four nerve agents caused differential degrees of inhibition in blood, brain regions and peripheral tissues. The tested oximes exhibited differential potency in reactivating nerve agent-inhibited ChE in various peripheral tissues, but did not affect ChE activity in the brain regions. There was no direct relation between blood and peripheral tissues in the reactivating efficacy of oxime treatments. ChE inhibited by sarin was the most susceptible to oxime reactivation while cyclosarin the least susceptible. There was no difference in the ChE reactivating potency between the dimethanesulfonate and dichloride salts of HI-6. MMB-4 significantly reactivated the ChE inhibited by these four nerve agents in blood and all three peripheral tissues of the guinea pig, and among all the oximes tested it was the most effective in vivo ChE reactivator against all four nerve agents.
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http://dx.doi.org/10.1016/j.cbi.2010.03.006DOI Listing
September 2010

Changes in extracellular striatal acetylcholine and brain seizure activity following acute exposure to nerve agents in freely moving guinea pigs.

Toxicol Mech Methods 2010 Mar;20(3):143-52

Pharmacology Branch, Research Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA.

Organophosphorus nerve agents irreversibly inhibit acetylcholinesterase (AChE) in the peripheral and central nervous systems, causing an increase in the concentration of acetylcholine (ACh) in the synapse or neuromuscular junction and subsequent adverse effects. In this study, in vivo microdialysis was utilized to collect samples from the striatum for monitoring changes in extracellular ACh levels along with cortical electroencephalographic (EEG) recordings for identifying seizure activity after acute subcutaneous (s.c.) exposure to 1.0 x LD(50) of the nerve agents sarin, soman, or one of two V-type agents (VX, or a Russian V-agent, designated VR) in unanesthetized freely moving guinea pigs. Based on EEG recordings, these animals were subsequently divided into groups that developed seizures (S) and those that did not develop seizures (NS). Maximum ACh levels in the striatum were observed at 60-70 min for sarin and soman S groups and 105 min for VX and VR S groups. In all NS groups the greatest increase in extracellular ACh occurred within 30 min after exposure, although in the sarin NS group a few sporadic increases of ACh from control occurred. Animals that developed seizures, regardless of the nerve agent, had significantly higher extracellular striatal ACh levels compared to the controls or those animals that did not develop seizures, yet both S and NS groups displayed similar levels of blood AChE inhibition. Regardless of the agent, all animals in the non-seizure groups survived 24 h, while lethality (25-42%) was observed only in animals that experienced seizure activity.
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http://dx.doi.org/10.3109/15376511003657439DOI Listing
March 2010

Evaluation of nine oximes on in vivo reactivation of blood, brain, and tissue cholinesterase activity inhibited by organophosphorus nerve agents at lethal dose.

Toxicol Mech Methods 2009 Sep;19(6-7):386-400

Pharmacology Branch, Research Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA.

The capability of several oximes (HI-6, HLö7, MMB-4, TMB-4, carboxime, ICD 585, ICD 692, ICD 3805, and 2-PAM) to reactivate in vivo AChE inhibited by the nerve agents sarin, cyclosarin, VX, or VR in blood, brain regions, and peripheral tissues in guinea pigs was examined and compared. Animals were injected subcutaneously with 1.0 LD(50) of sarin, cyclosarin, VR, or VX, and treated intramuscularly 5 min later with one of these compounds. Toxic signs and lethality were monitored, and tissue AChE activities were determined at 60 min after nerve agent. The animals exposed to sarin or cyclosarin, alone or with non-oxime treatment, some died within 60 min; however, when treated with an oxime, no animal died. For VR or VX, all animals survived for 60 min after exposure, with or without non-oxime or oxime therapy. These nerve agents caused differential degrees of inhibition: in whole blood sarin = cyclosarin > VR = VX; in brain regions sarin > cyclosarin > VX > VR; and in peripheral tissues sarin > VX > cyclosarin > VR. These oximes exhibited differential potency in reactivating nerve agent-inhibited AChE in various peripheral tissues, but not AChE activity in the brain regions. There was no difference in the AChE reactivating potency between the dichloride and dimethanesulfonate salts of HI-6. AChE inhibited by sarin was the most and cyclosarin the least susceptible to oxime reactivation. Overall, MMB-4 appeared to be, among all oximes tested, the most effective in vivo AChE reactivator against the broadest spectrum of nerve agents.
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http://dx.doi.org/10.1080/15376510903213892DOI Listing
September 2009

In vivo oxime administration does not influence Ellman acetylcholinesterase assay results.

Toxicol Mech Methods 2009 Sep;19(6-7):379-85

Pharmacology Branch, Research Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5400, USA.

Organophosphorus compounds (OPs) are potent inhibitors of acetylcholinesterase (AChE). Treatment for OP poisoning is by administration of atropine sulfate, an oxime, and diazepam. Oximes such as 2-PAM are used to reactivate OP-inhibited AChE so as to restore normal enzymatic function and serve as a true antidote. There are reports of non-enzymatic hydrolysis by oximes of acetylthiocholine in in vitro preparations in the widely used Ellman assay for AChE activity, which may confound the interpretation of AChE activity by producing elevated results. The purpose of this experiment was to determine if there is appreciable interference by therapeutic levels of oximes on the results of the Ellman assay in assessing AChE reactivation by oxime compounds in vivo. When therapeutic doses of oximes (2-PAM, HI-6, MMB-4, or MINA) were administered intramuscularly to guinea pigs and samples collected 60 min later, there was no statistical difference between oxime and saline control groups in measured AChE activity in various tissue samples, including blood. With appropriate dilution of samples prior to spectrophotometric assay, the Ellman assay is an acceptable method to measure in vivo oxime reactivation of inhibited AChE. Inclusion of an oxime control group to insure that this particular type of interference is not causing false readings in the assay is a prudent step.
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http://dx.doi.org/10.1080/15376510903234773DOI Listing
September 2009

Effects of 4-pyridine aldoxime on nerve agent-inhibited acetylcholinesterase activity in guinea pigs.

Arch Toxicol 2009 Dec 10;83(12):1083-9. Epub 2009 Sep 10.

Pharmacology Branch, Research Division, US Army Medical Research Institute of Chemical Defense, ATTN: MCMR-CDR-P, 3100 Ricketts Point Road, Aberdeen Proving Ground, Aberdeen, MD 21010-5400, USA.

Methoxime (MMB-4) is a leading candidate oxime acetylcholinesterase (AChE) reactivator to replace pralidoxime (2-PAM) for therapeutic treatment of nerve agent intoxication. 4-Pyridine aldoxime (4-PA) is a synthetic starting material, a breakdown product, and a probable metabolite of MMB-4. There is a possibility that 4-PA may adversely interact with the nerve agent, thereby affecting nerve agent toxicity and biological AChE activity. This study evaluated the effects of 4-PA on sarin (GB)-, cyclosarin (GF)-, and VX-induced toxicity and AChE activity in blood, brain, and peripheral tissues of guinea pigs. Animals were pretreated with atropine methyl nitrate (1.0 mg/kg, im) 15 min prior to subcutaneous administration with 1.0 x LD(50) of GB, GF, or VX and then treated 15 min after the administration of nerve agents with 4-PA (3.5, 7.0, or 14.0 mg/kg, im). The dose-response effects of 4-PA alone were also examined. Toxic signs and lethality were monitored, blood and tissues were collected, and AChE activities were determined at 60 min after nerve agent administration. Under the condition of this study, all animals exposed to nerve agents exhibited some degree of toxic signs such as salivation, lacrimation, rhinorrhea, and convulsions. 4-PA at the three doses tested neither induced toxic signs nor altered the toxicity of GB, GF, or VX at the 1.0 x LD(50) exposure dose. Additionally, it did not modify the AChE activity in blood, brain, and peripheral tissues by itself or affect the AChE activity inhibited by a 1.0 x LD(50) dose of these three nerve agents in guinea pigs.
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http://dx.doi.org/10.1007/s00204-009-0465-4DOI Listing
December 2009

Protection against sarin-induced seizures in rats by direct brain microinjection of scopolamine, midazolam or MK-801.

J Mol Neurosci 2010 Jan 19;40(1-2):56-62. Epub 2009 Aug 19.

Pharmacology Branch, Research Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA.

Control of seizure activity is critical to survival and neuroprotection following nerve agent exposure. Extensive research has shown that three classes of drugs, muscarinic antagonists, benzodiazepines, and N-methyl-D: -aspartate antagonists, are capable of moderating these seizures. This study began to map the neural areas in rat brain that respond to these three drug classes resulting in anticonvulsant effects. Drugs of each class (scopolamine, midazolam, MK-801) were evaluated for their ability to prevent sarin-induced seizures when injected into specific brain areas (lateral ventricle, anterior piriform cortex, basolateral amygdala, area tempestas). Animals were pretreated by microinjection with saline or a dose of drug from one of the three classes 30 min prior to receiving 150 microg/kg sarin, subcutaneously, followed by 2.0 mg/kg atropine methylnitrate, intramuscularly. Animals were then returned to their cages, where electroencephalographic activity was monitored for seizures. Anticonvulsant effective doses (ED(50)) were determined using an up-down dosing procedure over successive animals. Scopolamine provided anticonvulsant effects in each area tested, while midazolam was effective in each area except the lateral ventricle. MK-801 was only effective at preventing seizures when injected into the basolateral amygdala or area tempestas. The results show a unique neuroanatomical and pharmacological specificity for control of nerve agent-induced seizures.
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http://dx.doi.org/10.1007/s12031-009-9253-0DOI Listing
January 2010

Treatment with tertiary oximes prevents seizures and improves survival following sarin intoxication.

J Mol Neurosci 2010 Jan 13;40(1-2):63-9. Epub 2009 Aug 13.

Pharmacology Branch, Research Division, U.S. Army Medical Research Institute of Chemical Defense, ATTN: MCMR-CDR-P, Aberdeen Proving Ground, MD 21010-5400, USA.

The capability of the tertiary oximes, monoisonitrosoacetone (MINA) and diacetylmonoxime (DAM), to reactivate acetylcholinesterase (AChE) inhibited by sarin (GB) in the blood, brain, and peripheral tissues of guinea pigs was compared with that of the quaternary oximes 2-PAM, HLö7, and MMB-4. Animals were injected subcutaneously (s.c.) with 1.0 x LD(50) of GB and treated intramuscularly (i.m.) 5 min later with one of these oximes. Sixty minutes after GB exposure, tissues were collected for AChE analysis. At low doses, MINA and DAM produced significant increases in AChE activity in all brain areas examined, but no significant AChE reactivation in peripheral tissues or blood. At higher doses, MINA and DAM increased AChE activity in the brain, peripheral tissues, and blood. In contrast, the quaternary oximes produced significant reactivation in peripheral tissues and blood AChE, but no significant reactivation of brain AChE. In another study, animals were pretreated i.m. with pyridostigmine 30 min prior to s.c. challenge with 2.0 x LD(50) of GB and treated i.m. 1 min later with atropine sulfate (2.0 mg/kg), plus a varied dose of oximes. MINA and DAM prevented or terminated GB-induced seizure activity and protected against GB lethality in a dose-dependent fashion. In contrast, none of the quaternary oximes prevented or stopped GB-induced seizures. Thus, tertiary oximes reactivated AChE in the brain, improved survival, and terminated seizures following GB intoxication.
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http://dx.doi.org/10.1007/s12031-009-9259-7DOI Listing
January 2010

Comparison of the intramuscular, intranasal or sublingual routes of midazolam administration for the control of soman-induced seizures.

Basic Clin Pharmacol Toxicol 2009 Jan;104(1):27-34

Pharmacology Branch, Research Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD, USA.

This study evaluated the anticonvulsant effectiveness of midazolam to stop seizures elicited by the nerve agent soman when midazolam was administered by different routes (intramuscular, intranasal or sublingual) at one of two different times after the onset of seizure activity. Guinea pigs previously prepared with cortical electrodes to record brain electroencephalographic activity were pre-treated with pyridostigmine (0.026 mg/kg, intramuscularly) 30 min. before challenge with a seizure-inducing dose of the nerve agent soman (56 microg/kg, subcutaneously), and 1 min. later, they were administered 2.0 mg/kg atropine sulfate admixed with 25.0 mg/kg 2-PAM Cl (intramuscularly). Groups of animals were administered differing doses of midazolam by the intramuscular, intranasal or sublingual route at either the onset of seizure activity or 40 min. after the onset of seizure activity that was detected in the electroencephalographic record. When given immediately after seizure onset, the anticonvulsant ED50 of intramuscular midazolam was significantly lower than that of intranasal midazolam, which in turn was significantly lower than sublingual midazolam at that time. At the 40-min. treatment delay, the anticonvulsant ED50s of intramuscular or intranasal midazolam did not differ and both were significantly lower than the sublingual route. Higher doses of midazolam were required to stop seizures at the 40-min. treatment delay time compared to immediate treatment. The speed of seizure control for intramuscular or intranasal midazolam was the same while sublingual midazolam acted significantly slower. Midazolam was effective in treating soman-induced seizures when given by all three routes, but with differences in potency and speed of action.
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http://dx.doi.org/10.1111/j.1742-7843.2008.00326.xDOI Listing
January 2009

Kinetics of sarin (GB) following a single sublethal inhalation exposure in the guinea pig.

Inhal Toxicol 2007 Jun;19(8):667-81

U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland 21010-5424, USA.

To improve toxicity estimates from sublethal exposures to chemical warfare nerve agents (CWNA), it is necessary to generate mathematical models of the absorption, distribution, and elimination of nerve agents. However, current models are based on representative data sets generated with different routes of exposure and in different species and are designed to interpolate between limited laboratory data sets to predict a wide range of possible human exposure scenarios. This study was performed to integrate CWNA sublethal toxicity data in male Duncan Hartley guinea pigs. Specific goal was to compare uptake and clearance kinetics of different sublethal doses of sarin (either 0.1 x or 0.4 x LC50) in blood and tissues of guinea pigs exposed to agent by acute whole-body inhalation exposure after the 60-min LC50 was determined. Arterial catheterization allowed repeated blood sampling from the same animal at various time periods. Blood and tissue levels of acetylcholinesterase, butyrylcholinesterase, and regenerated sarin (rGB) were determined at various time points during and following sarin exposure. The following pharmacokinetic parameters were calculated from the graph of plasma or RBC rGB concentration versus time: time to reach the maximal concentration; maximal concentration; mean residence time; clearance; volume of distribution at steady state; terminal elimination-phase rate constant; and area under plasma concentration time curve extrapolated to infinity using the WinNonlin analysis program 5.0. Plasma and RBC t(1/2) for rGB was also calculated. Data will be used to develop mathematical model of absorption and distribution of sublethal sarin doses into susceptible tissues.
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http://dx.doi.org/10.1080/08958370701353296DOI Listing
June 2007

Anticonvulsants for nerve agent-induced seizures: The influence of the therapeutic dose of atropine.

J Pharmacol Exp Ther 2007 Jan 2;320(1):154-61. Epub 2006 Oct 2.

Pharmacology Branch, Research Division, United States Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Rd., Aberdeen Proving Ground, MD 21010-5400, USA.

Two guinea pig models were used to study the anticonvulsant potency of diazepam, midazolam, and scopolamine against seizures induced by the nerve agents tabun, sarin, soman, cyclosarin, O-ethyl S-(2-(diisopropylamino)ethyl)methylphosphonothioate (VX), and O-isobutyl S-(2-diethylamino)ethyl)-methyl phosphonothioate (VR). Animals instrumented for electroencephalogram recording were pretreated with pyridostigmine bromide (0.026 mg/kg i.m.) 30 min before challenge with 2 x LD50 (s.c.) of a nerve agent. In model A, atropine sulfate (2.0 mg/kg i.m.) and pyridine-2-aldoxime methylchloride (2-PAM; 25.0 mg/kg i.m.) were given 1 min after nerve agent challenge, and the tested anticonvulsant was given (i.m.) 5 min after seizure onset. In model B, a lower dose of atropine sulfate (0.1 mg/kg i.m.) was given along with 2-PAM 1 min after nerve agent challenge, and the anticonvulsant was given at seizure onset. With the lower dose of atropine, seizure occurrence increased to virtually 100% for all agents; the time to seizure onset decreased for sarin, cyclosarin, and VX; the signs of nerve agent intoxication were more severe; and coma resulted frequently with cyclosarin. The anticonvulsant ED50 doses for scopolamine or diazepam were, in general, not different between the two models, whereas the anticonvulsant ED50 values of midazolam increased 3- to 17-fold with the lower atropine dose. Seizure termination times were not systematically effected by the different doses of atropine. The order of anticonvulsant effectiveness within each model was scopolamine > or = midazolam > diazepam. The findings indicate that the dose of atropine given as antidotal therapy can significantly influence measures of nerve agent toxicity and responsiveness to anticonvulsant therapy.
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http://dx.doi.org/10.1124/jpet.106.111252DOI Listing
January 2007

Circadian rhythms of heart rate and locomotion after treatment with low-dose acetylcholinesterase inhibitors.

J Appl Toxicol 2006 Sep-Oct;26(5):410-8

Department of Research, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA.

This study tested the hypothesis that repeated exposure to low levels of sarin, pyridostigmine bromide (PB) or their combination, at doses equivalent to those possibly experienced by veterans of the 1991 Persian Gulf War, could lead to persistent or delayed autonomic effects and thus help to explain the cause of clinical findings in this population. Male Sprague-Dawley rats were treated for 3 weeks with: saline injection (0.5 ml kg(-1), s.c., 3 times weekly) with tap drinking water (control); saline injection with PB (80 mg l(-1) in drinking water); sarin injection (62.5 microg kg(-1), s.c., 0.5 x LD(50), 3 times weekly) with tap drinking water (sarin); or sarin injection with PB in drinking water (sarin + PB). At 2, 4 or 16 weeks post-treatment, heart rate (HR) and locomotor activity (LA) were studied by radiotelemetry. Two weeks posttreatment, HR in drug-treated animals was significantly lower than in controls. A decrease in low-frequency HR power spectrum (PS) was found at 00:00 h and 08:00 h with sarin + PB and at 00:00 h with sarin, while total power was enhanced with sarin + PB at 22:00 h. Minimal effects of drug treatments on HR and HR PS were detected at 4 and 16 weeks post-treatment. No significant differences in LA between control and other groups were found. Since no consistent long-term effects were found in any of the variables studied, these experiments do not support the hypothesis that repeated administration of low doses of PB and the nerve agent sarin can induce persistent or delayed alterations in autonomic function.
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http://dx.doi.org/10.1002/jat.1155DOI Listing
August 2007

Cerebral acetylcholine and choline contents and turnover following low-dose acetylcholinesterase inhibitors treatment in rats.

Arch Toxicol 2006 Nov 21;80(11):761-7. Epub 2006 Apr 21.

Research Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA.

Male Sprague-Dawley rats were treated for 3 weeks with (1) regular tap drinking water plus subcutaneous (s.c.) saline (0.5 ml/kg) injections three times/week, (2) pyridostigmine bromide (PB) in drinking water (80 mg/L) plus s.c. saline injections three times/week, (3) regular tap drinking water plus s.c. sarin (0.5 x LD(50)) injections three times/week, or (4) PB in drinking water plus s.c. sarin injections three times/week. Repeated doses of sarin, in the presence or absence of PB, were devoid of acute toxicity during the three-week treatment period. Two, 4, and 16 weeks post-treatment, animals were given an intravenous pulse injection of choline labeled with 4 deuterium atoms (D4Ch) followed, after 1 min, by microwave fixation of the brain in vivo. Tissue levels of endogenous acetylcholine (D0ACh), endogenous choline (D0Ch), D4Ch, and ACh synthesized from D4Ch (D4ACh) were measured by gas-chromatography mass-spectrometry in hippocampus, infundibulum, mesencephalon, neocortex, piriform cortex, and striatum. Ch uptake from blood and ACh turnover were estimated from D4Ch and D4ACh concentrations in brain tissue, respectively. Statistically significant differences among brain regions were found for D0Ch, D4Ch, D0ACh and D4ACh at 2, 4 and 16 weeks post-treatment. However, differences in the values of these parameters between control and drug treatments were found only for D0ACh and D0Ch at 2 and 4 weeks, but not at 16 weeks post-treatment. In conclusion, the results from these experiments do not support a delayed or persistent alteration in cholinergic function after exposure to low doses of PB and/or sarin.
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http://dx.doi.org/10.1007/s00204-006-0101-5DOI Listing
November 2006