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Organophosphoate (OP) chemicals are known to inhibit the enzyme acetylcholinesterase (AChE). Studying OP poisoning is difficult because common small animal research models have serum carboxylesterase, which contributes to animals' resistance to OP poisoning. Historically, guinea pigs have been used for this research; however, a novel genetically modified mouse strain (KIKO) was developed with nonfunctional serum carboxylase (Es1 KO) and an altered acetylcholinesterase (AChE) gene, which expresses the amino acid sequence of the human form of the same protein (AChE KI). KIKO mice were injected with 1xLD 50 of an OP nerve agent or vehicle control with or without atropine. After one to three minutes, animals were injected with 35 mg/kg of the currently fielded Reactivator countermeasure for OP poisoning. Postmortem brains were imaged on a Bruker RapifleX ToF/ToF instrument. Data confirmed the presence of increased acetylcholine in OP-exposed animals, regardless of treatment or atropine status. More interestingly, we detected a small amount of Reactivator within the brain of both exposed and unexposed animals; it is currently debated if reactivators can cross the blood-brain barrier. Further, we were able to simultaneously image acetylcholine, the primary affected neurotransmitter, as well as determine the location of both Reactivator and acetylcholine in the brain. This study, which utilized sensitive MALDI-MSI methods, characterized KIKO mice as a functional model for OP countermeasure development.

The identification of improved medical countermeasures against exposure to chemical warfare nerve agents (CWNAs), a class of organophosphorus compounds, is dependent on the choice of animal model used in preclinical studies. CWNAs bind to acetylcholinesterase and prevent the catalysis of acetylcholine, causing a plethora of peripheral and central physiologic manifestations, including seizure. Rodents are widely used to elucidate the effects of CWNA-induced seizure, albeit with a caveat: they express carboxylesterase activity in plasma. Carboxylesterase, an enzyme involved in the detoxification of some organophosphorus compounds, plays a scavenging role and decreases CWNA availability, thus exerting a protective effect. Furthermore, species-specific amino acid differences in acetylcholinesterase confound studies that use oximes or other compounds to restore its function after inhibition by CWNA. The creation of a human acetylcholinesterase knock-in/serum carboxylesterase knockout (C57BL/6-Ces1c tm1.1Loc AChE tm1.1Loc /J; a.k.a KIKO) mouse may facilitate better modeling of CWNA toxicity in a small rodent species. The current studies characterize the effects of exposure to soman, a highly toxic CWNA, and evaluate the efficacy of anti-seizure drugs in this newly developed KIKO mouse model. Data demonstrate that a combination of midazolam and ketamine reduces seizure duration and severity, eliminates the development of spontaneous recurrent seizures, and protects certain brain regions from neuronal damage in a genetically modified model with human relevance to organophosphorus compound toxicity. This new animal model and the results of this study and future studies using it will enhance medical countermeasures development for both defense and homeland security purposes.

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., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Poisoning via organophosphorus (OP) nerve agents occurs when the OP binds and inhibits the enzyme acetylcholinesterase (AChE). This enzyme is responsible for the metabolism of the neurotransmitter acetylcholine (ACh) which transmits signals between nerves and several key somatic regions. When AChE is inhibited, the signal initiated by ACh is not properly terminated. Excessive levels of ACh result in a cholinergic crisis, and in severe cases can lead to death. Current treatments for OP poisoning involve the administration of atropine, which blocks ACh receptors, and oximes, which reactivate AChE after inhibition. Efforts to improve the safety, efficacy, and broad spectrum utility of these treatments are ongoing and usually require the use of appropriate animal model systems. For OP poisoning, the guinea pig (Cavia porcellus) is a commonly used animal model because guinea pigs more closely mirror primate susceptibility to OP poisoning than do other animals such as rats and mice. This is most likely because among rodents and other small mammals, guinea pigs have a very low relative concentration of serum carboxylesterase, an enzyme known to bind OPs in vitro and to act as an endogenous bioscavenger in vivo. Although guinea pigs historically have been used to test OP poisoning therapies, it has been found recently that guinea pig AChE is substantially more resistant to oxime-mediated reactivation than human AChE. To examine the molecular basis for this difference, we reverse transcribed mRNA encoding guinea pig AChE, amplified the resulting cDNA, and sequenced this product. The nucleotide and deduced amino acid sequences of guinea pig AChE were then compared to the human version. Several amino acid differences were noted, and the predicted locations of these differences were mapped onto a structural model of human AChE. To examine directly how these differences affect oxime-mediated reactivation of AChE after inhibition by OPs, human and guinea pig red blood cell ghosts were prepared and used as sources of AChE, and the relative capacity of several different oximes to reactivate each OP-inhibited AChE were determined. The differences we report between human and guinea pig AChE raise additional concerns about the suitability of the guinea pig as an appropriate small animal model to approximate human responses to OP poisoning and therapies., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)