The use of bispyridinium non-oxime analogues for the restoration of nerve agent impaired neuromuscular transmission in rat hemidiaphragms - Structure optimization.

Organophosphate pesticide poisoning challenges health care systems worldwide. Furthermore, nerve agents remain a continuous threat. The treatment options for organophosphate poisoning have virtually been unchanged for decades, relying on symptomatic treatment and the use of oximes to indirectly restore neuromuscular function. Hence, compounds targeting directly nicotinic acetylcholine receptors (nAChRs) might substantially improve treatment options. The current study investigated a series of bispyridinium analogues with a trimethylene or 2,2'-diethyloxy linker in a rat hemidiaphragm model, using indirect field stimulation. Methyl- and ethyl-substituted bispyridinium analogues restored neuromuscular function up to 37 ± 17% (MB419, a 3-methyl analogue) at a stimulation frequency of 20Hz. The bispyridinium analogues with a 2- or 3-methyl group, or a 2- or 3-ethyl group, tended towards a higher restoration of neuromuscular function than those with a 4-methyl or 4-ethyl group, respectively. The current data can be used for future studies to optimize structure-based molecular modeling of compounds targeting the nAChR.

Restoration of nerve agent impaired neuromuscular transmission in rat diaphragm by bispyridinium non-oximes - Structure-activity relationships.

Organophosphate (OP) poisoning is currently treated with atropine, oximes and benzodiazepines. The nicotinic signs, i.e., respiratory impairment, can only be targeted indirectly via the use of oximes as reactivators of OP-inhibited acetylcholinesterase. Hence, compounds selectively targeting nicotinic acetylcholine receptors (nAChRs) might fundamentally improve current treatment options. The bispyridinium compound MB327 has previously shown some therapeutic effect against nerve agents in vitro and in vivo. Nevertheless, compound optimization was deemed necessary, due to limitations (e.g., toxicity and efficacy). The current study investigated a series of 4-tert-butyl bispyridinium compounds and of corresponding bispyridinium compounds without substituents in a rat diaphragm model using an indirect field stimulation technique. The length of the respective linker influenced the ability of the bispyridinium compounds to restore muscle function in rat hemidiaphragms. The current data show structure-activity relationships for a series of bispyridinium compounds and provide insight for future structure-based molecular modeling.

Modelling organophosphate intoxication in C. elegans highlights nicotinic acetylcholine receptor determinants that mitigate poisoning.

Organophosphate intoxication via acetylcholinesterase inhibition executes neurotoxicity via hyper stimulation of acetylcholine receptors. Here, we use the organophosphate paraoxon-ethyl to treat C. elegans and use its impact on pharyngeal pumping as a bio-assay to model poisoning through these neurotoxins. This assay provides a tractable measure of acetylcholine receptor mediated contraction of body wall muscle. Investigation of the time dependence of organophosphate treatment and the genetic determinants of the drug-induced inhibition of pumping highlight mitigating modulation of the effects of paraoxon-ethyl. We identified mutants that reduce acetylcholine receptor function protect against the consequence of intoxication by organophosphates. Data suggests that reorganization of cholinergic signalling is associated with organophosphate poisoning. This reinforces the under investigated potential of using therapeutic approaches which target a modulation of nicotinic acetylcholine receptor function to treat the poisoning effects of this important class of neurotoxins.

Comparative Aspects of Ricin Toxicity by Inhalation.

The pathogenesis of ricin toxicity following inhalation has been investigated in many animal models, including the non-human primate (predominantly the rhesus macaque), pig, rabbit and rodent. The toxicity and associated pathology described in animal models are broadly similar, but variation appears to exist. This paper reviews the published literature and some of our own unpublished data and describes some of the possible reasons for this variation. Methodological variation is evident, including method of exposure, breathing parameters during exposure, aerosol characteristics, sampling protocols, ricin cultivar, purity and challenge dose and study duration. The model species and strain used represent other significant sources of variation, including differences in macro- and microscopic anatomy, cell biology and function, and immunology. Chronic pathology of ricin toxicity by inhalation, associated with sublethal challenge or lethal challenge and treatment with medical countermeasures, has received less attention in the literature. Fibrosis may follow acute lung injury in survivors. There are advantages and disadvantages to the different models of pulmonary fibrosis. To understand their potential clinical significance, these factors need to be considered when choosing a model for chronic ricin toxicity by inhalation, including species and strain susceptibility to fibrosis, time it takes for fibrosis to develop, the nature of the fibrosis (e.g., self-limiting, progressive, persistent or resolving) and ensuring that the analysis truly represents fibrosis. Understanding the variables and comparative aspects of acute and chronic ricin toxicity by inhalation is important to enable meaningful comparison of results from different studies, and for the investigation of medical countermeasures.

From "crisis to recovery": A complete insight into the mechanisms of chlorine injury in the lung.

Chlorine (Cl2) gas is a toxic industrial chemical (TIC) that poses a hazard to human health following accidental and/or intentional (e.g. terrorist) release. By using a murine model of sub-lethal Cl2 exposure we have examined the airway hyper responsiveness, cellular infiltrates, transcriptomic and proteomic responses of the lung. In the "crisis" phase at 2 h and 6 h there is a significant decreases in leukocytes within bronchoalveolar lavage fluid accompanied by an upregulation within the proteome of immune pathways ultimately resulting in neutrophil influx at 24 h. A flip towards "repair" in the transcriptome and proteome occurs at 24 h, neutrophil influx and an associated drop in the lung function persisting until 14 d post-exposure and subsequent "recovery" after 28 days. Collectively, this research provides new insights into the mechanisms of damage, early global responses and processes of repair induced in the lung following the inhalation of Cl2.

Influence of Experimental End Point on the Therapeutic Efficacy of Essential and Additional Antidotes in Organophosphorus Nerve Agent-Intoxicated Mice.

The therapeutic efficacy of treatments for acute intoxication with highly toxic organophosphorus compounds, called nerve agents, usually involves determination of LD50 values 24 h after nerve agent challenge without and with a single administration of the treatment. Herein, the LD50 values of four nerve agents (sarin, soman, tabun and cyclosarin) for non-treated and treated intoxication were investigated in mice for experimental end points of 6 and 24 h. The LD50 values of the nerve agents were evaluated by probit-logarithmical analysis of deaths within 6 and 24 h of i.m. challenge of the nerve agent at five different doses, using six mice per dose. The efficiency of atropine alone or atropine in combination with an oxime was practically the same at 6 and 24 h. The therapeutic efficacy of the higher dose of the antinicotinic compound MB327 was slightly higher at the 6 h end point compared to the 24 h end point for soman and tabun intoxication. A higher dose of MB327 increased the therapeutic efficacy of atropine alone for sarin, soman and tabun intoxication, and that of the standard antidotal treatment (atropine and oxime) for sarin and tabun intoxication. The therapeutic efficacy of MB327 was lower than the oxime-based antidotal treatment. To compare the 6 and 24 h end points, the influence of the experimental end point was not observed, with the exception of the higher dose of MB327. In addition, only a negligible beneficial impact of the compound MB327 was observed. Nevertheless, antinicotinics may offer an additional avenue for countering poisoning by nerve agents that are difficult to treat, and synthetic and biological studies towards the development of such novel drugs based on the core bispyridinium structure or other molecular scaffolds should continue.

Cholinergic signaling at the body wall neuromuscular junction distally inhibits feeding behavior in Caenorhabditis elegans.

Complex biological functions within organisms are frequently orchestrated by systemic communication between tissues. In the model organism Caenorhabditis elegans, the pharyngeal and body wall neuromuscular junctions are two discrete structures that control feeding and locomotion, respectively. Separate, the well-defined neuromuscular circuits control these distinct tissues. Nonetheless, the emergent behaviors, feeding and locomotion, are coordinated to guarantee the efficiency of food intake. Here, we show that pharmacological hyperactivation of cholinergic transmission at the body wall muscle reduces the rate of pumping behavior. This was evidenced by a systematic screening of the effect of the cholinesterase inhibitor aldicarb on the rate of pharyngeal pumping on food in mutant worms. The screening revealed that the key determinants of the inhibitory effect of aldicarb on pharyngeal pumping are located at the body wall neuromuscular junction. In fact, the selective stimulation of the body wall muscle receptors with the agonist levamisole inhibited pumping in a lev-1-dependent fashion. Interestingly, this response was independent of unc-38, an alpha subunit of the nicotinic receptor classically expressed with lev-1 at the body wall muscle. This implies an uncharacterized lev-1-containing receptor underpins this effect. Overall, our results reveal that body wall cholinergic transmission not only controls locomotion but simultaneously inhibits feeding behavior.

Molecular determinants of binding of non-oxime bispyridinium nerve agent antidote compounds to the adult muscle nAChR.

Organophosphorus nerve agents (NAs) are the most lethal chemical warfare agents and have been used by state and non-state actors since their discovery in the 1930s. They covalently modify acetylcholinesterase, preventing the breakdown of acetylcholine (ACh) with subsequent loss of synaptic transmission, which can result in death. Despite the availability of several antidotes for OPNA exposure, none directly targets the nicotinic acetylcholine receptor (nAChR) mediated component of toxicity. Non-oxime bispyridinium compounds (BPDs) have been shown previously to partially counteract the effects of NAs at skeletal muscle tissue, and this has been attributed to inhibition of the muscle nAChR. Functional data indicate that, by increasing the length of the alkyl linker between the pyridinium moieties of BPDs, the antagonistic activity at nAChRs can be improved. Molecular dynamics simulations of the adult muscle nAChR in the presence of BPDs identified key residues likely to be involved in binding. Subsequent two-electrode voltage clamp recordings showed that one of the residues, εY131, acts as an allosteric determinant of BPD binding, and that longer BPDs have a greater stabilizing effect on the orthosteric loop C than shorter ones. The work reported will inform future design work on novel antidotes for treating NA exposure.

C. elegans pharyngeal pumping provides a whole organism bio-assay to investigate anti-cholinesterase intoxication and antidotes.

Inhibition of acetylcholinesterase by either organophosphates or carbamates causes anti-cholinesterase poisoning. This arises through a wide range of neurotoxic effects triggered by the overstimulation of the cholinergic receptors at synapses and neuromuscular junctions. Without intervention, this poisoning can lead to profound toxic effects, including death, and the incomplete efficacy of the current treatments, particularly for oxime-insensitive agents, provokes the need to find better antidotes. Here we show how the non-parasitic nematode Caenorhabditis elegans offers an excellent tool for investigating the acetylcholinesterase intoxication. The C. elegans neuromuscular junctions show a high degree of molecular and functional conservation with the cholinergic transmission that operates in the autonomic, central and neuromuscular synapses in mammals. In fact, the anti-cholinesterase intoxication of the worm's body wall neuromuscular junction has been unprecedented in understanding molecular determinants of cholinergic function in nematodes and other organisms. We extend the use of the model organism's feeding behaviour as a tool to investigate carbamate and organophosphate mode of action. We show that inhibition of the cholinergic-dependent rhythmic pumping of the pharyngeal muscle correlates with the inhibition of the acetylcholinesterase activity caused by aldicarb, paraoxons and DFP exposure. Further, this bio-assay allows one to address oxime dependent reversal of cholinesterase inhibition in the context of whole organism recovery. Interestingly, the recovery of the pharyngeal function after such anti-cholinesterase poisoning represents a sensitive and easily quantifiable phenotype that is indicative of the spontaneous recovery or irreversible modification of the worm acetylcholinesterase after inhibition. These observations highlight the pharynx of C. elegans as a new tractable approach to explore anti-cholinesterase intoxication and recovery with the potential to resolve critical genetic determinants of these neurotoxins' mode of action.

Establishment of a Novel Oral Murine Model of Ricin Intoxication and Efficacy Assessment of Ovine Ricin Antitoxins.

Ricin, produced from the castor beans of Ricinus communis, is a cytotoxin that exerts its action by inactivating ribosomes and causing cell death. Accidental (e.g., ingestion of castor beans) and/or intentional (e.g., suicide) exposure to ricin through the oral route is an area of concern from a public health perspective and no current licensed medical interventions exist to protect from the action of the toxin. Therefore, we examined the oral toxicity of ricin in Balb/C mice and developed a robust food deprivation model of ricin oral intoxication that has enabled the assessment of potential antitoxin treatments. A lethal oral dose was identified and mice were found to succumb to the toxin within 48 h of exposure. We then examined whether a despeciated ovine F(ab')2 antibody fragment, that had previously been demonstrated to protect mice from exposure to aerosolised ricin, could also protect against oral intoxication. Mice were challenged orally with an LD99 of ricin, and 89 and 44% of mice exposed to this otherwise lethal exposure survived after receiving either the parent anti-ricin IgG or F(ab')2, respectively. Combined with our previous work, these results further highlight the benefit of ovine-derived polyclonal antibody antitoxin in providing post-exposure protection against ricin intoxication.

Influence of experimental end point on the therapeutic efficacy of the antinicotinic compounds MB408, MB442 and MB444 in treating nerve agent poisoned mice - a comparison with oxime-based treatment.

Therapeutic efficacy of antidotal treatment of acute poisoning by nerve agents is generally assessed by the evaluation of LD50 values of nerve agents over 24 h following poisoning without or with a single administration of antidotal treatment. In this study, LD50 values of four nerve agents (sarin, soman, tabun and cyclosarin) for non-treated and treated poisoning were evaluated in mice for two experimental end points - 6 h and 24 h. While the efficacy of atropine or oxime-based antidotal treatment was the same regardless of the experimental end point, the therapeutic efficacy of all three newly developed bispyridinium non-oxime compounds (MB408, MB442, and MB444) was mostly slightly higher at the 6 h end point compared to the 24 h end point, although the therapeutic efficacy of MB compounds was not superior to oxime-based antidotal treatment. These results contrast with a study in guinea-pigs using a structurally-related compound, MB327, which showed a striking increase in protection at 6 h compared to 24 h. It is suggested that the disparity may be due to pharmacokinetic differences between the two animal species.

3-Quinuclidinyl-α-methoxydiphenylacetate: A multi-targeted ligand with antimuscarinic and antinicotinic effects designed for the treatment of anticholinesterase poisoning.

Racemic 3-quinuclidinyl-α-methoxydiphenylacetate (MB266) was synthesised. Its activity at muscarinic acetylcholine receptors (mAChRs), and muscle and neuronal nicotinic acetylcholine receptors (nAChRs), was compared to that of atropine and racemic 3-quinucidinyl benzilate (QNB) using a functional assay based on agonist-induced elevation of intracellular calcium ion concentration in CN21, Chinese Hamster Ovary (CHO) and SHSY5Y human cell lines. MB266 acted as an antagonist at acetylcholine receptors, displaying 18-fold selectivity for mAChR versus nAChR (compared to the 15,200-fold selectivity observed for QNB). Thus O-methylation of QNB reduced the affinity for mAChR antagonism and increased the relative potency at both muscle and neuronal nAChRs. Despite MB266 having a pharmacological profile potentially useful for the treatment of anticholinesterase poisoning, its administration did not improve the neuromuscular function in a soman-poisoned guinea-pig diaphragm preparation pretreated with the organophosphorus nerve agent soman. Consideration should be given to exploring the potential of MB266 for possible anticonvulsant action in vitro as part of a multi-targeted ligand approach.

Assessment of false transmitters as treatments for nerve agent poisoning.

Nerve agents inhibit acetylcholinesterase (AChE), leading to a build-up of acetylcholine (ACh) and overstimulation at cholinergic synapses. Current post-exposure nerve agent treatment includes atropine to treat overstimulation at muscarinic synapses, a benzodiazepine anti-convulsant, and an oxime to restore the function of AChE. Aside from the oxime, the components do not act directly to reduce the overstimulation at nicotinic synapses. The false transmitters acetylmonoethylcholine (AMECh) and acetyldiethylcholine (ADECh) are analogs of ACh, synthesised similarly at synapses. AMECh and ADECh are partial agonists, with reduced activity compared to ACh, so it was hypothesised the false transmitters could reduce overstimulation. Synthetic routes to AMECh and ADECh, and their precursors, monoethylcholine (MECh) and diethylcholine (DECh), were devised, allowing them to be produced easily on a laboratory-scale. The mechanism of action of the false transmitters was investigated in vitro. AMECh acted as a partial agonist at human muscarinic (M1 and M3) and muscle-type nicotinic receptors, and ADECh was a partial agonist only at certain muscarinic subtypes. Their precursors acted as antagonists at muscle-type nicotinic, but not muscarinic receptors. Administration of MECh and DECh improved neuromuscular function in the soman-exposed guinea-pig hemi-diaphragm preparation. False transmitters may therefore help reduce nerve agent induced overstimulation at cholinergic synapses.

Multiple metabolic pathways are predictive of ricin intoxication in a rat model.

INTRODUCTION: Exposure to ricin can be lethal and treatments that are under development have short windows of opportunity for administration after exposure. It is therefore essential to achieve early detection of ricin exposure to provide the best prognosis for exposed individuals. Ricin toxin can be detected in clinical samples via several antibody-based techniques, but the efficacy of these can be limited due to the rapid processing and cellular uptake of toxin in the body and subsequent low blood ricin concentrations. Other diagnostic tools that perform, in an orthogonal manner, are therefore desirable. OBJECTIVES: To determine time-dependent metabolic changes in Sprague-Dawley rats following intravenous exposure to ricin. METHODS: Sprague-Dawley rats were intravenously exposed to ricin and multiple blood samples were collected from each animal for up to 48 h following exposure in two independent studies. Plasma samples were analysed applying HILIC and C18 reversed phase UHPLC-MS assays followed by univariate and multivariate analysis. RESULTS: In Sprague-Dawley rats we have demonstrated that metabolic changes measured in blood can distinguish between rats exposed intravenously to ricin and controls prior to the onset of behavioral signs of intoxication after 24 h. A total of 37 metabolites were significantly altered following exposure to ricin when compared to controls. The arginine/proline, bile acid and triacylglyceride metabolic pathways were highlighted as being important with two triacylglycerides at 8 h post exposure giving an AUROC score of 0.94. At 16 h and 24 h the AUROC score increased to 0.98 and 1.0 with the number of metabolites in the panel increasing to 5 and 7, respectively. CONCLUSIONS: These data demonstrate that metabolites may be a useful tool to diagnose and detect ricin exposure, thus increasing the effectiveness of supportive therapy and future ricin-specific medical treatments.

Some benefit from non-oximes MB408, MB442 and MB444 in combination with the oximes HI-6 or obidoxime and atropine in antidoting sarin or cyclosarin poisoned mice.

The effect of three newly developed bispyridinium non-oxime compounds (MB408, MB442, and MB444) on the therapeutic efficacy of a standard antidotal treatment (atropine in combination with the oxime HI-6 or obidoxime) of acute poisoning by two nerve agents (sarin and cyclosarin) in mice was studied. The therapeutic efficacy of atropine in combination with an oxime with or without one of the bispyridinium non-oximes was evaluated by determination of the 24 h LD50 values of the nerve agents studied and by measurement of the survival time after supralethal poisoning. Addition of all tested non-oximes increased the therapeutic efficacy of atropine in combination with an oxime against sarin poisoning; however, the differences were not significant. The non-oximes also positively influenced the number of surviving mice 6 h after supralethal poisoning with sarin. In the case of cyclosarin, they were also slightly beneficial in the treatment of acute poisoning. The higher dose of MB444 was able to significantly increase the therapeutic efficacy of standard antidotal treatment of poisoning with cyclosarin. The benefit of each bispyridinium non-oxime compound itself was obviously dose-dependent. In summary, the addition of MB compounds to the standard antidotal treatment of acute nerve agent poisoning was beneficial for the antidotal treatment of sarin or cyclosarin poisoning, although their benefit at 24 h after poisoning was not significant, with the exception of the higher dose of MB444 against cyclosarin.

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

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

An imaging flow cytometry method to assess ricin trafficking in A549 human lung epithelial cells.

The endocytosis and trafficking of ricin in mammalian cells is an important area of research for those producing ricin anti-toxins and other ricin therapeutics. Ricin trafficking is usually observed by fluorescence microscopy techniques. This gives good resolution and leads to a detailed understanding of the internal movement of ricin within cells. However, microscopy techniques are often hampered by complex analysis and quantification techniques, and the inability to look at ricin trafficking in large populations of cells. In these studies we have directly labelled ricin and assessed if its trafficking can be observed using Imaging Flow Cytometry (IFC) both to the cytoplasmic region of cells and specifically to the Golgi apparatus. Using IDEAS® data analysis software the specific fluorescence location of the ricin within the cells was analysed. Then, using cytoplasmic masking techniques to quantify the number of cells with endocytosed cytoplasmic ricin or cells with Golgi-associated ricin, kinetic endocytosis curves were generated. Here we present, to the authors' knowledge, the first example of using imaging flow cytometry for evaluating the subcellular transport of protein cargo, using the trafficking of ricin toxin in lung cells as a model.

Production, Characterisation and Testing of an Ovine Antitoxin against Ricin; Efficacy, Potency and Mechanisms of Action.

Ricin is a type II ribosome-inactivating toxin that catalytically inactivates ribosomes ultimately leading to cell death. The toxicity of ricin along with the prevalence of castor beans (its natural source) has led to its increased notoriety and incidences of nefarious use. Despite these concerns, there are no licensed therapies available for treating ricin intoxication. Here, we describe the development of a F(ab')2 polyclonal ovine antitoxin against ricin and demonstrate the efficacy of a single, post-exposure, administration in an in vivo murine model of intoxication against aerosolised ricin. We found that a single dose of antitoxin afforded a wide window of opportunity for effective treatment with 100% protection observed in mice challenged with aerosolised ricin when given 24 h after exposure to the toxin and 75% protection when given at 30 h. Treated mice had reduced weight loss and clinical signs of intoxication compared to the untreated control group. Finally, using imaging flow cytometry, it was found that both cellular uptake and intracellular trafficking of ricin toxin to the Golgi apparatus was reduced in the presence of the antitoxin suggesting both actions can contribute to the therapeutic mechanism of a polyclonal antitoxin. Collectively, the research highlights the significant potential of the ovine F(ab')2 antitoxin as a treatment for ricin intoxication.

Interference of the T Cell and Antigen-Presenting Cell Costimulatory Pathway Using CTLA4-Ig (Abatacept) Prevents Staphylococcal Enterotoxin B Pathology.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds the receptors in the APC/T cell synapse and causes increased proliferation of T cells and a cytokine storm syndrome in vivo. Exposure to the toxin can be lethal and cause significant pathology in humans. The lack of effective therapies for SEB exposure remains an area of concern, particularly in scenarios of acute mass casualties. We hypothesized that blockade of the T cell costimulatory signal by the CTLA4-Ig synthetic protein (abatacept) could prevent SEB-dependent pathology. In this article, we demonstrate mice treated with a single dose of abatacept 8 h post SEB exposure had reduced pathology compared with control SEB-exposed mice. SEB-exposed mice showed significant reductions in body weight between days 4 and 9, whereas mice exposed to SEB and also treated with abatacept showed no weight loss for the duration of the study, suggesting therapeutic mitigation of SEB-induced morbidity. Histopathology and magnetic resonance imaging demonstrated that SEB mediated lung damage and edema, which were absent after treatment with abatacept. Analysis of plasma and lung tissues from SEB-exposed mice treated with abatacept demonstrated significantly lower levels of IL-6 and IFN-γ (p < 0.0001), which is likely to have resulted in less pathology. In addition, exposure of human and mouse PBMCs to SEB in vitro showed a significant reduction in levels of IL-2 (p < 0.0001) after treatment with abatacept, indicating that T cell proliferation is the main target for intervention. Our findings demonstrate that abatacept is a robust and potentially credible drug to prevent toxic effects from SEB exposure.

Evaluation of the benefit of the bispyridinium compound MB327 for the antidotal treatment of nerve agent-poisoned mice.

The potency of the bispyridinium non-oxime compound MB327 [1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium) diiodide] to increase the therapeutic efficacy of the standard antidotal treatment (atropine in combination with an oxime) of acute poisoning with organophosphorus nerve agents was studied in vivo. The therapeutic efficacy of atropine alone - or atropine in combination with an oxime, MB327, or both an oxime and MB237 - was evaluated by the determination of LD50 values of several nerve agents (tabun, sarin and soman) in mice with and without treatment. The addition of MB327 increased the therapeutic efficacy of atropine alone, and atropine in combination with an oxime, against all three nerve agents, although differences in the LD50 values only reached statistical significance for sarin. In conclusion, the addition of the compound MB327 to the standard antidotal treatment of acute poisonings with nerve agents was beneficial regardless of the chemical structure of the nerve agent, although at the dose employed, MB327 in combination with atropine, or atropine and an oxime, provided only a modest increase in protection ratio. These results from mice, and previous ones from guinea-pigs, provide consistent evidence for additional, albeit modest, efficacy resulting from the inclusion of the antinicotinic compound MB327 in standard antidotal therapy. Given the typically steep probit slope for the dose-lethality relationship for nerve agents, such modest increases in protection ratio could provide significant survival benefit.