Chemical, biological, radiological, and nuclear mass casualty medicine: a review of lessons from the Salisbury and Amesbury Novichok nerve agent incidents.

On March 4, 2018, two casualties collapsed on a park bench in Salisbury, Wiltshire, UK. They were later discovered to have been the victims of an attempted murder using the Soviet-era Novichok class of nerve agent. The casualties, along with three further critically ill patients, were cared for in Salisbury District Hospital's Intensive Care Unit. Before the COVID-19 pandemic, the Salisbury and Amesbury incidents were the longest-running major incidents in the history of the UK National Health Service. This narrative review seeks to reflect on the lessons learned from these chemical incidents, with a particular focus on hospital and local organisational responses.

Organophosphorus compounds and oximes: a critical review.

Organophosphorus (OP) pesticides and nerve agents still pose a threat to the population. Treatment of OP poisoning is an ongoing challenge and burden for medical services. Standard drug treatment consists of atropine and an oxime as reactivator of OP-inhibited acetylcholinesterase and is virtually unchanged since more than six decades. Established oximes, i.e. pralidoxime, obidoxime, TMB-4, HI-6 and MMB-4, are of insufficient effectiveness in some poisonings and often cover only a limited spectrum of the different nerve agents and pesticides. Moreover, the value of oximes in human OP pesticide poisoning is still disputed. Long-lasting research efforts resulted in the preparation of countless experimental oximes, and more recently non-oxime reactivators, intended to replace or supplement the established and licensed oximes. The progress of this development is slow and none of the novel compounds appears to be suitable for transfer into advanced development or into clinical use. This situation calls for a critical analysis of the value of oximes as mainstay of treatment as well as the potential and limitations of established and novel reactivators. Requirements for a straightforward identification of superior reactivators and their development to licensed drugs need to be addressed as well as options for interim solutions as a chance to improve the therapy of OP poisoning in a foreseeable time frame.

Organophosphorus nerve agent poisoning: managing the poisoned patient.

Organophosphorus (OP) nerve agent poisoning made the headlines in 2018 with the nerve agent 'Novichok' poisonings in Salisbury, England. This event highlighted a gap in the knowledge of most clinicians in the UK. In response, this special article aims to enlighten and signpost anaesthetists and intensivists towards the general management of OP nerve agent poisoned patients. Drawing on a broad range of sources, we will discuss what OP nerve agents are, how they work, and how to recognise and treat OP nerve agent poisoning. OP nerve agents primarily act by inhibiting the enzyme acetylcholinesterase, causing an acute cholinergic crisis; death usually occurs through respiratory failure. The antimuscarinic agent atropine, oximes (to reactivate acetylcholinesterase), neuroprotective drugs, and critical care remain the mainstays of treatment. The risk to medical staff from OP poisoned patients appears low, especially if there is a thorough decontamination of the poisoned patient and staff wear appropriate personal protective equipment. The events in Salisbury in the past year were shocking, and the staff at Salisbury District General Hospital performed admirably in treating those affected by Novichok nerve agent poisoning. We eagerly anticipate their future clinical publications so that the medical community might learn from their valuable experiences.

Oxime K203: a drug candidate for the treatment of tabun intoxication.

For over 60 years, researchers across the world have sought to deal with poisoning by nerve agents, the most toxic and lethal chemical weapons. To date, there is no efficient causal antidote with sufficient effect. Every trialed compound fails to fulfil one or more criteria (e.g. reactivation potency, broad reactivation profile). In this recent contribution, we focused our attention to one of the promising compounds, namely the bis-pyridinium reactivator K203. The oxime K203 is very often cited as the best reactivator against tabun poisoning. Herein, we provide all the available literature data in comprehensive and critical review to address whether K203 could be considered as a new drug candidate against organophosphorus poisoning with the stress on tabun. We describe its development from the historical point of view and review all available in vitro as well as in vivo data to date. K203 is easily accessible by a relatively simple two-step synthesis. It is well accommodated in the enzyme active gorge of acetylcholinesterase providing suitable interactions for reactivation, as shown by molecular docking simulations. According to a literature survey, in vitro data for tabun-inhibited AChE are extraordinary. However, in vivo efficiency remains unconvincing. The K203 toxicity profile did not show any perturbations compared to clinically used standards; on the other hand versatility of K203 does not exceed currently available oximes. In summary, K203 does not seem to address current issues associated with the organophosphorus poisoning, especially the broad profile against all nerve agents. However, its reviewed efficacy entitles K203 to be considered as a backup or tentative replacement for obidoxime and trimedoxime, currently only available anti-tabun drugs.

Poisoning with Soman, an Organophosphorus Nerve Agent, Alters Fecal Bacterial Biota and Urine Metabolites: a Case for Novel Signatures for Asymptomatic Nerve Agent Exposure.

The experimental pathophysiology of organophosphorus (OP) chemical exposure has been extensively reported. Here, we describe an altered fecal bacterial biota and urine metabolome following intoxication with soman, a lipophilic G class chemical warfare nerve agent. Nonanesthetized Sprague-Dawley male rats were subcutaneously administered soman at 0.8 (subseizurogenic) or 1.0 (seizurogenic) of the 50% lethal dose (LD50) and evaluated for signs of toxicity. Animals were stratified based on seizing activity to evaluate effects of soman exposure on fecal bacterial biota and urine metabolites. Soman exposure reshaped fecal bacterial biota by altering Facklamia, Rhizobium, Bilophila, Enterobacter, and Morganella genera of the Firmicutes and Proteobacteria phyla, some of which are known to hydrolyze OP chemicals. However, analogous changes were not observed in the bacterial biota of the ileum, which remained the same irrespective of dose or seizing status of animals after soman intoxication. However, at 75 days after soman exposure, the bacterial biota stabilized and no differences were observed between groups. Interestingly, in considering just the seizing status of animals, we found that the urine metabolomes were markedly different. Leukotriene C4, kynurenic acid, 5-hydroxyindoleacetic acid, norepinephrine, and aldosterone were excreted at much higher rates at 72 h in seizing animals, consistent with early multiorgan involvement during soman poisoning. These findings demonstrate the feasibility of using the dysbiosis of fecal bacterial biota in combination with urine metabolome alterations as forensic evidence for presymptomatic OP exposure temporally to enable administration of neuroprotective therapies of the future.IMPORTANCE The paucity of assays to determine physiologically relevant OP exposure presents an opportunity to explore the use of fecal bacteria as sentinels in combination with urine to assess changes in the exposed host. Recent advances in sequencing technologies and computational approaches have enabled researchers to survey large community-level changes of gut bacterial biota and metabolomic changes in various biospecimens. Here, we profiled changes in fecal bacterial biota and urine metabolome following a chemical warfare nerve agent exposure. The significance of this work is a proof of concept that the fecal bacterial biota and urine metabolites are two separate biospecimens rich in surrogate indicators suitable for monitoring OP exposure. The larger value of such an approach is that assays developed on the basis of these observations can be deployed in any setting with moderate clinical chemistry and microbiology capability. This can enable estimation of the affected radius as well as screening, triage, or ruling out of suspected cases of exposures in mass casualty scenarios, transportation accidents involving hazardous materials, refugee movements, humanitarian missions, and training settings when coupled to an established and validated decision tree with clinical features.

A primer on nerve agents: what the emergency responder, anesthesiologist, and intensivist needs to know.

PURPOSE: The purpose of this review article is to familiarize first responders, anesthesiologists, and intensivists with the medical management of patients exposed to nerve agents. SOURCE: This review is based on the current medical literature available to the general medical community. PRINCIPAL FINDINGS: Nerve agents are some of the deadliest substances known to humanity. Though they kill primarily via muscle paralysis, which leads to respiratory arrest, these agents affect virtually every organ system in the body. Their primary mechanism of action is the body-wide inhibition of cholinesterases. This inhibition leads to the accumulation of acetylcholine, stimulating both nicotinic and muscarinic receptors. After decontamination, the primary treatment is with atropine to control muscarinic symptoms and with oximes to reactivate the cholinesterases and treat the nicotinic symptoms. Atropine doses can be much higher than conventionally used. Seizures are generally best treated with benzodiazepines. Patients with substantial exposure may require ventilatory and intensive care unit support for prolonged periods of time. CONCLUSION: While it is unlikely that most medical practitioners will ever encounter nerve agent poisoning, it is critical to be aware of the presenting symptoms and how best to treat patients exposed to these deadly agents. History has shown that rapid medical treatment can easily mean the difference between life and death for a patient in this situation.

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.

Warfare Nerve Agents and Paraoxonase-1 as a Potential Prophylactic Therapy against Intoxication.

Nerve agents are a class of lethal neurotoxic chemicals used in chemical warfare. In this review, we have discussed a brief history of chemical warfare, followed by an exploration of the historical context surrounding nerve agents. The article explores the classification of these agents, their contemporary uses, their toxicity mechanisms, and the disadvantages of the current treatment options for nerve agent poisoning. It then discusses the possible application of enzymes as prophylactics against nerve agent poisoning, outlining the benefits and drawbacks of paraoxonase- 1. Finally, the current studies on paraoxonase-1 are reviewed, highlighting that several challenges need to be addressed in the use of paraoxonase-1 in the actual field and that its potential as a prophylactic antidote against nerve agent poisoning needs to be evaluated. The literature used in this manuscript was searched using various electronic databases, such as PubMed, Google Scholar, Web of Science, Elsevier, Springer, ACS, Google Patent, and books using the keywords chemical warfare agent, butyrylcholinesterase, enzyme, nerve agent, prophylactic, and paraoxonase-1, with the time scale for the analysis of articles between 1960 to 2023. The study has suggested that concerted efforts by researchers and agencies must be made to develop effective countermeasures against NA poisoning and that paraoxonase-1 has suitable properties for the development of efficient prophylaxis against NA poisoning.

Early diagnosis of nerve agent exposure with a mobile test kit and implications for medical countermeasures: a trigger to react.

Recent uses of nerve agents underline the need of early diagnosis as trigger to react (initiating medical countermeasures, avoiding cross-contamination). As organophosphorus (OP) pesticide poisoning exerts the same pathomechanism, that is, inhibition of the pivotal enzyme acetylcholinesterase (AChE), a portable cholinesterase (ChE) test kit was applied in an emergency room for rapid diagnosis of OP poisoning. OP nerve agents or pesticides result in the inhibition of AChE. As AChE is also expressed on erythrocytes, patient samples are easily available. However, in most clinics only determination of plasma butyrylcholinesterase (BChE) is established which lacks a pathophysiological correlate, shows higher variability in the population and behaves different regarding inhibition by OP and reactivation by oximes. The ChE test kit helped to diagnose atypical cases of OP poisoning, for example, missing of typical muscarinic symptoms, and resulted in administration of pralidoxime, the oxime used in Serbia. The ChE test kit also allows an initial assessment whether an oxime therapy is successful. In one case report, AChE activity increased after oxime administration indicating therapeutic success whereas BChE activity did not. With only BChE at hand, this therapeutic effect would have been missed. As inhibition of AChE or BChE activity is determined, the CE-certified device is a global diagnostic tool for all ChE inhibitors including carbamates which might also be misused as chemical weapon. The ChE test kit is a helpful point-of-care device for the diagnosis of ChE inhibitor poisoning. Its small size and easy menu-driven use advocate procurement where nerve agent and OP pesticide exposure are possible.

Efficient detoxification of nerve agents by oxime-assisted reactivation of acetylcholinesterase mutants.

The recent advancements in crystallography and kinetics studies involving reactivation mechanism of acetylcholinesterase (AChE) inhibited by nerve agents have enabled a new paradigm in the search for potent medical countermeasures in case of nerve agents exposure. Poisonings by organophosphorus compounds (OP) that lead to life-threatening toxic manifestations require immediate treatment that combines administration of anticholinergic drugs and an aldoxime as a reactivator of AChE. An alternative approach to reduce the in vivo toxicity of OP centers on the use of bioscavengers against the parent organophosphate. Our recent research showed that site-directed mutagenesis of AChE can enable aldoximes to substantially accelerate the reactivation of OP-enzyme conjugates while dramatically slowing down rates of OP-conjugate dealkylation (aging). Therefore, this review focuses on oxime-assisted catalysis by AChE mutants that provides a potential means for degradation of organophosphates in the plasma before reaching the cellular target site. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.

Butyrylcholinesterase, a stereospecific in vivo bioscavenger against nerve agent intoxication.

Human butyrylcholinesterase (E.C. 3.1.1.8) purified from blood plasma has previously been shown to provide protection against up to five and a half times the median lethal dose of an organophosphorus nerve agent in several animal models. In this study the stoichiometric nature of the protection afforded by human butyrylcholinesterase against organophosphorus nerve agents was investigated in guinea pigs. Animals were administered human butyrylcholinesterase (26.15 mg/kg ≡ 308 nmol/kg) by the intravascular or intramuscular route. Animals were subsequently dosed with either soman or VX in accordance with a stage-wise adaptive dose design to estimate the modified median lethal dose in treated animals. Human butyrylcholinesterase (308 nmol/kg) increased the median lethal dose of soman from 154 nmol/kg to 770 nmol/kg. Comparing the molar ratio of agent molecules to enzyme active sites yielded a stoichiometric protective ratio of 2:1 for soman, likely related to the similar stereoselectivity the enzyme has compared to the toxic target, acetylcholinesterase. In contrast, human butyrylcholinesterase (308 nmol/kg) increased the median lethal dose of VX from 30 nmol/kg to 312 nmol/kg, resulting in a stoichiometric protective ratio of only 1:1, suggesting a lack of stereoselectivity for this agent.

Pharmacokinetics of Three Oximes in a Guinea Pig Model and Efficacy of Combined Oxime Therapy.

Organophosphorus nerve agents (NA) inhibit acetylcholinesterase (AChE) which results in the over-stimulation of both the central and peripheral nervous systems, creating a toxic syndrome that can be lethal if left untreated (Cannard, 2006). It is standard practice to treat Sarin (GB) intoxication with an oxime, an antimuscarinic such as atropine and an anticonvulsant. Three common oximes are available: HI-6, Pralidoxime (2-PAM) and Obidoxime (Obi), all possess a nucleophile that can break the NA-AChE covalent bond. However, each oxime's efficacy profile against various agents is different (Thiermann and Worek, 2018). In an effort to broaden therapeutic efficacy against a range of possible NA's, consideration should be given to the use of two oximes in combination. Using a guinea pig model, the first arm of this study was to determine the pharmacokinetics (PK) of HI-6 DMS, 2-PAM chloride and Obi chloride (at autoinjector equivalent doses) following intramuscular (i.m.) co-administration along with atropine to replicate either a single isometrically scaled dose (referred to in this study as a single autoinjector equivalent) of 2-PAM (and equimolar doses of Obi and HI-6) or double doses (referred to in this study as two autoinjector equivalents). The second arm of the study evaluated the efficacy of Obi and 2-PAM individually at a single or double autoinjector dose and also in combination against GB exposure. Pharmacokinetic profiles of each oxime were evaluated for both arms of the study and no significant change in parameters were reported. Improved cholinesterase reactivation was observed in a dose dependent manner with combined therapy showing similar reactivation to individual oximes alone at a two autoinjector equivalent dose. Seizure activity was reduced when combined oxime therapy was administered. This improvement was also reflected in the Racine seizure index score assigned at the end of the experimental period. To the best of our knowledge, this study is the first to evaluate and compare the pharmacokinetics of three oximes and the combination of two oximes (2-PAM and Obi) administered in naïve animals or those exposed to GB. Combined oxime therapy (Obi and 2-PAM) resulted in improved seizure control, increased cholinesterase reactivation peripherally and centrally and improved behavioral signs (Racine score). This study provides evidence that combination of oximes is effective, does not result in adverse events and that the pharmacokinetics of each oxime are not affected when administered in combination.

A catalytic bioscavenger with improved stability and reduced susceptibility to oxidation for treatment of acute poisoning with neurotoxic organophosphorus compounds.

Organophosphorus (OP)1 nerve agents pose a severe toxicological threat, both after dissemination in military conflicts and by terrorists. Hydrolytic enzymes, which may be administered into the blood stream of victims by injection and can decompose the circulating nerve agent into non-toxic metabolites in vivo, could offer a treatment. Indeed, for the phosphotriesterase found in the bacterium Brevundimonas diminuta (BdPTE),2 engineered versions with improved catalytic efficiencies have been described; yet, their biochemical stabilities are insufficient for therapeutic use. Here, we describe the application of rational protein design to develop novel mutants of BdPTE that are less susceptible to oxidative damage. In particular, the replacement of two unpaired cysteine residues by more inert amino acids led to higher stability while maintaining high catalytic activity towards a broad spectrum of substrates, including OP pesticides and V-type nerve agents. The mutant BdPTE enzymes were produced in Escherichia coli, purified to homogeneity, and their biochemical and enzymological properties were assessed. Several candidates both revealed enhanced thermal stability and were less susceptible to oxidative stress, as demonstrated by mass spectrometry. These mutants of BdPTE may show promise for the treatment of acute intoxications by nerve agents as well as OP pesticides.

Temporal Effects of 2% Pilocarpine Ophthalmic Solution on Human Pupil Size and Accommodation.

INTRODUCTION: First responders and those who work with organophosphate (OP) compounds can experience ocular symptoms similar to those caused by exposure to low levels of nerve agents. This study was designed to examine the efficacy of a safe, clinically available, simulant that reproduces ocular symptoms associated with low-level OP exposure. Among these ocular symptoms are a constriction of the pupils (miosis), decreased visual acuity, and changes in accommodation. MATERIALS AND METHODS: Volunteers aged 18-40 were assigned to groups receiving either a two-drop or three-drop dose of FDA approved 2% pilocarpine ophthalmic solution. Baseline visual performance measurements were taken before eye drop instillation and a timer was started following the first drop of pilocarpine. Once eye drops were administered, visual performance including distant and near vision, pupil size, and accommodation were measured every 5 minutes for 2 hours. RESULTS: Both groups experienced significant miosis in excess of 90 minutes. Visual acuity was significantly reduced because of accommodative changes. The three-drop group experienced longer lasting combined effects when compared to the two-drop group. CONCLUSIONS: 2% pilocarpine ophthalmic solution can safely simulate major ocular symptoms of OP exposure for behavioral research studies for at least 60 minutes.

Magnetic resonance imaging analysis of long-term neuropathology after exposure to the nerve agent soman: correlation with histopathology and neurological dysfunction.

Nerve agents (NAs) produce acute and long-term brain injury and dysfunction, as evident from the Japan and Syria incidents. Magnetic resonance imaging (MRI) is a versatile technique to examine such chronic anatomical, functional, and neuronal damage in the brain. The objective of this study was to investigate long-term structural and neuronal lesion abnormalities in rats exposed to acute soman intoxication. T2-weighted MRI images of 10 control and 17 soman-exposed rats were acquired using a Siemens MRI system at 90 days after soman exposure. Quantification of brain tissue volumes and T2 signal intensity was conducted using the Inveon Research Workplace software and the extent of damage was correlated with histopathology and cognitive function. Soman-exposed rats showed drastic hippocampal atrophy with neuronal loss and reduced hippocampal volume (HV), indicating severe damage, but had similar T2 relaxation times to the control group, suggesting limited scarring and fluid density changes despite the volume decrease. Conversely, soman-exposed rats displayed significant increases in lateral ventricle volumes and T2 times, signifying strong cerebrospinal fluid expansion in compensation for tissue atrophy. The total brain volume, thalamic volume, and thalamic T2 time were similar in both groups, however, suggesting that some brain regions remained more intact long-term after soman intoxication. The MRI neuronal lesions were positively correlated with the histological markers of neurodegeneration and neuroinflammation 90 days after soman exposure. The predominant MRI hippocampal atrophy (25%) was highly consistent with massive reduction (35%) of neuronal nuclear antigen-positive (NeuN+ ) principal neurons and parvalbumin-positive (PV+ ) inhibitory interneurons within this brain region. The HV was significantly correlated with both inflammatory markers of GFAP+ astrogliosis and IBA1+ microgliosis. The reduced HV was also directly correlated with significant memory deficits in the soman-exposed cohort, confirming a possible neurobiological basis for neurological dysfunction. Together, these findings provide powerful insight on long-term region-specific neurodegenerative patterns after soman exposure and demonstrate the feasibility of in vivo neuroimaging to monitor neuropathology, predict the risk of neurological deficits, and evaluate response to medical countermeasures for NAs.

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.

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.