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Highly toxic organophosphorous nerve agents (OPAs) have been used in several armed conflicts and terror attacks in the last few decades. A new method for retrospective determination of alkyl methylphosphonic acid (AMPA) metabolites in urine after exposure to VX, GB and GF nerve agents was developed. This method enables a rapid, sensitive and selective determination of trace levels of the nerve agent biomarkers ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA) in urine. The new technique involves a unique combination of two solid phase extraction (SPE) cartridges: a Ba/Ag/H cartridge for urine interference removal, and a ZrO2 cartridge for selective reconstitution and enrichment of the AMPAs. Extraction of AMPAs from the ZrO2 cartridge was accomplished with a 1% ammonium hydroxide (NH4OH) solution and was followed by analysis via liquid chromatography–mass spectrometry (LC–MS). The limits of quantitation (LOQs) were in the range of 10–100 pg/mL with recoveries of 64–71% (± 5–19%) after fast sample preparation and a total LC–MS analysis cycle time of 15 min and 13 min, respectively. This method was successfully applied in vivo in a rabbit that was exposed to 0.5 LD50 (7.5 µg/kg, i.v.) sarin for retrospective monitoring of the IMPA metabolite in urine. For the first time, IMPA was determined in rabbit urine samples for 15 days post-exposure, which is longer than any reported post-exposure method for AMPAs. To the best of our knowledge, this new method is the most sensitive and rapid for AMPA determination in urine by LC–MS/MS analysis.

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.

The highly toxic nerve agent sarin (o-isopropyl methyl-phosphonofluoridate, GB) has been used in several armed conflicts and terror attacks in recent decades. Due to its inherent high sensitivity, liquid chromatography–mass spectrometry (LC–MS/MS) has the potential to detect ultratrace levels of fluoride-regenerated G and V agents after appropriate chemical derivatization. A new method for the retrospective determination of exposure to sarin was developed. The method is based on sarin regeneration from blood using the fluoride-induced technique followed by derivatization with 2-[(dimethylamino)methyl]phenol (2-DMAMP) and LC–ESI–MS/MS (MRM) analysis. The validated method presents good linear response in the concentration range of 5–1000 pg/mL with a limit of quantitation (LOQ) of 5 pg/mL, 13.8% accuracy, 16.7% precision and a total recovery of 62% ± 9%. This new analytical approach has several advantages over existing GC/GC–MS-based methods in terms of sensitivity, specificity and simplicity, in addition to a short LC–MS cycle time of 12 min. The method was successfully applied in an in vivo experiment for retrospective determination of sarin in a rabbit exposed to 0.1 LD50 sarin (1.5 µg/kg, i.v.). GB-2-DMAMP was easily determined in samples drawn up to 11 days after exposure. The high S/N ratio (500) observed for the GB-2-DMAMP signal in the 11day sample poses the potential for an extended time frame of months for analysis with this new method for the retrospective detection of sarin exposure. To the best of our knowledge, this is the first report on LC–MS/MS trace analysis of regenerated GB from biological matrices.

Exposure to the chemical warfare nerve agent VX is extremely toxic, causing severe cholinergic symptoms. If not appropriately treated, death ultimately ensues. Based on our previously described whole-body vapor exposure system, we characterized in detail the clinical outcome, including respiratory dynamics, typical of whole-body exposure to lethal doses of VX vapor in freely moving rats. We further evaluated the efficacy of two different antidotal regimens, one comprising a single and the other repeated administration of antidotes, in countering the toxic effects of the exposure. We show that a 15 min exposure to air VX concentrations of 2.34–2.42 mg/m3 induced a late (15–30 min) onset of obvious cholinergic signs, which exacerbated over time, albeit without convulsions. Marked eye pathology was observed, characterized by pupil constriction to pinpoint, excessive lacrimation with red tears (chromodacryorrhea) and corneal damage. Respiratory distress was also evident, characterized by a three–fourfold increase in Penh values, an estimate of lung resistance, and by lung and diaphragm histological damage. A single administration of TAB (the oxime TMB-4, atropine and the anticholinergic and antiglutamatergic benactyzine) at the onset of clinical signs afforded only limited protection (66% survival), with clinical deterioration including weight loss, chromodacryorrhea, corneal damage, increased airway resistance and late death. In contrast, a combined therapy of TAB at the onset of clinical signs and repeated administration of atropine and toxogonin (ATOX) every 3–5 h, a maximum of five i.m. injections, led to 100% survival and a prompt recovery, accompanied by neither the above-described signs of eye pathology, nor by bronchoconstriction and respiratory distress. The necessity of recurrent treatments for successful elimination of VX vapor toxicity strongly supports continuous penetration of VX following termination of VX vapor exposure, most likely from a VX reservoir formed in the skin due to the exposure. This, combined with the above-described eye and respiratory pathology and absence of convulsions, are unique features of whole-body VX vapor exposure as compared to whole-body vapor exposure to other nerve agents, and should accordingly be considered when devising optimal countermeasures and medical protocols for treatment of VX vapor exposure.

Long-term retrospective monitoring of exposure to organophosphorus nerve agents is challenging. We recently developed two highly sensitive analytical methods for regenerated sarin (GB) nerve agent in blood and its primary metabolite, isopropyl-methylphosphonic acid (IMPA), in urine. These methods were implemented in a toxicokinetics study carried out with sarin injected (i.v.) to rabbits at doses corresponding to 0.1, 0.5 or 0.9 LD50. The time frame for monitoring regenerated sarin from blood was 70 days for 0.1 LD50 and 0.5 LD50 and 77 days for 0.9 LD50, where rapid elimination occurred in the first 8 days with an initial average half-life of 1.2 days, followed by a second, slower elimination, with a terminal average half-life of 8.4 days. The time frame for monitoring IMPA in urine was 7, 15 and 16 days for 0.1 LD50, 0.5 LD50 and 0.9 LD50 intoxications, respectively. Rapid elimination of IMPA in urine occurred after exposure, with an average half-life of ~ 0.8 days on days 2–6. For the first time, a slower elimination route for IMPA, with an average half-life of ~ 4 days from day 6 onwards, was revealed. Both IMPA and regenerated sarin pharmacokinetics exhibit linearity with dose. The overlaid pharmacokinetic profiles of regenerated sarin in blood along with IMPA in urine emphasize the dominance of IMPA with a rapid decay in urine in the first week and the slower long-term decay of protein-bound sarin later in blood. To our knowledge, the two new sensitive methods exhibit the longest monitoring time frame reported in biological samples.

To date, the only treatments developed for poisoning by organophosphorus compounds, the most toxic chemical weapons of mass destruction, have exhibited limited efficacy and versatility. The available causal antidotes are based on reactivation of the enzyme acetylcholinesterase (AChE), which is rapidly and pseudo-irreversibly inhibited by these agents. In this study, we developed a novel series of monoquaternary reactivators combining permanently charged moieties tethered to position 6- of 3-hydroxypyridine-2-aldoxime reactivating subunit. Highlighted representatives (21, 24, and 27; also coded as K1371, K1374, and K1375, respectively) that contained 1-phenylisoquinolinium, 7-amino-1-phenylisoquinolinium and 4-carbamoylpyridinium moieties as peripheral anionic site ligands, respectively, showed efficacy superior or comparable to that of the clinically used standards. More importantly, these reactivators exhibited wide-spectrum efficacy and were minutely investigated via determination of their reactivation kinetics in parallel with molecular dynamics simulations to study their mechanisms of reactivation of the tabun-inhibited AChE conjugate. To further confirm the potential applicability of these candidates, a mouse in vivo assay was conducted. While K1375 had the lowest acute toxicity and the most suitable pharmacokinetic profile, the oxime K1374 with delayed elimination half-life was the most effective in ameliorating the signs of tabun toxicity. Moreover, both in vitro and in vivo, the versatility of the agents was substantially superior to that of clinically used standards. Their high efficacy and broad-spectrum capability make K1374 and K1375 promising candidates that should be further investigated for their potential as nerve agents and insecticide antidotes.

The inhibition of acetylcholinesterase is regarded as the primary toxic mechanism of action for chemical warfare agents. Recently, there have been numerous reports suggesting that metabolic processes could significantly contribute to toxicity. As such, we applied a multi-omics pipeline to generate a detailed cascade of molecular events temporally occurring in guinea pigs exposed to VX. Proteomic and metabolomic profiling resulted in the identification of several enzymes and metabolic precursors involved in glycolysis and the TCA cycle. All lines of experimental evidence indicated that there was a blockade of the TCA cycle at isocitrate dehydrogenase 2, which converts isocitrate to α-ketoglutarate. Using a primary beating cardiomyocyte cell model, we were able to determine that the supplementation of α-ketoglutarate subsequently rescued cells from the acute effects of VX poisoning. This study highlights the broad impacts that VX has and how understanding these mechanisms could result in new therapeutics such as α-ketoglutarate. Electronic supplementary material The online version of this article (10.1007/s00204-020-02820-4) contains supplementary material, which is available to authorized users.

We present a simple method for chiral separation and analysis of organophosphorus nerve agents and apply it to monitor the enantioselective blood elimination kinetics of sarin in-vitro. The method is implemented in standard reverse phase LC-MS operating conditions, relieving the user of the dedicated operating conditions frequently demanded in chiral LC-MS analysis. The method consists of formation of diastereomers by a rapid derivatization with (R)-2-(1 aminoethyl) phenol, followed by LC-MS/MS analysis. Derivatization enantioselectivity was studied by comparing the reaction of optically pure sarin and racemic sarin, proving no substantial enantiomeric preference in the reaction and demonstrating the enantiomeric discrimination abilities of the technique. Enantioselective sarin elimination pathways were probed in-vitro by following the fast elimination kinetics of the two sarin enantiomers as well as its hydrolysis metabolite (isopropyl methyl-phosphonic acid, IMPA) in whole blood and plasma compared to water. Sarin enantiomers showed the known marked differences in elimination kinetics with rapid elimination of the (+) enantiomer and slower elimination of the (-) enantiomer in whole blood and plasma as well as dose-dependent kinetics (faster elimination at lower concentrations). We found that small amounts of acetonitrile in plasma prevent the rapid elimination of the (+) enantiomer, resulting in similar, slower elimination kinetics for both enantiomers.

VX, a potent inhibitor of cholinesterase (ChE), is considered as one of the most toxic, persistent and least volatile nerve agents. VX is absorbed in various environmental surfaces and is gradually released long after its initial dispersal. Its toxicity is mainly caused by disrupting central and peripheral cholinergic nervous system activity, leading to potential long-term detrimental effects on health. The primary objective of the present study was to assess the threshold VX dose leading to minimal physiological alterations following prolonged VX exposure. Characterization of such a threshold is crucial for dealing with unresolved operative dilemmas such as when it is safe enough to resettle a population that has been evacuated from a VX-contaminated area. Rats, continuously exposed to various doses of VX (0.225-45 µg/kg/day) for 4 weeks via implanted mini-osmotic pumps, showed a dose-dependent and continuous decrease in ChE activity in whole blood, brain and muscles, ranging between 20 and 100%. Exposure to 13.5 µg/kg/day led to a stable low ChE activity level (~ 20%), accompanied by transient and negligible electrocorticogram spectral power transformations, especially in the theta and alpha brain wave frequencies, and a significant decrease in total brain M2 receptor density. These changes were neither accompanied by observable signs of intoxication nor by changes in motor function, circadian rhythm or TSPO level (a reliable marker of brain damage). Following exposure to lower doses of 2.25 and 0.225 µg/kg/day, the only change measured was a reduction in ChE activity of 60 and 20%, respectively. Based on these results, we delineate ChE inhibition as the physiological measure most susceptible to alterations following prolonged VX exposure, and determine for the first time the threshold sub-acute VX dose for minimal physiological effects (up to 20% reduction in ChE activity) in the rat as 0.225 µg/kg/day.

A major challenge in organophosphate compound (OP) and OP nerve agent (OPNA) research has been in the identification and utilization of reliable biomarkers for rapid, sensitive, and efficient detection of OP exposure. Albumin has been widely studied as a biomarker for retrospective verification of exposure to OPNAs, including soman (GD), by detecting the phosphonylation of specific amino acid residues. The aim of the present study was to identify binding sites between GD and rabbit serum albumin in vitro and in vivo. A nano-liquid chromatography coupled with a quadrupole-orbitrap mass spectrometry (nLC-Q-Orbitrap-MS) was used to examine the GD-modified adducts of rabbit albumin. A total of 11 GD-modified sites were found in rabbit serum albumin across three experimental models. The following five GD-modified rabbit albumin sites, which were all lysine residues, were established in vivo: K188, K329, K162, K233, and K525. Two of these five lysine residues, K188 in peptide EK*ALISAAQER and K162 in peptide YK*AILTECCEAADK, were stable for at least 7 days in vivo. Molecular simulation of the GD–albumin interaction provided theoretical evidence for reactivity of the identified lysine residues. The findings suggest that these modifiable lysine residues are potential biomarkers of GD exposure for retrospective analysis by Q-Orbitrap-MS.

Irreversible inhibition of acetylcholinesterase (AChE) by organophosphates leads to many failures in living organism and ultimately in death. Organophosphorus compounds developed as nerve agents such as tabun, sarin, soman, VX and others belong to the most toxic chemical warfare agents and are one of the biggest threats to the modern civilization. Moreover, misuse of nerve agents together with organophosphorus pesticides (e.g. malathion, paraoxon, chlorpyrifos, etc.) which are annually implicated in millions of intoxications and hundreds of thousand deaths reminds us of insufficient protection against these compounds. Basic treatments for these intoxications are based on immediate administration of atropine and acetylcholinesterase reactivators which are currently represented by mono- or bis-pyridinium aldoximes. However, these antidotes are not sufficient to ensure 100 % treatment efficacy even they are administered immediately after intoxication, and in general, they possess several drawbacks. Herein, we have reviewed new efforts leading to the development of novel reactivators and proposition of new promising strategies to design novel and effective antidotes. Structure-activity relationships and biological activities of recently proposed acetylcholinesterase reactivators are discussed and summarized. Among further modifications of known oximes, the main attention has been paid to dual binding site ligands of AChE as the current mainstream strategy. We have also discussed new chemical entities as potential replacement of oxime functional group.

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.

The potent human toxicity of organophosphorus (OP) nerve agents calls for the development of effective antidotes. Standard treatment for nerve agent poisoning with atropine and an oxime has a limited efficacy. An alternative approach is the development of catalytic bioscavengers using OP-hydrolyzing enzymes such as paraoxonases (PON1). Recently, a chimeric PON1 mutant, IIG1, was engineered toward the hydrolysis of the toxic isomers of soman and cyclosarin with high in vitro catalytic efficiency. In order to investigate the suitability of IIG1 as a catalytic bioscavenger, an in vivo guinea pig model was established to determine the protective effect of IIG1 against the highly toxic nerve agent cyclosarin. Prophylactic i.v. injection of IIG1 (1 mg/kg) prevented systemic toxicity in cyclosarin (~2LD50)-poisoned guinea pigs, preserved brain acetylcholinesterase (AChE) activity, and protected erythrocyte AChE activity partially. A lower IIG1 dose (0.2 mg/kg) already prevented mortality and reduced systemic toxicity. IIG1 exhibited a high catalytic efficiency with a homologous series of alkylmethylfluorophosphonates but had low efficiency with the phosphoramidate tabun and was virtually ineffective with the nerve agent VX. This quantitative analysis validated the model for predicting in vivo protection by catalytic bioscavengers based on their catalytic efficiency, the level of circulating enzyme, and the dose of the intoxicating nerve agent. The in vitro and in vivo results indicate that IIG1 may be considered as a promising candidate bioscavenger to protect against the toxic effects of a range of highly toxic nerve agents.

Despite six decades of extensive research in medical countermeasures against nerve agent poisoning, a broad spectrum acetylcholinesterase (AChE) reactivator is not yet available. One current approach is directed toward synthesizing oximes with high affinity and reactivatability toward butyrylcholinesterase (BChE) in plasma to generate an effective pseudocatalytic scavenger. An interim solution could be the administration of external AChE or BChE from blood products to augment pseudocatalytic scavenging with slower but clinically approved oximes to decrease nerve agent concentrations in the body. We here semiquantitatively investigate the ability of obidoxime and HI-6 to decrease the inhibitory activity of VX with human AChE and BChE from whole blood, erythrocyte membranes, erythrocytes, plasma, clinically available fresh frozen plasma and packed red blood cells. The main findings are that whole blood showed a VX concentration-dependent decrease in inhibitory activity with HI-6 being more potent than obidoxime. Using erythrocytes and erythrocyte membranes again, HI-6 was more potent compared to obidoxime. With freshly prepared plasma, obidoxime and HI-6 showed comparable results for the decrease in VX. The use of the clinically available blood products revealed that packed red blood cells showed similar kinetics as fresh erythrocytes. Fresh frozen plasma resulted in a slower and incomplete decrease in inhibitory plasma compared to freshly prepared plasma. In conclusion, the administration of blood products in combination with available oximes augments pseudocatalytic scavenging and might be useful to decrease the body load of persistent, highly toxic nerve agents.

The organophosphorus nerve agents sarin, soman, cyclosarin and tabun phosphylate a tyrosine residue on albumin in human blood. These adducts may offer relatively long-lived biological markers of nerve agent exposure that do not 'age' rapidly, and which are not degraded by therapy with oximes. Sensitive methods for the detection of these adducts have been developed using liquid chromatography-tandem mass spectrometry. Adducts of all four nerve agents were detected in the blood of exposed guinea pigs being used in studies to improve medical countermeasures. The tyrosine adducts with soman and tabun were detected in guinea pigs receiving therapy 7 days following subcutaneous administration of five times the LD(50) dose of the respective nerve agent. VX also forms a tyrosine adduct in human blood in vitro but only at high concentrations.

The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat/K M) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k cat/K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat/K M values >107 M-1 min-1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships.

The hypothesis that acetylcholinesterase (AChE) inhibition is the mechanism of toxicity of organophosphorus (OP) compounds was examined by mathematically modeling the in vivo lethal effects of OP compounds and determining the amount of variation in OP toxicity that is explained by AChE inhibition. Mortality dose–response curves for several OP compounds (i.e., VX, soman, cyclosarin, sarin, tabun, diisopropylfluorophosphate and paraoxon) exhibited steep probit slopes (> 9.6) in guinea pigs. Steep probit slopes were also observed when the mortality dose–response curves for soman were examined in mice, rats, rabbits and non-human primates. The consistently steep probit slopes of the dose–response curves for highly toxic OP compounds suggested that these compounds have a single specific mechanism of toxicity regardless of the OP compound or the species in which it was tested. Regression analysis indicated that 93% of the 3,280-fold variation in the median lethal doses (i.e., LD50) of OP compounds in rats was explained by the variation in their in vitro rate constants for inhibition of AChE. Conversely, 91% of the 23-fold variation in the ability of the oximes pralidoxime and obidoxime to protect against the toxicity of OP compounds in guinea pigs was explained by the variation in the in vitro ability of oximes to reactivate OP-inhibited AChE. The best explanation for this variety of observations was that the primary mechanism of in vivo toxicity for highly toxic OP compounds is the inhibition of AChE, and the residual unexplained variation in OP toxicity that might be explained by other mechanisms represents

The utility was explored of a new approach to detect retrospectively exposure to nerve agents by means of conversion of the inhibitor moiety bound to the active site of the enzyme BuChE in plasma with fluoride ions into a phosphofluoridate which is subsequently analyzed by means of gas chromatography (GC). This quantifiesor=0.01% inhibition of BuChE and identifies the structure of the inhibitor except for the original leaving group. A three-tiered approach was followed involving the five classical nerve agents GA, GB, GF, GD, and VX, as well as the active metabolite of parathion, i.e., paraoxon: in vivo experiments in rhesus monkeys after iv administration of a sign-free dose of agent and concomitant in vitro experiments in plasma of rhesus monkeys and humans should allow an assessment of in vivo retrospectivity in humans. A systematic investigation was performed in order to find a single set of reaction conditions which yields a maximum amount of phosphofluoridate for all nerve agents. Fluoride-induced reactivation at 25 degrees C at a final concentration of 250 mM KF during 15 min in a pH-range between 4 and 6 appears to be effective. The in vitro decrease with time in reactivatibility of inhibited BuChE in plasma from humans and rhesus monkeys was largely due to aging of the phosphyl moiety, except for VX where spontaneous reactivation was a major cause. The decrease followed first-order except for a biphasic course in the case of GF in human and rhesus monkey plasma as well as of GD in rhesus plasma. In vitro half-lifes in human plasma ranged between ca. 14 h for GB and ca. 63 h for GA. A comparison of the in vivo data from rhesus monkeys and the in vitro data is complicated by the observation that the in vivo decrease with time of fluoride-reactivated phosphofluoridate is biphasic for all nerve agents. The terminal in vivo phase pertains to a small fraction of the amount of initially regenerated phosphofluoridate but is responsible for a considerable degree of retrospectivity, ranging between 14 and 56 days for GF and GB, respectively. The new procedure can be used in a variety of practical applications, e.g., (i) biomonitoring in health surveillance at exposure levels that are several orders of magnitude lower than presently possible; (ii) diagnosis in case of alleged exposure to nerve agents in time of war or after terrorist attacks; (iii) in forensic cases against suspected terrorists that have handled organophosphate anticholinesterases; and (iv) in research applications such as investigations on lowest observable effect levels of exposure to nerve agents.

The toxicokinetics of the four stereoisomers of the nerve agent C(+/-)P(+/-)-soman was analysed in cerebrospinal fluid (CSF) and blood in anaesthetized, spontaneously breathing pigs during a 90-min period after injection of soman. The pigs were challenged with different intravenous (i.v.) doses of C(+/-)P(+/-)-soman corresponding to 0.75-3.0 LD50 (4.5, 9.0 and 18 microg/kg in a bolus injection and 0.45 microg/kg per min as a slow infusion). Artificial ventilatory assistance was given if, after soman intoxication, the respiratory rate decreased below 19 breaths/min. Blood samples were taken from a femoral artery and CSF samples from an intrathecal catheter. The concentrations of the soman isomers were determined by gas chromatography coupled with high resolution mass spectrometry. All four isomers of soman were detected in both blood and CSF samples. The relatively non-toxic C(+/-)P(+) isomers disappeared from the blood stream and CSF within the first minute, whereas the levels of the highly toxic C(+/-)P(-) isomers could be followed for longer, depending on the dose. Concurrently with the soman analyses in blood and CSF, cholinesterase (ChE) activity and cardiopulmonary parameters were measured. C(+/-)P(-) isomers showed approx. 100% bioavailability in CSF when C(+/-)P(+/-)-soman was given i.v. as a bolus injection. In contrast, C(+/-)P(-) isomers displayed only 30% bioavailability in CSF after slow i.v. infusion of soman. The ChE activity in blood decreased below 20% of baseline in all groups of pigs irrespective of the soman dose. The effect of soman intoxication on the respiratory rate, however, seems to be dose-dependent and the reason for ventilatory failure and death. Artificial ventilation resulted in survival of the pigs for the time-period studied.

The direct reaction of seven pyridinium oximes with the nerve agents sarin, soman, and tabun was followed by a spectrophotometric method. The half-lives (t 1/2) of the oximes, the first- and second-order rate constants (k 1, k 2), and the maximal reaction velocity (\(\roundv \) max) were calculated according to changes in the absorbance of the zwitterion (betaine) peak. In all cases the reaction velocity of the nerve agents with any of the oximes was highest with tabun, followed by sarin and then soman. Comparing the reaction rates of three therapeutically used oximes with the same nerve agent, the highest rate was obtained for soman with obidoxime, for sarin with 2-PAM, and for tabun with HI 6. The maximal reaction velocities reveal that the detoxification of the nerve agents by direct reaction with oximes and the subsequent decomposition of the phosphonyl oxime in vivo do not substantially contribute to the therapeutic effect of these antidotes.

Oxime-assisted reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) is a crucial step in the post-inhibitory treatment of OP intoxication. The limited efficacy of oxime reactivators for all OP nerve agents and pesticides led to the development of various novel oximes and their thorough kinetic investigations. Hence, in the present investigation, we have tested 10 structurally different pyridinium oxime-based reactivators for their in vitro potency to reactivate paraoxon- and DFP-inhibited electric eel AChE. From structure activity relationship point of view, various oximes such as mono-quaternary (2-PAM, K100, K024) and bis-quaternary symmetric (obidoxime, TMB-4) and asymmetric (K027, K048, K203, K618, K628) oximes bearing different connecting linkers (oxybismethylene, trimethylene, propane, butane, butene, and xylene) have been studied. The observed kinetic data demonstrate that not only the position of oxime group is decisive for the increased reactivation ability of oximes, but the role of connecting linker is also significant. Oximes with aliphatic linkers are superior reactivators than the oximes with unsaturated and aromatic linkers. The optimal chain length for plausible reactivation ability for paraoxon- and DFP-inhibited AChE is 3 or 4 carbon–carbon connecting linker between prydinium rings.

Great efforts have been undertaken in the last decades to develop new oximes to reactivate acetylcholinesterase inhibited by organophosphorus compounds (OP). So far, a broad-spectrum oxime effective against structurally diverse OP is still missing, and alternative approaches, e.g. stoichiometric and catalytic scavengers, are under investigation. Fresh frozen plasma (FFP) has been used in human OP pesticide poisoning which prompted us to investigate the in vitro kinetics of OP nerve agent degradation by FFP. Degradation was rapid and calcium-dependent with the G-type nerve agents tabun, sarin, soman and cyclosarin with half-lives from 5 to 28 min. Substantially longer and calcium-independent degradation half-lives of 23–33 h were determined with the V-type nerve agents CVX, VR and VX. However, at all the tested conditions, the degradation of V-type nerve agents was several-fold faster than spontaneous hydrolysis. Albumin did not accelerate the degradation of nerve agents. In conclusion, the fast degradation of G-type nerve agents by FFP might be a promising tool, but would require transfusion shortly after poisoning. FFP does not seem to be suitable for detoxifying relevant agent concentrations in case of human poisoning by V-type nerve agents.

Organophosphorus nerve agents are the most toxic chemical warfare agents that are known to have been produced, stockpiled and weaponised. Their development, production, stockpiling and use are prohibited under the terms of the Chemical Weapons Convention and, together with their precursors, are subject to strict controls and verification procedures. The detection and identification of biological markers of exposure to nerve agents are required for three main purposes: confirmation of exposure for forensic purposes in cases of alleged use; diagnosis to guide appropriate medical countermeasures in the event of an exposure; and occupational health monitoring of workers in defence laboratories and demilitarisation facilities. Biomarkers of nerve agents fall into two main groups, free metabolites and adducts to proteins. These are reviewed together with analytical methods for their identification. Examples are provided of applications in cases of human exposure.

Multiple oximes have been synthesized and evaluated for use as countermeasures against chemical warfare nerve agents. The current U.S. military and civilian oxime countermeasure, 2-[(hydroxyimino)methyl]-1-methylpyridin-1-ium chloride (2-PAM), is under consideration for replacement with a more effective acetylcholinesterase reactivator, 1,1'-methylenebis{4-hydroxyiminomethyl}pyridinium dimethanesulfonate (MMB-4). Kinetic data in the scientific literature for MMB-4 are limited; therefore, a physiologically based pharmacokinetic (PBPK) model was developed for a structurally related oxime, 1,1'-trimethylenebis{4-hydroximinomethyl}pyridinium dibromide. Based on a previous model structure for the organophosphate diisopropylfluorophosphate, the model includes key sites of acetylcholinesterase inhibition (brain and diaphragm), as well as fat, kidney, liver, rapidly perfused tissues and slowly perfused tissues. All tissue compartments are diffusion limited. Model parameters were collected from the literature, predicted using quantitative structure-property relationships or, when necessary, fit to available pharmacokinetic data from the literature. The model was parameterized using rat plasma, tissue and urine time course data from intramuscular administration, as well as human blood and urine data from intravenous and intramuscular administration; sensitivity analyses were performed. The PBPK model successfully simulates rat and human data sets and has been evaluated by predicting intravenous mouse and intramuscular human data not used in the development of the model. Monte Carlo analyses were performed to quantify human population kinetic variability in the human evaluation data set. The model identifies potential pharmacokinetic differences between rodents and humans, indicated by differences in model parameters between species. The PBPK model can be used to optimize the dosing regimen to improve oxime therapeutic efficacy in a human population.

Sarin, a potent organophosphate cholinesterase inhibitor, induces an array of toxic effects including convulsions. Many antidotal treatments contain anticonvulsants to block seizure activity and the ensuing brain damage. Magnesium sulfate (MGS) is used to suppress eclamptic seizures in pregnant women with hypertension and was shown to block kainate-induced convulsions. Magnesium sulfate was evaluated herein as an anticonvulsant against sarin poisoning and its efficacy was compared with the potent anticonvulsants midazolam (MDZ) and caramiphen (CRM). Rats were exposed to a convulsant dose of sarin (96 μg/kg, im) and 1 min later treated with the oxime TMB4 and atropine to increase survival. Five minutes after initiation of convulsions, MGS, CRM, or MDZ were administered. Attenuation of tonic–clonic convulsions was observed following all these treatments. However, radio-telemetric electro-corticography (ECoG) monitoring demonstrated sustained seizure activity in MGS-injected animals while this activity was completely blocked by MDZ and CRM. This disrupted brain activity was associated with marked increase in brain translocator protein levels, a marker for brain damage, measured 1 week following exposure. Additionally, histopathological analyses of MGS-treated group showed typical sarin-induced brain injury excluding the hippocampus that was partially protected. Our results clearly show that MGS demonstrated misleading features as an anticonvulsant against sarin-induced seizures. This stems from the dissociation observed between overt convulsions and seizure activity. Thus, the presence or absence of motor convulsions may be an unreliable indicator in the assessment of clinical status and in directing adequate antidotal treatments following exposure to nerve agents in battle field or terror attacks.

Organophosphates are a group of pesticides and chemical warfare nerve agents that inhibit acetylcholinesterase, the enzyme responsible for hydrolysis of the excitatory neurotransmitter acetylcholine. Numerous structural variants exist for this chemical class, and data regarding their toxicity can be difficult to obtain in a timely fashion. At the same time, their use as pesticides and military weapons is widespread, which presents a major concern and challenge in evaluating human toxicity. To address this concern, a quantitative structure-activity relationship (QSAR) was developed to predict pentavalent organophosphate oxon human acetylcholinesterase bimolecular rate constants. A database of 278 three-dimensional structures and their bimolecular rates was developed from 15 peer-reviewed publications. A database of simplified molecular input line entry notations and their respective acetylcholinesterase bimolecular rate constants are listed in Supplementary Material, Table I. The database was quite diverse, spanning 7 log units of activity. In order to describe their structure, 675 molecular descriptors were calculated using AMPAC 8.0 and CODESSA 2.7.10. Orthogonal projection to latent structures regression, bootstrap leave-random-many-out cross-validation and y-randomization were used to develop an externally validated consensus QSAR model. The domain of applicability was assessed by the William's plot. Six external compounds were outside the warning leverage indicating potential model extrapolation. A number of compounds had residuals2 or-2, indicating potential outliers or activity cliffs. The results show that the HOMO-LUMO energy gap contributed most significantly to the binding affinity. A mean training R (2) of 0.80, a mean test set R (2) of 0.76 and a consensus external test set R (2) of 0.66 were achieved using the QSAR. The training and external test set RMSE values were found to be 0.76 and 0.88. The results suggest that this QSAR model can be used in physiologically based pharmacokinetic/pharmacodynamic models of organophosphate toxicity to determine the rate of acetylcholinesterase inhibition.

The reactivation of organophosphorus compound (OP)-inhibited acetylcholinesterase (AChE) by oximes is inadequate in case of different OP nerve agents. This fact led to the synthesis of numerous novel oximes by different research groups in order to identify more effective reactivators. In the present study, we investigated the reactivation kinetics of a homologous series of bispyridinium bis-oximes bearing a (E)-but-2-ene linker with tabun-, sarin-, and cyclosarin-inhibited human AChE. In part, marked differences in affinity and reactivity of the investigated oximes toward OP-inhibited human AChE were recorded. These properties depended on the position of the oxime groups and the inhibitor. None of the tested oximes was equally effective against all used OPs. In addition, the data indicate that a (E)-but-2-ene linker decreased in most cases the reactivating potency in comparison to oximes bearing an oxybismethylene linker, e.g., obidoxime and HI-6. The results of this study give further insight into structural requirements for oxime reactivators, underline the necessity to investigate the kinetic interactions of oximes and AChE with structurally different OP inhibitors, and point to the difficulty to develop an oxime reactivator which is efficient against a broad spectrum of OPs.

The purpose of this study was to compare the in vitro reactivation of the various molecular forms of soman-inhibited acetylcholinesterase by oximes such as HI-6, toxogonin and PAM, in striated muscle tissue from three species-rat, monkey and human. To simulate the various in vivo conditions the oxime was present either 5 min before and after (Pre-Post) or 5 min after (Post) exposure to the nerve agent soman. In the Pre-Post mode the oxime effects would result from a combination of not only shielding of acetylcholinesterase from soman inhibition but also from immediate reactivation of soman-inhibited acetylcholinesterase. In the Post experimental group the increase in soman-inhibited acetylcholinesterase activity was due to reactivation. HI-6 (Pre-Post) increased significantly the activity of soman-inhibited acetylcholinesterase in the rat, human and monkey muscle. HI-6 (Post) was a highly effective reactivator of soman-inhibited acetylcholinesterase in the rat muscle and moderately so in the human and monkey muscle. Toxogonin (Pre-Post) and toxogonin (Post) were effective in increasing soman-inhibited acetylcholinesterase activity in rat muscle but were relatively ineffective in the human and monkey muscle. PAM (Pre-Post) and PAM (Post) were ineffective in increasing soman-inhibited acetylcholinesterase activity in muscle from all species examined. Effectiveness of oxime-induced reactivation of soman-inhibited acetylcholinesterase could be estimated from the total acetylcholinesterase activity which appears to reflect the results found with the individual molecular forms of acetylcholinesterase. In addition, SAD-128, a non-oxime bispyridinium compound, appeared to enhance significantly the HI-6 induced reactivation of soman-inhibited acetylcholinesterase in human but not rat striated muscle.

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.

Pretreatment of rats and guinea pigs with the specific carboxylesterase inhibitor 2-(o-cresyl)-4H-1 ∶ 3 ∶ 2-benzodioxaphosphorin-2-oxide (CBDP) reduces the LD50 of the nerve agent C(±)P(±)-soman in these species to the same range as in primates. This suggests that such CBDP-pretreated animals can be used in investigations that are relevant for prophylaxis and therapy of intoxication with C(±)P(±)-soman in primates including humans. In order to test this hypothesis we have studied the toxicokinetics of the toxic C(±)P(−)-isomers of soman in artificially respirated and CBDP-pretreated rats and guinea pigs at intravenous doses corresponding to 6 × LD50. A comparison of the areas under the curve (AUCs) of the blood levels of C(±)P(−)-soman in pretreated and non-pretreated animals at the same absolute dose shows extreme nonlinearity with dose, indicating that CBDP occupies highly reactive binding sites which are no longer available for sequestration of the soman isomers. The AUCs of C(±)P(−)-soman at equitoxic doses of 6× LD50 are reduced by pretreatment with CBDP from 1683 to 464 ng.min.ml−1 in rats and from 978 to 176 ng.min.ml−1 in guinea pigs, which is in the range of the AUC in non-pretreated marmosets at an equitoxic dose (419 ng.min.ml−1). The blood levels of the C(±)P(−)-isomers in marmosets and CBDP rats are rather similar during the first 7 min, but persist in CBDP rats for 2 h longer at toxicologically relevant levels than in marmosets. The levels of C(±)P(−)-soman in CBDP-pretreated guinea pigs are substantially lower than in marmosets for an initial period of 80 min. Nevertheless, they drop below toxicologically relevant levels approximately 50 min later than in marmosets. Evidently, one should be cautious in considering CBDP, pretreated rats and guinea pigs as substitute primates.

A strategy is described to raise high-affinity antibodies directed against the organophosphorus nerve agent VX [O-ethyl S-(2-diisopropylamino)ethyl)methyl phosponothionate]. Ten chemical derivatives of VX (haptens) have been synthesized. Their structures differ principally from VX structure by substitution of S-atom by an O-atom or CH2-group and by introduction of a reactive group (carboxylic acid, arylamine or primary amine) on the O-ethyl side chain. None of these haptens, except one, exhibit potential toxicity as tested by their inhability to inhibit acetylcholinesterase (E.C. 3.1.1.7.). After coupling with a protein carrier, they were injected intradermally to rabbits. Nine of these immunogenic conjugates led to the appearance of antibodies able to bind VX in a competitive solid phase immunoassay. The apparent titer and affinity of the antisera differed greatly depending on the hapten used. The highest affinity (9 nM) was observed with the VX derivative bearing O-S substitution and O-ethyl-carboxylic side chains. The antibodies appear specific for VX, since cross-reactivity with other nerve agents (Soman, Sarin or Tabun) was low. However, two haptens elicited antibodies with affinity to Soman or Sarin in the micromolar range. Antibodies were able to neutralize VX inhibition of acetylcholinesterase in vitro but not in vivo.

The toxicity and efficacy of two oximes, HLö-7 and pyrimidoxime, were evaluated in mice and compared to those obtained with HI-6. HLö-7 and pyrimidoxime produced 24 h LD50 values of 356 and 291 mg/kg (i.p.), respectively. In combination with atropine (17.4 mg/kg, i.p.), HLö-7 was a very efficient therapy against poisoning by 3 x LD50 dose of soman, sarin and GF and 2 x LD50 dose of tabun with ED50 values of 12.4, 0.31, 0.32 and 25.2 mg/kg, respectively. In contrast, pyrimidoxime was a relatively poor therapy which resulted in ED50 values of greater than 150, 5.88, 100 and 71 mg/kg against poisoning by soman, sarin, GF and tabun, respectively. HLö-7 produced significant (p less than 0.05) reactivation of phosphorylated acetylcholinesterase, in vivo, resulting in 47, 38, 27 and 10% reactivation of sarin, GF, soman and tabun inhibited mouse diaphragm acetylcholinesterase, respectively. HLö-7 also antagonized sarin-induced hypothermia in mice suggesting that it reactivated central acetylcholinesterase. The potential of HLö-7 as a replacement oxime for the treatment of nerve agent poisoning is discussed.

Organophosphorus nerve agents inhibit the activity of cholinesterases by phosphylation of the active site serine. In addition, sarin, cyclosarin, soman and tabun have been shown to phosphylate a tyrosine residue in albumin. Therapies against nerve agent poisoning include the use of oximes to reactivate inhibited cholinesterases by displacement of the phosphyl moiety and hence detectable levels of adducts with cholinesterases may be reduced. Adducts with tyrosine have been shown to be persistent in the guinea pig in the presence of oxime therapy. Plasma samples obtained from an animal study aimed at improving therapy against nerve agent poisoning were used to compare the suitability of tyrosine and butyrylcholinesterase (BuChE) adducts as biomarkers of nerve agent exposure after treatment with therapeutic oximes. Under the terms of the project licence, these samples could be collected only on death of the animal, which occurred within hours of exposure or when culled at 23 or 24 days. Tyrosine adducts were detected in all samples collected following intra-muscular administration of twice the LD50 dose of the respective nerve agent. Aged BuChE adducts were detected in samples collected within a few hours after administration of soman and tabun, but not after 23 or 24 days. No BuChE adducts were detected in animals exposed to sarin and cyclosarin where samples were collected only after 23 or 24 days.

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.

The development of a specific and sensitive immunologic ELISA detection system for methylphosphonoflouridic acid. 1,2,2-trimethylpropylester (soman) by the use of monoclonal antibodies (MAbs) is described. The monoclonal antibodies F71D7, F71H10, F71B12 and F71H9 originally produced against the soman derivative methyl phosphonic acid, p-aminophenyl 1,2,2-trimethylpropyldiester (MATP) also reacted with soman in a previously developed, direct competitive ELISA. After optimizing the ELISA system by varying the reaction mixture and the solvents for the organophosphate, 5.0 x 10(-7) mol/l soman (80% purity), e.g. 2.5 ng or 2 ng pure soman per 25 microliters test buffer, could be detected after a total test duration of 40 min. A shortening of the incubation time to 10 min resulted in a drop of sensitivity to 1.8 x 10(-6) mol/l soman. Various alcohols which may be used as extraction media for soman from various materials (isopropanol, ethanol and methanol) were shown to inhibit peroxidase activity and thereby reduce the sensitivity of the test. However, the influence of alcohols decreased with the shortening of incubation time. All monoclonal antibodies showed little cross reactivity to sarin and no cross reactivity to tabun and VX. Judging on the reactivity of the MAbs with MATP and soman oxidazed by 1,2-dihydrobenzol, some reactivity with some other (non-toxic) soman analogues containing the same pinacolyl group can be expected. There was no evidence for stereoselectivity of the MAbs tested. Finally, soman could be detected in different biological samples like human serum, goat serum, rabbit serum, chicken serum, milk, and tap water in concentrations between 1.3 x 10(-6) and 2.0 x 10(-6) mol/l.

The toxicity of a combination of isopropyl methylphosphonofluoridate (sarin; GB) and cyclohexyl methylphosphonofluoridate (GF) and the efficacy of various oxime reactivators in combination with atropine against the combined GB/GF challenge were evaluated in mice. The 24-h s.c. LD50 of the GB/GF combination was 1.15 mumol/kg (1.10-1.21; 95% confidence limits). Mice administered GB/GF displayed typical signs of nerve agent poisoning such as tremors and convulsions, with death most likely due to anoxia subsequent to respiratory arrest. The GB/GF LD50 value was comparable to the s.c. LD50 of 1.35 and 1.21 mumol/kg for GF and GB in mice, respectively. Combining the two nerve agents did not result in potentiation of the toxicity. In combination with atropine sulfate (17.4 mg/kg, i.p.), which alone did not reduce mortality, the oximes tested, 2-PAM, obidoxime and HI-6, were all effective when administered 5 min before 3 x LD50 dose of GB/GF with 24-h ED50 values of 102.5, 18.22 and 1.96 mumol/kg, respectively. Use of the GB/GF combination does not appear to confer any unique toxicity profile and appears to be easily treated with the standard therapy of a cholinolytic and oxime.

The well-documented efficacy of HI 6 dichloride in reactivating acetylcholinesterase (AChE) inhibited by nerve agents is curtailed by its poor water-solubility at temperatures below 10 degrees C. This drawback can be circumvented by using HI 6 dimethanesulfonate, which has been developed in our laboratory. Since investigations on the efficacy of this new entity are lacking, it has been proposed that some bridging experiments be performed, aimed at demonstrating reactivator equivalence in vitro. The reactivating properties of the two salts were compared on human erythrocyte AChE inhibited with paraoxon, sarin, cyclosarin and agent VX. The comparison was extended to cynomolgus erythrocytes exposed in vitro for sarin and VX. Finally, mouse diaphragm preparations were circumfused with sarin, cyclosarin and VX and reactivation by each of the HI 6 salts was examined in muscle homogenates. AChE activity in erythrocyte suspensions was monitored by a modified Ellman procedure, muscle AChE by a radiometric assay. In all models tested no differences between the HI 6 salts could be detected ( P=0.05). From these data, equipotency in AChE reactivation by the two HI 6 salts can be anticipated.