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Abstract: The low effectiveness of atropine and oxime treatment in soman poisoning may be enhanced by carbamates pre-treatment. For ethical reasons medical countermeasures can only be tested in animal models despite the fact of substantial species differences. With this kinetic in vitro study the interactions between pyridostigmine, physostigmine and soman with human, Rhesus monkey, swine and guinea pig erythrocyte AChE were investigated. In addition, the effect of the carbamates on the residual activity and enzyme recovery after soman inhibition was examined with erythrocyte and intercostal muscle AChE from these species with a dynamic in vitro model with real-time determination of AChE activity. Only small to moderate species differences of the inhibition and decarbamylation kinetics were recorded. It was possible to show that with erythrocyte and muscle AChE a similar level of protection by carbamates and reactivation after discontinuation of the carbamates and soman could be observed. Thus, these data indicate that carbamate pre-treatment is expected to protect a critical level of muscle AChE and confirm the presumption that erythrocyte AChE may serve as a surrogate for synaptic AChE. Hence, these and previous data fortify the notion that erythrocyte AChE is a proper tool for in vitro kinetic studies as well as for therapeutic monitoring in experimental and clinical studies. [Copyright &y& Elsevier]

Abstract: Objectives: Reactivation of inhibited acetylcholinesterase (AChE) with oximes is a causal therapy of intoxication with organophosphorus compounds (OPs). Maximal oxime effects are expected when effective doses are administered as soon as possible and as long as reactivation can be anticipated. An obidoxime plasma level in the range of 10–20μM was estimated as appropriate. The achievement of this target was assessed in 34 severely OP-poisoned patients. Methods: After admission to the intensive care unit (ICU) the obidoxime regimen (250mg i.v. as bolus, followed by 750mg/24h) was started and maintained as long as reactivation was possible. Plasma concentrations of obidoxime were determined by HPLC. Results: A total amount of 2269±1726mg obidoxime was infused over 65h±55h resulting in a steady state plasma concentration of 14.5±7.3μM. Obidoxime was eliminated with t 1/2(1) 2.2 and t 1/2(2) 14h. The volumes of distribution amounted to 0.32±0.1L/kg (V (1)) and 0.28±0.12 (V (2))L/kg. Postmortem examination of tissue in one patient showed obidoxime accumulation in cartilage, kidney and liver and pointed to brain concentrations similar to plasma concentration. Conclusions: Using the suggested obidoxime regimen, the targeted plasma concentration could be achieved. Obidoxime was eliminated biphasically and was well tolerated. This result allows the recommendation of using this definite regimen for adults also in case of mass casualties. [Copyright &y& Elsevier]

Abstract: The easy accessibility to highly toxic OP (organophosphorus)-type chemical warfare agents (nerve agents) underlines the necessity for an effective medical treatment. Acute OP toxicity is primarily caused by inhibition of acetylcholinesterase (AChE, EC 3.1.1.7). Reactivators (oximes) of inhibited AChE are a mainstay of treatment. However, the commercially available compounds, obidoxime and pralidoxime, are considered rather ineffective against various nerve agents, including tabun. OP-type chemical warfare agents include an asymmetrical P-atom and consist of at least two stereoisomers. Previous studies with the nerve agents sarin and soman showed marked differences between (−)- and (+)-P isomers regarding AChE inhibition and stability in biological matrices. Hence, stereoselectivity is a key parameter for the development of optimized treatment. In the present study, the tabun enantiomers were isolated by semi-preparative liquid-chromatography (LC) with offline analysis by GC–PCI-MS and final characterization of optical purity (99.98% (−)-tabun and 99.83% (+)-tabun) and specific optical rotation. The inhibition and reactivation kinetics of the tabun enantiomers were determined with human and swine AChE and the aging kinetics with human AChE. The results show a large difference in the inhibitory potency between (−)- and (+)-tabun. The determination of reactivation and aging kinetics indicates that both reactions are at least in part determined by the residual (−)-tabun contamination (0.17%) of the (+)-tabun preparation. These data provide further insight into the kinetic interactions between tabun enantiomers and AChE and may contribute to the development of more effective treatment options. [Copyright &y& Elsevier]

Abstract: Developing improved medical countermeasures against chemical warfare agents (nerve agents) is urgently needed but time-consuming and costly. Here we introduce a robot-assisted liquid handling system with warming, cooling and incubating facilities to screen the detoxifying properties of biological and chemical materials against nerve agents. Two biological tests were established and plasma from various species, DFPase and three cyclodextrins were used as test materials. In test 1, plasma was mixed with sarin or VX and the inhibitory potency of the incubate was determined with human acetylcholinesterase (AChE) at 0, 30 and 60min. In test 2, test materials and nerve agents were mixed and incubated. Between 0 and 40min samples were taken and incubated for 3min with AChE and the residual AChE inhibition was determined to enable the semi-quantitative evaluation of the detoxification kinetics. The automated assays proved to be highly reproducible. It was possible to pre-select detoxifying reagents with test 1 and to determine more detailed detoxifying kinetics with test 2. In conclusion, the automated assay may be considered as a versatile tool for the high-throughput screening of potential detoxifying materials against different nerve agents. With this two-step assay it is possible to screen effectively for detoxifying materials in a high-throughput system. [Copyright &y& Elsevier]

Abstract: Thirty-four adult patients with severe organophosphorus compounds (OP) poisoning requiring artificial ventilation were enrolled in a clinical study and received atropine and obidoxime (250mg i.v., followed by 750mg/24h) as antidotal treatment. Here, we re-analyzed the cholinesterase status (red blood cell acetylcholinesterase (RBC-AChE) activity, reactivatability of RBC-AChE, and plasma butyrylcholinesterase (Pl-BChE) activity) in relation to the neuromuscular transmission (NMT) data. When RBC-AChE activity ranged between 100% and 30% NMT was unimpaired after tetanic stimulation with frequencies up to 50Hz. A further decrease in RBC-AChE activity was accompanied by a marked disturbance of NMT, being strongly impaired at AChE activities <5% of normal. Higher stimulation frequencies (>30Hz) facilitated the discrimination of the types of impairment. The neuromuscular transmission was the best quantified by using the ratio of the ninth to the first amplitude, while the standard method was less discriminative. At RBC-AChE levels higher than 40% of normal weaning from the ventilator may be considered. Completely aged RBC-AChE as indicated by loss of reactivatability loses its guidance function. Then, steadily increasing Pl-BChE activity suggests lack of circulating poison. One-week later, neuromuscular transmission may be largely normal and patients could be weaned from the respirator if other complications are not withstanding. [Copyright &y& Elsevier]

Abstract: Sulfur mustard (SM) is a strong vesicant agent which has been used in several military conflicts. Large stockpiles still exist to the present day. SM is believed to be a major threat to civilian populations because of the persistent asymmetric threat by non-state actors, such as terrorist groups, its easy synthesis and handling and the risk of theft from stockpiles. Following an asymptomatic interval of several hours, acute SM exposure produces subepidermal skin blisters, respiratory tract damage, eye lesions and bone marrow depression. Iranian victims of SM exposure during the Iran–Iraq (1984–1988) war were treated at intensive care units of 3 Munich hospitals. All 12 patients were injured following aerial attacks with SM filled bombs, which exploded in a distance between 5 and 30m. All patients soon noted an offensive smell of garlic, addle eggs or oil roasted vegetables. No individual protective equipment was used. Eye itching and skin blistering started 2h after SM exposure. Some patients complained of nausea, dizziness and hoarseness. 4h after exposure, most patients started vomiting. Eye symptoms worsened and most patients suffered from temporary blindness due to blepharospasm and lid oedema. Additionally, pulmonary symptoms such as productive cough occurred. Patients were transferred to Munich 4–17 days after SM exposure. On admission all patients showed significant skin blistering and pigmentation. Conjunctivitis and photophobia were the major eye symptoms. Pulmonary symptoms, including productive cough were persistent. Bronchoscopy revealed massive inflammation of the trachea with signs of necrosis. 3 patients needed tracheotomy. Chest X-ray did not yield abnormal observations. This presentation summarizes the experience of treating SM victims in Munich and discusses therapeutic implications. [Copyright &y& Elsevier]

Abstract: The widespread use of organophosphorus compounds (OPs) as pesticides and the frequent misuse of OP nerve agents in military conflicts or terrorist attacks emphasize the high clinical relevance of OP poisoning. The toxic symptomatology is caused by inhibition of acetylcholinesterase (AChE). A mainstay of standard antidotal treatment is atropine for antagonizing effects mediated by over stimulation of muscarinic ACh-receptors and oxime to reactivate OP-inhibited AChE. For therapeutic monitoring of oxime treatment in OP poisoning, measurement of erythrocyte AChE is suitable because erythrocyte AChE is an easily accessible surrogate for synaptic AChE. However, measurement of erythrocyte AChE is not standard practice. In contrast, determination of plasma butyrylcholinesterase (BChE) activity is in routine use for monitoring the benefit of oxime therapy. As oxime efficacy is limited with certain OPs (e.g. dimethoate, tabun, soman) alternative therapeutic approaches, e.g. the application of scavengers (BChE) which may sequester OPs before they reach their physiological target, are under investigation. To assess the eligibility of BChE as laboratory parameter and (pseudo catalytic or stoichiometric) scavenger in OP poisoning we initiated an in vitro study under standardized experimental conditions with the objective of determination of kinetic constants for inhibition, reactivation and aging of plasma BChE. It could be shown that, due to limited efficacy of obidoxime, pralidoxime, HI 6 and MMB4 with OP-inhibited BChE, plasma BChE activity is an inappropriate parameter for therapeutic monitoring of oxime treatment in OP poisoning. Furthermore, oxime-induced reactivation is too slow to accomplish a pseudo catalytic function, so that administered BChE may be merely effective as a stoichiometric scavenger. [Copyright &y& Elsevier]

Abstract: The availability of highly toxic OP-type chemical warfare agents (nerve agents) and the exertion of organophosphorus compounds during military conflicts and terrorist attacks against civilians in the past underlines the necessity of an effective treatment regimen of OP-poisoning. Presently, standard treatment includes administration of an antimuscarinic agent (e.g. atropine) and a reactivator of inhibited AChE (oxime), but is considered to be rather ineffective with certain nerve agents due to low oxime effectiveness of the currently available oximes, obidoxime and pralidoxime. The evaluation of new oximes as antidotes relies on the implementation of animal experiments for ethical reasons and is complicated by a limited extrapolation of animal data to humans. The development of a reliable animal model might accelerate the evaluation of new substances and their approval as antidotes, whereas, the pig as higher mammalian species seems to be promising as model animal. A dynamic in vitro model, which allows the calculation of AChE activities at different scenarios was developed to facilitate the definition of effective oxime concentrations and the optimization of oxime treatment of OP poisoning of humans and may furthermore be helpful by designing animal experiments. The model is based on a combination of enzyme kinetics (inhibition, reactivation, aging) of AChE with OP, toxicokinetics and oxime pharmacokinetics. By considering species-specific kinetic data this dynamic model was used for the calculation of AChE activities in humans and pigs after percutaneous exposure with 5× LD50 VR (Russian VX) and treatment with HI 6, a promising new reactivator of OP-inhibited AChE. Due to a low affinity of HI 6 with VR-inhibited pig AChE the oxime dose that causes maximal reactivation of VR-inhibited pig AChE is conspicuously higher compared to humans. Therefore, the design of animal experiments in consideration of calculated data based on species-specific kinetic values may lead to a more reliable extrapolation of animal data to humans and may reduce the number of necessary animal experiments. [Copyright &y& Elsevier]

Abstract: The increasing threat of nerve agent use for terrorist purposes against civilian and military population calls for effective therapeutic preparedness. At present, administration of atropine and an oxime are recommended, although effectiveness of this treatment is not proved in clinical trials. Here, monitoring of intoxications with organophosphorus (OP) pesticides may be of help, as their actions are closely related to those of nerve agents and intoxication and therapy follow the same principles. To this end, the clinical course of poisoning and the effectiveness of antidotal therapy were investigated in patients requiring artificial ventilation being treated with atropine and obidoxime. However, poisoning with OP pesticides shows extremely heterogeneous pictures of cholinergic crisis frequently associated with clinical complications. To achieve valuable information for the therapy of nerve agent poisoning, cases resembling situations in nerve agent poisoning had to be extracted: (a) intoxication with OPs forming reactivatable OP–AChE-complexes with short persistence of the OP in the body resembling inhalational sarin intoxication; (b) intoxication with OPs resulting rapidly in an aged OP–AChE-complex resembling inhalational soman intoxication; (c) intoxications with OPs forming a reactivatable AChE–OP complex with prolonged persistence of the OP in the body resembling percutaneous VX intoxication. From these cases it was concluded that sufficient reactivation of nerve agent inhibited non-aged AChE should be possible, if the poison load was not too high and the effective oximes were administered early and with an appropriate duration. When RBC–AChE activity was higher than some 30%, neuromuscular transmission was relatively normal. Relatively low atropine doses (several milligrams) should be sufficient to cope with muscarinic symptoms during oxime therapy. [Copyright &y& Elsevier]

Abstract: Organophosphorus compounds (OP) are in wide spread use as pesticides and highly toxic OP may be used as chemical warfare agents (nerve agents). OP inhibit acetylcholinesterase (AChE), therefore, standard treatment includes AChE reactivators (oximes) in combination with antimuscarinic agents. In the last decades, the efficacy of oximes has been investigated in various animal models, mostly in rodents. However, extrapolating animal data to humans is problematical because of marked differences between rodents and humans concerning the toxicokinetics of nerve agents, the pharmacokinetics of antidotes and the AChE enzyme kinetics. In order to improve the understanding of species differences and to enable a more reliable extrapolation of animal data to humans a study was initiated to investigate the effect of highly toxic nerve agents, i.e. VX, Russian VX (VR) and Chinese VX (CVX), with human and pig erythrocyte AChE. Hereby, the rate constants for the inhibition of AChE by these OP (k i) and for the spontaneous dealkylation (k a) and reactivation (k s) of OP-inhibited AChE as well as for the oxime-induced reactivation of OP-inhibited AChE by the oximes obidoxime, 2-PAM, HI 6, HLö 7 and MMB-4 were determined. Compared to human AChE pig AChE showed a lower sensitivity towards the investigated OP. Furthermore, a slower spontaneous dealkylation and reactivation of pig AChE was recorded. The potency of the investigated oximes was remarkably lower with OP-inhibited pig AChE. These data may contribute to a better understanding of species differences and may provide a kinetic basis for extrapolation of data from pig experiments to humans. [Copyright &y& Elsevier]

Abstract: The rife use of organophosphorus compounds (OP) as pesticides and the exertion of highly toxic OP-type chemical warfare agents (nerve agents) during military conflicts and terrorist attacks in the past emphasize the necessity of the development of effective therapeutic countermeasures. Presently, standard treatment of poisoning by OP includes administration of atropine as an antimuscarinic agent and of oximes, e.g. obidoxime or pralidoxime, as reactivators of OP-inhibited acetylcholinesterase (AChE), but is considered to be rather ineffective with certain nerve agents. The evaluation of new oximes as antidotes is only possible by implementation of animal experiments for ethical reasons and therefore complicated by a limited extrapolation of animal data to humans due to marked species differences. A computer simulation based on combination of AChE kinetic data (inhibition, reactivation, aging) with OP toxicokinetics and oxime pharmacokinetics allows the calculation of AChE activities at different scenarios and may facilitate to define effective oxime concentrations and to optimize oxime dosage in OP poisoning. On the base of species-specific kinetic data this model was used to calculate AChE activities in humans and pigs after percutaneous exposure to 5×LD50 VX and treatment with HI 6. Due to marked species differences between human and pig AChE the HI 6 dose that is necessary to cause a comparable reactivation of VX-inhibited pig AChE is conspicuously higher. Hence, designing animal experiments with the aid of computer modeling may reduce the number of animal experiments and allow a more reliable extrapolation of animal data to humans. [Copyright &y& Elsevier]

• hiPSC-CM were used to study the cardiac effects of glycosides derived from oleanders. • Rate corrected field potential duration and beat arrest were assessed in a microelectrode array system. • Cardiac effects increased in the order thevetin A < digoxin < peruvoside = digitoxigenin < oleandrin < neriifolin in the present model. The ingestion of Nerium oleander and Thevetia peruviana are common causes for poisoning in Southeast Asia. All parts of the oleander shrub contain cardiac glycosides of the cardenolide type. These glycosides act via inhibition of a Na+/K+-ATPase which might cause severe arrhythmia and subsequent death in oleander-poisoned patients. The current study uses human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) in a microelectrode array (MEA) system to assess the cardiac effects of neriifolin, oleandrin, digitoxigenin, peruvoside and thevetin A from the oleander plant. Digoxin was used as established reference compound. All tested compounds showed a corrected field potential duration (FPDc) shortening and was the lowest for 600 nM digitoxigenin with −36.9 ± 1.2 %. Next to the dose-dependent pro-arrhythmic potential, a complete beat arrest of the spontaneously beating hiPSC-CM was observed at a concentration of 300 nM for neriifolin, 600 nM for oleandrin and 1000 nM for digitoxigenin and peruvoside. Thevetin A did not cause arrhythmia up to a final concentration of 1000 nM. Thus, it was possible to establish a cardiac effect rank order of the tested substances: neriifolin > oleandrin > digitoxigenin = peruvoside > digoxin > thevetin A. [ABSTRACT FROM AUTHOR]

STUDY OBJECTIVE: Measurement of acetylcholinesterase (AChE) is recommended in the management of organophosphorus poisoning, which results in 200,000 deaths worldwide annually. The Test-mate ChE 400 is a portable field kit designed for detecting occupational organophosphorus exposure that measures RBC AChE and plasma cholinesterase (PChE) within 4 minutes. We evaluate Test-mate against a reference laboratory test in patients with acute organophosphorus self-poisoning. METHODS: This was a cross-sectional comparison study of 14 patients with acute organophosphorus poisoning between May 2007 and June 2008. RBC AChE and PChE were measured in 96 and 91 samples, respectively, with the Test-mate ChE field kit and compared with a reference laboratory, using the limits of agreement method (Bland and Altman), [kappa] statistics, and Spearman's correlation coefficients. RESULTS: There was good agreement between the Test-mate ChE and the reference laboratory for RBC AChE. The mean difference (Test-mate-reference) was -0.62 U/g hemoglobin, 95% limits of agreement -10.84 to 9.59 U/g hemoglobin. Good agreement was also observed between the categories of mild, moderate, and severe RBC AChE inhibition (weighted [kappa] 0.85; 95% confidence interval [CI] 0.83 to 0.87). Measurement of PChE also showed good agreement, with a mean difference (Test-mate-reference) of +0.06 U/mL blood, 95% limits of agreement -0.41 to 0.53 U/mL blood. Spearman's correlation coefficients were 0.87 (95% CI 0.81 to 0.91) for RBC AChE and 0.76 (95% CI 0.66 to 0.84) for PChE. Analysis for within-subject correlation of subjects did not change the limits of agreement. CONCLUSION: The Test-mate ChE field kit reliably provides rapid measurement of RBC AChE in acute organophosphorus poisoning. [ABSTRACT FROM AUTHOR]

Highlights: [•] MB327 was used in a smooth muscle model for the first time and compared to oximes and atropine. [•] MB327 showed fully reversible smooth muscle relaxing effect at lower concentrations than all tested oximes. [•] Antimuscarinic potency of MB327 may contribute to its therapeutic effect in organophosphorous compound poisoning. [Copyright &y& Elsevier]

Abstract: Sulfur mustard (SM; bis(2-chloroethyl)sulphide; HD) is a blister inducing agent causing DNA damage and subsequently, cell death, mostly by apoptosis in basal keratinocytes. Despite intensive investigations on the cellular mechanism, there are, as of now, no causal therapeutics to prevent or antagonize SM-related damage to cells and tissues. In order to develop treatment strategies against vesication, it is important to distinguish apoptosis from necrosis in SM treated human keratinocytes. DNA fragmentation is a hallmark of apoptosis and regulated by a cascade of enzymes (endonucleases, DNase I, NUC 18), which finally cut the chromatin into specific formations of 180–200 base pairs, the nucleosomes. A feasible way to monitor apoptosis is the detection of nucleosomes by means of the Cell Death Detection ELISAplus® (CDDE). In contrast, during necrosis DNA fragmentation is at random and delivers larger fragments, which therefore are significantly less in number and predominantly occur in cell culture supernatant. To monitor necrosis, we measured the release of intracellular adenylate kinase (AK) into cell culture supernatant by means of the ToxiLight® Bioluminescence Assay (TL). With combination of the Cell Death Detection ELISAplus® and the ToxiLight® Bioluminescence Assay, we acquired more comprehensive information on cell survival and mechanisms of cell death, following an SM exposure. To validate the assay we tested common apoptosis- and necrosis-inducing agents like SM 300μM for 30min, Lewisite (L) 60μM for 5min and Triton X-100 0.1%. The results show that it is possible to differentiate between the two modes of cell death and to quantify their extent. This assay is highly effective in quantifying apoptosis and necrosis caused by cytotoxic agents and in estimating protective effects of potential active pharmaceutical ingredients. [Copyright &y& Elsevier]

Abstract: Although the three most commonly used large mammal species in the safety assessment of drugs remain the dog, the macaque and the marmoset, swine, especially minipigs, have also been widely used over the years in many toxicological studies. Swine present a number of interesting biological and physiological characteristics. Similarities in skin properties with humans have led to extensive in vitro and in vivo studies. There is a specific interest in cardiovascular research, as well as in anaesthesiology and critical care medicine due to common features of swine and human physiology. Although knowledge of swine brain structure and functions remains incomplete, data does exist. The multiple blood sampling that is necessary in pharmacokinetic and toxicokinetic studies are possible, as well as multiparametric monitoring and interventions with equipment used in human clinical settings. Practicality (handling), scientific (stress reduction) and ethical (invasive monitoring) reasons have led research teams to incorporate anaesthesia into their paradigms which makes the analysis of data increasingly difficult. Although not substantiated by scientific data, the swine appears to have an intermediate position in the scale of public perception between non-human primates and animals commonly referred to as pets (i.e. dogs and cats) and rodents. The benefits of the swine model justify the use of these animals in the design of more effective medical countermeasures against known chemical warfare agents (nerve agents, vesicants and lung damaging agents). Exposure to organophosphorus (OP) pesticides represents a severe health issue in developing countries, while OP intoxication with the more lethal military nerve agents is not only of military concern but also a terrorist threat. Tailoring therapeutic regimens to the reality of OP poisoning is of the utmost importance when little experimental data and sparse human clinical data are available in the decision making process. We will present some of the advantages and disadvantages of the swine model in OP countermeasures elaborating on two examples. First, we will present the issues related to the use of anaesthesia during experimental OP poisoning and second we will show how results from experiments with swine can be integrated into a kinetic-based dynamic model to evaluate oxime efficacy. A better knowledge of OP poisoning in swine (comparative toxicokinetics, pharmacokinetics and biochemistry) is definitely necessary before accepting it as a first choice non-rodent model. However, there exists a large amount of data in the model on anaesthesia and different types of shock favouring their use for evaluation of complex situations such as the anaesthesia of OP poisoned patients and combined injuries. [Copyright &y& Elsevier]

The bispyridinium compound MB327 has been shown previously to have a positive pharmacological effect against poisoning with organophosphorous compounds (OPCs). The mechanism by which it exerts its therapeutic effect seems to be directly mediated by the nicotinic acetylcholine receptor (nAChR). In the present study, the development of mass spectrometry based binding assays (MS Binding Assays) for characterization of the binding site of MB327 at the nAChR from Torpedo californica is described. MS Binding Assays follow the principle of radioligand binding assays, but do not, in contrast to the latter, require a radiolabeled reporter ligand, as the readout is in this case based on mass spectrometric detection. For [ 2 H 6 ]MB327, a deuterated MB327 analogue employed as reporter ligand in the MS Binding Assays, an LC-ESI–MS/MS method was established allowing for its fast and reliable quantification in samples resulting from binding experiments. Using centrifugation for separation of non-bound [ 2 H 6 ]MB327 from target-bound [ 2 H 6 ]MB327 in saturation and autocompetition experiments (employing native MB327 as competitor) enabled reliable determination of specific binding. In this way, the affinities for [ 2 H 6 ]MB327 ( K d = 15.5 ± 0.9 μmol L −1 ) and for MB327 ( K i = 18.3 ± 2.6 μmol L −1 ) towards the nAChR could be determined for the first time. The almost exactly matching affinities for MB327 and [ 2 H 6 ]MB327 obtained in the MS Binding Assays are in agreement with potencies previously found in functional studies. In summary, our results demonstrate that the established MS Binding Assays represent a promising tool for affinity determination of test compounds towards the binding site of MB327 at the nAChR. [ABSTRACT FROM AUTHOR]

The primary toxic mechanism of organophosphorus compounds, i.e. nerve agents or pesticides, is based on the irreversible inhibition of acetylcholinesterase. In consequence of the impaired hydrolysis, the neurotransmitter acetylcholine accumulates in cholinergic synapses and disturbs functional activity of nicotinic and muscarinic acetylcholine receptors by overstimulation and subsequent desensitization. The resulting cholinergic syndrome will become acute life-threatening, if not treated adequately. The current standard treatment, consisting of administration of a competitive mAChR antagonist ( e.g. atropine) and an oxime ( e.g. obidoxime, pralidoxime), is not sufficient in the case of soman or tabun intoxications. Consequently, alternative therapeutic options are necessary. An innovative approach comprises the use of compounds selectively targeting nAChRs, especially positive allosteric modulators, which increase the population of the conducting receptor state. MB327 (1,1′-(propane-1,3-diyl)bis(4- tert -butylpyridinium) di(iodide)) is able to restore soman-blocked muscle-force in preparations of various species including human and was recently identified as “resensitizer”. In contrast to the well-studied MB327, the pharmacological efficacy of the 2- and 3- tert -butylpyridinium propane regioisomers is unknown. As a first step, MB327 and its 3-regioisomer (PTM0001) and 2-regioisomer (PTM0002) were pharmacologically characterized using [ 3 H]epibatidine binding assays, functional studies by solid supported membranes based electrophysiology, and in vitro muscle-force investigations of soman-poisoned rat hemidiaphragm preparations by indirect field stimulation technique. The results obtained from targets of different complexity (receptor, muscle tissue) showed that the pharmacological profiles of the 2- and 3-regioisomers were relatively similar to those of MB327. Furthermore, high concentrations showed inhibitory effects, which might critically influence the application as an antidote. Thus, more effective drugs have to be developed. Nevertheless, the combination of the methods presented is an effective tool for clarifying structure-activity relationships. [ABSTRACT FROM AUTHOR]

Organophosphorus compounds (OPC), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). Inhibited AChE results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nAChR) in the postsynaptic membrane is provoked. Direct targeting of nAChR to reduce receptor desensitisation might be an alternative therapeutic approach. For drug discovery, functional properties of potent therapeutic candidates need to be investigated in addition to affinity properties. Solid supported membrane (SSM)-based electrophysiology is useful for functional characterisation of ligand-gated ion channels like nAChRs, as charge translocations via capacitive coupling of the supporting membrane can be measured. By varying the agonist (carbamoylcholine) concentration, different functional states of the nAChR were initiated. Using plasma membrane preparations obtained from Torpedo californica electric organ, functional properties of selected nAChR ligands and non-oxime bispyridinium compounds were investigated. Depending on overall-size, the bispyridinium compounds enhanced or inhibited cholinergic signals induced by 100 μM carbamoylcholine. Applying excessive concentrations of the agonist carbamoylcholine provoked desensitisation of the nAChRs, whereas addition of bispyridinium compounds bearing short alkyl linkers exhibited functional recovery of previously desensitised nAChRs. The results suggest that these non-oxime bispyridinium compounds possibly interacted with nAChR subtypes in a manner of a positive allosteric modulator (PAM). The described newly developed functional assay is a valuable tool for the assessment of functional properties of potential compounds such as nAChR modulating ligands, which might be a promising approach in the therapeutically treatment of OPC-poisonings. [ABSTRACT FROM AUTHOR]

Highlights: [•] We investigated bispyridinium compounds in binding experiments with nAChRs. [•] We compared [3H]epibatidine affinity profiles to the inhibition of human AChE. [•] nAChR affinity and AChE inhibition depended on linker length. [•] Divalent cations enhanced [3H]epibatidine binding. [ABSTRACT FROM AUTHOR]

Abstract: The standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes is not sufficiently effective against all types of nerve agents. Alternative therapeutic strategies are required and bispyridinium non-oximes, acting as nicotinic antagonists, were identified as promising compounds. A previous study showed that the di(methanesulfonate) salt of the bispyridinium compound MB327 could restore soman-impaired neuromuscular function in vitro and improve survival of sarin, soman and tabun poisoned guinea pigs in vivo. Here, by using the indirect field stimulation technique, the ability of MB327 to counteract soman-impaired neuromuscular transmission was investigated in human intercostal muscle and rat diaphragm preparations. MB327 restored muscle force in a concentration-dependent manner in both species without reactivating soman-inhibited acetylcholinesterase. The therapeutic effect of MB327 could be washed out, indicating a direct effect at the nicotinic receptor level. Also the ability of MB327 to restore respiratory muscle function could be demonstrated for the first time in rat and human tissue. In combination with previous in vitro and in vivo findings MB327 may be considered a promising compound for the treatment of nerve agent poisoning and further studies are needed to identify optimized drug combinations, concentrations and dosing intervals to provide an effective therapy for OP poisoning. [Copyright &y& Elsevier]

Abstract: The chemical weapon nerve agent known as Russian VX (VR) is a potent organophosphorus (OP) compound that is much less studied than its VX analogue with respect to toxicity, as well as to the effectiveness of several known countermeasures against it. An anaesthetized domestic swine model was utilized to assess several approaches in mitigating its toxicity, including the utility of cooling VR treated skin to increase the therapeutic window for treatment. The 6h LD50 for VR topically applied on the ear was 100μg/kg. Treatment of VR exposed animals (5×LD50) with pralidoxime (2PAM) very poorly regenerated inhibited blood cholinesterase activity, but was partially effective in preventing signs of OP poisoning and increasing survival. In contrast, treatment with the Hagedorn oxime HI-6 reactivated cholinesterase, eliminated all signs of poisoning and prevented death. Decontamination with the Reactive Skin Decontaminant Lotion (RSDL) 15min after VR exposure was completely effective in preventing death. Cooling of the VR exposure sites for 2 or 6h prevented signs of OP poisoning and death during the cooling period. However, these animals died very quickly after the cessation of cooling, unless they were treated with oxime or decontaminated with RSDL. Blood analyses showed that cooling of agent exposure sites delayed the entry of VR into the bloodstream. Medical treatment with HI-6 and to a lesser extent 2PAM, or decontamination with RSDL are effective in protecting against the toxic effects of cutaneous exposure to VR. Immobilizing this agent (and related compounds) within the dermal reservoir by cooling the exposure sites, dramatically increases the therapeutic window in which these medical countermeasures are effective. [Copyright &y& Elsevier]

Abstract: Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes lacks efficacy with different nerve agents. A direct pharmacologic intervention at the nicotinic acetylcholine receptor (nAChR) was proposed as an alternative therapeutic approach and promising in vitro and in vivo results were obtained with the bispyridinium compound SAD-128. In addition, a number of SAD-128 analogues improved neuromuscular transmission of soman-poisoned diaphragms in vitro. We investigated the interaction of six of these SAD-128 analogues with the orthosteric binding site of the human α7 nAChR and Torpedo californica nAChR with a high-throughput assay using radioactive ligands. The determined affinity constants indicate a weak interaction of three test compounds (K i in the micromolar range) with both receptors, but no interaction could be recorded with the other three test compounds. The six SAD-128 analogues showed a low intrinsic inhibitory potency with human acetylcholinesterase (IC50 >400μM). In conclusion, the results of the present study do not indicate a correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission and it is assumed that other mechanisms contribute to the therapeutic effect of the tested compounds. [Copyright &y& Elsevier]

Abstract: An important factor for successful therapy of poisoning with organophosphorus compounds (OP) is the rapid restoration of blocked respiratory muscle function. To achieve this goal, oximes are administered for reactivation of inhibited acetylcholinesterase (AChE). Unfortunately, clinically used oximes, e.g. obidoxime and pralidoxime, are of limited effectiveness in poisoning with different OP nerve agents requiring the search for alternative oximes, e.g. HI 6. In view of substantial species differences regarding reactivation properties of oximes, the effect of HI 6 was investigated with sarin, tabun and soman exposed human intercostal muscle. Muscle force production by indirect field stimulation and the activity of the human muscle AChE was assessed. 30μM HI 6 resulted in an almost complete recovery of sarin blocked muscle force and in an increase of completely inhibited muscle AChE activity to approx. 30% of control. In soman or tabun exposed human intercostal muscle HI 6 (50 and 100μM) had no effect on blocked muscle force or on inhibited human muscle AChE activity. In addition, HI 6 up to 1000μM had no effect on soman blocked muscle force indicating that this oxime has no direct, pharmacological effect in human tissue. These results emphasize that sufficient reactivation of AChE is necessary for a beneficial therapeutic effect on nerve agent blocked neuromuscular transmission. [Copyright &y& Elsevier]

Abstract: The organophosphate (OP) nerve agent VX is a weaponized chemical warfare agent that has also been used by terrorists against civilians. This contact poison produces characteristic signs of OP poisoning, including miosis, salivation, mastication, dysrhythmias and respiratory distress prior to death. Although successful treatment of OP poisoning can be obtained through decontamination and/or oxime reactivation of agent-inhibited cholinesterase, medical countermeasures that increase the therapeutic window for these measures would be of benefit. An anaesthetized swine model was utilized to examine the effects of lethal VX exposure to the skin, followed by cooling the exposure site prior to decontamination or treatment. The cooling was simply accomplished by using crushed ice in grip-seal plastic bags applied to the exposure sites. Cooling of skin exposed to lethal doses of VX significantly increased the window of opportunity for successful decontamination using the Reactive Skin Decontaminant Lotion® (RSDL®) or treatment with the oxime antidotes HI-6 and 2PAM. Analyses of blood VX levels showed that cooling acted to slow or prevent the entry of VX into the bloodstream from the skin. If the exposure site is known, the simple and non-invasive application of cooling provides a safe means with which to dramatically increase the therapeutic window in which decontamination and/or antidote treatment against VX are life-saving. [Copyright &y& Elsevier]

Abstract: In the present study, we report the first in vivo toxicokinetic study of tabun (O-ethyl-N,N-dimethylphosphoramidocyanidate). The toxicokinetics of the enantiomers of tabun were investigated in anesthetized swine after intravenous administration of 3×LD50 (161.4μg/kg) tabun. Blood samples were taken for gas chromatographic–mass spectrometric determination of the tabun enantiomers and for measurement of the activity of red blood cell acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BChE). The tabun enantiomers could be quantified in swine blood to a minimum concentration of 3.0pg/ml (18.5pM) and could be detected to a minimum concentration of 1.0pg/ml (6.2pM). The concentration–time profiles of both tabun enantiomers were best described by a bi-exponential equation. The elimination of (+)-tabun and (−)-tabun were comparable in the initial phase. In the terminal phase a remarkable difference was found, with terminal half lives of 11.5min for (+)-tabun and 23.1min for (−)-tabun. (+)-Tabun showed a markedly longer persistence in vivo than (+)-enantiomers of other G-type nerve agents and could be detected in all swine at least up to 30min post-injection, (−)-tabun at least up to 90min post-injection. These results demonstrate a rather rapid elimination of tabun enantiomers in vivo and may provide a toxicokinetic basis for the further development and optimization of medical countermeasures against this nerve agent. [ABSTRACT FROM AUTHOR]

Abstract: Chemical warfare agents (nerve agents) are still available and present a real threat to the population. Numerous in vitro and in vivo studies showed that various nerve agents, e.g. tabun and cyclosarin, are resistant towards standard therapy with atropine and oxime. Based on these facts we applied a modified biological assay for the easy, semi-quantitative testing of the detoxifying properties of the β-cyclodextrin derivative CD-IBA. Cyclosarin, sarin, tabun and VX were incubated with CD-IBA for 1–50min at 37°C, then an aliquot was added to erythrocyte acetylcholinesterase (AChE) and the percentage of AChE inhibition was determined. The validity of the assay was confirmed by concomitant quantification of tabun by GC–MS. Different concentrations of cyclosarin were detoxified by CD-IBA in a concentration-dependent velocity. The ability to detoxify various nerve agents decreased in the order cyclosarin>sarin>tabun≫VX. Hereby, no detoxification of VX could be detected. Sarin was detoxified in a biphasic reaction with a fast reduction of inhibitory potential in the first phase and a slower detoxification in the second phase. CD-IBA detoxified tabun in a one phase decay and, compared to cyclosarin and sarin, a longer half-life was determined with tabun. The modified biological assay is appropriate for the initial semi-quantitative screening of candidate compounds for the detoxification of nerve agents. The β-cyclodextrin derivative CD-IBA demonstrated its ability to detoxify different nerve agents. [Copyright &y& Elsevier]

Abstract: Sulfur mustard (SM) is a strong alkylating agent, which produces subepidermal blisters, erythema and inflammation after skin contact. Despite the well-described SM-induced gross and histopathological changes, the exact underlying molecular mechanisms of these events are still a matter of research. As part of an international effort to elucidate the components of cellular signal transduction pathways, a large body of data has been accumulated in the last decade of SM research, revealing deeper insight into SM-induced inflammation, DNA damage response, cell death signaling, and wound healing. SM potentially alkylates nearly every constituent of the cell, leading to impaired cellular functions. However, SM-induced DNA alkylation has been identified as a major trigger of apoptosis. This includes monofunctional SM-DNA adducts as well as DNA crosslinks. As a consequence, DNA replication is blocked, which leads to cell cycle arrest and DNA single and double strand breaks. The SM-induced DNA damage results in poly(ADP-ribose) polymerase (PARP) activation. High SM concentrations induce PARP overactivation, thus depleting cellular NAD+ and ATP levels, which in consequence results in necrotic cell death. Mild PARP activation does not disturb cellular energy levels and allows apoptotic cell death or recovery to occur. SM-induced apoptosis has been linked both to the extrinsic (death receptor, Fas) and intrinsic (mitochondrial) pathway. Additionally, SM upregulates many inflammatory mediators including interleukin (IL)-1α, IL-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α) and others. Recently, several investigators linked NF-κB activation to this inflammatory response. This review briefly summarizes the skin toxicity of SM, its proposed toxicodynamic actions and strategies for the development of improved medical therapy. [Copyright &y& Elsevier]

Abstract: Sulphur mustard (SM) is a chemical warfare agent that causes erythema and blistering of the skin with a latency of several hours. Although SM is known for almost 200 years the cellular mechanisms involved in the damaging process are not fully understood. There is evidence that changes in nitric oxide (*NO), reactive oxygen species (ROS) and reactive nitrogen species (RNS) might be involved in the damaging process. Aim: To find out more about the pathophysiology of SM, we investigated the initial formation of biochemical markers of nitrosative and oxidative stress as well as activation (translocation from plasma-membrane) and upregulation of eNOS and iNOS, respectively. Methods: Human immortalized keratinocytes (HaCaT cell line) were exposed to SM (100μM or 300μM) for 30min. Cells were fixed after 1h, 3h or 6h of incubation in SM-free medium and immunostained. Live cell experiments were performed using the NO-sensitive dye DAF2-DA. In order to assess cell viability after BH4 supplementation, we analyzed apoptosis using CDD-ELISA. Results: SM significantly increased biochemical markers of nitrosative and oxidative stress already 1h after exposure. Moreover, the NO producing enzymes eNOS and iNOS showed concentration- and time-dependent changes in their activation or expression levels. Initially, live cell imaging experiments could not confirm NO production after SM exposure. Only when cells were supplemented with tetrahydrobiopterine, stable NO production was detectable. Apoptotic activity was increased due to SM exposure and could be reduced after BH4 treatment. Conclusion: Our data point towards concentration- and time-dependent formation of iNOS and activation of eNOS due to translocation from plasma-membrane. Live cell experiments yielded first indications of catalytic decoupling of NOS that could be reversed by supplementation with tetrahydrobiopterin (BH4). Addition of BH4 1h after SM exposure significantly decreased apoptosis compared to the unsupplemented control. [Copyright &y& Elsevier]

Abstract: Previous kinetic studies investigating the interactions between human acetylcholinesterase (AChE), structurally different organophosphorus compounds (OP) and oximes did not reveal a conclusive structure–activity relationship of the different reactions. The only exception was for a homologous series of methylphosphonofluoridates bearing C1–C4 O-n- or O-i-alkyl residues. Hence, it was tempting to investigate the kinetic interactions between different pentylsarin analogues, human AChE and two oximes, obidoxime and HI 6, in order to increase the understanding of structure–activity relationship between highly toxic OP and human AChE. The rate constants for the inhibition of human erythrocyte AChE by four pentylsarin compounds (k i), for the spontaneous dealkylation (aging, k a) and reactivation (k s) of inhibited AChE as well as for the oxime-induced reactivation of inhibited AChE by obidoxime and HI 6 reflected by the dissociation constant (K D) and the reactivity constant (k r) were determined. All pentylsarin analogues had a high inhibitory potency towards AChE. Inhibited AChE was subject to spontaneous reactivation which outweighed aging substantially. Pentylsarin-inhibited AChE could be reactivated by oximes, HI 6 being more potent than obidoxime. The determination of inhibition, reactivation and aging kinetics of pentylsarin analogues with human AChE extends the database on interactions between AChE and methylphosphonofluoridate homologues with C1–C4 n- and i-alkyl residues demonstrating a structure–activity relationship depending on the chain length with certain differences regarding inhibition and post-inhibitory reactions. Unfortunately, no structure–activity relationship could be observed for the oxime-induced reactivation of inhibited AChE. In view of previous results with numerous structurally different organophosphates, organophosphonates and phosphoramidates it has to be concluded that up to now kinetic studies did not provide decisive information for the development of more effective oxime-based reactivators. [Copyright &y& Elsevier]

Abstract: Treatment regimen of poisonings by organophosphorus (OP) compounds usually includes oxime therapy. The treatment options in soman poisoning are very limited due to rapid aging of the inhibited acetylcholinesterase (AChE), when the enzyme species is considered as irreversibly inhibited and resistant towards reactivation by oximes. Hence, oxime treatment probably comes too late in realistic scenarios. As an alternative, protecting part of the enzyme by reversible inhibition prior to soman exposure has been proposed. One means of protecting against soman poisoning is the prophylactic use of certain reversible inhibitors (carbamates) of AChE. The question whether there is a possibility of an interaction between pre-treating carbamates and oximes at AChE arises. Therefore we studied the effects of the oximes obidoxime, HI 6 and MMB-4 on the rate of decarbamylation for physostigmine- and pyridostigmine-inhibited human erythrocyte AChE both in a dynamically working in vitro model and a static cuvette system. Our results show that HI 6 increased the rate of decarbamylation for both physostigmine- and pyridostigmine-inhibited enzyme in both systems, the observed effect by HI 6 increasing with higher doses. Obidoxime had a slightly accelerating effect on the pyridostigmine-inhibited enzyme. MMB-4 applied to pyridostigmine-inhibited AChE in the static system only showed no difference to the experiments made in absence of oxime. No oxime showed a tendency to retard the rate of decarbamylation. [Copyright &y& Elsevier]

Abstract: Organophosphorus compound-based (OP) chemical warfare agents (nerve agents) represent a continuing threat to military forces and the civilian population. OPs act primarily by inhibiting acetylcholinesterase (AChE), the standard treatment for which includes AChE reactivators (oximes) in combination with antimuscarinic drugs. In the last decades, the efficacy of oximes has been investigated mostly in small animal models. In order to increase the predictive value of animal studies it is desirable to measure numerous physiological and biochemical parameters. This is difficult in small animals. Large animal models fulfil these requirements and swine are increasingly being used in toxicology studies. Swine breeds generally show considerable variability in different characteristics which may be minimised by the use of specially bred minipigs which have a known genetic background and health status. A comparative study was, therefore, initiated to investigate the kinetic properties of the White Landrace pig and Göttingen minipig AChE in respect of inhibition by the pesticide paraoxon; the nerve agents cyclosarin, VX and VR; the reactivation of inhibited AChE by oximes (obidoxime, pralidoxime and HI 6); and the aging and spontaneous reactivation of inhibited AChE. The determination of the respective kinetic constants found similarities between pig and minipig AChE which showed marked differences in comparison with human AChE values. This has to be considered in designing meaningful models for the investigation of oxime efficacy in pig or minipig experiments. The generated data indicate comparable kinetic properties of pig and minipig AChE and may provide a kinetic basis for extrapolation of data from pig studies to humans. [Copyright &y& Elsevier]

Abstract: Erectile function is critically dependent upon the activation of the endothelial nitric oxide synthase (eNOS) in the smooth muscle cells of penile corpus cavernosum tissue. Nebivolol is a β1-selective β-adrenoceptor blocker (β-ARB) with additional vasodilating properties, which have been attributed to eNOS-activation. Our study investigated whether nebivolol is able to increase eNOS activity in erectile tissue. Murine penile tissue was incubated in an organ bath under control conditions and in the presence of nebivolol or metoprolol. Immunofluorescence staining was performed using specific antibodies against eNOS-activation or eNOS-serine 1177 phosphorylation. Corpus cavernosum smooth muscle tissue was identified using a smooth muscle actin antibody. In addition, slices of murine erectile tissue were incubated with diaminofluorescein (DAF), a specific fluorescence marker for NO-liberation. Under control conditions and after application of metoprolol, we observed a small eNOS-activation and serine 1177-phosphorylation in murine corpus cavernosum tissue. A significant increase in eNOS-activation and serine 1177-phosphorylation of eNOS was observed only in the presence of nebivolol (10 μM). These alterations of the eNOS protein induced after application of nebivolol were associated with a time-dependent increase in DAF fluorescence in murine erectile tissue. We conclude that β-adrenoceptor blockers differentially influence erectile tissue. Since cardiovascular diseases are often associated with the development of erectile dysfunction, the nebivolol-induced eNOS-activation in corpus cavernosum may be beneficial when treating patients suffering from cardiovascular disease. [Copyright &y& Elsevier]

Abstract: Organophosphorus (OP) pesticides or nerve agents cause severe intoxication by inhibition of acetylcholinesterase, finally resulting in death due to respiratory failure. The phrenic nerve diaphragm preparation is considered as the classic model to investigate the effect of OP intoxications and oxime treatment at the neuromuscular junction. However, this preparation is unsuitable for larger species or for muscle strips from biopsies where no nerve is available for stimulation. An alternative technique is the indirect field stimulation of muscles containing intramuscular nerve branches only. The proposed method by Wolthuis et al. [Wolthuis, O.L., Vanwersch, R.A.P., Van Der Wiel, H.J., 1981. The efficacy of some bis-pyridinium oximes as antidotes to soman in isolated muscles of several species including man. Eur. J. Pharmacol. 70, 355–369] was modified and experimentally reevaluated in isolated mouse diaphragms. To confirm that electrical field stimulation technique induced muscle contraction only via the neuromuscular endplate the nicotinic antagonists pancuronium or d-tubocurarine (1μM) were given. In the presence of a nicotinic antagonist hardly any contraction was blocked after indirect field stimulation technique with very short pulses (5μs, <0.6A), in contrast to direct muscle stimulation (broader pulse width, or higher amplitude >0.6A). During paraoxon circumfusion (20min, 1μmol/l) muscle force generation by indirect stimulation was almost completely blocked. Restoration of paralyzed muscle function to 80% of initial values could be achieved after paraoxon wash out (20min) and circumfusion with obidoxime (1μmol/l, 20min). This data correspond quite well to data shown earlier when using conventional nerve stimulation techniques. [Copyright &y& Elsevier]

Abstract: The pertinent threat of using organophosphorus compound (OP)-type chemical warfare agents (nerve agents) during military conflicts and by non-state actors requires the continuous search for more effective medical countermeasures. OP inhibit acetylcholinesterase (AChE) and therefore standard treatment of respective poisoning includes AChE reactivators (oximes) in combination with antimuscarinic agents. Hereby, standard oximes, 2-PAM and obidoxime, are considered to be rather insufficient against various nerve agents. Numerous experimental oximes have been investigated in the last decades by in vitro and in vivo models. Recently, we studied the reactivating potency of several oximes with human AChE inhibited by structurally different OP and observed remarkable differences depending on the OP and oxime. In order to investigate structure–activity relationships we determined the various kinetic constants (inhibition, reactivation, aging) for a series of sarin analogues bearing a methyl, ethyl, n-propyl, n-butyl, i-propyl, i-butyl, cyclohexyl or pinacolyl group with human AChE and BChE. The rate constants for the inhibition of human erythrocyte AChE and plasma BChE by these OP (k i), for the spontaneous dealkylation (k a) and reactivation (k s) of OP-inhibited AChE and BChE as well as for the oxime-induced reactivation of OP-inhibited AChE and BChE by the oximes obidoxime, 2-PAM, HI 6, HLö 7 and MMB-4 were determined. With compounds bearing a n-alkyl group the inhibition rate constant increased with chain length. A relation between chain length and spontaneous reactivation velocity was also observed. In contrast, no structure–activity dependence could be observed for the oxime-induced reactivation of AChE and BChE inhibited by the compounds tested. In general, OP-inhibited AChE and BChE were susceptible towards reactivation by oximes. HLö 7 was the most potent reactivator followed by HI 6 and obidoxime while 2-PAM and MMB-4 were rather weak reactivators. These data indicate a potential structure–activity relationship concerning inhibition and spontaneous reactivation but not for oxime-induced reactivation. [Copyright &y& Elsevier]