Physostigmine (alone and together with adjunct) pretreatment against soman, sarin, tabun and VX intoxication

A pretreatment for organophosphorus (OP) anticholinesterase (e.g., soman) intoxication should prevent lethality and convulsions (CNV) at 2 LD50s and be behavioral-decrement-free when given alone. Behavioral-deficit-free pretreatment regimens (PRGs) for guinea pigs consisted of Physostigmine (0.15 mg/kg, im) and adjunct. Adjuncts [mg/kg, im] tested were akineton [0.25], aprophen [8], trihexyphenidyl [2], atropine [16], azaprophen [5], benactyzine [1.25], cogentin [4], dextromethorphan [7.5], ethopropazine [12], kemadrin [1], memantine [5], promethazine [5], scopolamine [0.08] and vontrol [2]. PRGs were given 30 min before soman (60 micrograms/kg, sc; 2 LD50s) or other OP agents. Animals were then observed and graded for signs of intoxication, including CNV at 7 time points and at 24 hr. Physostigmine alone reduced the incidence of CNV and lethality induced by 2 LD50s of soman by 42 and 60%, respectively. All of the PRGs tested abolished lethality and 12 shortened recovery time to 2 hr or less. Also, PRGs including azaprophen or atropine prevented CNV. When selected PRGs were tested against intoxication by sarin, tabun or VX, the efficacy was generally superior to that for soman. The data show that several PRGs are effective against soman intoxication in guinea pigs.     

Behavioral efficacy of diazepam against nerve agent exposure in rhesus monkeys.

The possibility that nerve agents will be used on the battlefield is real. The traditional therapy against nerve agent exposure consists of pyridostigmine pretreatment and atropine-pralidoxime chloride therapy administered after nerve agent exposure. This therapy regimen is extremely effective in preventing mortality in laboratory animals exposed to multilethal concentrations of nerve agent, yet these animals often display convulsions, brain damage, and behavioral incapacitation. We report here that the addition of diazepam to the traditional therapy for nerve agent (soman) exposure not only decreases the incidence of convulsions, but also attenuates the cognitive impairments of rhesus monkeys trained on a Serial Probe Recognition (SPR) task. Monkeys which received diazepam treatment required only 6 days before their performance on the SPR task returned to presoman exposure levels, compared to nondiazepam-treated monkeys which required 15 days. Moreover, only 1 out of the 5 monkeys which received diazepam treatment suffered tonic-clonic convulsions; in contrast all 5 monkeys which did not receive diazepam treatment experienced severe convulsive episodes. These results suggest that diazepam would be an excellent adjunct to traditional nerve agent therapy to facilitate behavioral recovery from nerve agent intoxication that might be encountered by US military personnel on the battlefield or accidental organophosphate poisoning encountered in industrial or agricultural accidents.     

Medical countermeasure against respiratory toxicity and acute lung injury following inhalation exposure to chemical warfare nerve agent VX

To develop therapeutics against lung injury and respiratory toxicity following nerve agent VX exposure, we evaluated the protective efficacy of a number of potential pulmonary therapeutics. Guinea pigs were exposed to 27.03 mg/m(3) of VX or saline using a microinstillation inhalation exposure technique for 4 min and then the toxicity was assessed. Exposure to this dose of VX resulted in a 24-h survival rate of 52%. There was a significant increase in bronchoalveolar lavage (BAL) protein, total cell number, and cell death. Surprisingly, direct pulmonary treatment with surfactant, liquivent, N-acetylcysteine, dexamethasone, or anti-sense syk oligonucleotides 2 min post-exposure did not significantly increase the survival rate of VX-exposed guinea pigs. Further blocking the nostrils, airway, and bronchioles, VX-induced viscous mucous secretions were exacerbated by these aerosolized treatments. To overcome these events, we developed a strategy to protect the animals by treatment with atropine. Atropine inhibits muscarinic stimulation and markedly reduces the copious airway secretion following nerve agent exposure. Indeed, post-exposure treatment with atropine methyl bromide, which does not cross the blood-brain barrier, resulted in 100% survival of VX-exposed animals. Bronchoalveolar lavage from VX-exposed and atropine-treated animals exhibited lower protein levels, cell number, and cell death compared to VX-exposed controls, indicating less lung injury. When pulmonary therapeutics were combined with atropine, significant protection to VX-exposure was observed. These results indicate that combinations of pulmonary therapeutics with atropine or drugs that inhibit mucous secretion are important for the treatment of respiratory toxicity and lung injury following VX exposure.     

Immuno- and neurotoxicological investigation of combined subacute exposure with the carbamate pesticide propoxur and cadmium in rats

In the present study, the effects of subchronic per os exposures to cadmium chloride (CdCl(2)), and a carbamate insecticide, propoxur (Pr), were investigated in male Wistar rats on general toxicological (body weight gain, relative organ weights) haematological (RBC, WBC, Ht, MCV, cell content of the femoral bone marrow) immune function (plaque forming cell (PFC) assay, delayed type hypersensitivity (DTH) reaction) and neurotoxicological (spontaneous and stimulus-evoked cortical activity, nerve conduction velocity) parameters. The animals were treated for 4, 8 and 12 weeks with 6.43 mg/kg CdCl(2), 8.51 mg/kg Pr, or with a combination of 6.43 mg/kg CdCl(2)+0.851 mg/kg Pr or 8.51 mg/kg Pr+1.61 mg/kg CdCl(2). Cadmium exposure affected the relative thymus, liver, and adrenal weight, RBC count, haematocrit and MCV, and there was an increase in nerve conduction velocity and a decrease in the cortical evoked potential latency. Pr induced a decrease in thymus weight, had some effect on the liver weight but none on the electrophysiological parameters. A significant interaction between Cd and Pr was detected by the following parameters: RBC, Ht, PFC, and nerve conduction velocity. The results indicate that combined exposures in humans may result in a shift in the apparent detection limits and/or in the LOEL of the single substances. The latter raises the necessity to reconsider exposure limits in situations where the risk of combined exposure is high.     

Evaluation of a two-drug combination pretreatment against organophosphorus exposure

A pretreatment combination of physostigmine and azaprophen (6-methyl-6-azabicyclo[3.2.1]octan-3-ol-2,2-diphenylpropionate), a novel cholinolytic, was evaluated for its ability to minimize soman-induced incapacitation and lethality in guinea pigs. This was accomplished by using response surface methodology to model and analyze the combination, varying physostigmine from 0 to 194 micrograms/kg, azaprophen from 0 to 5 mg/kg, and soman from 30 to 150 micrograms/kg. One hundred percent survival was achieved against 5 LD50 of soman using as little as 100 micrograms/kg of physostigmine in the presence of 5 mg/kg azaprophen. Both survival and soman-induced incapacitation were similarly affected by this pretreatment combination. For both endpoints, greater efficacy was achieved with the combination than could be achieved with either component alone (therapeutic synergism). This suggests that such a pretreatment combination may prove very efficacious against soman-induced lethality and incapacitation in higher species.     

Oximes as pretreatment before acute exposure to paraoxon

Organophosphates, useful agents as pesticides, also represent a serious danger due to their high acute toxicity. There is indication that oximes, when administered before organophosphate exposure, can protect from these toxic effects. We have tested at equitoxic dosage (25% of LD₀₁) the prophylactic efficacy of five experimental (K‐48, K‐53, K‐74, K‐75, K‐203) and two established oximes (pralidoxime and obidoxime) to protect from mortality induced by the organophosphate paraoxon. Mortalities were quantified by Cox analysis and compared with those observed after pretreatment with a strong acetylcholinesterase inhibitor (10‐methylacridine) and after the FDA‐approved pretreatment compound pyridostigmine. All nine tested substances statistically significantly reduced paraoxon‐induced mortality. Best protection was conferred by the experimental oxime K‐48, reducing the relative risk of death (RR) to 0.10, which was statistically significantly superior to pyridostigmine (RR = 0.31). The other oximes reduced the RR to 0.13 (obidoxime), 0.20 (K‐203), 0.21 (K‐74), 0.24 (K‐75) and 0.26 (pralidoxime), which were significantly more efficacious than 10‐methylacridine (RR = 0.65). These data support the hypothesis that protective efficacy is not primarily due to cholinesterase inhibition and indicate that the tested experimental oximes may be considered promising alternatives to the established pretreatment compound pyridostigmine.     

Assessment of nerve agent exposure: existing and emerging methods

The perceived threat of the use of nerve agents by terrorists against civilian targets implies the need for methods for point-of-care (POC) diagnosis. This review presents an overview of methods that are currently available for the assessment of exposure to nerve agents. Since these methods are mostly MS based, they require complex and expensive equipment and well-trained personnel and, consequently, they are not very suitable for rapid POC diagnosis. However, new technologies are emerging that allow, among others, immunochemical detection of acetylcholinesterase inhibited by nerve agents. Also, lab-on-a-chip methodologies are under development. It is anticipated that MS methods will be suitable for POC diagnosis within a few years, due to the miniaturization of equipment and the emergence of methodologies that enable mass spectrometric analysis with little sample pretreatment and that are potentially fieldable, such as direct analysis in real time and desorption electrospray ionization MS.     

Neurotoxicologic studies of two carbamate pesticides in subacute animal experiments

Two pesticides of the carbamate type, Carbaryl (1 naphthyl N methylcarbamate) and Arprocarb (2-isopropoxyphenyl-N-methylcarbamate) were tested for neurotoxicologic effects in subacute experiments using male white rats of the Wistar R strain. The authors examined the process of learning and performance of a previously learned task in a maze, and studied EEG patterns under resting conditions and under rhythmic light stimulation. The cholinesterase activity of the plasma, of erythrocytes and of various parts of the brain were recorded, body and organ weights were determined and histologic examinations were performed. In the neurotoxicologic examinations mild, but permanent and increasing, functional deviations of the nervous system were found, which could be readily demonstrated by the methods used. Carbaryl caused mild inhibition of the cholinesterase activity in various parts of the brain; the blood cholinesterase activity was practically unchanged. Protein content of the brain parts increased significantly after treatment with both compounds. No alterations were found in body and organ weights, respectively (except that of the adrenals), and no change was observed in the histologic examinations. Although the authors do not regard the agents as dangerous neurotoxic agents, they emphasize the necessity of observing all necessary precautions because subacute poisoning may occur. After exposure to these reversible cholinesterase inhibitors, the presence of a normal value for blood cholinesterase activity does not preclude the possibility of poisoning.     

Simulation of cholinesterase status at different scenarios of nerve agent exposure

The ongoing threat of homicidal use of organophosphorus-type chemical warfare agents ("nerve agents") during military conflicts and by terrorists underlines the necessity for effective medical countermeasures. Standard treatment with atropine and the established acetylcholinesterase (AChE) reactivators, obidoxime and pralidoxime, is considered to be ineffective with certain nerve agents due to low oxime effectiveness. From obvious ethical reasons only animal experiments can be used to evaluate new oximes as nerve agent antidotes. However, the extrapolation of data from animal to humans is hampered by marked species differences. Since reactivation of OP-inhibited AChE is considered to be the main mechanism of action of oximes, human erythrocyte AChE can be exploited to test the efficacy of new oximes. Recently, a dynamic computer model was developed which allows the calculation of AChE activities at different scenarios by combining enzyme kinetics (inhibition, reactivation, aging) with OP toxicokinetics and oxime pharmacokinetics. Now, this computer model was further extended by including the pharmaco- and enzyme kinetics of carbamate pretreatment. Simulations were performed for intravenous and percutaneous nerve agent exposure and intramuscular oxime treatment in the presence and absence of pyridostigmine pretreatment using published data. The model presented may serve as a tool for evaluating the impact of carbamate pretreatment on oxime-induced reactivation of inhibited AChE, for defining effective oxime concentrations and for optimizing oxime treatment. In addition, this model may be useful for the development of meaningful therapeutic strategies in animal experiments.     

Neuropsychological sequelae from acute poisoning and long-term exposure to carbamate and organophosphate pesticides

This research examines the effects of different degrees of pesticide exposure on neuropsychological performance. Exposures varied from acute poisoning coupled with chronic exposure to low or high levels of chronic exposure (defined by years of exposure). A cross-sectional neuropsychological and biochemical study was conducted in greenhouse farmers from southern Spain: data from 24 acutely poisoned workers and 40 non-poisoned but chronically (low or high) exposed sprayers were compared to 26 controls. We examined performance on 21 neuropsychological tests that assessed attention, memory, praxis, gnosis, motor coordination, naming and reasoning and also examined values of plasmatic cholinesterase. Results indicated statistically significant neuropsychological deficits in the acute poisoning and high chronic exposure groups after controlling for confounds, whereas similar performance was seen in the low chronic exposed subjects and controls. Subjects who were acutely poisoned performed worse than the other groups on perceptual, visuomotor, visual memory and mood state domains. Both the acutely poisoned and the chronically high exposed subjects obtained significantly lower scores in the perceptual, verbal memory and visuomotor domains. Levels of butyrylcholinesterase were related to the seasonal sprayer activity except in the case of acutely poisoned subjects. Conclusions: Both acutely poisoned long-term workers and chronically high (> 10 years) exposed workers exhibited similar disturbances in perception and visuo-motor processing, in the absence of any related acute effect of butyrylcholinesterase inhibition. In the case of acutely poisoned subjects, verbal and perceptive learning and recall and constructive abilities were also impaired. These results point to the need for follow-up studies to assess the possible sequelae of chronic and acute exposure to pesticides and their interactions.     

Quantification of nerve agent VX-butyrylcholinesterase adduct biomarker from an accidental exposure

The lack of data in the open literature on human exposure to the nerve agent O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothioate (VX) gives a special relevance to the data presented in this study in which we report the quantification of VX-butyrylcholinesterase adduct from a relatively low-level accidental human exposure. The samples were analyzed by gas chromatography-high resolution mass spectrometry using the fluoride ion regeneration method for the quantification of multiple nerve agents including VX. Six human plasma samples from the same individual were collected after the patient had been treated once with oxime immediately after exhibiting signs of exposure. Detection limits of approximately 5.5 pg/mL plasma were achieved for the G-analogue of VX (G-VX). Levels of the G-VX ranged from 81.4 pg/mL on the first day after the exposure to 6.9 pg/mL in the sample taken 27 days after the exposure. Based on the reported concentration of human butyrylcholinesterase in plasma of approximately 80 nM, it can be calculated that inhibition levels of >or= 0.05% of BuChE can be accurately quantified. These data further indicate that the fluoride ion regeneration method is a potentially powerful tool that can be used to assess low-level exposure to VX.     

Non-enzymatic pretreatment of nerve agent (soman) poisoning: a brief state-of-the-art review

The rapid onset of toxic signs following nerve agent intoxication and the apprehension that current therapy (atropine, oxime, diazepam) may not prevent brain damage, requires supportive pretreatment. Since the current pretreatment drug pyridostigmine fails in protecting brain-AChE, more effective pretreatment is necessary.A main focus of present-day pretreatment research is on bioscavengers, another is on centrally active reversible AChE-inhibitors combined with drugs showing anti-cholinergic, anti-glutamatergic, neuroprotective and non-sedating GABA-ergic activity. Strategies aimed at improving efficacy of pharmacological pretreatment will briefly be discussed. Galantamine, given as a pretreatment or stand-alone therapy, emerged as one of the best medical countermeasures against nerve agent poisoning in guinea pigs. Other preclinical studies demonstrated effective pretreatment consisting of physostigmine combined with procyclidine, scopolamine or bupropion (all single injections), against nerve agent poisoning in guinea pigs. A long sign-free pretreatment with physostigmine (Alzet pump), combined with single injection of procyclidine just before soman poisoning, enhanced the efficacy of a post-poisoning therapy consisting of 3 autoinjector equivalents of HI-6, atropine and diazepam, considerably.     

Protection against nerve agent poisoning by a noncompetitive nicotinic antagonist

The acute toxicity of organophosphorus (OP) nerve agents arises from accumulation of acetylcholine (ACh) and overstimulation of ACh receptors. The mainstay of current pharmacotherapy is the competitive muscarinic antagonist, atropine. Nicotinic antagonists have not been used due to the difficulties of administering a dose of a competitive neuromuscular blocker sufficient to antagonise the effects of excessive ACh, but not so much that it paralyses the muscles. An alternative approach would be to use a noncompetitive antagonist whose effects would not be overcome by increasing ACh concentrations. This study demonstrates that the compound 1,1′-(propane-1,3-diyl)bis(4-tert-butylpyridinium), which blocks open nicotinic ion channels noncompetitively, is able to reverse the neuromuscular paralysis after nerve agent poisoning in vitro and to protect guinea pigs against poisoning by nerve agents when used as part of a therapeutic drug combination including a muscarinic antagonist. In contrast to the oxime HI-6, this compound was equally effective in protecting against poisoning by sarin or tabun. Further studies should identify more effective compounds with this action and optimise doses for protection against nerve agent poisoning in vivo.     

Curcumin pretreatment protects against acute acrylonitrile-induced oxidative damage in rats

Acrylonitrile (AN) is widely used in the manufacturing of fibers, plastics and pharmaceuticals. Free radical-mediated lipid peroxidation is implicated in the toxicity of AN. The present study was designed to examine the ability of curcumin, a natural polyphenolic compound, to attenuate acute AN-induced lipid peroxidation in the brain and liver of rats. Male Sprague–Dawley rats were orally administered curcumin at doses of 0 (olive oil control), 50 or 100 mg/kg bodyweight daily for 7 consecutive days. Two hours after the last dose of curcumin, rats received an intraperitoneal injection of 50 mg AN/kg bodyweight. Acute exposure to AN significantly increased the generation of lipid peroxidation products, reflected by high levels of malondialdehyde (MDA) both in the brain and liver. These increases were accompanied by a significant decrease in reduced glutathione (GSH) content and a significant reduction in catalase (CAT) activity in the same tissues. No consistent changes in superoxide dismutase (SOD) activity were observed between the control and AN-treatment groups in both tissues. Pretreatment with curcumin reversed the AN-induced effects, reducing the levels of MDA and enhancing CAT activity and increasing reduced GSH content both in the brain and liver. Furthermore, curcumin effectively prevented AN-induced decrease in cytochrome c oxidase activity in both liver and brain. These results establish that curcumin pretreatment has a beneficial role in mitigating AN-induced oxidative stress both in the brains and livers of exposed rats and these effects are mediated independently of cytochrome P450 2E1 inhibition. Accordingly, curcumin should be considered as a potential safe and effective approach in attenuating the adverse effects produced by AN-related toxicants.     

Production and characterization of antibodies directed against organophosphorus nerve agent VX

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.     

Erythropoetin as a novel agent with pleiotropic effects against acute lung injury

Current pharmacotherapy for acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is not optimal, and the biological and physiological complexity of these severe lung injury syndromes requires consideration of combined-agent treatments or agents with pleiotropic action. In this regard, exogenous erythropoietin (EPO) represents a possible candidate since a number of preclinical studies have revealed beneficial effects of EPO administration in various experimental models of ALI. Taken together, this treatment strategy is not a single mediator approach, but it rather provides protection by modulating multiple levels of early signaling pathways involved in apoptosis, inflammation, and peroxidation, potentially restoring overall homeostasis. Furthermore, EPO appears to confer vascular protection by promoting angiogenesis. However, only preliminary studies exist and more experimental and clinical studies are necessary to clarify the efficacy and potentially cytoprotective mechanisms of EPO action. In addition to the attempts to optimize the dose and timing of EPO administration, it would be of great value to minimize any potential toxicity, which is essential for EPO to fulfill its role as a potential candidate for the treatment of ALI in routine clinical practice. The present article reviews recent advances that have elucidated biological and biochemical activities of EPO that may be potentially applicable for ALI/ARDS management.     

Protection of guinea pigs against soman poisoning with ferrocene carbamate

The protective effect of ferrocene carbamate pretreatment against soman poisoning was studied in guinea pigs. At doses corresponding to 1/20 x and 1/10 × LD₅₀ of this carbamate a 20% and 45% decrease of the acetylcholinesterase in blood and brain, respectively, was obtained. In combination with additional pretreatment, diazepam, and therapy, HI-6 and atropine, the protective ratios (LD₅₀ of soman in treated animals/LD₅₀ of soman in untreated animals) were around 20 and 40, respectively. Animals pretreated with the high dose of the ferrocene carbamate that survived 10 x and 15xLD_50s of soman showed no remaining signs of poisoning after 24 h. Thus, the ferrocene carbamate afforded a better protection against soman than physostigmine. The explanation for this could be due to the properties of the ferrocene carbamate, not correlated to its cholinesterase inhibiting activity. This hypothesis is discussed.     

Effects of the antituberculous agent ethambutol on myelinated nerve

The action of the antituberculous agent ethambutol on the myelinated nerve was studied in two ways: (1) Resting and compound action potentials of the desheathed sciatic nerve were measured; (2) Na⁺ and K⁺ currents through the node of Ranvier were investigated by means of the voltage clamp. Reversible dose-dependent decreases of the compound action potential and of the conduction velocity were observed when ethambutol was applied in concentrations of 1–7 mM for 15 min; prolongation up to 60 min caused irreversible changes in membrane function and a decrease in Ca content. Ethambutol induced a hyperpolarizing change of the resting potential. When the nerve was depolarized in Ca²⁺-free solutions, ethambutol repolarized the nerve membrane. Ethambutol instantly reduced sodium and potassium currents through the nodal membrane. Presumably ethambutol acts primarily as a Ca²⁺ chelating cation on negative charges at the nerve membrane.     

Improvements of the fluoride reactivation method for the verification of nerve agent exposure

One of the most appropriate biomarkers for the verification of organophosphorus nerve agent exposure is the conjugate of the nerve agent to butyrylcholinesterase (BuChE). The phosphyl moiety of the nerve agent can be released from the BuChE enzyme by incubation with fluoride ions, after which the resulting organophosphonofluoridate can be analyzed with gas chromatography-mass spectrometry (GC-MS). This paper describes recent improvements of the fluoride-induced reactivation in human plasma or serum samples by enhancing the sample preparation with new solid-phase extraction cartridges and the MS analysis with large volume injections. Analysis is performed with thermal desorption GC with either mass selective detection with ammonia chemical ionization or high-resolution MS with electron impact ionization. The organophosphorus chemical warfare agents analyzed in this study are O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate, ethyl methylphosphonofluoridate, isopropyl methylphosphonofluoridate (sarin, GB), O-ethyl N,N-dimethylphosphoramidocyanidate, ethyl N,N-dimethylphosphoramidofluoridate, and cyclohexyl methylphosphonfluoridate. Detection limits of approximately 10 pg/mL plasma were achieved for all analytes, which corresponds to 0.09% inhibition with GB on a sample with normal BuChE levels.