Cutaneous challenge with chemical warfare agents in the SKH-1 hairless mouse (II): effects of some currently used skin decontaminants (RSDL and Fuller's earth) against liquid sulphur mustard and VX exposure.

Using the hairless mouse screening model presented in the companion paper(1) the aim of this study was to assess two skin decontaminating systems: Fuller's earth (FE) and Reactive Skin Decontamination Lotion (RSDL) against two extremely toxic chemical warfare agents that represent a special percutaneous hazard, sulphur mustard (SM) and O-ethyl-S-(2[di-isopropylamino]ethyl)methyl-phosphonothioate (VX). Five minutes after being exposed on the back to either 2 µL of neat sulphur mustard or 50 µg.kg(-1) of diluted VX, mice were decontaminated. Both systems were able to reduce blisters 3 days after SM exposure. However, RSDL was found to be more efficient than FE in reducing the necrosis of the epidermis and erosion. In the case of VX exposure, RSDL, whatever the ratio of decontaminant to toxicant used (RSDL 10, 20, 50), was not able to sufficiently prevent the inhibition of plasma cholinesterases taken as a surrogate marker of exposure and toxicity. Only FE reduced significantly the ChE inhibition. Some of these observations are different from our previous results obtained in domestic swine and these changes are thus discussed in the perspective of using SKH-1 hairless mice for the initial in vivo screening of decontaminants.

Cutaneous challenge with chemical warfare agents in the SKH-1 hairless mouse. (I) Development of a model for screening studies in skin decontamination and protection.

Exposure to lethal chemical warfare agents (CWAs) is no longer only a military issue due to the terrorist threat. Among the CWAs of concern are the organophosphorus nerve agent O-ethyl-S-(2[di-isopropylamino]ethyl)methyl-phosphonothioate (VX) and the vesicant sulfur mustard (SM). Although efficient means of decontamination are available, most of them lose their efficacy when decontamination is delayed after exposure of the bare skin. Alternatively, CWA skin penetration can be prevented by topical skin protectants. Active research in skin protection and decontamination is thus paramount. In vivo screening of decontaminants or skin protectants is usually time consuming and may be expensive depending on the animal species used. We were thus looking for a suitable, scientifically sound and cost-effective model, which is easy to handle. The euthymic hairless mouse Crl: SKH-1 (hr/hr) BR is widely used in some skin studies and has previously been described to be suitable for some experiments involving SM or SM analogs. To evaluate the response of this species, we studied the consequences of exposing male anaesthetized SKH-1 mice to either liquid VX or to SM, the latter being used in liquid form or as saturated vapours. Long-term effects of SM burn were also evaluated. The model was then used in the companion paper (Taysse et al.(1)).

Pharmacokinetic analysis of pralidoxime after its intramuscular injection alone or in combination with atropine-avizafone in healthy volunteers.

BACKGROUND AND PURPOSE: Treatment of organophosphate poisoning with pralidoxime needs to be improved. Here we have studied the pharmacokinetics of pralidoxime after its intramuscular injection alone or in combination with avizafone and atropine using an auto-injector device. EXPERIMENTAL APPROACH: The study was conducted in an open, randomized, single-dose, two-way, cross-over design. At each period, each subject received either intramuscular injections of pralidoxime (700 mg), or two injections of the combination: pralidoxime (350 mg), atropine (2 mg), avizafone (20 mg). Pralidoxime concentrations were quantified using a validated LC/MS-MS method. Two approaches were used to analyse these data: (i) a non-compartmental approach; and (ii) a compartmental modelling approach. KEY RESULTS: The injection of pralidoxime combination with atropine and avizafone provided a higher pralidoxime maximal concentration than that obtained after the injection of pralidoxime alone (out of bioequivalence range), while pralidoxime AUC values were equivalent. Pralidoxime concentrations reached their maximal value earlier after the injection of the combination. According to Akaike and to goodness of fit criteria, the best model describing the pharmacokinetics of pralidoxime was a two-compartment with a zero-order absorption model. When avizafone and atropine were injected with pralidoxime, the best model describing pralidoxime pharmacokinetics becomes a two-compartment with a first-order absorption model. CONCLUSIONS AND IMPLICATIONS: The two approaches, non-compartmental and compartmental, showed that the administration of avizafone and atropine with pralidoxime results in a faster absorption into the general circulation and higher maximal concentrations, compared with the administration of pralidoxime alone.

Bioavailability of diazepam after intramuscular injection of its water-soluble prodrug alone or with atropine-pralidoxime in healthy volunteers.

BACKGROUND AND PURPOSE: The aim of this study was to assess the relative bioavailability of diazepam after administration of diazepam itself or as a water-soluble prodrug, avizafone, in humans. EXPERIMENTAL APPROACH: The study was conducted in an open, randomized, single-dose, three-way, cross-over design. Each subject received intramuscular injections of avizafone (20 mg), diazepam (11.3 mg) or avizafone (20 mg) combined with atropine (2 mg) and pralidoxime (350 mg) using a bi-compartmental auto-injector (AIBC). Plasma concentrations of diazepam were quantified using a validated LC/MS-MS assay, and were analysed by both a non-compartmental approach and by compartmental modelling. KEY RESULTS: The maximum concentration (C(max)) of diazepam after avizafone injection was higher than that obtained after injection of diazepam itself (231 vs. 148 ng.mL(-1)), while area under the curve (AUC) values were equal. Diazepam concentrations reached their maximal value faster after injection of avizafone. Injection of avizafone with atropine-pralidoxime (AIBC) had no effect on diazepam C(max) and AUC, but the time to C(max) was increased, relative to avizafone injected alone. According to the Akaike criterion, the pharmacokinetics of diazepam after injection as a prodrug was best described as a two-compartment with zero-order absorption model. When atropine and pralidoxime were injected with avizafone, the best pharmacokinetic model was a two-compartment with a first-order absorption model. CONCLUSION AND IMPLICATIONS: Diazepam had a faster entry to the general circulation and achieved higher C(max) after injection of prodrug than after the parent drug. Administration of avizafone in combination with atropine and pralidoxime by AIBC had no significant effect on diazepam AUC and C(max).

In vitro selection and efficacy of topical skin protectants against the nerve agent VX.

Against highly toxic chemicals that are quickly absorbed in the skin, topical formulations could adequately complement specific protective suits and equipments. In this work, we evaluated in vitro and compared the skin protection efficacy against the nerve agent VX of four different topical formulations: oil-in-water and water-in-oil emulsions, a perfluorinated-based cream and a hydrogel. Semi-permeable silicone membrane, pig-ear and human abdominal split-thickness skin samples mounted in diffusion cells were compared as in vitro permeation tests. The results showed that silicone membrane could be used instead of skin samples to screen for potentially effective formulations. However, the results indicated that due to potentially significant interactions between formulations and skin, relevant ranking of formulations according to their protective efficacy could require tests with skin samples. The main phase of emulsions, water or oil, was not found to be critical for skin protective efficacy against VX. Instead, specific film-forming ingredients such as perfluorinated-based polymers and silicones could significantly affect the skin protective efficacy of formulations. We showed that a hydrogel containing specific hydrophilic polymers was by far the most effective of the formulations evaluated against VX skin permeation in vitro.

[Interest of Ineurope syringe for nerve agent intoxication]. / Intérêt de la seringue Ineurope devant une intoxication par neurotoxique de guerre.

Chemical weapons represent an ever-growing threat, not only for military forces but also for civilian populations. Nerve agents such as those used in terrorist attacks by the Aum sect in Tokyo are among the deadliest of those non conventional weapons. The French military health service has developed a new auto-injector presenting as a self-usable dual-chamber syringe and successfully obtained a new drug approval to provide this new emergency treatment for the military and civilians. After a short review of the pathophysiology and clinical presentation of acute nerve agent, the authors report the development and the process of new drug application. They finally suggest a clinical guideline for practical use in case of terrorist attack.

Evaluation of in vitro tests to assess the efficacy of formulations as topical skin protectants against organophosphorus compounds.

Prevention of exposure to the neurotoxic organophosphorus compounds (OP) is a major concern both for pesticide users and soldiers. Skin barrier creams are being developed to complement or replace uncomfortable chemical protective suits. Quick evaluation of formulations efficacy mainly relies on in vitro tests which lead to consistent, complementary and relevant results. The objectives of this work were to determine the consistency of results from in vitro tests and importance of the formulation composition in the skin protective efficacy. The efficacy of three formulations, i.e. oil-in-water and water-in-oil emulsions and perfluorinated compounds-based cream, was evaluated against the OP paraoxon in vitro. Our results indicated that the least effective formulations could be quickly identified by performing in vitro permeation tests with silicone membrane and by evaluating interfacial interactions between formulations and OP. Among the tested formulations, the perfluorinated compounds-based cream could have a broader spectrum of efficacy than emulsions against OP and other toxic chemicals.

An unexpected plasma cholinesterase activity rebound after challenge with a high dose of the nerve agent VX.

Organophosphorus chemical warfare agents (nerve agents) are to be feared in military operations as well as in terrorist attacks. Among them, VX (O-ethyl-S-[2-(diisopropylamino)ethyl] methylphosphonothioate) is a low volatility liquid that represents a percutaneous as well as an inhalation hazard if aerosolized. It is a potent irreversible cholinesterase (ChE) inhibitor that causes severe signs and symptoms, including respiratory dysfunction that stems from different mechanisms. VX-induced pulmonary oedema was previously reported in dogs but mechanisms involved are not well understood, and its clinical significance remains to be assessed. An experimental model was thus developed to study VX-induced cardiovascular changes and pulmonary oedema in isoflurane-anaesthetized swine. In the course of this study, we observed a fast and unexpected rebound of plasma ChE activity following inhibition provoked by the intravenous injection of 6 and 12 microg kg(-1) of VX. In whole blood ChE activity, the rebound could stay unnoticed. Further investigations showed that the rebound of plasma esterase activity was neither related to spontaneous reactivation of ChE nor to VX-induced increase in paraoxonase/carboxylesterase activities. A bias in Ellman assay, haemoconcentration or severe liver cytolysis were also ruled out. All in all, these results suggest that the rebound was likely due to the release of butyrylcholinesterase into the blood stream from ChE producing organs. Nature of the organ(s) and mechanisms involved in enzyme release will need further investigations as it may represent a mechanism of defence, i.e. VX scavenging, that could advantageously be exploited.

Percutaneous penetration and distribution of VX using in vitro pig or human excised skin validation of demeton-S-methyl as adequate simulant for VX skin permeation investigations.

The organophosphorus (OP) chemical warfare V agent O-ethyl-S-[2(di-isopropylamino)ethyl] methyl phosphonothioate (VX), is a highly toxic compound which mainly penetrates the body via percutaneous pathways. Hence, the following prerequisite: to ascertain compound absorption and percutaneous profile distribution with a view to further assessing the efficacy of topical skin protectants. We first selected the most appropriate receptor fluid to carry out in vitro VX absorption experiments, namely: Hanks's Balanced Salt Solution (HBSS). After a 24-h topical exposure time lapse, we measured altogether the percentage of applied dose unabsorbed and absorbed, penetration rate, lag time, permeability coefficient (K(p)), and dose of VXeq present in skin. To such an end, we used full-thickness and split-thickness pig-ear or human abdominal skin membranes. Further, we scrutinised the potential use of two specific molecules as suitable surrogates for VX percutaneous penetration analyses: thus, we compared the present VX toxicokinetic parameters to earlier findings from our research unit, with respect to OP insecticides demethon-S-methyl (DSM) and paraoxon (POX). Within the framework of our study, we wish to highlight the following evidence: (a) pig-ear skin proves a relevant model to predict in vitro human abdominal skin, taking into account a 2-fold higher skin permeability to VXeq; (b) both full or split-thickness skin membranes may be used indiscriminately to gauge penetration rate and absorbed dose; (c) DSM applied on full-thickness pig-ear skin is the most relevant model to mimic the in vitro VX absorption through full-thickness skin model.

[Methods and models for percutaneous absorption studies of organophosphates]. / Méthodes et modèles d'étude de l'absorption percutanée des composés organophosphorés.

Organophosphates are highly toxic compounds, mainly penetrating the organism through the percutaneous route. In this context, having topical skin protectant capable of limitating their percutaneous penetration is of crucial importance for exposed people. Knowledge of the toxicokinetic parameters corresponding to the percutaneous penetration of these compounds is a key step for the development of these protective formulations. The different experimental approaches, in vivo and ex vivo, used to predict percutaneous penetration of these toxics are presented. The relevance and reliability of cutaneous experimental models are analysed.

Percutaneous penetration and absorption of parathion using human and pig skin models in vitro and human skin grafted onto nude mouse skin model in vivo.

This study determined and compared the percutaneous penetration and absorption of an organophosphorus (OP) pesticide, parathion (PA), using three experimental skin models: namely the human abdominal- and pig-ear skin in vitro models and the Human Skin grafted onto a nude mouse (HuSki) in vivo model. The percentage of topically applied dose absorbed and the doses present in the stratum corneum and skin were systematically determined at 24 h under similar experimental conditions. The three experimental skin models were first compared. Then, the advantages of the HuSki model for in vivo PA skin absorption studies were evaluated compared with the pig in vivo model previously used by others. Lastly, the relevance of each skin model to predict the permeability of human skin to PA in vivo was assessed by comparing our results with previously published in vivo human volunteer values. It was demonstrated that (a) pig-ear skin is relevant for predicting the in vitro human abdominal skin absorption taking into account a 2-3 times higher skin permeability to PA, (b) using ethanol as the vehicle, the absorption of PA was 4-5 times higher in the HuSki model than in the pig model but supports the usefulness of the HuSki model to easy mass balance studies, (c) both human in vitro and HuSki models closely predict the in vivo human volunteer absorption at 24 h when acetone is used as a vehicle but the HuSki model overcomes the known limitations of in vitro models for studying the fate of PA in the different skin layers after topical application.

[Overview on neurogenesis induced by organophosphate poisoning: results and perspectives]. / Neurogénèse réparatrice après intoxication par un neurotoxique organophosphoré. Bilan et perspectives.

Nerve agents could be potentially used as during a conflict or a terrorist attack. If an emergency treatment is not provided after poisoning, irreversible brain damages and behavioural sequels are expected to appear. In vivo cell therapy seems to be a promising approach for delayed treatment to contribute to brain repair. A mobilization of endogenous neural progenitors would be the basis of such an approach. After migrating, these progenitors would engraft in damaged brain regions and subsequently differentiate into functional neurons. In this review, after a few reminders regarding nerve agent poisoning and the emergency treatment of such an intoxication, progresses in terms of cell therapy and its potential application to nerve agent-induced brain lesions are summarized.

In vitro percutaneous penetration of organophosphorus compounds using full-thickness and split-thickness pig and human skin.

Organophosphorus compounds (OPs), such as pesticides and chemical warfare agents like sarin (GB), soman (GD) and VX, are highly toxic compounds. The OP vapours and their liquid forms are readily absorbed through the skin, therefore, protecting the skin of people who are potentially exposed to these agents is crucial. The development of effective countermeasures relies on a better knowledge of the percutaneous penetration of such molecules. The purpose of this present study is to determine the in vitro percutaneous penetration parameters of two pesticides DSM and DFP, as potential simulants of V and G agents, respectively, using four in vitro systems: full-thickness and split-thickness human abdominal and pig-ear skin membranes mounted on static diffusion cells. Based on the toxicokinetic parameters of the percutaneous penetration of DSM and DFP, we demonstrated that (a) pig-ear skin is a relevant model to predict the in vitro human skin permeability taking into account a 2-fold difference between these two species (b) both full and split-thickness skin membranes could be used indiscriminately, (c) DSM and DFP would be appropriate surrogates for V and G agents to perform skin permeation studies.

Swine models in the design of more effective medical countermeasures against organophosphorus poisoning.

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.

[Stem-cell engraftment as delayed therapy to repair organophosphate-induced brain damage]. / Utilisation potentielle des greffes de cellules souches pour le traitement différé des lésions cérébrales induites par les neurotoxiques organophosphorés.

Amongst organophosphate compounds, both pesticides and warfare neurotoxics are probably the most representative. These compounds are irreversible acetylcholinesterase inhibitors. Usual clinical signs observed after acute poisoning are mainly respiratory distress, convulsions and seizures. Following acute poisoning, an emergency treatment must be provided as soon as possible (maximum delay of 1 hour post-poisoning), to prevent irreversible brain damage and patient death. At the present time, there is no efficient delayed treatment which could be provided if this 1 hour latency is overpassed. However, neurogenesis by stem cell engraftment, eventually complemented by gene therapy strategy, could be a potential therapeutic approach to repair organophosphate-induced brain damage. Main stem cell engraftement strategies successfully used for brain damage of various origins are reviewed in this Article.

[Are acute toxicity testing and the three Rs rule reconcilable? Example of the lethal dose 50 determination]. / Les études de toxicité aiguë chez l'animal et la règle des trois R sont-elles conciliables? Exemple de la détermination des doses létales 50%

Toxicity assessment and demonstration of innocuousness of chemical compounds have been part of the research studies conducted in the fields of pharmacy, agriculture and chemical industry for years. Acute systemic toxicity studies are an important element of the safety evaluation. They remain compulsory for regulatory purposes and important for the public opinion that does not accept the risk anymore. Evolutions of the ethics in animal experiments foster a necessary reduction of the number of animals involved in this type of experiments, following the well-known principle of the three Rs rule of Russell and Burch (1959) (Reduction, refinement and replacement). These two views seem in contradiction. Using the example of acute toxicity testing and focusing on the now very criticized parameter lethal dose 50, we will present approaches, including statistical ones, that a toxicologist can use, when free to choose, to keep on conducting the indispensable in vivo studies while abiding by ethical recommendations.

Acute exposure to a low or mild dose of soman: biochemical, behavioral and histopathological effects.

Effects of low to mild doses of soman on central and blood cholinesterase (ChE) activities and anxiety behavior were studied in mice 30 min, 24 h and 7 days after poisoning. At these two latter time points, histopathological consequences of soman intoxication were also studied. The 30-microg/kg dose of soman produced 30 min after intoxication, about 35% of central ChE inhibition, and an anxiolytic effect without toxic signs or histopathological changes. The 50-microg/kg dose of soman produced at the same time, about 56% of central ChE inhibition, slight clinical signs of poisoning without convulsions, an anxiogenic effect with a slight hypolocomotion but no brain damage. A mild dose of soman (90 microg/kg) produced at this same time point about 80% of central ChE inhibition, and led to ataxia and tremors in every mouse and to convulsions in some of them. Thirty minutes and 24 h after poisoning, the behavioral tests revealed neither anxiolytic nor anxiogenic responses despite a clear hypolocomotion. Only mice that experienced long-lasting convulsions developed neuropathological changes. The functional implication of our results, as well as the biological relevance of blood vs. brain ChE levels, as an index of intoxication severity are discussed.

Delta activity as an early indicator for soman-induced brain damage: a review.

The organophosphorus (OP) compound soman is known to produce long-lasting epileptic seizure activity and associated brain damage. The present paper reviews the findings of five recent studies that tentatively established correlations between the development of soman-induced neuropathology and some subtle changes in the electrocortigraphic (ECoG) power spectrum. It is important to note that the reported experiments have been performed independently by three different teams (France, The Netherlands, USA) in various animal models (rat, guinea-pig, cynomolgus monkey) through different protocols of intoxication, pharmacological environments, and methods for ECoG spectral analysis. Despite these disparities, the five studies show that a suistained shift of ECoG power toward the lowest frequency range, i.e. the delta band, occurs within the first hours of soman-induced seizures. This early ECoG spectral change is concurrent with the first neuropathological changes in brain and is almost constantly followed, days or weeks later, by at least minimal neuropathology. Moreover the relative contribution of delta activity to the ECoG power spectrum still remains abnormally high for 1-3 days after seizure onset, i.e. within the phase of damage maturation. On the other hand, somnan-induced neuropathology was not observed in non-seizuring animals in which the delta activity was not increased above the pre-soman baseline. Similarly, no brain damage was ever shown in seizuring subjects in which the initial delta change eventually normalized after the curative administration of efficient anticonvulsant drugs such as the non-competitive antagonists of the NMDA receptor. These results, in agreement with previously published observations, strongly suggest that an increase of the relative power in the delta band might be a real-time marker of the ongoing development of soman-induced, seizure-related cerebral lesions and a reliable predictor for the final neuronal losses to come. Therefore, the monitoring of delta activity during the 24-72 h period that follows soman exposure may potentially be a useful tool to follow "on-line" the progression of brain damage and to control the neuroprotective activity of'a medication. Moreover since the method is non-invasive in man and since the above-presented results have been partly found in primates, the applicability of spectral analysis as a prognostic means in human OP poisoning ought to be seriously considered.

Subchronic administration of various pretreatments of nerve agent poisoning. I. Protection of blood and central cholinesterases, innocuousness towards blood-brain barrier permeability.

PYR, a reversible AChE inhibitor, is the current pretreatment against OP intoxication. However, PHY in the presence or absence of SCO on one side, and HUP on the other side, could be considered as potential substitutes for PYR. In the present study, the effects of the subchronic administration of these different current or potential pretreatments on the BBB permeability for blood-borne albumin and on the activity of the blood and central cholinesterases are comparatively evaluated in guinea-pigs. Altogether, although some marginal disruptions of BBB are detected, the different current or potential pretreatments studied seem to have a total innocuousness on the permeability of the BBB for proteins. Finally, at the light of its particular inhibitory effects on blood and central cholinesterases, HUP, compared to the other drugs, seems to be the optimal candidate to be used as pretreatment against OP poisoning.

Subchronic administration of various pretreatments of nerve agent poisoning. II. Compared efficacy against soman toxicity.

OP nerve agents, such as soman, are potent irreversible inhibitors of central and peripheral acetylcholinesterases. Pretreatment of OP poisoning relies on the subchronic administration of a reversible acetylcholinesterase inhibitor. In the present study, the protective effects against soman toxicity of such compounds i.e. pyridostigmine, physostigmine (alone or associated with scopolamine) or huperzine are compared in guinea-pigs instrumented for EEG recording. Each medication is given via a subcutaneous mini-osmotic pump for 6 days at a delivery rate providing about 30% maximal inhibition of red cell acetylcholinesterase activity. The animals then receive iterative injections of soman (1/3 LD50) every 10 min. With pyridostigmine, reflecting a decreased overall tolerance to the poisoning, the cumulative doses of soman producing either tremors and convulsions or seizures are lower than those found in non-pretreated intoxicated controls. On the other hand, physostigmine does not afford satisfactory protection against the early mortality after intoxication. On this specific point, physostigmine + scopolamine and huperzine, although they do not prevent the appearance of seizures, give best results. The effects of each pretreatment on acetylcholinesterase, butyrylcholinesterase and carboxylesterase (these two latter enzymes may act as endogenous scavengers of OP compounds) are also examined in vitro and in the blood of each animal during subchronic administration. Huperzine appears as a selective inhibitor of red cell acetylcholinesterase activity while pyridostigmine or physostigmine additionally inhibit plasmatic butyrylcholinesterase. Considerations about huperzine or physostigmine + scopolamine as the most appropriate candidate for the pretreatment of OP poisoning are given.