In Vitro Interaction of Organophosphono- and Organophosphorothioates with Human Acetylcholinesterase.

The implementation of the Chemical Weapons Convention (CWC) in 1997 was a milestone in the prohibition of chemical warfare agents (CWA). Yet, the repeated use of CWA underlines the ongoing threat to the population. Organophosphorus (OP) nerve agents still represent the most toxic CWA subgroup. Defensive research on nerve agents is mainly focused on the "classical five", namely tabun, sarin, soman, cyclosarin and VX, although Schedule 1 of the CWC covers an unforeseeable number of homologues. Likewise, an uncounted number of OP pesticides have been produced in previous decades. Our aim was to determine the in vitro inhibition kinetics of selected organophosphono- and organophosphorothioates with human AChE, as well as hydrolysis of the agents in human plasma and reactivation of inhibited AChE, in order to derive potential structure-activity relationships. The investigation of the interactions of selected OP compounds belonging to schedule 1 (V-agents) and schedule 2 (amiton) of the CWC with human AChE revealed distinct structural effects of the P-alkyl, P-O-alkyl and N,N-dialkyl residues on the inhibitory potency of the agents. Irrespective of structural modifications, all tested V-agents presented as highly potent AChE inhibitors. The high stability of the tested agents in human plasma will most likely result in long-lasting poisoning in vivo, having relevant consequences for the treatment regimen. In conclusion, the results of this study emphasize the need to investigate the biological effects of nerve agent analogues in order to assess the efficacy of available medical countermeasures.

Molecular modeling studies on the interactions of 7-methoxytacrine-4-pyridinealdoxime, 4-PA, 2-PAM, and obidoxime with VX-inhibited human acetylcholinesterase: a near attack conformation approach.

7-methoxytacrine-4-pyridinealdoxime (7-MEOTA-4-PA, named hybrid 5C) is a compound formerly synthesized and evaluated in vitro, together with 4-pyridine aldoxime (4-PA) and commercial reactivators of acetylcholinesterase (AChE). This compound was designed with the purpose of being a prophylactic reactivator, capable of interacting with different subdomains of the active site of AChE. To investigate these interactions, theoretical results from docking were first compared with experimental data of hybrid 5C, 4-PA, and two commercial oximes, on the reactivation of human AChE (HssAChE) inhibited by VX. Then, further docking studies, molecular dynamics simulations, and molecular mechanics Poisson-Boltzmann surface area calculations, were carried out to investigate reactivation performances, considering the near attack conformation (NAC) approach, prior to the nucleophilic substitution mechanism. Our results helped to elucidate the interactions of such molecules with the different subdomains of the active site of HssAChE. Additionally, NAC poses of each oxime were suggested for further theoretical studies on the reactivation reaction.

Efficacy of atropine sulfate/obidoxime chloride co-formulation against sarin exposure in guinea pigs.

The efficacy and pharmacokinetics of the aqueous co-formulation contents of the Trobigard™ (atropine sulfate, obidoxime chloride) auto-injector were evaluated in a sarin exposed guinea pig model. Two subcutaneous (sc) sarin challenge doses were evaluated in guinea pigs instrumented with brain and heart electrodes for electroencephalogram (EEG) and electrocardiogram (ECG). Sarin challenge doses were chosen to reflect exposure subclasses with sublethal (moderate to severe clinical signs) and lethal consequences. The level of protection of intramuscular human equivalent doses of the co-formulation was defined by (1) the mitigation of signs and symptoms at a sublethal level and (2) the increase of survival time at the supralethal sarin dose levels. Pharmacokinetics of both atropine sulfate and obidoxime were proportional at 1 and 3 human equivalent doses, and only a small increase in heart rate was observed briefly as a side effect. At both sarin challenge doses, 54 µg/kg and 84 µg/kg, the co-formulation treatment was effective against sarin-induced effects. Survival rates were improved at both sarin challenge levels, whereas clinical signs and changes in EEG activity could not in all cases be effectively mitigated, in particular at the supralethal sarin challenge dose level. Reactivation of sarin inhibited cholinesterase was observed in blood, and higher brain cholinesterase activity levels were associated with a better clinical condition of the co-formulation treated animals. Although the results cannot be directly extrapolated to the human situation, pharmacokinetics and the effects over time related to plasma levels of therapeutics in a freely moving guinea pig could aid translational models and possibly improve prediction of efficacy in humans.

Synthesis, Biological Evaluation, and Docking Studies of Novel Bisquaternary Aldoxime Reactivators on Acetylcholinesterase and Butyrylcholinesterase Inhibited by Paraoxon.

Nerve agents and oxon forms of organophosphorus pesticides act as strong irreversible inhibitors of two cholinesterases in the human body: acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8), and are therefore highly toxic compounds. For the recovery of inhibited AChE, antidotes from the group of pyridinium or bispyridinium aldoxime reactivators (pralidoxime, obidoxime, HI-6) are used in combination with anticholinergics and anticonvulsives. Therapeutic efficacy of reactivators (called "oximes") depends on their chemical structure and also the type of organophosphorus inhibitor. Three novel oximes (K131, K142, K153) with an oxime group in position four of the pyridinium ring were designed and then tested for their potency to reactivate human (Homo sapiens sapiens) AChE (HssACHE) and BChE (HssBChE) inhibited by the pesticide paraoxon (diethyl 4-nitrophenyl phosphate). According to the obtained results, none of the prepared oximes were able to satisfactorily reactivate paraoxon-inhibited cholinesterases. On the contrary, extraordinary activity of obidoxime in the case of paraoxon-inhibited HssAChE reactivation was confirmed. Additional docking studies pointed to possible explanations for these results.

Unbinding of fluorinated oxime drug from the AChE gorge in polarizable water: a well-tempered metadynamics study.

Despite the fact that fluorination makes a drug more lipophilic, the molecular level understanding of protein-fluorinated drug interactions is very poor. Due to their enhanced ability to penetrate the blood brain barrier, they are suitable for reactivation of organophosphorus inactivated acetylcholinesterase (AChE) in the central nervous system. We systematically studied the unbinding of fluorinated obidoxime (FOBI) and non-fluorinated obidoxime (OBI) from the active site gorge of the serine hydrolase AChE in mean field polarizable water by employing all atom molecular dynamics simulations. It is observed that the unbinding process is strongly influenced by cation-π, hydrogen bond (HB) and water bridge interactions. The FOBI drug interacts more strongly with the protein residues than OBI and this is also verified from quantum mechanical calculations. Distinct unbinding pathways for FOBI and OBI are observed as evident from the 1D and 2D potential of mean force of the unbinding profiles. The present study suggests that the FOBI drug is held more firmly in the gorge of AChE in comparison to OBI and may lead to higher reactivation efficiency of the inactivated enzyme.

Synthesis and in vitro reactivation study of isonicotinamide derivatives of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide as reactivators of Sarin and VX inhibited human acetylcholinesterase (hAChE).

Previously (Karade et al., 2014), we have reported the synthesis and in vitro evaluation of bis-pyridinium derivatives of pyridine-3-yl-(2-hydroxyimino acetamide), as reactivators of sarin and VX inhibited hAChE. Few of the molecules showed superior in vivo protection efficacy (mice model) (Kumar et al., 2014; Swami et al., 2016) in comparison to 2-PAM against DFP and sarin poisoning. Encouraged by these results, herein we report the synthesis and in vitro evaluation of isonicotinamide derivatives of pyridine-3-yl-(2-hydroxyimino acetamide) (4a-4d) against sarin and VX inhibited erythrocyte ghost hAChE. Reactivation kinetics of these compounds was studied and the determined kinetic parameters were compared with that of commercial reactivators viz. 2-PAM and obidoxime. In comparison to 2-PAM and obidoxime, oxime 4a and 4b exhibited enhanced reactivation efficacy toward sarin inhibited hAChE while oxime 4c showed far greater reactivation efficacy toward VX inhibited hAChE. The acid dissociation constant and IC50 values of these oximes were determined and correlated with the observed reactivation potential.

Unbinding free energy of acetylcholinesterase bound oxime drugs along the gorge pathway from metadynamics-umbrella sampling investigation.

Because of the pivotal role that the nerve enzyme, acetylcholinesterase plays in terminating nerve impulses at cholinergic synapses. Its active site, located deep inside a 20 Å gorge, is a vulnerable target of the lethal organophosphorus compounds. Potent reactivators of the intoxicated enzyme are nucleophiles, such as bispyridinium oxime that binds to the peripheral anionic site and the active site of the enzyme through suitable cation-π interactions. Atomic scale molecular dynamics and free energy calculations in explicit water are used to study unbinding pathways of two oxime drugs (Ortho-7 and Obidoxime) from the gorge of the enzyme. The role of enzyme-drug cation-π interactions are explored with the metadynamics simulation. The metadynamics discovered potential of mean force (PMF) of the unbinding events is refined by the umbrella sampling (US) corrections. The bidimensional free energy landscape of the metadynamics runs are further subjected to finite temperature string analysis to obtain the transition tube connecting the minima and bottlenecks of the unbinding pathway. The PMF is also obtained from US simulations using the biasing potential constructed from the transition tube and are found to be consistent with the metadynamics-US corrected results. Although experimental structural data clearly shows analogous coordination of the two drugs inside the gorge in the bound state, the PMF of the drug trafficking along the gorge pathway point, within an equilibrium free energy context, to a multistep process that differs from one another. Routes, milestones and subtlety toward the unbinding pathway of the two oximes at finite temperature are identified.

Zn(II)/pyridyloxime complexes as potential reactivators of OP-inhibited acetylcholinesterase: in vitro and docking simulation studies.

In order to investigate the ability of metal complexes to act as reactivators of organophosphorus compounds (OP)-inhibited acetylcholinesterase (AChE), we have synthesized and crystallographically characterized three novel mononuclear Zn(II) complexes formulated as [ZnCl2{(4-py)CHNOH}2] (1), [ZnBr2{(4-py)CHNOH}2] (2) and [Zn(O2CMe)2{(4-py)CHNOH}2]∙2MeCN (3∙2MeCN), where (4-py)CHNOH is 4-pyridinealdoxime. Their reactivation potency was tested in vitro with a slight modification of the Ellman's method using Electric eel acetylcholinesterase and the insecticide paraoxon (diethyl 4-nitrophenyl phosphate) as inhibitor. The activity of the already reported complex [Zn2(O2CPh)2{(4-py)CHNOH}2]·2MeCN (4·2MeCN) and of the clinically used drug obidoxime 1,1'-[oxybis(methylene)]bis{4-[(E)- (hydroxyimino)methyl]pyridinium} was also examined. The results of the in vitro experiments demonstrate moderate reactivation of the metal complexes compared to the drug obidoxime. On the other hand, it is clearly shown that the metal complex is the responsible molecular entity for the observed activity, as the reactivation efficacy of the organic ligand (4-pyridinealdoxime) is found to be inconsequential. Docking simulation studies were performed in the light of predicted complex-enzyme interactions using the paraoxon-inhibited enzyme along with the four Zn(II) complexes and obidoxime as a reference reactivator. The results showed that the three mononuclear metal complexes possess the required characteristics to be accommodated into the active site of AChE, while the entrance of the dinuclear Zn(II) compound is unsuccessful. An interesting outcome of docking simulations is the fact that the mononuclear compounds accommodate into the active site of AChE in a similar mode as obidoxime.

In vitro reactivation kinetics of paraoxon- and DFP-inhibited electric eel AChE using mono- and bis-pyridinium oximes.

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

[Studying kinetics of oxime-induced reactivation of malathion-inhibited cholinesterase].

The kinetics of oxime-induced reactivation of malathion-inhibited cholinesterase has been experimentally studied in vitro. It is shown that oximes do not restore the activity of inhibited butyrylcholinesterase. Acetylcholinesterase reactivation peak (5-mins long) was found to take place upon introduction of dipyroxime (32.5%), pralidoxime (18%), carboxyme (16%) at a concentration of 2.5 x 10(-4) mol/l or toxogonine (26%) at a concentration of 5 x 10(-4) mol/l. Toxogonine demonstrated the maximum affinity to phosphorylated enzyme, while dipyroxime is characterized by a high reactivity with respect to oxime. Significant reactivating ability of these preparations (kR -2300 mol(-1) min(-1) makes them promising solution for the treatment of malathion intoxication.

A common mechanism for resistance to oxime reactivation of acetylcholinesterase inhibited by organophosphorus compounds.

Administration of oxime therapy is currently the standard approach used to reverse the acute toxicity of organophosphorus (OP) compounds, which is usually attributed to OP inhibition of acetylcholinesterase (AChE). Rate constants for reactivation of OP-inhibited AChE by even the best oximes, such as HI-6 and obidoxime, can vary >100-fold between OP-AChE conjugates that are easily reactivated and those that are difficult to reactivate. To gain a better understanding of this oxime specificity problem for future design of improved reactivators, we conducted a QSAR analysis for oxime reactivation of AChE inhibited by OP agents and their analogues. Our objective was to identify common mechanism(s) among OP-AChE conjugates of phosphates, phosphonates and phosphoramidates that result in resistance to oxime reactivation. Our evaluation of oxime reactivation of AChE inhibited by a sarin analogue, O-methyl isopropylphosphonofluoridate, or a cyclosarin analogue, O-methyl cyclohexylphosphonofluoridate, indicated that AChE inhibited by these analogues was at least 70-fold more difficult to reactivate than AChE inhibited by sarin or cyclosarin. In addition, AChE inhibited by an analogue of tabun (i.e., O-ethyl isopropylphosphonofluoridate) was nearly as resistant to reactivation as tabun-inhibited AChE. QSAR analysis of oxime reactivation of AChE inhibited by these OP compounds and others suggested that the presence of both a large substituent (i.e., ≥ the size of dimethylamine) and an alkoxy substituent in the structure of OP compounds is the common feature that results in resistance to oxime reactivation of OP-AChE conjugates whether the OP is a phosphate, phosphonate or phosphoramidate.

Detoxification of tabun at physiological pH mediated by substituted ß-cyclodextrin and glucose derivatives containing oxime groups.

The ability of 13 ß-cyclodextrin and 2 glucose derivatives containing substituents with oxime groups as nucleophilic components to accelerate the degradation of tabun at physiological pH has been evaluated. To this end, a qualitative and a quantitative enzymatic assay as well as a highly sensitive enantioselective GC-MS assay were used. In addition, an assay was developed that provided information about the mode of action of the investigated compounds. The results show that attachment of pyridinium-derived substituents with an aldoxime group in 3- or 4-position to a ß-cyclodextrin ring affords active compounds mediating tabun degradation. Activities differ depending on the structure, the number, and the position of the substituent on the ring. Highest activity was observed for a ß-cyclodextrin containing a 4-formylpyridinium oxime residue in 6-position of one glucose subunit, which detoxifies tabun with a half-time of 10.2 min. Comparison of the activity of this compound with that of an analog in which the cyclodextrin ring was replaced by a glucose residue demonstrated that the cyclodextrin is not necessary for activity but certainly beneficial. Finally, the results provide evidence that the mode of action of the cyclodextrin involves covalent modification of its oxime group rendering the scavenger inactive after reaction with the first tabun molecule.

Assessment of acetylcholinesterase activity using indoxylacetate and comparison with the standard Ellman's method.

Assay of acetylcholinesterase (AChE) activity plays an important role in diagnostic, detection of pesticides and nerve agents, in vitro characterization of toxins and drugs including potential treatments for Alzheimer's disease. These experiments were done in order to determine whether indoxylacetate could be an adequate chromogenic reactant for AChE assay evaluation. Moreover, the results were compared to the standard Ellman's method. We calculated Michaelis constant Km (2.06 × 10(-4) mol/L for acetylthiocholine and 3.21 × 10(-3) mol/L for indoxylacetate) maximum reaction velocity V(max) (4.97 × 10(-7) kat for acetylcholine and 7.71 × 10(-8) kat for indoxylacetate) for electric eel AChE. In a second part, inhibition values were plotted for paraoxon, and reactivation efficacy was measured for some standard oxime reactivators: obidoxime, pralidoxime (2-PAM) and HI-6. Though indoxylacetate is split with lower turnover rate, this compound appears as a very attractive reactant since it does not show any chemical reactivity with oxime antidots and thiol used for the Ellman's method. Thus it can be advantageously used for accurate measurement of AChE activity. Suitability of assay for butyrylcholinesterase activity assessment is also discussed.

Benefits of butyrylcholinesterase reactivability testing in organophosphate poisoning.

Organophosphate (OP) poisoning continues to represent an important medical issue through its high prevalence among toxic pathologies and through its severity. In diagnosing this toxicological disorder, the most frequently utilized and available laboratory test remains the assessment of plasma cholinesterase - butyrylcholinesterase (BChE) - activity. Despite the reluctance of many researchers on the usefulness of serum BChE for kinetic analysis in OP intoxications, we have tested a recently proposed protocol, which is safe, non-expensive, easy to perform, appropriate to distinguish between an aged cholinesterase and a still reactivable one. Our aim was to validate the usefulness of this protocol, studying a series of 23 consecutive patients acutely intoxicated with OP, admitted in a regional Emergency Hospital, over a 1-year period. Introducing the proposed test in the routine of monitoring OP-intoxicated patients has resulted in the identification of a pattern with a funnel aspect, consequence of the initial possibility to increment the degree of BChE activity. This funnel shape defines the presence of reactivability, while its absence demonstrates the lack of obidoximes effect, due to cholinesterase's ageing process. This method consisted in an advantage for the diagnosis, having the potential of improving prognostic evaluation and therapeutic orientation in OP intoxications.

Incorporation of obidoxime into human serum albumin nanoparticles: optimisation of preparation parameters for the development of a stable formulation.

Intoxication with organophosphorous nerve agents such as paraoxon requires immediate administration of antidotes such as oximes. However, the oximes lack sufficient activity in the central nervous system as they are unable to rapidly penetrate the blood-brain barrier (BBB) in therapeutically relevant concentrations. Human serum albumin (HSA) nanoparticles represent a promising drug carrier system for the transport of drugs across the BBB. This study focussed on the development of an obidoxime-loaded nanoparticles prepared by desolvation using an incorporation technique. The nanoparticle preparation parameters, i.e. drug amount, pH value, ethanol volume and crosslinking degree, were optimised. The in vitro release study showed a sustained release profile, indicating the suitability of the developed formulation for the transport of oximes across the BBB.

Characterization of the anticholinergic properties of obidoxime; functional examinations of the rat atria and the urinary bladder.

Obidoxime, a well-known bis-pyridinium reactivator, is often the preferred antidote of organophosphorus poisoning caused by pesticides and tabun. It is also considered to be an allosteric modulator of muscarinic receptors, preferably M2 sub-type. This study compared the effect of obidoxime and atropine in vivo and in vitro on the cholinergic stimulation of the rat heart (M2) and the urinary bladder (M3). The results showed that obidoxime exerts anti-muscarinic effects, that may play an important role in the treatment of organophosphourus poisoning, and that the muscarinic receptor inhibition profile shows M2 receptor selectivity. This anti-muscarinic effect is much smaller that the effect of atropine and might be due to the allosteric inhibition of the receptors. The results also indicate that the acetylcholinesterase inhibition and the muscarinic receptor antagonism occur at different concentrations and dose levels.

Adsorption of obidoxime onto human serum albumin nanoparticles: drug loading, particle size and drug release.

The standard treatment of poisoning by organophosphorous compounds such as paraoxon includes the administration of oximes. Due to their inability to rapidly cross the blood-brain barrier (BBB) in therapeutically relevant concentrations, these drugs possess insufficient activity in the central nervous system. Since human serum albumin (HSA) nanoparticles enable the delivery of a variety of drugs across the BBB into the brain, in the present study the antidote obidoxime was bound to these particles by adsorption. The resulting sorption isotherms showed a best fit to Langmuir isotherms indicating that obidoxime adsorbs to HSA nanoparticles forming a monolayer. A maximum drug loading of 93.5 microg obidoxime/mg of nanoparticles at pH 8.3 was calculated. At higher concentrations the particle diameter increased significantly with obidoxime concentration leading to instable particle systems. The in vitro release of obidoxime from HSA nanoparticles showed a rapid release of the drug from the nanoparticles within 3 h.

Kinetic analysis of interactions of different sarin and tabun analogues with human acetylcholinesterase and oximes: is there a structure-activity relationship?

The repeated misuse of highly toxic organophosphorus compound (OP) based chemical warfare agents in military conflicts and terrorist attacks poses a continuous threat to the military and civilian sector. The toxic symptomatology of OP poisoning is mainly caused by inhibition of acetylcholinesterase (AChE, E.C. 3.1.1.7) resulting in generalized cholinergic crisis due to accumulation of the neurotransmitter acetylcholine (ACh) in synaptic clefts. Beside atropine as competitive antagonist of ACh at muscarinic ACh receptors oximes as reactivators of OP-inhibited AChE are a mainstay of standard antidotal treatment. However, human AChE inhibited by certain OP is rather resistant to oxime-induced reactivation. The development of more effective oxime-based reactivators may fill the gaps. To get more insight into a potential structure-activity relationship between human AChE, OPs and oximes in vitro studies were conducted to investigate interactions of different tabun and sarin analogues with human AChE and the oximes obidoxime and HI 6 by determination of various kinetic constants. Rate constants for the inhibition of human AChE by OPs, spontaneous dealkylation and reactivation as well as reactivation by obidoxime and HI 6 of OP-inhibited human AChE were determined. The recorded kinetic data did not allow a general statement concerning a structure-activity relationship between human AChE, OP and oximes.

A comparison of neuroprotective efficacy of the oxime K203 and its fluorinated analogue (KR-22836) with obidoxime in Tabun-poisoned rats.

The ability of the newly developed bispyridinium compound K203 and its fluorinated analogue KR-22836 to reduce tabun-induced acute neurotoxic signs and symptoms was compared with the currently available reactivator of acetylcholinesterase-obidoxime. Tabun-induced neurotoxicity and the neuroprotective effects of all tested oximes in combination with atropine in rats poisoned with tabun at a sublethal dose (200 µg/kg intramuscularly (i.m.); 80% of LD(50) value) were monitored by a functional observational battery at 24 hr after tabun challenge. The results indicate that all tested oximes combined with atropine were able to survive tabun-poisoned rats 24 hr after tabun challenge while one non-treated tabun-poisoned rat died within 24 hr after tabun poisoning. All tested oximes combined with atropine were able to decrease tabun-induced neurotoxicity in the case of sublethal poisoning but they did not eliminate all tabun-induced acute neurotoxic signs and symptoms. While the ability to reduce tabun-induced acute neurotoxicity of obidoxime and K203 was similar, the neuroprotective efficacy of KR-22836 was slightly higher compared to other tested oximes. Thus, the newly developed fluorinated analogue of K203, called KR-22836, is able to slightly increase the neuroprotective effectiveness of antidotal treatment of acute tabun poisonings compared to K203 and currently available obidoxime.

Suitability of human butyrylcholinesterase as therapeutic marker and pseudo catalytic scavenger in organophosphate poisoning: a kinetic analysis.

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.