Effects of organophosphates on precision-cut kidney slices.

Organophosphate (OP) poisoning, both accidental and with suicidal intent, is a global medical challenge. While the primary toxicity of these pesticides is based on the inhibition of acetylcholinesterase (AChE), case reports describe patients developing OP-mediated renal insufficiency. We set out to investigate possible pathomechanisms utilizing rat precision-cut kidney slices (PCKS). Depending on the method of investigation, PCKS were observed for a maximum of 10 days. PCKS exposed to OP compounds (malaoxon, malathion, paraoxon, parathion) showed a dose-dependent loss of viability and a reduction of total protein content over the course of 10 days. A concentration of 500 µM OP showed the most differences between OP compounds. After two days of incubation parathion showed a significantly lower level of viability than malathion. The respective effects of paraoxon and malaoxon were not significantly different from control. However, effects of OP were only observed in concentrations exceeding those that were needed to achieve significant AChE inhibition in rat kidney tissue. In addition, we observed histological changes, without inducing LDH leakage. Overall, results suggest that OP exert effects in kidney tissue, that exceed those expected from the sole inhibition of AChE and vary between compounds. Without signs of necrosis, findings call for studies that address other possible pathomechanisms including inflammatory response, oxidative stress or activation of apoptosis to further understand nephrotoxicity of OP compounds. Monitoring oxon concentration over time, we demonstrated reduced enzyme inhibiting properties in the presence of PCKS, suggesting interactions between OP compound and kidney tissue.

Investigation of cardiac glycosides from oleander in a human induced pluripotent stem cells derived cardiomyocyte 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.

A case report of cholinesterase inhibitor poisoning: cholinesterase activities and analytical methods for diagnosis and clinical decision making.

Suicidal ingestion of organophosphorus (OP) or carbamate (CM) compounds challenges health care systems worldwide, particularly in Southeast Asia. The diagnosis and treatment of OP or CM poisoning is traditionally based on the clinical appearance of the typical cholinergic toxidrome, e.g. miosis, salivation and bradycardia. Yet, clinical signs might be inconclusive or even misleading. A current case report highlights the importance of enzymatic assays to provide rapid information and support clinicians in diagnosis and rational clinical decision making. Furthermore, the differentiation between OP and CM poisoning seems important, as an oxime therapy will most probably not provide benefit in CM poisoning, but-as every pharmaceutical product-it might result in adverse effects. The early identification of the causing agent and the amount taken up in the body are helpful in planning of the therapeutic regimen including experimental strategies, e.g. the use of human blood products to facilitate scavenging of the toxic agent. Furthermore, the analysis of biotransformation products and antidote levels provides additional insights into the pathophysiology of OP or CM poisoning. In conclusion, cholinesterase activities and modern analytical methods help to provide a more effective treatment and a thorough understanding of individual cases of OP or CM poisoning.

In vitro pharmacological characterization of the bispyridinium non-oxime compound MB327 and its 2- and 3-regioisomers.

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 [3H]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.

Development of MS Binding Assays targeting the binding site of MB327 at the nicotinic acetylcholine receptor.

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 [2H6]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 [2H6]MB327 from target-bound [2H6]MB327 in saturation and autocompetition experiments (employing native MB327 as competitor) enabled reliable determination of specific binding. In this way, the affinities for [2H6]MB327 (Kd=15.5±0.9µmolL-1) and for MB327 (Ki=18.3±2.6µmolL-1) towards the nAChR could be determined for the first time. The almost exactly matching affinities for MB327 and [2H6]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.

Functional analysis of Torpedo californica nicotinic acetylcholine receptors in multiple activation states by SSM-based electrophysiology.

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.

Investigations of kinetic interactions between lipid emulsions, hydroxyethyl starch or dextran and organophosphorus compounds.

CONTEXT: Numerous studies demonstrated a limited efficacy of clinically used oximes in case of poisoning by various organophosphorus compounds. A broad spectrum oxime antidote covering all organophosphorus nerve agents and pesticides is still missing and effective (bio-)scavengers have not yet been marketed. OBJECTIVE: The interactions of the available and clinically approved hydroxyethyl starch, dextran and lipid emulsions with organophosphorus nerve agents and pesticides were investigated in order to provide an in vitro base for the evaluation of these compounds in human organophosphorus poisoning. MATERIALS AND METHODS: The degradation kinetics of organophosphorus compounds by the glucose derivatives and lipid emulsions were investigated with an acetylcholinesterase inhibition assay. RESULTS: The incubation of organophosphorus compounds with TRIS-Ca(2+) buffer resulted in a time-dependent degradation of the nerve agents with half-lives of 42 min for cyclosarin, 49 min for sarin, 99 min for tabun, 107 min for soman 19 h for malaoxon and 54 h for VX. In contrast, incubation with all tested compounds resulted in a stabilisation of the organophosphorus compounds. DISCUSSION: Our results suggest that binding of lipophilic organophosphorus compounds could result in a reduced spontaneous and enzyme-induced degradation of the toxic compounds. CONCLUSION: High dose lipid emulsions and glucose derivatives stabilised organophosphorus compounds in vitro.

Affinities of bispyridinium non-oxime compounds to [(3)H]epibatidine binding sites of Torpedo californica nicotinic acetylcholine receptors depend on linker length.

The toxicity of organophosphorus nerve agents or pesticides arises from accumulation of acetylcholine and overstimulation of both muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs) due to inhibition of acetylcholinesterase (AChE). Standard treatment by administration of atropine and oximes, e.g., obidoxime or pralidoxime, focuses on antagonism of mAChRs and reactivation of AChE, whereas nicotinic malfunction is not directly treated. An alternative approach would be to use nAChR active substances to counteract the effects of accumulated acetylcholine. Promising in vitro and in vivo results were obtained with the bispyridinium compounds SAD-128 (1,1'-oxydimethylene bis(4-tert-butylpyridinium) dichloride) and MB327 (1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)), which were partly attributed to their interaction with nAChRs. In this study, a homologous series of unsubstituted and 4-tert-butyl-substituted bispyridinium compounds with different alkane linker lengths was investigated in competition binding experiments using [(3)H]epibatidine as a reporter ligand. Additionally, the effect of the well-characterised MB327 on the [(3)H]epibatidine equilibrium dissociation (KD) constant in different buffers was determined. This study demonstrated that divalent cations increased the affinity of [(3)H]epibatidine. Since quaternary ammonium molecules are known to inhibit AChE, the obtained affinity constants of the tested bispyridinium compounds were compared with the inhibition of human AChE. In competition experiments, bispyridinium derivatives of longer linker length displaced [(3)H]epibatidine and inhibited AChE strongly. Bispyridinium compounds with short linkers, at most, have an allosteric interaction with the [(3)H]epibatidine binding sites and barely inhibited AChE. In dependence on alkane linker length, the bispyridinium compounds seemed to interact at different binding sites. However, the exact binding sites of the bispyridinium compounds responsible for the positive pharmacological effects have still not been identified, making predictive drug design difficult.

Effect of MB327 and oximes on rat intestinal smooth muscle function.

Organophosphorous compounds (OP) are highly toxic compounds. Great efforts have been undertaken in the last decades to develop new reactivators of OP-inhibited acetylcholinesterase. So far, a broad-spectrum oxime bearing efficacy against all OP is still missing and alternative approaches are presently under investigation. Previous experiments demonstrated that the bispyridinium non-oxime MB327 was able to improve OP-impaired muscle force in human, rat and guinea pig respiratory muscles and to increase survival in soman, sarin and tabun poisoned guinea pigs. Recent studies indicate that MB327 exhibits a high affinity to muscarinic acetylcholine receptors but up to now, only scarce information is available on the effects of MB327 in isolated organs. Now, the antimuscarinic effect of MB327 was compared to that of established oximes and atropine in a rat jejunum smooth muscle model. MB327 showed a fully reversible smooth muscle relaxing effect at lower concentrations (EC50≈ 6 µM) than all tested oximes. In fact, MB327 exhibited an antimuscarinic smooth muscle relaxing effect at concentrations which were shown to improve OP-impaired skeletal muscle force. Hence, it may be assumed that the antimuscarinic potency of MB327 may contribute to its therapeutic effect in OP poisoning.

Functionalized cyclodextrins bearing an alpha nucleophile--a promising way to degrade nerve agents.

Organophosphorus nerve agents are irreversible inhibitors of acetylcholinesterase. Current treatment of nerve agent poisoning has limited efficacy and more efficient medical countermeasures need to be developed. A promising approach is to design chemical scavengers more stable during storage and less immunogenic than bioscavengers. Furthermore, they could be produced at lowest production costs. Cyclodextrins are attractive cyclic oligosaccharides that can be used to develop chemical scavengers of organophosphorus nerve agents. Their abilities to form inclusion and non-inclusion complexes with organic substrates are useful to trap chemical warfare agents. Selective introduction of an α-nucleophile residue on the secondary face of ß-cyclodextrin allowed to obtain supramolecular derivatives active against organophosphorus compounds. The degradation activity of these monosubstituted cyclodextrins was determined against paraoxon and chemical warfare agents. These tests showed that the structure of the scavengers mainly influences the interaction between the organophosphorus substrate, or its reaction products, and the cyclodextrin moiety. All the tested G-type agents were efficiently degraded. According to the binding modes of cyclosarin, some oligosaccharidic scavengers led to an enantioselective degradation of this nerve agent. These promising derivatives open the way to further investigations of new structural modifications to reach more sophisticated and efficient scavengers for prophylactic and curative medical applications.

Competition radioligand binding assays for the investigation of bispyridinium compound affinities to the human muscarinic acetylcholine receptor subtype 5 (hM(5) ).

Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes lacks efficacy with some nerve agents. Promising in vitro and in vivo results were obtained with the bispyridinium compound SAD-128 which was partly attributed to its interaction with nicotinic acetylcholine receptors. Previous studies indicate that bispyridinium compounds interact with muscarinic acetylcholine receptors as well. The muscarinic M(5) receptor is not well investigated compared to other subtypes, but could be important in the search for new drugs for treating nerve agent poisoning. A set of bispyridinium compounds structurally related to SAD-128 were tested in competition binding experiments with recombinant human M(5) muscarinic acetylcholine receptors. Five of the six investigated bispyridinium compounds interacted with the orthosteric binding site, with affinities in the low micromolar range. These data indicate that interaction of bispyridinium compounds with muscarinic receptors may contribute to their therapeutic efficacy.

Restoration of soman-blocked neuromuscular transmission in human and rat muscle by the bispyridinium non-oxime MB327 in vitro.

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.

Restoration of nerve agent inhibited muscle force production in human intercostal muscle strips with HI 6.

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. 30 µM 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 100 µM) had no effect on blocked muscle force or on inhibited human muscle AChE activity. In addition, HI 6 up to 1000 µM 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.

Interaction of bispyridinium compounds with the orthosteric binding site of human α7 and Torpedo californica nicotinic acetylcholine receptors (nAChRs).

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 > 400 µM). 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.

Immobilization of Russian VX skin depots by localized cooling: implications for decontamination and medical countermeasures.

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 100 µg/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) 15 min 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.

The therapeutic use of localized cooling in the treatment of VX poisoning.

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.

In vitro kinetic interactions of pyridostigmine, physostigmine and soman with erythrocyte and muscle acetylcholinesterase from different species.

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.

Pre- and post-treatment effect of physostigmine on soman-inhibited human erythrocyte and muscle acetylcholinesterase in vitro.

Standard treatment of organophosphorus (OP) poisoning includes administration of an antimuscarinic (e.g., atropine) and of an oxime-based reactivator. However, successful oxime treatment in soman poisoning is limited due to rapid aging of phosphylated acetylcholinesterase (AChE). Hence, the inability of standard treatment procedures to counteract the effects of soman poisoning resulted in the search for alternative strategies. Recently, results of an in vivo guinea pig study indicated a therapeutic effect of physostigmine given after soman. The present study was performed to investigate a possible pre- and post-treatment effect of physostigmine on soman-inhibited human AChE given at different time intervals before or after perfusion with soman by using a well-established dynamically working in vitro model for real-time analysis of erythrocyte and muscle AChE. The major findings were that prophylactic physostigmine prevented complete inhibition of AChE by soman and resulted in partial spontaneous recovery of the enzyme by de-carbamylation. Physostigmine given as post-treatment resulted in a time-dependent reduction of the protection from soman inhibition and recovery of AChE. Hence, these date indicate that physostigmine given after soman does not protect AChE from irreversible inhibition by the OP and that the observed therapeutic effect of physostigmine in nerve agent poisoning in vivo is probably due to other factors.

Effect of different buffers on kinetic properties of human acetylcholinesterase and the interaction with organophosphates and oximes.

Acetylcholinesterase (AChE) is the primary target of organophosphorus compounds (OP). The investigation into interactions between AChE, OP and oximes in vitro may be affected by the experimental conditions, e.g. by the buffer system. Hence, it was tempting to investigate the Michaelis-Menten kinetics and the inhibition and reactivation kinetics of paraoxon-ethyl, sarin, soman and VX in the presence of phosphate, MOPS, Tyrode and TRIS buffer with human AChE. Compared to phosphate buffer, the inhibition and reactivation kinetics of human erythrocyte AChE were markedly changed by TRIS and in part by MOPS, whereas Tyrode showed similar results to phosphate buffer. These results indicate an effect of the tested buffers on the properties of AChE, and an interaction between OP and oximes has to be considered for the design of in vitro studies and may impair the comparison of data from different laboratories. In view of the comparability of human in vitro kinetic data determined with phosphate buffer with data from human OP poisoning, it seems to be a suitable buffer for the investigation into interactions between AChE, OP and oximes.

Toxicokinetics of tabun enantiomers in anaesthetized swine after intravenous tabun administration.

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 3xLD(50) (161.4mug/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.