On-site analysis of acetylcholinesterase and butyrylcholinesterase activity with the ChE check mobile test kit-Determination of reference values and their relevance for diagnosis of exposure to organophosphorus compounds.

Poisoning by organophosphorus compounds (OP) still poses a major medical challenge. Diagnosis of clinical signs of OP poisoning is still the most important parameter for the initiation of specific treatment. However, in case of unspecific signs and of delayed onset of cholinergic crisis a rapid, reliable and on-site analysis of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity would be of great value. Recently the ChE check mobile, a CE-certified ready to use kit for the determination of whole blood AChE and BChE activities, was developed. Here, we evaluated whole blood AChE and BChE reference values with samples taken from 181 male and 61 female volunteers and analyzed them on-site with the ChE check mobile test kit. The analysis of the data revealed a large inter-individual variability (BChE>AChE), only a small sex difference for AChE but a significant difference for BChE activities. The now available normal range values enable an evaluation of determined AChE and BChE activities in case of suspected exposure to OP nerve agents and pesticides. However, the large inter-individual variability of AChE and BChE activities calls for the determination of pre-exposure values in specific subpopulations in order to enable the diagnosis of low-level OP exposure.

Kinetic prerequisites of oximes as effective reactivators of organophosphate-inhibited acetylcholinesterase: a theoretical approach.

The standard treatment of poisoning by organophosphorus compounds (OP) includes the reversible muscarine receptor antagonist atropine and oximes for the reactivation of OP-inhibited acetylcholinesterase (AChE). There is an ongoing discussion on the benefit of oxime therapy in OP pesticide poisoning, and experimental data indicate a limited efficacy of oximes against various nerve agents. Oxime effectiveness can be quantified in vitro by determination of the reactivity (k(r)) and affinity constants (1/K(D)). These constants can be used to calculate reactivation velocities and oxime concentrations necessary for the reactivation of a desired fraction of inhibited AChE. Model calculations indicate that a k(r) > 0.1 min(-1) and K(D) < 100 µM are minimal requirements for oxime effectiveness when reactivation is performed in the absence of free OP. In addition, the findings demonstrate that selective increase of either reactivity or affinity of an oxime would be insufficient. Hereby, it has to be taken into account that an increase of affinity to OP-inhibited AChE is generally accompanied by an increased affinity to native AChE and subsequent reduction in oxime tolerance. Hence, future developments of more effective oximes should consider kinetic demands by attempting to achieve a certain level of reactivity and affinity, preferentially towards OP-inhibited AChE.

Kinetic analysis of interactions of paraoxon and oximes with human, Rhesus monkey, swine, rabbit, rat and guinea pig acetylcholinesterase.

Previous in vitro studies showed marked species differences in the reactivating efficiency of oximes between human and animal acetylcholinesterase (AChE) inhibited by organophosphorus (OP) nerve agents. These findings provoked the present in vitro study which was designed to determine the inhibition, aging, spontaneous and oxime-induced reactivation kinetics of the pesticide paraoxon, serving as a model compound for diethyl-OP, and the oximes obidoxime, pralidoxime, HI 6 and MMB-4 with human, Rhesus monkey, swine, rabbit, rat and guinea pig erythrocyte AChE. Comparable results were obtained with human and monkey AChE. Differences between human, swine, rabbit, rat and guinea pig AChE were determined for the inhibition and reactivation kinetics. A six-fold difference of the inhibitory potency of paraoxon with human and guinea pig AChE was recorded while only moderate differences of the reactivation constants between human and animal AChE were determined. Obidoxime was by far the most effective reactivator with all tested species. Only minor species differences were found for the aging and spontaneous reactivation kinetics. The results of the present study underline the necessity to determine the inhibition, aging and reactivation kinetics in vitro as a basis for the development of meaningful therapeutic animal models, for the proper assessment of in vivo animal data and for the extrapolation of animal data to humans.

Reactivation of organophosphate-inhibited human, Cynomolgus monkey, swine and guinea pig acetylcholinesterase by MMB-4: a modified kinetic approach.

Treatment of poisoning by highly toxic organophosphorus compounds (OP, nerve agents) is a continuous challenge. Standard treatment with atropine and a clinically used oxime, obidoxime or pralidoxime is inadequate against various nerve agents. For ethical reasons testing of oxime efficacy has to be performed in animals. Now, it was tempting to investigate the reactivation kinetics of MMB-4, a candidate oxime to replace pralidoxime, with nerve agent-inhibited acetylcholinesterase (AChE) from human and animal origin in order to provide a kinetic basis for the proper assessment of in vivo data. By applying a modified kinetic approach, allowing the use of necessary high MMB-4 concentrations, it was possible to determine the reactivation constants with sarin-, cyclosarin-, VX-, VR- and tabun-inhibited AChE. MMB-4 exhibited a high reactivity and low affinity towards OP-inhibited AChE, except of tabun-inhibited enzyme where MMB-4 had an extremely low reactivity. Species differences between human and animal AChE were low (Cynomolgus) to moderate (swine, guinea pig). Due to the high reactivity of MMB-4 a rapid reactivation of inhibited AChE can be anticipated at adequate oxime concentrations which are substantially higher compared to HI-6. Additional studies are necessary to determine the in vivo toxicity, tolerability and pharmacokinetics of MMB-4 in humans in order to enable a proper assessment of the value of this oxime as an antidote against nerve agent poisoning.

Comparative study of oxime-induced reactivation of erythrocyte and muscle AChE from different animal species following inhibition by sarin or paraoxon.

Standard treatment of acute poisoning by organophosphorus compounds (OP) includes administration of an antimuscarinic (e.g. atropine) and of an oxime-based reactivator of OP-inhibited acetylcholinesterase (AChE). A recently introduced dynamically working in vitro model with real-time determination of membrane-bound AChE activity was shown to be a very versatile and promising model to investigate oxime-induced reactivation kinetics of OP-inhibited enzyme. In this assay, human AChE from erythrocytes or muscle tissue was immobilized on a particle filter. This bioreactor was continuously perfused with substrate and chromogen and AChE activity was analyzed on-line in a flow-through detector. The model has been successfully adopted to Rhesus monkey, swine and guinea pig erythrocytes and intercostal muscle AChE. In addition, the basic kinetic constants of inhibition, aging, spontaneous- and oxime-induced-reactivation of erythrocyte AChE from these species were determined with a standard static model. The major findings were, in part substantial species differences in the inhibition (sarin, paraoxon) and reactivation kinetics (obidoxime, HI 6) of erythrocyte AChE, but comparable kinetics of inhibition and reactivation between erythrocyte and muscle AChE. Hence, these data provide further support of the assumption that erythrocyte AChE is an adequate surrogate of muscle (synaptic) AChE and admonish that major species differences have to be considered for the design and evaluation of therapeutic animal models.

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.

Evaluation of medical countermeasures against organophosphorus compounds: the value of experimental data and computer simulations.

Despite extensive research for more than six decades on medical countermeasures against poisoning by organophosphorus compounds (OP) the treatment options are meagre. The presently established acetylcholinesterase (AChE) reactivators (oximes), e.g. obidoxime and pralidoxime, are insufficient against a number of nerve agents and there is ongoing debate on the benefit of oxime treatment in human OP pesticide poisoning. Up to now, the therapeutic efficacy of oximes was mostly evaluated in animal models but substantial species differences prevent direct extrapolation of animal data to humans. Hence, it was considered essential to establish relevant experimental in vitro models for the investigation of oximes as antidotes and to develop computer models for the simulation of oxime efficacy in different scenarios of OP poisoning. Kinetic studies on the various interactions between erythrocyte AChE from various species, structurally different OP and different oximes provided a basis for the initial assessment of the ability of oximes to reactivate inhibited AChE. In the present study, in vitro enzyme-kinetic and pharmacokinetic data from a minipig model of dimethoate poisoning and oxime treatment were used to calculate dynamic changes of AChE activities. It could be shown that there is a close agreement between calculated and in vivo AChE activities. Moreover, computer simulations provided insight into the potential and limitations of oxime treatment. In the end, such data may be a versatile tool for the ongoing discussion of the pros and cons of oxime treatment in human OP pesticide poisoning.

Structure-activity analysis of aging and reactivation of human butyrylcholinesterase inhibited by analogues of tabun.

hBChE [human BChE (butyrylcholinesterase)] naturally scavenges OPs (organophosphates). This bioscavenger is currently in Clinical Phase I for pretreatment of OP intoxication. Phosphylated ChEs (cholinesterases) can undergo a spontaneous time-dependent process called 'aging' during which the conjugate is dealkylated, leading to creation of an enzyme that cannot be reactivated. hBChE inhibited by phosphoramidates such as tabun displays a peculiar resistance to oxime-mediated reactivation. We investigated the basis of oxime resistance of phosphoramidyl-BChE conjugates by determining the kinetics of inhibition, reactivation (obidoxime {1,1'-(oxybis-methylene) bis[4-(hydroxyimino) methyl] pyridinium dichloride}, TMB-4 [1,3-trimethylene-bis(4-hydroxyiminomethylpyridinium) dibromide], HLö 7 {1-[[[4-(aminocarbonyl) pyridinio]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl] pyridinium dimethanesulfonate)}, HI-6 {1-[[[4-(aminocarbonyl) pyridinio] methoxy] methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride monohydrate} and aging, and the crystal structures of hBChE inhibited by different N-monoalkyl and N,N-dialkyl tabun analogues. The refined structures of aged hBChE conjugates show that aging proceeds through O-dealkylation of the P(R) enantiomer of N,N-diethyl and N-propyl analogues, with subsequent formation of a salt bridge preventing reactivation, similarly to a previous observation made on tabun-ChE conjugates. Interestingly, the N-methyl analogue projects its amino group towards the choline-binding pocket, so that aging proceeds through deamination. This orientation results from a preference of hBChE's acyl-binding pocket for larger than 2-atoms linear substituents. The correlation between the inhibitory potency and the N-monoalkyl chain length is related to increasingly optimized interactions with the acyl-binding pocket as shown by the X-ray structures. These kinetics and X-ray data lead to a structure-activity relationship that highlights steric and electronic effects of the amino substituent of phosphoramidate. This study provides the structural basis to design new oximes capable of reactivating phosphoramidyl-hBChE conjugates after intoxication, notably when hBChE is used as pretreatment, or to design BChE-based catalytic bioscavengers.

Kinetic analysis of reactivation and aging of human acetylcholinesterase inhibited by different phosphoramidates.

The high number of fatalities due to poisoning by organophosphorus compound-based (OP) pesticides and the availability of highly toxic OP-type chemical warfare agents (nerve agents) emphasize the necessity for an effective medical treatment. Acute OP toxicity is mainly caused by inhibition of acetylcholinesterase (AChE, EC 3.1.1.7). Reactivators (oximes) of inhibited AChE are a mainstay of treatment. However, human AChE inhibited by certain OP, e.g. the phosphoramidates tabun and fenamiphos, is rather resistant towards reactivation by oximes while AChE inhibited by others, e.g. the phosphoramidate methamidophos is easily reactivated by oximes. To get more insight into a potential structure-activity relationship human AChE was inhibited by 16 different tabun analogues and the time-dependent reactivation by 1mM obidoxime, TMB-4, MMB-4, HI 6 or HLö 7, the reactivation kinetics of obidoxime and the kinetics of aging and spontaneous reactivation were investigated. A clear structure-activity relationship of aging, spontaneous and oxime-induced reactivation kinetics could be determined with AChE inhibited by N-monoalkyl tabun analogues depending on the chain length of the N-alkyl residue. N,N-dialkyl analogues bearing ethyl and n-propyl residues were completely resistant towards reactivation while N,N-di-i-propyl tabun was highly susceptible towards reactivation by oximes. AChE inhibited by phosphonoamidate analogues of tabun, bearing a N,N-dimethyl and N,N-diethyl group, could be reactivated and had comparable reactivation kinetics with obidoxime. These results in conjunction with previous data with organophosphates and organophosphonates emphasizes the necessity for kinetic studies as basis for future work on structural analysis with human AChE and for the development of effective broad-spectrum oximes.