Photometric microplate assay for estimation of the efficacy of paraoxon-inhibited acetylcholinesterase reactivation

Photometric microplate assay was performed for testing of paraoxon-inhibited acetylcholinesterase (AChE) using three reactivators for reactivation purposes: obidoxime, pralidoxime, and HI-6. 3-D graphs (percent of reactivation vs. concentration of reactivator and vs. time of reactivator effecting) were constructed for each reactivator to compare their efficacy. The best results were obtained using obidoxime where reactivation was near to 80%. Suitability of photometric microplates for following of reactivation procedures is discussed.     

Synthesis,in vitro screening and molecular docking of isoquinolinium-5-carbaldoximes as acetylcholinesterase and butyrylcholinesterase reactivators

Abstract

The series of symmetrical and unsymmetrical isoquinolinium-5-carbaldoximes was designed and prepared for cholinesterase reactivation purposes. The novel compounds were evaluated for intrinsic acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) inhibition, when the majority of novel compounds resulted with high inhibition of both enzymes and only weak inhibitors were selected for reactivation experiments on human AChE or BChE inhibited by sarin, VX, or paraoxon. The AChE reactivation for all used organophosphates was found negligible if compared to the reactivation ability of obidoxime. Importantly, two compounds were found to reactivate BChE inhibited by sarin or VX better to obidoxime at human attainable concentration. One compound resulted as better reactivator of NEMP (VX surrogate)-inhibited BChE than obidoxime. The in vitro results were further rationalized by molecular docking studies showing future directions on designing potent BChE reactivators.     

Synthesis of monooxime-monocarbamoyl bispyridinium compounds bearing (E)-but-2-ene linker and evaluation of their reactivation activity against tabun- and paraoxon-inhibited acetylcholinesterase

Six AChE monooxime-monocarbamoyl reactivators with an (E)-but-2-ene linker were synthesized using modification of currently known synthetic pathways. Their potency to reactivate AChE inhibited by the nerve agent tabun and insecticide paraoxon was tested in vitro. The reactivation efficacies of pralidoxime, HI-6, obidoxime, K048, K075 and the newly prepared reactivators were compared. According to the results obtained, one reactivator seems to be promising against tabun-inhibited AChE and two reactivators against paraoxon-inhibited AChE. The best results were obtained for bisquaternary substances with at least one oxime group in position four.     

A Comparison of the Ability of a New Bispyridinium Oxime—1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane Dibromide and Currently used Oximes to Reactivate Nerve Agent-inhibited Rat Brain Acetylcholinesterase by In Vitro Methods

The efficacy of a new bispyridinium oxime 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane dibromide, called K048, and currently used oximes (pralidoxime, obidoxime, the oxime HI-6) to reactivate acetylcholinesterase inhibited by various nerve agents (sarin, tabun, cyclosarin, VX) was tested by in vitro methods. The new oxime K048 was found to be a more efficacious reactivator of nerve agent-inhibited acetylcholinesterase than pralidoxime (in the case of VX, tabun and cyclosarin), obidoxime (cyclosarin and tabun) and HI-6 (tabun) but it did not reach the efficacy of currently used oximes for the reactivation of acetylcholinesterase inhibited by sarin. Thus, the oxime K048 seems to be a relatively efficacious broad spectrum acetylcholinesterase reactivator and, therefore, it could be useful for the treatment of a nerve agent-exposed population if information about detection of the type of nerve agent is not available.     

Pyridinium-2-carbaldoximes with quinolinium carboxamide moiety are simultaneous reactivators of acetylcholinesterase and butyrylcholinesterase inhibited by nerve agent surrogates

The pyridinium-2-carbaldoximes with quinolinium carboxamide moiety were designed and synthesised as cholinesterase reactivators. The prepared compounds showed intermediate-to-high inhibition of both cholinesterases when compared to standard oximes. Their reactivation ability was evaluated in vitro on human recombinant acetylcholinesterase (hrAChE) and human recombinant butyrylcholinesterase (hrBChE) inhibited by nerve agent surrogates (NIMP, NEMP, and NEDPA) or paraoxon. In the reactivation screening, one compound was able to reactivate hrAChE inhibited by all used organophosphates and two novel compounds were able to reactivate NIMP/NEMP-hrBChE. The reactivation kinetics revealed compound 11 that proved to be excellent reactivator of paraoxon-hrAChE better to obidoxime and showed increased reactivation of NIMP/NEMP-hrBChE, although worse to obidoxime. The molecular interactions of studied reactivators were further identified by in silico calculations. Molecular modelling results revealed the importance of creation of the pre-reactivation complex that could lead to better reactivation of both cholinesterases together with reducing particular interactions for lower intrinsic inhibition by the oxime.     

New quaternary pyridine aldoximes as casual antidotes against nerve agents intoxications

In this work, the ability of four newly synthesized oximes--K005 (1,3-bis(2-hydroxyiminomethylpyridinium) propane dibromide), K027 (1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide), K033 (1,4-bis(2-hydroxyiminomethylpyridinium) butane dibromide) and K048 (1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide) to reactivate acetylcholinesterase (AChE, EC 3.1.1.7) inhibited by nerve agents is summarized. Reactivation potency of these compounds was tested using standard in vitro reactivation test. Tabun, sarin, cyclosarin and VX agent were used as appropriate testing nerve agents. Rat brain AChE was used as a source of the enzyme. Efficacies of new reactivators to reactivate tabun-, sarin-, cyclosarin- and VX-inhibited AChE were compared with the currently used AChE reactivators (pralidoxime, obidoxime and HI-6). Oxime K048 seems to be promising reactivator of tabun-inhibited AChE. Its reactivation potency is significantly higher than that of HI-6 and pralidoxime and comparable with the potency of obidoxime. The best reactivator of sarin-inhibited AChE seems to be oxime HI-6. None of the new AChE reactivators reached comparable reactivation potency. The same results were obtained for cyclosarin-inhibited AChE. However, oxime K033 is also potent reactivator of AChE inhibited by this nerve agent. In the case of VX inhibition, obidoxime and new oximes K027 and K048 seem to be the best AChE reactivators. None from the currently tested AChE reactivators is able to reactivate AChE inhibited by all nerve agents used and, therefore, the search for new potential broad spectrum AChE reactivators is needed.     

A comparison of reactivating efficacy of newly developed oximes (K074, K075) and currently available oximes (obidoxime, HI-6) in soman, cyclosarin and tabun-poisoned rats

The potency of newly developed oximes (K074, K075) and commonly used oximes (obidoxime, HI-6) to reactivate nerve agent-inhibited acetylcholinesterase was evaluated in rats poisoned with soman, tabun or cyclosarin at a lethal dose corresponding to their LD(50) value. In vivo determined percentage of reactivation of soman-inhibited blood and brain acetylcholinesterase in poisoned rats showed that only the oxime HI-6 was able to reactivate soman-inhibited acetylcholinesterase in the peripheral (blood) as well as central (brain) compartment. In vivo determined percentage of reactivation of tabun-inhibited blood and brain acetylcholinesterase in poisoned rats showed that obidoxime is the most efficacious reactivator of tabun-inhibited acetylcholinesterase among studied oximes in the peripheral compartment (blood) while K074 seems to be the most efficacious reactivator of tabun-inhibited acetylcholinesterase among studied oximes in the central compartment (brain). In vivo determined percentage of reactivation of cyclosarin-inhibited blood and brain acetylcholinesterase in poisoned rats showed that HI-6 is the most efficacious reactivator of cyclosarin-inhibited acetylcholinesterase among studied oximes. Due to their reactivating effects, both newly developed K oximes can be considered to be promising oximes for the antidotal treatment of acute tabun poisonings while the oxime HI-6 is still the most promising oxime for the treatment of acute soman and cyclosarin poisonings.     

A comparison of reactivating efficacy of newly developed oximes (K074, K075) and currently available oximes (obidoxime, HI-6) in cyclosarin-and tabun-poisoned rats

The potency of newly developed oximes (K074, K075) and commonly used oximes (obidoxime, HI-6) to reactivate nerve agent-inhibited acetylcholinesterase was evaluated in rats poisoned with tabun or cyclosarin at a lethal dose corresponding to the LD50 value. In vivo determined percentage of reactivation of tabun-inhibited blood and brain acetylcholinesterase showed that obidoxime is the most efficacious reactivator of tabun-inhibited acetylcholinesterase among studied oximes in the peripheral compartment (blood) although the differences between obidoxime and newly developed oximes were not significant. On the other hand, one of the newly developed oximes (K074) seems to be a significantly more efficacious reactivator of tabun-inhibited acetylcholinesterase in the central compartment (brain) than the other studied oximes. In addition, the oxime HI-6 is unable to sufficiently reactivate tabun-inhibited acetylcholinesterase in rats. In vivo determined percentage of reactivation of cyclosarin-inhibited blood and brain acetylcholinesterase in poisoned rats showed that HI-6 is the most efficacious reactivator of cyclosarin-inhibited acetylcholinesterase among the studied oximes in the peripheral (blood) as well as central (brain) compartment although the differences between the oxime HI-6 and other tested oximes in the brain were not significant. Due to their reactivating effects, both newly developed K-oximes can be considered to be promising oximes for the antidotal treatment of acute tabun poisoning while the oximes HI-6 is still the most promising oxime for the treatment of acute cyclosarin poisonings due to its high potency in reactivating cyclosarin-inhibited acetylcholinesterase in the peripheral as well as central compartment.     

Synthesis of monooxime-monocarbamoyl bispyridinium compounds bearing (E)-but-2-ene linker and evaluation of their reactivation activity against tabun- and paraoxon-inhibited acetylcholinesterase

Six AChE monooxime-monocarbamoyl reactivators with an (E)-but-2-ene linker were synthesized using modification of currently known synthetic pathways. Their potency to reactivate AChE inhibited by the nerve agent tabun and insecticide paraoxon was tested in vitro. The reactivation efficacies of pralidoxime, HI-6, obidoxime, K048, K075 and the newly prepared reactivators were compared. According to the results obtained, one reactivator seems to be promising against tabun-inhibited AChE and two reactivators against paraoxon-inhibited AChE. The best results were obtained for bisquaternary substances with at least one oxime group in position four.     

A comparison of the therapeutic and reactivating efficacy of newly developed bispyridinium compounds (K206, K269) with currently available oximes against tabun in rats and mice

The potency of newly developed bispyridinium compounds (K206, K269) in reactivating tabun-inhibited acetylcholinesterase and eliminating tabun-induced lethal toxic effects was compared with commonly used oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies which determined percentage of reactivation of tabun-inhibited blood and tissue AChE in poisoned rats showed that the reactivating efficacy of both newly developed oximes is comparable with obidoxime and trimedoxime in blood but lower than the reactivating potency of trimedoxime and obidoxime in the diaphragm and brain. Nevertheless, the differences in reactivating efficacy of obidoxime, trimedoxime and K206 was not significant while the potency of K269 to reactivate tabun-inhibited acetylcholinesterase was significantly lower. Both newly developed oximes were also found to be relatively efficacious in elimination of the lethal toxic effects in tabun-poisoned mice. Their therapeutic efficacy corresponds to the therapeutic potency of obidoxime. The oxime HI-6, relatively efficacious against soman, did not seem to be an adequately effective oxime in reactivation of tabun-inhibited AChE and to counteract lethal effects of tabun. Both newly developed oximes (K206, K269) are significantly more efficacious in reactivating tabun-inhibited AChE in rats and to eliminate lethal toxic effects of tabun in mice than the oxime HI-6 but their reactivating and therapeutic potency does not prevail over the effectiveness of currently available obidoxime and trimedoxime and, therefore, they are not suitable for their replacement of commonly used oximes for the treatment of acute tabun poisoning.     

Obidoxime antagonizes the neuromuscular failure induced by neostigmine and diisopropyl fluorophosphate via different mechanisms

The efficacies and mechanisms of obidoxime in antagonizing the neuromuscular failure induced by neostigmine and diisopropyl fluorophosphate (DFP) were studied in mouse phrenic nerve/diaphragm preparations. Obidoxime antagonized neostigmine-induced tetanic fade (EC₅₀: 300 µM) by inhibiting the regenerative and sustained depolarization during repetitive stimulation. The antagonism was associated with a depression and shortening of single endplate potentials (EPPs) and miniature EPPs (MEPPs). In contrast, the neuromuscular failure induced irreversibly after treatment with DFP and followed by washout was restored by obidoxime at concentrations (EC₅₀: 0.6 µM) 500-fold lower than that against neostigmine. The regenerative depolarization was abolished with no depression of single EPPs and MEPPs, and the antagonistic action persisted after washout of obidoxime. The EC₅₀ of obidoxime was proportionately increased in the presence of increasing concentrations of DFP. Nevertheless, the EC₅₀ against DFP, at a concentration (30 µM) 15-fold in excess of that which caused tetanic fade, was still 10-fold lower than that which antagonized neostigmine. In both cases, the amplitudes of train EPPs were increased. It is concluded that obidoxime antagonizes neostigmine-induced neuromuscular failure by a curare-like action but antagonizes DFP by an enzyme reactivation.     

A comparison of tabun-inhibited rat brain acetylcholinesterase reactivation by three oximes (HI-6, obidoxime, and K048) in vivo detected by biochemical and histochemical techniques

Tabun belongs to the most toxic nerve agents. Its mechanism of action is based on acetylcholinesterase (AChE) inhibition at the peripheral and central nervous systems. Therapeutic countermeasures comprise administration of atropine with cholinesterase reactivators able to reactivate the inhibited enzyme. Reactivation of AChE is determined mostly biochemically without specification of different brain structures. Histochemical determination allows a fine search for different structures but is performed mostly without quantitative evaluation. In rats intoxicated with tabun and treated with a combination of atropine and HI-6, obidoxime, or new oxime K048, AChE activities in different brain structures were determined using biochemical and quantitative histochemical methods. Inhibition of AChE following untreated tabun intoxication was different in the various brain structures, having the highest degree in the frontal cortex and reticular formation and lowest in the basal ganglia and substantia nigra. Treatment resulted in an increase of AChE activity detected by both methods. The highest increase was observed in the frontal cortex. This reactivation was increased in the order HI-6 < K048 < obidoxime; however, this order was not uniform for all brain parts studied. A correlation between AChE activity detected by histochemical and biochemical methods was demonstrated. The results suggest that for the mechanism of action of the nerve agent tabun, reactivation in various parts of the brain is not of the same physiological importance. AChE activity in the pontomedullar area and frontal cortex seems to be the most important for the therapeutic effect of the reactivators. HI-6 was not a good reactivator for the treatment of tabun intoxication.     

Internal standard high-performance liquid chromatography method for the determination of obidoxime in urine of organophosphate-poisoned patients

Obidoxime is an antidote approved for reactivation of inhibited acetylcholinesterase in organophosphate poisoning. HPLC methods were described for its determination in blood or aqueous solutions but not for the determination in urine. Since data for renal obidoxime excretion ranged from 2.2 to 84% of administered dose in healthy volunteers depending on the route of administration and little is known about pharmacokinetics of obidoxime in severely intoxicated patients we developed an internal standard (HI 6) reversed-phase HPLC method for determining obidoxime in urine. The mobile phase consisted of methanol, the counter ion 1-heptane sulfonic acid and tetrabutylammonium phosphate, the stationary phase involved a 5 μm reversed-phase column (125×4 mm). Obidoxime was detected spectrophotometrically at 288 nm. The limit of quantification (LOQ) was 1 μM, the limit of detection (LOD) 0.5 μM. Linear calibration curves were obtained in a concentration range from 1 to 1000 μM. Intra- and inter-day precision C.V.s were below 4%. Accuracy was 95.9% in the LOQ range. Using this method, we were able to quantify obidoxime in urine of an organophosphate poisoned patient. Based on this data we calculated that 58% of the administered dose was excreted in the urine.     

Synthesis and in vitro evaluation of bis-quaternary 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide derivatives as reactivators against sarin and VX inhibited human acetylcholinesterase (hAChE)

A series of bis-quaternary pyridinium derivatives 3a–3i of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide (2) have been synthesized. The synthesized pyridinium compounds have an amide group in conjugation to the oxime moiety. These compounds were evaluated in vitro for their reactivation efficacy against organophosphorus (OP) nerve agents (NAs) (sarin and VX) inhibited human erythrocyte ghost acetylcholinesterase (hAChE) and compared with the reactivation efficacy of 2-PAM and obidoxime. The pK_a values of the synthesized compounds were found closer to the pK_a values of 2- and 4-pyridinium oxime reactivators such as 2-PAM and obidoxime. Some of the compounds have shown better reactivation efficacy than 2-PAM, and obidoxime against sarin and VX inhibited AChE.     

A comparison of the reactivating and therapeutic efficacy of newly developed bispyridinium oximes (K250, K251) with commonly used oximes against tabun in rats and mice

The potency of newly developed bispyridinium compounds (K250, K251) in reactivating tabun-inhibited acetylcholinesterase and reducing tabun-induced lethal toxic effects was compared with currently available oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies determined percentage of reactivation of tabun-inhibited blood and tissue AChE in poisoned rats and showed that the reactivating efficacy of both newly developed oximes is comparable with the oxime HI-6 but it is significantly lower than the reactivating effects of obidoxime and trimedoxime, especially in diaphragm and brain. Both newly developed oximes were also found to be able to slightly reduce lethal toxic effects in tabun-poisoned mice. Their therapeutic efficacy is higher than the potency of the oxime HI-6 but it is lower than the therapeutic effects of trimedoxime and obidoxime. Thus, the reactivating and therapeutic potency of both newly developed oximes (K250, K251) does not prevail over the effectiveness of currently available oximes and, therefore, they are not suitable for their replacement for the treatment of acute tabun poisoning.     

A comparison of the ability of a new bispyridinium oxime--1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane dibromide and currently used oximes to reactivate nerve agent-inhibited rat brain acetylcholinesterase by in vitro methods

The efficacy of a new bispyridinium oxime 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane dibromide, called K048, and currently used oximes (pralidoxime, obidoxime, the oxime HI-6) to reactivate acetylcholinesterase inhibited by various nerve agents (sarin, tabun, cyclosarin, VX) was tested by in vitro methods. The new oxime K048 was found to be a more efficacious reactivator of nerve agent-inhibited acetylcholinesterase than pralidoxime (in the case of VX, tabun and cyclosarin), obidoxime (cyclosarin and tabun) and HI-6 (tabun) but it did not reach the efficacy of currently used oximes for the reactivation of acetylcholinesterase inhibited by sarin. Thus, the oxime K048 seems to be a relatively efficacious broad spectrum acetylcholinesterase reactivator and, therefore, it could be useful for the treatment of a nerve agent-exposed population if information about detection of the type of nerve agent is not available.     

A comparison of tabun-inhibited rat brain acetylcholinesterase reactivation by three oximes (HI-6, obidoxime,and K048) in vivo detected by biochemical and histochemical techniques

Tabun belongs to the most toxic nerve agents. Its mechanism of action is based on acetylcholinesterase (AChE) inhibition at the peripheral and central nervous systems. Therapeutic countermeasures comprise administration of atropine with cholinesterase reactivators able to reactivate the inhibited enzyme. Reactivation of AChE is determined mostly biochemically without specification of different brain structures. Histochemical determination allows a fine search for different structures but is performed mostly without quantitative evaluation. In rats intoxicated with tabun and treated with a combination of atropine and HI-6, obidoxime, or new oxime K048, AChE activities in different brain structures were determined using biochemical and quantitative histochemical methods. Inhibition of AChE following untreated tabun intoxication was different in the various brain structures, having the highest degree in the frontal cortex and reticular formation and lowest in the basal ganglia and substantia nigra. Treatment resulted in an increase of AChE activity detected by both methods. The highest increase was observed in the frontal cortex. This reactivation was increased in the order HI-6 < K048 < obidoxime; however, this order was not uniform for all brain parts studied. A correlation between AChE activity detected by histochemical and biochemical methods was demonstrated. The results suggest that for the mechanism of action of the nerve agent tabun, reactivation in various parts of the brain is not of the same physiological importance. AChE activity in the pontomedullar area and frontal cortex seems to be the most important for the therapeutic effect of the reactivators. HI-6 was not a good reactivator for the treatment of tabun intoxication.     

Monoquaternary pyridinium salts with modified side chain-synthesis and evaluation on model of tabun- and paraoxon-inhibited acetylcholinesterase

Acetylcholinesterase reactivators are crucial antidotes for the treatment of organophosphate intoxication. Eighteen monoquaternary reactivators of acetylcholinesterase with modified side chain were developed in an effort to extend the properties of pralidoxime. The known reactivators (pralidoxime, HI-6, obidoxime, trimedoxime, methoxime) and the prepared compounds were tested in vitro on a model of tabun- and paraoxon-inhibited AChE. Monoquaternary reactivators were not able to exceed the best known compounds for tabun poisoning, but some of them did show reactivation better or comparable with pralidoxime for paraoxon poisoning. However, extensive differences were found by a SAR study for various side chains on the non-oxime part of the reactivator molecule.     

Changes of rat plasma total low molecular weight antioxidant level after tabun exposure and consequent treatment by acetylcholinesterase reactivators

These experiments were performed on a rat model. The rats were divided into eight groups and consequently exposed to either a saline solution (control), atropine or a combination of atropine and tabun. The reactivation efficacy of the oximes was estimated on the rats exposed to tabun, atropine and a reactivator of AChE. The oximes HI-6, obidoxime, trimedoxime, K203 and KR-22836 were used as representative compounds of commonly available and new AChE reactivators. Besides the positive effect of the administered reactivators on blood AChE activity, the sizable modulation of low molecular weight antioxidant (LMWA) levels was also determined. The LMWA levels in the the animals treated with the oxime reactivators were decreased in comparison with the animals treated by atropine alone. It was found that the levels of LMWA returned to the level found in the control animals when either trimedoxime, K203 or KR-22836 were administered. The principle of oxime reactivator function and a novel insight into AChE activity regulation and oxidative stress is discussed.     

A comparison of the potency of trimedoxime and other currently available oximes to reactivate tabun-inhibited acetylcholinesterase and eliminate acute toxic effects of tabun

Tabun (O-ethyl-N,N-dimethyl phosphoramidocyanidate) belongs to highly toxic organophosphorus compounds misused as chemical warfare agents for military as well as terroristic purposes. It differs from other highly toxic organophosphates by its chemical structure and by the fact that tabun-inhibited acetylcholinesterase is extraordinarily difficult to reactivate. The potency of trimedoxime and other commonly used oximes (pralidoxime, obidoxime, the oxime HI-6) to reactivate tabun-inhibited acetylcholinesterase and to eliminate tabun-induced acute effects was evaluated using in vitro and in vivo methods. In vitro calculated kinetic parameters of reactivation of tabun-inhibited acetylcholinesterase from rat brain homogenate and in vivo determined percentage of reactivation of tabun-inhibited blood and tissue acetylcholinesterase in poisoned rats show that trimedoxime seems to be the most efficacious reactivator in the case of tabun poisonings. Trimedoxime was also found to be the most efficacious oxime in the elimination of acute lethal toxic effects in tabun-poisoned rats and mice. The oxime HI-6, so efficacious against soman, does not seem to be sufficiently effective oxime to reactivate tabun-inhibited acetylcholinesterase and to counteract acute lethal effects of tabun.