C Basic & Clinical Pharmacology & Toxicology 2006, 98, 389–394.Printed in Denmark . All rights reserved

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ISSN 1742-7835

Russian VX: Inhibition and Reactivation ofAcetylcholinesterase Compared with VX Agent

Kamil Kuca1, Daniel Jun1, Jiri Cabal1, Martina Hrabinova1, Lucie Bartosova1 and Veronika Opletalova2

1Department of Toxicology, Faculty of Military Health Sciences, University of Defence, 500 01 Hradec Kralove,and 2Department of Pharmaceutical Chemistry and Drug Control, Charles University in Prague, Faculty of Pharmacy

in Hradec Kralove, 500 05 Hradec Kralove, Czech Republic

(Received June 30, 2005; Accepted October 26, 3005)

Abstract: Organophosphorus compounds such as nerve agents inhibit, practically irreversibly, cholinesterases by theirphosphorylation in the active site of these enzymes. Current antidotal treatment used in the case of acute nerve agentintoxications consists of combined administration of anticholinergic drug (usually atropine) and acetylcholinesterase(AChE, EC 3.1.1.7) reactivator (HI-6, obidoxime, pralidoxime), which from a chemical view is a derivative from the groupof pyridinium or bispyridinium aldoximes (commonly called ‘‘oxime’’). Oximes counteract acetylcholine increase, resultingfrom AChE inhibition. In the human body environment these compounds are powerful nucleophiles and are able to breakdown the bond between AChE and nerve agent molecule. This process leads to renewal of enzyme functionality – to itsreactivation. The usefulness of oxime in the reactivation process depends on its chemical structure and on the nerve agentwhereby AChE is inhibited. Due to this fact, selection of suitable reactivator in the treatment of intoxications is veryimportant. In our work, we have compared differences in the in vitro inhibition potency of VX and Russian VX on rat,pig and human brain, and subsequently we have tested reactivation of rat brain cholinesterase inhibited by these agentsusing oxime HI-6, obidoxime, pralidoxime, trimedoxime and methoxime. The results showed that no major differences inthe reactivation process of both VX and Russian VX-inhibited cholinesterase. The similarity in reactivation was causedby analogous chemical structure of either nerve agent; and that oxime HI-6 seems to be the most effective reactivatortested, which confirms that HI-6 is currently the most potent reactivator of AChE inhibited by nerve agents. The resultsobtained in our study should be considered in the future development of new AChE reactivators.

Organophosphorus compounds are serine esterases andproteases inhibitors widely used in agriculture as insecti-cides and acaricides and in industry and technology assoftening agents and additives to lubricants (Petroianu et

al. 2004). Due to high toxicity of some derivatives from thisgroup, they are also used as a special group of chemicalwarfare agents namely nerve agents. Sarin, soman, cyclosar-in, tabun or agent VX are among the most known membersof the nerve agents group (Bajgar 2004a).

These compounds are powerful inhibitors of mammalianas well as insect acetylcholinesterase (AChE, EC 3.1.1.7),the enzyme splitting neuromediator acetylcholine intocholinergic synapses leading to a creation of choline andacetic acid (Patocka et al. 2004). In the case of AChE inhi-bition, acetylcholine accumulates at nerve cholinergicsynapses and hyperstimulates post-synaptic cholinergic re-ceptors. Afterwards, cholinergic crisis occurs and the intoxi-cated person can die within few minutes (Sevelova et al.2005).

Organophosphorus compounds pose an increasing threatin the world due to their possible use in war conflicts (Gulf

Author for correspondende: Kamil Kuca, Department of Toxi-cology, Faculty of Military Health Sciences, University of Defence,Trebesska 1575, 500 01 Hradec Kralove, Czech republic (faxπ420 495 518 094, e-mail kucakam/pmfhk.cz, kucakam/seznam.cz).

war 1990–1991) or in terrorist acts (The sect Aum ShinriKyo, Japan, 1994, 1995) (Bajgar 2004b).

Anticholinergics and AChE reactivators are used fortreatment of nerve agent intoxications. Generally, antichol-inergics (mainly atropine) are used for relieving muscarinicsigns and symptoms whereas AChE reactivators (called ox-imes) are used for reactivation of nerve agent-inhibitedAChE (van Helden et al. 1996). Pralidoxime, obidoxime andHI-6 are common AChE reactivators incorporated into an-tidotal equipment against nerve agent intoxications in somecountries (Kassa 2002).

Many scientific papers deal with reactivation of AChEinhibited by sarin, tabun, cyclosarin, soman and VX (Kucaet al. 2003 & 2005; Bartosova-Sevelova et al. 2004). How-ever, there is no information dealing with reactivation ofRussian VX-inhibited AChE (Maxwell et al. 1997; Koplov-itz 2000) (table 1, fig. 1).

Fig. 1. Structure of VX agent and Russian VX agent.

KAMIL KUCA ET AL.390

Table 1.

Physico-chemical properties of RussianVX.

Boiling point 86æ at 1 mtorrVapor density Heavier than airVapor pressure 0.00062 mmHg at 25æLiquid density 1.003Volatility 8.9 mg/m3

Flash point 150 æ

Table 2.

IC50 of VX agent and Russian VX agent.

Nerve agent Species IC50 [M]

VX agent Rat 1.62 ¡ 10ª9

Russian VX agent Rat 1.00 ¡ 10ª8

Russian VX agent Pig 5.88 ¡ 10ª9

Russian VX agent Human 1.00 ¡ 10ª8

In this work, we have compared differences in the in vitro

inhibition potency of VX and Russian VX on rat, pig andhuman brain homogenate, and differences in the reacti-vation of rat brain cholinesterase inhibited by these agentsusing currently used AChE reactivators – HI-6, obidoxime,pralidoxime, trimedoxime and methoxime.

As it is generally known, percentage ratio of cholinester-ase activities between AChE and butyrylcholinesterase (Bu-ChE; 3.1.1.8) in rat brain is 80% to 20% (Giacobini &Holmstedt 1958).

AChE reactivation in the brain (Kassa 2002) or brainparts (Bajgar 1991) was determined to be an important fac-tor limiting death or survival of animals intoxicated withparticular nerve agent (Bajgar 1991). The reactivation by10–20% is suitable for survival of experimental animals in-toxicated with nerve agents (Bajgar 1991 & 2004b). Theability of monoquaternary oxime 2-pralidoxime to pen-etrate the blood brain barrier was confirmed by Sakuradaet al. (2003), and Cassel et al. (1997) for bisquaternary oxi-me HI-6. For all these mentioned reasons, we have usedbrain homogenate as the appropriate source of the cholin-esterases.

Table 3.

Parameters characterizing the reactivation process (nΩ3).

KR kR kr

AChE reactivator Nerve agent [mM] [minª1] [minª1 Mª1]

Pralidoxime Russian VX agent 1259∫106 0.05∫0.0020 394∫29Trimedoxime Russian VX agent 100∫7 0.06∫0.0042 550∫74Obidoxime Russian VX agent 251∫33 0.2∫0.0101 356∫20Methoxime Russian VX agent 40∫5 0.22∫0.0208 510∫34HI-6 Russian VX agent 25∫3 0.3∫0.0157 2308∫209Pralidoxime VX agent 127∫13 0.05∫0.0080 370∫61Trimedoxime VX agent 159∫26 0.13∫0.0061 818∫70Obidoxime VX agent 562∫88 0.33∫0.0286 587∫84Methoxime VX agent 431∫59 0.15∫0.0168 341∫32HI-6 VX agent 130∫17 0.19∫0.0123 1469∫145

KR – dissociation constant of inhibited enzyme-reactivator complex; kR – the first-order rate constant of reactivation; kr – the second-orderrate constant of reactivation.

Materials and Methods

Chemicals. AChE reactivators pralidoxime (1-methyl-2-hydroxyimi-nomethylpyridinium chloride) and obidoxime (ToxogoninA; 1,3-bis(4-hydroxyiminomethylpyridinium)-2-oxa-propane dichloride)were purchased from Leciva (Czech Republic) and Merck (Ger-many), respectively. Methoxime (MMC-4; 1,1-bis(4-hydroxyimino-methylpyridinium)-methane dibromide), trimedoxime (TMB-4; 1,3-bis(4-hydroxyiminomethylpyridinium)-propane dibromide) and HI-6 (1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridini-um)-2-oxa-propane dichloride) were synthesized at our departmentearlier. Purity of all tested AChE reactivators was tested using TLC(DC-Alufolien Cellulose F; mobile phase n-butanol:acetic acid:wat-er – 5:1:2; Dragendorff Reagent) and NMR (Varian Gemini 300,Palo Alto, CA, USA) (Palecek et al. 2005). Russian VX (R-VX;R33; O-isobutyl S-(2-diethylaminoethyl)methyl phosphothioate)and VX (O-ethyl S-(2-diisopropylaminoethyl)methyl phosphothio-ate) were obtained from the Military Technical Institute (Brno,Czech Republic). All other chemicals used in this experiment wereof analytical grade and were purchased from Sigma Aldrich (CzechRepublic).

Source of the cholinesterases. Rats and pigs were obtained from Bio-Test Konarovice (Czech Republic). Human brains (nucleus caud-atus) were obtained from the Faculty of Medicine in Hradec Kralo-ve (Charles University in Prague, Czech Republic) Preparation ofthe homogenate was done as follows. The brains were washed witha saline and homogenized in distilled water (10% w/v) by the useof an Ultra-Turrax instrument (Janke-Kunkel, Germany) at 20.000r.p.m. for 1 min. Aliquots (2 ml) of the homogenate were stored atª35 æ in a freezer. They were thawed immediately prior to use.

In vitro experiments. Inhibition efficacies of VX and Russian VXand subsequent reactivation of cholinesterase inhibited by theseagents were tested in vitro on brain homogenates inhibited by ap-propriate nerve agent using standard reactivation test with electro-metric instrumentation (Kuca & Kassa 2003).

Inhibition of cholinesterases. Brain homogenate (0.5 ml) from ratswere treated with solution (0.5 ml) of VX or Russian VX in appro-priate concentration for 30 min. Pig and human homogenates weretreated in the same way with only Russsian VX. Afterwards, thereaction solution was adjusted to 23 ml with 0.3 M sodium chloride.Then, 0.02 M solution of acetylcholine iodide (2 ml) was added tothe mixture. The liberated acetic acid was titrated with 0.01 M so-dium hydroxide on an RTS 822 titrator (Radiometer, Denmark) inthe pH-stat mode (pH 8.0) at room temperature (25 æ). The slope ofthe linear part of the time dependence of the sodium hydroxide used

391VX VERSUS RUSSIAN VX – INHIBITION AND REACTIVATION

Fig. 2. Inhibition of cholinesterases from different species by Rus-sian VX agent (source of cholinesterases – rat, pig, human brain;time of inhibition 30 min.; temperature 25 æ; pH 8.0).

represents the activity of the inhibited enzyme (in fact, the initialrate of the enzymatic reaction).

Reactivation of cholinesterases. Rat brain homogenate (0.5 ml) wastreated with aqueous solution of appropriate nerve agent (0.5 ml)for 30 min., which resulted in about 95% inhibition of the enzymes.Reactivation of the inhibited cholinesterases was performed im-mediately after inhibition. A solution (1 ml) of the reactivator inappropriate concentration (from 10ª7 to 10ª2 M) was added to theenzyme. After 10 min. reactivation at 25 æ, the mixture was adjustedto 23 ml with 0.3 M sodium chloride solution. Then, 0.02 M solu-tion of acetylcholine iodide (2 ml) was added and immediately after-wards the activity of the reactivated enzymes was determined anal-ogously as described in the previous experiments.

Fig. 3. Inhibition of cholinesterases by VX agent and Russian VXagent (source of cholinesterases – rat brain; time of inhibition 30min.; temperature 25 æ; pH 8.0).

Fig. 4. Concentration-reactivation relationship of tested oximes –Russian VX agent (source of cholinesterases – rat brain; time ofinhibition 30 min.; time of reactivation 10 min.; temperature 25 æ;pH 8.0).

Results

Inhibition of cholinesterases.

Values of the constant IC50, characterizing the inhibitionpotency of tested nerve agents, are summarized in table 2.Typical sigmoidal curves characterizing the inhibition pro-cess were obtained. The IC50 values for VX and RussianVX differ by one order of magnitude in rat brain (fig. 3).In the case of species differences, in all cases almost similarcourse of inhibition was obtained (fig. 2).

Reactivation of inhibited cholinesterases.

The calculated kinetic constants describing the reactivationprocess obtained in our experiments are shown in table 3.The affinity of HI-6 towards VX agent-inhibited, as well as

Fig. 5. Concentration-reactivation relationship of pralidoxime –Russian VX agent versus VX agent (source of cholinesterases – ratbrain; time of inhibition 30 min.; time of reactivation 10 min.; tem-perature 25 æ; pH 8.0).

KAMIL KUCA ET AL.392

Fig. 6. Concentration-reactivation relationship of trimedoxime –Russian VX agent versus VX agent (source of cholinesterases – ratbrain; time of inhibition 30 min.; time of reactivation 10 min.; tem-perature 25 æ; pH 8.0).

Russian VX agent-inhibited enzymes, characterized by theKR value, is the highest of the all oximes tested. Rate con-stant kR (the first-order rate constant which characterizesthe ability of oxime to interrupt covalent bond between en-zyme and phosphate) is the highest for obidoxime in thecase of VX agent-inhibited cholinesterases reactivation. Inthe case of Russian VX agent-inhibited cholinesterases reac-tivation, HI-6 showed the highest reactivation potency.

Rate constant kr (the second-order rate constant) charac-terizes the velocity of the whole reactivation process. Itsvalue is calculated from the ratio between kR and KR. Ac-cording to this constant, in both cases (VX agent and Rus-sian VX agent reactivation), oxime HI-6 was the most po-tent reactivator tested.

As can be clearly seen from the dependence between re-activator concentration and percentage of reactivationshown in fig. 4, all oximes tested were able to reactivateRussian VX agent-inhibited enzymes. There are some dif-ferences in the course of reactivation curves. In the caseof methoxime and HI-6, maximal reactivation potency wasobtained at lower concentration (0.0001 M) in comparison

Fig. 7. Concentration-reactivation relationship of obidoxime – Rus-sian VX agent versus VX agent (source of cholinesterases – ratbrain; time of inhibition 30 min.; time of reactivation 10 min.; tem-perature 25 æ; pH 8.0).

Fig. 8. Concentration-reactivation relationship of methoxime –Russian VX agent versus VX agent (source of cholinesterases – ratbrain; time of inhibition 30 min.; time of reactivation 10 min.; tem-perature 25 æ; pH 8.0).

with obidoxime (0.001 M), pralidoxime (0.005 M) and tri-medoxime (0.01 M). According to the above metioned re-sults (just 10% of reactivators penetrate blood-brain bar-rier), it is (Sakurada et al. 2003) physiologically impossibleto attain these concentrations in brain.

Differencies between Russian VX agent and VX agent inthe course of the reactivation process for individual reactiv-ators are shown in fig. 5–9. According to these results, thereare little difference between both nerve agents for all oximestested, except trimedoxime. In the case of trimedoxime, thedifference was achieved at high concentration, which couldnot be administered to human beings.

Discussion

Our study shows that there are almost no differences in theinhibition of rat, pig and human brain human homogenateby Russian VX agent. Difference in measured IC50 valuesfor VX agent and Russian VX agent shows that VX agent ishalf-fold more toxic in comparison with Russian VX agent.

Calculated kinetic parameters of oximes studied for thereactivation of VX-inhibited cholinesterases closely corre-spond to their potency to reactivate Russian VX-inhibited

Fig. 9. Concentration-reactivation relationship of HI-6 – RussianVX agent versus VX agent (source of cholinesterases – rat brain;time of inhibition 30 min.; time of reactivation 10 min.; temperature25 æ; pH 8.0).

393VX VERSUS RUSSIAN VX – INHIBITION AND REACTIVATION

cholinesterases in vitro. The differences obtained were evensmaller compared to those published by Kassa & Cabal(1999a & b) for sarin and cyclosarin. These compounds dif-fer only in the alkoxy group on the phosphorus atom ofnerve agent (sarin – isopropyl; cyclosarin – cyclohexyl).

The oxime HI-6, characterized by high affinity to VX andRussian VX-inhibited cholinesterases and by the highestvelocity of reactivation, is able to significantly reactivateboth nerve agent-inhibited cholinesterases at low concen-trations. According to our results, methoxime seems to beanother very promising reactivator of VX and Russian VX-inhibited cholinesterases. Unfortunately, its potency to re-activate other nerve agent-inhibited cholinesterases is not ashigh as compared with HI-6 (Cabal et al. 2004; Sevelova et

al. 2004). Results obtained in this study suggest HI-6 toother oximes tested (pralidoxime, trimedoxime, methoximeand obidoxime) for future human use because of higherprobability of HI-6 to achieve bioavailable concentrationvalues in body compartments. The other oximes studied(pralidoxime, trimedoxime, obidoxime and methoxime) arealso able to significantly reactivate VX agent and RussianVX agent-inhibited cholinesterases but only at relativelyhigh concentrations that do not correspond to in vivo rec-ommended doses (Kuca & Kassa 2003).

Contrary to the promising cholinesterases reactivators,pralidoxime was found to be the worst of all oximes tested.According to our present results and previous in vitro andin vivo results obtained for all nerve agents, pralidoxime ispoor nerve agent reactivator (Kassa & Cabal 1999a & b;Kassa 2002; Bajgar 2004; Cabal et al. 2004; Kuca & Kassa2004). Although this reactivator is taken as gold standardof antidotal mean (Petroianu et al. 2005), Due to this fact,there is no reason for involving this reactivator as the anti-dotal mean in some armies. This reactivator should be re-placed by another reactivator with sufficient potency to re-activate broader spectra of nerve agents intoxications, forexample HI-6. However, oxime HI-6 is not able to reactivatetabun and pesticides intoxications (Worek et al. 1996; Cabalet al. 2004). Owing to this fact, new reactivators withbroader reactivation ability are still synthesized and tested(Pang et al. 2003; Yang et al. 2003; Kuca et al. 2004; Chen-namaneni et al. 2005; Kim et al. 2005).

Therefore, further research in this field is needed to findnew oximes able to sufficiently reactivate nerve agent-inhib-ited cholinesterases regardless of the nerve agent used.

AcknowledgementsThe authors wish to thank to Mrs. Jana Uhlirova for her

skilful technical assistance. The experiments described herewere carried out in compliance with the current law of theCzech Republic. This work was supported by grants of theMinistry of Defence (Czech Republic) OBVTUO2003008and Charles University (Czech Republic) No. 126/2005/B-BIO/FaF.

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