The Reactivating and Therapeutic Efficacy of Oximes to Counteract Russian VX Poisonings

Abstract

Russian VX (O-isobutyl-S-(2-diethylaminoethyl)methylphosphonothioate) is the structural analogue of VX agent. It differs from VX agent (O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothioate) by two alkyl groups. The potency of currently available oximes (pralidoxime, obidoxime, HI-6) to reactivate Russian VX–inhibited acetylcholinesterase and to eliminate Russian VX–induced acute toxic effects was evaluated using in vivo methods. In vivo determined percentage of reactivation of Russian VX–inhibited blood and brain acetylcholinesterase in poisoned rats shows that HI-6 seems to be the most efficacious reactivator of Russian VX–inhibited acetylcholinesterase among currently used oximes in the peripheral compartment, whereas no difference between reactivating efficacy of all tested oximes was observed in the central compartment. The oxime HI-6 was also found to be the most efficacious oxime in the elimination of acute lethal toxic effects in Russian VX–poisoned mice among all studied oximes. Thus, the oxime HI-6 seems to be the most suitable oxime for the antidotal treatment of acute poisonings with Russian VX as in the case of VX, sarin, cyclosarin, and soman poisonings.

Highly toxic organophosphorus compounds called nerve agents are considered to be the most dangerous chemical warfare agents. The most important representatives of nerve agents are tabun, sarin, soman, cyclosarin, and VX. Despite of the entry into force in April 1997 of the Chemical Weapons Convention forbidding the development, production, stockpiling, and the use of chemical warfare agents, the world has seen a rapid proliferation of such agents. Their acute toxic effects are based on phosphonylation of the acetylcholinesterase (AChE; EC 3.1.1.7), leading to the irreversible inhibition of this enzyme and subsequent overstimulation of postsynaptic cholinergic receptors due to the accumulation of the neurotransmitter acetylcholine (ACh) in synapses of the central and peripheral nervous systems (Lotti 2000; Marrs 1993). The current standard antidotal treatment of poisoning with nerve agents usually consists of a muscarinic cholinergic receptor antagonist to block the overstimulation of cholinergic receptors by ACh and an oxime to reactivate nerve agent–inhibited AChE (Dawson 1994; Marrs 1993; Taylor 1996). Generally, anticholinergics (mainly atropine) are used for relieving of muscarinic signs and symptoms, whereas AChE reactivators (called oximes) are used for reactivation of nerve agent–inhibited AChE (van Helden et al. 1996). Pralidoxime, obidoxime, and HI-6 are common AChE reactivators introduced into antidotal means against nerve agent intoxications in many countries (Kassa 2002a).

There are many articles demonstrating the reactivation of AChE inhibited by sarin, tabun, cyclosarin, soman, and VX (Bartosova-Sevelova et al. 2004; Kuca, Patocka, and Cabal 2003; Kuca et al. 2005; Worek et al. 2004). However, there is a lack of information describing reactivation of Russian VX–inhibited AChE (Koplovitz 2000; Maxwell, Brecht, and Koplovitz 1997). Russian VX (O-isobutyl-S-(2-diethylaminoethyl)methylphosphonothioate) is the structural analogue of VX agent (Figure 1). It differs from VX agent (O-ethyl-S-(2-diisopropylaminoethyl)methylphosphonothioate) by two alkyl groups (Kuca et al. 2006). Due to the differences in the chemical structure of Russian VX and VX agent, Russian VX is more persistent in the environment and therefore poses a greater threat (Crenshaw et al. 2001). The aim of this work was to demonstrate the differences in the potency of currently used AChE reactivators (HI-6, obidoxime, pralidoxime) to reactivate blood and brain AChE in rats poisoned with Russian VX and in the potency of all tested oximes to eliminate Russian VX–induced acute lethal effects in mice.

MATERIALS AND METHODS

Animals

Male albino Wistar rats weighing 200 to 230 g and NMRI mice weighing 16 to 18 g were purchased from Biotest Konarovice (Czech Republic). Rats and mice represent the experimental animals fully acceptable for the study on the efficacy of antidotes to counteract nerve agent poisonings (Bajgar 2004; Dawson 1994; Kassa 2002a). They were kept in an air-conditioned room with the light from 07:00 to 19:00 h and were allowed access to standard food and tap water ad libitum. The animals were divided into groups of eight animals. Handling of the experimental animals was done under the supervision of the Ethics Committee of the Faculty of Military Health Sciences, Czech Republic.

Chemicals

AChE reactivators pralidoxime (2-PAM; 1-methyl-2-hydroxyiminomethylpyridinium chloride) and obidoxime (Toxogonin; 1,3-bis(4-hydroxyiminomethylpyridinium)-2-oxa-propane dichloride) were purchased from Leciva (Czech Republic) and Merck (Germany), respectively. The oxime HI-6 (1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoyl-pyridinium)-2-oxa-propane dichloride) was synthesized at our Department of Toxicology according to the previously published procedure (Hagedorn, Stark, and Lorenz 1972). Purity of all tested AChE reactivators was tested using thin-layer chromatography (TLC) (DC-Alufolien Cellulose F; mobile phase n-butanol: acetic acid:water 5:1:2; Dragendorff Reagent) and nuclear magnetic resonance (NMR) (Varian Gemini 300; Palo Alto CA, USA) (Palecek et al. 2005). Russian VX was obtained from the Military Technical Institute, Brno (Czech Republic) where it was synthesized according to the documented procedure (Smicek 2005). All other chemicals used in this experiment were of analytical grade and were purchased from Sigma Aldrich (Czech Republic). All substances were administered intramuscularly (i.m.) at a volume of 1 mL/kg body weight (bw) for rats and at a volume of 10 mL/kg bw for mice.

In Vivo Experiments

Before starting the evaluation of reactivating and therapeutic efficacy of oximes, the acute toxicity of tested oximes was evaluated in rats and mice by the assessment of their LD₅₀ values and their 95% confidence limits using probit-logarithmical analysis of death occuring within 24 h after i.m. administration of each oxime at five different doses with eight animals per dose (Tallarida and Murray 1987).

The ability of oximes to reactivate Russian VX-inhibited AChE in vivo was determined as follows. To evaluate the reactivating efficacy of the oximes, the rats were injected i.m. with either atropine (21 mg/kg) alone or atropine (21 mg/kg) in combination with one of the oximes studied in equieffective, human-relevant dose (2% LD₅₀) 1 min after the rats received Russian VX i.m. at a dose of 14.0 μg/kg (LD₅₀). The control rats were administered i.m. with saline at the same volume instead of Russian VX and antidotes. The rats were decapitated and exsanguinated to obtain the blood 30 min following Russian VX poisoning. The brains were removed and homogenized in distilled water to determine AChE activity by a spectrophotometric method (Ellman et al. 1961). The reactivation rate was calculated using the AChE activity values: 1− [((oxime + atropine) − (saline))/((atropine control) − (saline))] × 100 (Clement, Hansen, and Boulet 1992). The AChE activity was expressed as μkat/kg or μkat/L (μmol substrate hydrolyzed/kg wet tissue or L blood/s).

The potency of oximes in combination with atropine to eliminate Russian VX-induced acute lethal effects in mice was determined as follows. The LD₅₀ value of Russian VX and its 95% confidence limit were assessed using probit-logarithmical analysis of death occuring within 24 h after i.m. administration of Russian VX at five different doses with eight mice per dose (Tallarida and Murray 1987). Then, Russian VX–poisoned mice were treated i.m. with one of tested oximes at equieffective doses (2% LD₅₀) in combination with atropine (21 mg/kg) at 1 min after i. m. challenge of Russian VX. The effectiveness of tested antidotal mixtures was evaluated by the assessment of the LD₅₀ values and their 95% confidence limits using probit-logarithmical analysis of death occuring within 24 h after i.m. administration of Russian VX at five different doses with eight mice per dose. The efficacy of tested antidotal mixtures was expressed as protective ratio (LD₅₀ value of Russian VX in protected mice/LD₅₀ value of Russian VX in unprotected mice). Statistical significance was determined by the use of Student’s t test and differences were considered significant when p < 0.05. Statistical evaluation was determined with the relevant computer programs (Tallarida and Murray 1987).

RESULTS

The acute toxicity of all tested oximes is shown in Table 1. Our results clearly demonstrate that the oxime HI-6 is markedly less toxic compared to pralidoxime and obidoxime. Therefore, the therapeutic dose of the oxime HI-6 corresponding to 2% LD₅₀ is significantly higher than the dose of pralidoxime and obidoxime. There is an advantage of the oxime HI-6 for the antidotal treatment of poisonings with nerve agents, generally.

The ability of oximes to reactivate Russian VX-inhibited AChE in rat blood and brain in vivo is shown in Table 2. All studied oximes were able to significantly reactivate Russian VX-inhibited AChE in the blood (p < .05) but only the oxime HI-6 restored its activity completely (100%). Although some differences in the reactivating efficacy of all tested oximes in the blood were found, the potency of all oximes tested to reactivate Russian VX–inhibited AChE in the brain was similar regardless of the type of oxime. The oxime HI-6 was demonstrated to be the most efficacious reactivator of Russian VX–inhibited AChE in the peripheral compartment, but its potency to reactivate Russian VX-inhibited AChE in the brain was comparable with other studied oximes.

The potency of oximes in combination with atropine to eliminate Russian VX–induced acute lethal effects in mice is shown in Table 3. Although the oxime HI-6 was able to decrease the acute toxicity of Russian VX more than seven times, the potency of the other currently available oximes (obidoxime and pralidoxime) to eliminate acute toxic effects of Russian VX was much lower. Obidoxime decreased the acute toxicity of Russian VX almost three times and pralidoxime less than two times. Thus, the oxime HI-6 is significantly more efficacious to eliminate lethal toxic effects of Russian VX in comparison with obidoxime as well as pralidoxime (p < .05) (Table 3). Russian VX–poisoned mice showed a wide spectrum of clinical signs of poisoning including muscarinic (lacrymation, salivation, chewing, miosis) and nicotinic (tremor, tonic-clonic convulsions) signs within a few minutes. In the case of antidotal treatment, the clinical signs appeared later and their intensity was diminished compared to untreated poisoning regardless of the type of oxime.

DISCUSSION

At the present time, the oxime HI-6 is considered to be the most promising reactivator of AChE inhibited by some nerve agents (Dawson 1994; Kassa 2002a). This knowledge corresponds to the results demonstrated in this study. They show the differences in therapeutic and reactivating efficacies among studied oximes. The oxime HI-6 was found to be more efficacious to eliminate acute lethal effects of Russian VX in mice and to reactivate Russian VX-inhibited AChE in the peripheral compartment (blood) of poisoned rats. On the other hand, the potency of all studied oximes to reactivate Russian VX–inhibited AChE in the central compartment (brain) was similar regardless of the type of oxime because Russian VX–induced inhibition of brain AChE in poisoned rats was relatively low (about 15% of control values) and because the penetration of oximes across brain-blood barrier is limited due to their chemical structure (the presence of quaternary nitrogen in their molecule) (Cassel et al. 1997; Sakurada et al. 2003). The activity of blood AChE corresponds to the activity of AChE in the peripheral nervous system and shows the severity of clinical signs caused by overstimulation of peripheral muscarinic and nicotinic cholinergic receptors. On the other hand, the activity of central AChE shows the severity of centrally mediated clinical signs of intoxication caused by the overstimulation of central muscarinic and nicotinic cholinergic receptors (Bajgar 2004; Marrs 1993; Taylor 1996).

The acute toxicity of Russian VX is mostly caused by the inhibition of peripheral AChE because Russian VX agent, as a structural analogue of VX agent, penetrates very poorly through the blood-brain barrier due to its chemical structure (Marrs 1993; Bajgar 2004). Therefore, the acute clinical signs after administration of VX or Russian VX are mostly caused by inhibition of peripheral AChE and subsequent accumulation of ACh on peripheral muscarinic and nicotinic receptors. The reactivation of Russian VX–inhibited peripheral AChE is more important for the therapeutic efficacy of oximes than the reactivation of central AChE. Therefore, the oxime HI-6 can be more efficacious in eliminating acute lethal effects of Russian VX, although there is not a difference among the potency of all oximes studied to reactivate Russian VX–inhibited AChE in the brain central compartment.

The data demonstrating the therapeutic and reactivating potency of all oximes studied against Russian VX are similar to the data previously published for VX agent (Kassa 2002b; Kuca and Kassa 2004). The similarity in efficacy of all oximes to reactivate Russian VX– and VX-inhibited AChE is probably due to analogous chemical structure of both nerve agents (Figure 1). In addition, our in vivo data closely correspond with previously published in vitro data describing the reactivating potency of some commonly used oximes to reactivate VX- and Russian VX–inhibited AChE in the rat brain homogenate. The oxime HI-6, characterized by high affinity to VX- and Russian VX-inhibited AChE and by the highest velocity of reactivation, was at low concentrations able to significantly reactivate AChE inhibited by both nerve agents (Kuca et al. 2006). The other studied oximes (pralidoxime and obidoxime) were also able to significantly reactivate VX- and Russian VX–inhibited AChE, but only at relatively high concentrations that do not correspond to in vivo recommended doses (Kuca and Kassa 2003; Kuca et al. 2006). Therefore, the results obtained in in vitro as well as in vivo studies indicate that HI-6 may be more effective than the other oximes tested (pralidoxime and obidoxime) for future human use because of the higher probability to achieve therapeutically sufficient concentration values in peripheral as well as central compartments for the oxime HI-6 compared to pralidoxime and obidoxime.