Abstract
The mechanism of intoxication with organophosphorus compounds, including highly toxic nerve agents and less toxic pesticides, is based on the formation of irreversibly inhibited acetylcholinesterase, which causes cumulation of neuromediator acetylcholine in synaptic clefts and subsequent overstimulation of cholinergic receptors, that is followed by a generalized cholinergic crisis. Nerve agent poisoning is conventionally treated using a combination of a cholinolytic (atropine mostly) to counteract the accumulation of acetylcholine and acetylcholinesterase reactivators (pralidoxime or obidoxime) to reactivate inhibited acetylcholinesterase. In this study of cyclosarin poisoning treatment, oximes of different chemical structures (obidoxime, HI-6, BI-6, and HS-6) were tested in vitro on rat brain acetylcholinesterase (enzyme source: rat brain homogenate), and afterwards, they were tested in vivo in equimolar doses, in mice and rats. The HI-6 oxime appeared to be the most effective oxime in vitro and in vivo.
Sarin, tabun, soman, cyclosarin, and VX are among the best known nerve agents. These compounds cause longterm inactivation of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) by phosphorylation of serine residue at the active site of the enzyme (Bajgar 2004).
The presently used antidotes against nerve agent intoxications, such as pralidoxime or obidoxime (causal antidotes) in combination with atropine (functional antidote), do not appear universally suitable as antidotes against poisonings with all kinds of nerve agents (Kassa 2002). For example, pralidoxime has no reactivation ability in treatment of cyclosarin- and tabun-inhibited AChE. Obidoxime is not able to reactivate AChE inhibited by cyclosarin and its ability to reactivate tabun inhibited AChE is poor (Cabal et al. 2004; Kassa and Cabal 1999; Bartosova et al. 2004).
Cyclosarin (GF-agent; O-cyclohexylmethylphosphonofluoridate) belongs to the group of nerve agents considered as potential chemical warfare agents (Clement 1992; Koplovitz et al. 1992; Lundy et al. 1992). During the Persian Gulf War, it was believed that Iraq might have GF in its arsenal (Gee 1992). Unfortunately, as mentioned above, currently available oximes for medical first aid (pralidoxime and obidoxime) are not potent reactivators of cyclosarin-inhibited AChE.
Owing to this fact, we have tested during last 3 years more than 50 structurally different AChE reactivators using our in vitro screening test (10−3 M concentration of the oxime only) (Kuca and Patocka 2004; Kuca, Patocka, and Cabal 2003). According to our results, the oximes HS-6, BI-6, and HI-6 seem to be the most potent reactivators of cyclosarin-inhibited AChE. In this work, we compared their potency to reactivate in vitro cyclosarin-inhibited AChE in the concentration range of 10−8 to 10−2 M, and, afterwards, we tested their therapeutic efficacy in vivo. Obidoxime, a typical member of the AChE reactivator group, was used as the standard reactivator for comparison.
MATERIALS AND METHODS
Animals
Adult albino female mice weighing 28 to 30 g and Wistar rats weighing 200 to 220 g were used throughout this study. They were housed six in a cage, in a temperature-controlled (20°C to 24°C) environment with 12-h light/dark cycles (lights on from 0600 to 1800 h) and they had free access to food and water except during the experimental period. The animals were divided into groups (six animals in each group). The animals used in this study were handled under the supervision of the Ethics Committee of the Medical Faculty of Charles University and the Faculty of Military Health Sciences in Hradec Kralove, Czech Republic.
Chemicals
Cyclosarin (88.9%) was provided by the Military Technical Institute Zemianske Kostolany, Slovakia. HS-6 (1-(2-hydroxyiminomethylpyridinium)-3-(3-carbamoylpyridinium)-2-oxa-propane dichloride) oxime was a gift of Dr. Stojiljkovic, Serbia and Montenegro. Obidoxime (1,3-bis(4-hydroxyiminom-ethylpyridinium)-2-oxa-propane dibromide) was purchased from Merck (Germany). HI-6 (1-(2-hydroxyiminomethylpyri-dinium)-3-(4-carbamoylpyridinium)-2-oxa-propane dichloride) and BI-6 (1-(2-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)-but-2-ene dibromide) were synthesized earlier at our laboratory. All other chemicals and drugs were obtained from commercial sources and were of reagent grade.
In Vitro Experiments
A standard in vitro method was used for the evaluation of reactivation potencies of the abovementioned oximes (Kuca and Kassa 2003). General conditions were as follows. Rat brain homogenate diluted in distilled water (10%, w/v) was used as a source of the cholinesterases (ChEs). Measurement was taken at 25°C, pH 7.6, and concentration of the AChE reactivators in a range of 10−7 to 10−2 M. Time of AChE inhibition with nerve agent was 30 min. Time of reactivation of inhibited AChE with reactivator was 10 min. The activity of ChEs was determined by pH static titration of acetic acid released from acetylcholine iodide using an autotitrator (Radiometer TTT 80 Titrator, Copenhagen, Denmark).
Animal Experiments
In our experiment, four oximes, HS-6, HI-6, BI-6, obidoxime, each in combination with atropine, were tested as an antidotal treatment of cyclosarin intoxication. Dose of each oxime administered for treatment of cyclosarin intoxication was 100 μmol/kg of body weight; atropine at 21.0 mg/kg of body weight was given as well to both mice and rats. The dose of antidotes was chosen to compare therapeutic ratios with previous results. This oxime therapy combined with atropine was administered intramuscularly (i.m.) 1 min after cyclosarin challenge (i.m.). Cyclosarin-induced toxicity in mice and rats after i.m. administration was evaluated by the assesment of LD50 value at 24 h.
Data Analysis
Cyclosarin-induced toxicity was evaluated by the assessment of LD50 values and their 95% confidence limits that were calculated by probit analysis of deaths occuring within 24 h after administration of cyclosarin at five different doses with six animals per dose (Tallarida and Murray 1987). The differences between LD50 values were considered to be significant when p < 0.05 using Student’s t test (Roth, Josifko, and Trcka 1962). The efficacy of tested treatment was expressed as a therapeutic ratio (LD50 value of cyclosarin in treated animals/LD50 value of cyclosarin in nontreated animals).
RESULTS
In Vitro
Data characterizing in vitro reactivation potency of tested oximes are summarized in Table 1 and Figure 1. As can be seen, reactivation potency of obidoxime is too low. Reactivation potency of other oximes tested decreases as follows HI-6 > BI-6 > HS-6.
Curves characterizing the relationship between concentration of oximes and their ability to reactivate AChE show that HI-6 and BI-6 have similar concentration-responses during the reactivation process. Both reactivators have their maximum reactivation capability at the concentration 10−4 M, which could be attainable in in vivo experiments. On the other hand, oxime HS-6 does not achieve as high reactivation potency as HI-6 and BI-6 do, and moreover, its maximum reactivation potency occurs at higher concentration of 10−3 M.
In Vivo
A comparison of the therapeutic efficacy of oximes is presented in Table 2. Atropine was able to decrease the cyclosarin-induced acute toxicity only slightly in both mice and rats. Obidoxime and HI-6 in combination with atropine were chosen as the standard therapeutic regimens in evaluating of therapeutic potency of BI-6 and HS-6 (HI-6 and obidoxime are available in Czech Armed Forces as ANTIVA and COMBOPEN, respectively). All the oximes were able to reduce the acute toxicity of cyclosarin by at least 1.5-fold. Obidoxime was the least effective oxime in both mice and rats. The therapeutic efficacy of BI-6 was only slightly higher than that of HS-6 in mice but very significantly higher in rats. HI-6 in combination with atropine was the most effective therapeutic regimen in both mice and rats.
DISCUSSION
As it can be seen from obtained results, all four tested oximes were potent reactivators of cyclosarin-inhibited AChE using in vivo methods, only obidoxime was not effective in vivo. Moreover, the reactivation effectiveness was greater for all than that of obidoxime. This is significant because the same results were obtained for two different species.
Results obtained using in vivo results are not always similar with in vitro results (Lucic et al. 1997). In order to get a better comparison between in vitro and in vivo methods we tested all oximes in equimolar doses in in vivo experiments. Our results show that our in vivo results are in very good agreement with our in vitro results. In the future, for the comparison of in vitro and in vivo results, the dose of oximes administered in vivo should be equimolar.
Previous studies with cyclosarin-poisoned animals suggested a poor efficacy of the marketed oximes obidoxime and pralidoxime, whereas the newer oximes HI-6 and HLo 7 proved to be effective antidotes (Clement 1992; Kassa and Bajgar 1995; Koplovitz et al. 1992; Lundy et al. 1992; Sevelova, Kuca, and Krejcova-Kunesova 2005). Because aging should not be the major cause of its refractory property toward oximes (half-time for aging >4 h), this phenomenon is worthy of investigation in the future. Accordingly, in this study it was found that therapeutic regimen consisting of HI-6 and atropine showed the highest therapeutic ratio. In addition, the safety factor of HI-6 is considerably greater than the conventional oximes (pralidoxime or obidoxime) (Kassa and Cabal 1999).
Oxime BI-6 was also found to be potent reactivator of cyclosarin-inhibited AChE. Similar results were obtained earlier by Kassa and Cabal (1999). However, its potency to reactivate cyclosarin-inhibited ChEs does not exceed the HI-6 potency.
In conclusion, according to our results, HI-6 (available in Czech Armed Forces as ANTIVA) remains the most effective oxime in treatment of cyclosarin intoxication. Unfortunately, this AChE reactivator is not currently available as a one-shot antidote for soldiers on the battlefield. Implementation of this reactivator as a first aid antidote desires further investigation.
Footnotes
Figure and Tables
Acknowledgements
The authors are grateful to Mrs. M. Hrabinova and Mrs. J. Uhlirova for their skilful technical assistance. This work was supported by the Grant of Ministry of Defence of Czech Republic no. ONVLA-JEP20031.