A COMPARISON OF THE POTENCY OF NEWLY DEVELOPED OXIMES ( K 347 , K 628 ) AND CURRENTLY AVAILABLE OXIMES ( OBIDOXIME , HI-6 ) TO COUNTERACT ACUTE NEUROTOXIC EFFECTS OF TABUN IN RATS

Organophosphorus nerve agents are considered to be the most dangerous chemical warfare agents. Their acute toxic effects are based on the phosphonylation of acetylcholinesterase (AChE, EC 3.1.1.7), leading to the irreversible inhibition of its active site and subsequent overstimulation of postsynaptic cholinergic receptors due to the accumulation of the neurotransmitter acetylcholine in the synapses of the central and peripheral nervous systems (21, 22). The standard antidotal treatment of nerve agent poisoning usually includes an anticholinergic agent to block the overstimulation of cholinergic receptors and an oxime to reactivate nerve agent-inhibited AChE (4, 29). Compounds with a nucleophilic oximate anion have been discovered and are considered to be able to reactivate nerve agent-inhibited AChE by dephosphonylating the enzyme active site and restoring its activity. However, some nerve agents were found to be resistant to standard antidotal treatment. One of the most resistant nerve agent is tabun (O-ethyl-N,N-dimethyl phosphoramidocyanidate), the deleterious effects of which are extraordinarily difficult to antagonize because of the changes in hydrogen bonding and the conformational changes of AChE-tabun complex prior to an aging process in the AChE active site (1, 6). Tabun can produce centrally-mediated seizure activity that rapidly progresses to status epilepticus and contributes to profound brain damage (22, 29). The exposure of experimental animals to tabun in convulsions-inducing doses may result in irreversible lesions in the central nervous system (CNS) that can be manifested as behavioral effects in survivors that have convulsed (9). Therefore, the ability of antidotes to block the acute neurotoxic effects of tabun and prevent development of irreversible lesions in CNS is important for successful antidotal treatment. Generally, the oximes exert more potent effects in the peripheral compartment compared to central compartment due to their poor penetration into CNS. Nevertheless, the penetration of oximes into CNS and subsequent reactivation of nerve agent-inhibited AChE in the brain were demonstrated (3, 28). Although the rate of this reactivation is lower compared to that in the peripheral system, its role in CNS is important for survival from nerve agent exposure (11, 22). Generally, the commonly used reactivators of phosphonylated AChE based on monopyridinium (e.g. pralidoxime) and bispyridinium oximes (e.g. obidoxime, the oxime HI-6)


Introduction
Organophosphorus nerve agents are considered to be the most dangerous chemical warfare agents.Their acute toxic effects are based on the phosphonylation of acetylcholinesterase (AChE, EC 3.1.1.7),leading to the irreversible inhibition of its active site and subsequent overstimulation of postsynaptic cholinergic receptors due to the accumulation of the neurotransmitter acetylcholine in the synapses of the central and peripheral nervous systems (21,22).
The standard antidotal treatment of nerve agent poisoning usually includes an anticholinergic agent to block the overstimulation of cholinergic receptors and an oxime to reactivate nerve agent-inhibited AChE (4,29).Compounds with a nucleophilic oximate anion have been discovered and are considered to be able to reactivate nerve agent-inhibited AChE by dephosphonylating the enzyme active site and restoring its activity.However, some nerve agents were found to be resistant to standard antidotal treatment.One of the most resistant nerve agent is tabun (O-ethyl-N,N-dimethyl phosphoramidocyanidate), the deleterious effects of which are extraordinarily difficult to antagonize because of the changes in hydrogen bonding and the conformational changes of AChE-tabun complex prior to an aging process in the AChE active site (1,6).
Tabun can produce centrally-mediated seizure activity that rapidly progresses to status epilepticus and contributes to profound brain damage (22,29).The exposure of experimental animals to tabun in convulsions-inducing doses may result in irreversible lesions in the central nervous system (CNS) that can be manifested as behavioral effects in survivors that have convulsed (9).Therefore, the ability of antidotes to block the acute neurotoxic effects of tabun and prevent development of irreversible lesions in CNS is important for successful antidotal treatment.Generally, the oximes exert more potent effects in the peripheral compartment compared to central compartment due to their poor penetration into CNS.Nevertheless, the penetration of oximes into CNS and subsequent reactivation of nerve agent-inhibited AChE in the brain were demonstrated (3,28).Although the rate of this reactivation is lower compared to that in the peripheral system, its role in CNS is important for survival from nerve agent exposure (11,22).
Generally, the commonly used reactivators of phosphonylated AChE based on monopyridinium (e.g.pralidoxime) and bispyridinium oximes (e.g.obidoxime, the oxime  are not able to counteract sufficiently the acute toxic effects of tabun because of their low reactivating efficacy (13,18,26,30).Therefore, the replacement of commonly used oximes (pralidoxime, obidoxime, HI-6) with a more effective oxime has been a long-standing goal for the treatment of tabun poisoning (5,24).New oximes, K347 (1-benzyl-2-hydroxyiminomethylpyridinium bromide) and K628 [4-carbamoyl-4'-hydroxyiminomethyl-1,1'-(1,3-phenylenedimethyl)bispyridinium dibromide] (Fig. 1) have been synthesized at our Department of Toxicology (25) to improve the efficacy of antidotal treatment.The evaluation of their potency to reactivate tabun-inhibited AChE using in vitro methods showed that the reactivating efficacy of both newly developed oximes roughly corresponds to the effectiveness of obidoxime and it is better than the potency of HI-6 to reactivate tabun-inhibited AChE (25).The aim of this study was to compare the neuroprotective efficacy of two newly deve-loped oximes (K347, K628) with currently available oximes (obidoxime, the oxime HI-6) in combination with an anticholinergic drug atropine in tabun-poisoned rats.The tabun-induced neurotoxic signs were determined using a functional observational battery, a non-invasive and relatively sensitive type of neurological examination for a wide range of neurobiological functions including measurements of sensory, motor and autonomic nervous functions (7).

Materials and Methods
Male albino Wistar rats weighing 200-230 g were purchased from VELAZ (Prague, Czech Republic).They were kept in an air-conditioned room (22 ± 2 o C and 50 ± 10 % relative humidity, with lights from 7.00 to 19.00 hr) and allowed access to standard food and tap water ad libitum.The rats were divided into groups of 8 animals.Handling of the experimental animals was performed in compliance with relevant laws and institutional guidelines and under the supervision of the Ethics Committee of the Faculty of Military Health Sciences in Hradec Kralove (Czech Republic).
Tabun was obtained from the Military Technical Institute in Brno (Czech Republic) and was 96 % pure as assayed by acidimetric titration.All oximes studied were of 98.5 % purity and were synthesized at the Department of Toxicology of the Faculty of Military Health Sciences in Hradec Kralove (Czech Republic).Their purities were analyzed using HPLC.All other drugs and chemicals of analytical grade were obtained commercially and used without further purification.All substances were administered intramuscularly (i.m.) at a volume of 1 mL/kg body weight (b.w.).
Tabun was administered at a sublethal dose (220 μg/kg b.w.-80 % LD 50 ).One minute following tabun poisoning, the rats were treated with atropine (21 mg/kg b.w.) in combination with the oxime HI-6, obidoxime, K347 or K628 at equitoxic doses corresponding to 5 % of their LD 50 values.The neurotoxicity of tabun was monitored using the functional observational battery at 24 hr following tabun poisoning.The evaluated markers of tabun-induced neurotoxicity in experimental animals were compared with the parameters obtained from control rats given saline instead of tabun and antidotes at the same volume.
The functional observational battery consists of 47 measurements of sensory, motor and autonomic nervous functions.Some of them are scored (Table 1), the others are measured in absolute units (7,8).The first evaluation was obtained when tabun-poisoned rats were in the home cage.The observer evaluated each animal`s posture, palpebral closure and involuntary motor movements.Then, each rat was removed from the home cage and briefly hand-held.The exploratory activity, piloerection and other skin abnormalities were noted.Salivation and nose secretion were also registered and scored.Then, the rats were placed on a flat surface which served as an open field.A timer was started for 3 min during which the frequency of rearing responses was recorded.At the same time, gait characteristics were noted and ranked, and arousal, stereotypy and bizarre behaviors and abnormal posture were evaluated.At the end of the third min, the number of fecal boluses and urine pools on the adsorbent pad was registered.Reflex testing comprising recording each rat`s response to the frontal approach of the blunt end of a pen, a touch of the pen to the posterior flank and an auditory clic stimulus was also used.The response to a pinch on the tail and the ability of pupils to constrict in response to light were then assessed.These measures were followed by a test for the aerial righting reflex and by the measurements of forelimb and hindlimb grip strength, body weight, body temperature and finally hindlimb landing foot splay.The whole battery of tests required approximately 6-8 min per rat.The observer of the behavior did not know about the design of the experiments.Motor activity data were collected shortly after finishing the functional observational battery, using an apparatus for testing of a spontaneous motor activity of laboratory animals (constructed at the Faculty of Military Health Sciences, Hradec Kralove, Czech Republic).The animals were placed for a short period (10 min) in the measuring cage and their movements (total, horizontal and vertical activity) were recorded.
Data collected with the functional observational battery and motor activity assessment include categorial, ordinal and continuous values.Statistical analyses were performed on a PC with a special interactive programme NTX (7).The categorial and ordinal values were formulated as contingency tables and judged consecutively by the Chi-squared test of homogeneity, the Concordance-Discordance test and the Kruskal-Wallis test, respectively.The continual data were assessed by successive statistical tests: the CI for Delta, the Barlett test for Equality of Variance, the Williams test and the Test for Distribution Functions (27).The differences were considered significant when p < 0.05.

Results
All tabun-poisoned rats survived till the end of experiment (24 hr following the intoxication) regardless of the type of oxime used.Non-treated tabun-poisoned rats survived for 24 hr after tabun challenge, too.
The results of the experiments related to the measurement of tabun-induced neurotoxicity at 24 hr following tabun poisoning are divided into three parts (activity and neuromuscular measures, sensorimotor and excitability measures and autonomic measures -23) and summarized in Tab.2a-c.Observation of neurotoxic signs indicated that many functional disorders in the tabun-poisoned rats lasted for at least 24 hr.Tabun produced passive behavior of rats during handling and retention, miosis and a decrease in muscular tone at 24  0,00 0,00 0,00 0,00 0,00 0,00 5 tremors 0,00 0,00 0,00 0,00 0,00 0,00 6 clonic movements 0,00 0,00 0,00 0,00 0,00 0,00 7 tonic movements 0,00 0,00 0,00 0,00 0,00 0,00 8 gait 0,00 and rearing activity were significantly decreased and gait was somewhat impaired.The pupils of the tabun-poisoned rats did not constrict in response to light because of tabuninduced miosis.A significant decrease in limb grip strength, food receiving, body temperature and spontaneous horizontal as well as vertical motor activity were also observed at 24 hr following tabun challenge (Tab.2a-c).While the potency of the oxime HI-6 to eliminate tabun-induced acute neurotoxic effects was negligible, both newly developed oximes (K347, K628) in combination with atropine were able to prevent some tabun-induced signs of neurotoxicity observed at 24 hr following tabun challenge with the ex-ception of a decrease in muscular tone, exploratory and rearing activity, miosis, gait impairment, a decrease in hindlimb grip strength, food receiving and spontaneous horizontal as well as vertical motor activity (Tab.2a-c).Obidoxime in combination with atropine was able additionally to eliminate gait impairment and a decrease in spontaneous vertical motor activity (Tab.2a).
Based on the structure-activity relationship study, there are five most important structural factors influencing the affinity of the AChE reactivators toward nerve agent-inhibited AChE and subsequent oxime reactivity: the presence of the quaternary nitrogen in the reactivator molecule, the length of the connection chain between two pyridinium rings, the presence of the oxime group, the position of the oxime group at the pyridinium ring and the number of oxime groups in the reactivator structure.The above mentioned data on a reactivator structure allowed us to postulate requirements for the structural parameters of new reactivators of tabun-inhibited AChE (19).For tabun poisonings, at least one oxime in position four on the heteroaromatic ring is necessary for substantial reactivating, therapeutic and neuroprotective potency whilst an oxime in position two has a low or no capability to counteract acute toxicity of tabun (19).Additionally, the optimal linker length suitable for tabun intoxication varies from 3 to 4 carbon-carbon bonds (2).
Our results demonstrate that both newly developed oximes (K347, K628) are partly able to reduce tabun-induced acute neurotoxic signs and symptoms.The difference between the neuroprotective efficacy of both newly developed oximes is not significant although K347 is a monopyridinium oxime and K628 is a bispyridinium oxime.Thus, the difference between the penetration of monopyridinium and bispyridiniun oximes into CNS is not enough to cause the significant difference between their neuroprotective effectiveness.
The results confirm that there is not a single, broad-spectrum oxime suitable for the antidotal treatment of poisonings with all organophosphorus agents (16).The neuroprotective efficacy of both newly developed oximes (K347, K628) is slightly lower than the neuroprotective efficacy of obido-xime and it is markedly higher compared to the oxime HI-6.Their neuroprotective efficacy corresponds to their potency in reactivating tabun-inhibited AChE (25).As the neuroprotective potency of both newly developed oximes is slightly lower compared to the effectiveness of the currently available obidoxime, they are not a suitable replacement for obidoxime in the treatment of acute tabun poisonings.
hr following tabun administration.Exploratory The values of tabun-induced sensorimotor and excitability neurotoxic markers measured at 24 hr following tabun challenge by the functional observational battery (scored values).Statistical significance: *p < 0.05 (comparison with the control values).