A COMPARISON OF THE NEUROPROTECTIVE EFFICACY OF PHARMACOLOGICAL PRETREATMENT AND ANTIDOTAL TREATMENT IN SOMAN-POISONED RATS

Despite of the entry into force in April 1997 of the Chemical Weapons Convention forbidding the production, storage and use of chemical warfare agents, the world has seen a rapid proliferation of such agents, especially nerve agents. Therefore, inactivation of extremely toxic organophosphorus compounds (nerve agents) has become a subject of major importance. The international control of their proliferation is thwarted by the ease of their synthesis and by similarity between their chemical precursors and widely used pest-control agents. Their harmful effect is related to their potency to irreversibly inhibit mammalian acetylcholinesterase (AChE, EC 3.1.1.7), the enzyme responsible for the regulation of neurotransmitter acetylcholine (ACh) concentration at cholinergic synapses (23). The inhibition of AChE induces a major increase in ACh level in the cholinergic nervous system producing muscle fasciculations, respiratory distress and epileptic fits leading to the generalized seizures. In surviving animals, the seizures lead to severe incapacitation and to irreversible brain damage with lesions especially in hippocampus, piriform cortex (17,21) and other cortical structures (13). The current antidotal treatment of nerve agent-induced acute poisoning usually consists of anticholinergic drugs to antagonize the effects of ACh excess at cholinergic receptor sites and oximes to reactivate nerve agent-inhibited AChE (4,7,20). Unfortunately, some organophosphates were found to be resistant to standard antidotal treatment. One of the most resistant organophosphorus compound is soman (pinacolyl methylphosphonofluoridate). Its deleterious effects are extraordinarily difficult to counteract because of rapid aging of soman-inhibited AChE (2). The relatively unsatisfactory treatment available for acute nerve agent poisoning has prompted studies of pretreatment possibilities that allow survival and increase re-


Introduction
Despite of the entry into force in April 1997 of the Chemical Weapons Convention forbidding the production, storage and use of chemical warfare agents, the world has seen a rapid proliferation of such agents, especially nerve agents. Therefore, inactivation of extremely toxic organophosphorus compounds (nerve agents) has become a subject of major importance. The international control of their proliferation is thwarted by the ease of their synthesis and by similarity between their chemical precursors and widely used pest-control agents. Their harmful effect is related to their potency to irreversibly inhibit mammalian acetylcholinesterase (AChE, EC 3.1.1.7), the enzyme responsible for the regulation of neurotransmitter acetylcholine (ACh) concentration at cholinergic synapses (23). The inhibition of AChE induces a major increase in ACh level in the cholinergic nervous system producing muscle fasciculations, respiratory distress and epileptic fits leading to the generalized seizures. In surviving animals, the seizures lead to severe incapacitation and to irreversible brain damage with lesions especially in hippocampus, piriform cortex (17,21) and other cortical structures (13).
The current antidotal treatment of nerve agent-induced acute poisoning usually consists of anticholinergic drugs to antagonize the effects of ACh excess at cholinergic receptor sites and oximes to reactivate nerve agent-inhibited AChE (4,7,20). Unfortunately, some organophosphates were found to be resistant to standard antidotal treatment. One of the most resistant organophosphorus compound is soman (pinacolyl methylphosphonofluoridate). Its deleterious effects are extraordinarily difficult to counteract because of rapid aging of soman-inhibited AChE (2).
The relatively unsatisfactory treatment available for acute nerve agent poisoning has prompted studies of pretreatment possibilities that allow survival and increase re-sistance of organisms exposed to nerve agents. Currently used method of protection against nerve agent poisoning is the use of pyridostigmine bromide, a reversible carbamate AChE inhibitor (1). Prophylactic effect of pyridostigmine can result from its reversible inhibition of AChE. It binds a small fraction of AChE in the periphery and reversibly shields it from irreversible inhibition by the nerve agents (3). However, pyridostigmine-induced increase in the level of ACh can itself cause symptoms of poisoning. Therefore, it would be useful to counteract the effects of the accumulated ACh using anticholinergic drugs. In addition, the combination of pyridostigmine with anticholinergic drugs allows to increase the dose of pyridostigmine because the anticholinergic drugs are able to counteract side cholinergic effects of pyridostigmine (8,10). One of these mixtures, pyridostigmine in combination with benactyzine (BNZ) and trihexyphenidyle (THP), designated PANPAL, has been developed in the Czech Republic and introduced to the Czech Army (22). Another mixture, pyridostigmine in combination with biperiden, has been developed in Bulgaria (18).
The aim of this study was to compare the neuroprotective effects of both pharmacological pretreatment mixtures with or without antidotal treatment consisting of HI-6 and atropine in soman-poisoned rats. The soman-induced neurotoxic signs were determined using a Functional observational battery, a non-invasive neurological examination containing measurements of sensory, motor and autonomic nervous functions.

Methods
Animals used in our experiments were male albino Wistar rats weighing 180-220 g purchased from Konárovice (Czech Republic). They were kept in an air-conditioned room and allowed to access to standard food and tap water ad libitum. The rats were divided into groups of eight animals (n=8). Handling of the experimental animals was done under the supervision of the Ethics Committee of the Medical Faculty of Charles University and Purkyne Military Medical Academy in Hradec Králové (Czech Republic).
Soman of 98.5% purity was obtained from Zemianské Kostolany (Slovak Republic). Its purity was assayed by acidimetric titration. The oxime HI-6 was synthesised at the Department of Toxicology of the Military Medical Academy and was 98% pure. Its purity was analysed using HPLC. All other chemicals and drugs of analytical grade were obtained commercially and used without further purification.
Pyridostigmine (0.75 mg/kg of body weight) in combination with BNZ (16 mg/kg of body weight) and THP (6.3 mg/kg of body weight) or pyridostigmine (0.75 mg/kg of body weight) in combination with bideriden (2.5 mg/kg of body weight) was administered intramuscularly (i.m.) as solution in distilled water (0.1ml/100g of body weight) 30 min before i.m. soman challenge at a lethal dose (54 µg/kg b.w. -LD 50 ). Antidotal treatment (HI-6 in combination with atropine) was carried out by i.m. injection 1 min following soman administration. The doses of HI-6 (15.6 mg/kg of body weight) and anticholinergic drug atropine (10 mg/kg of body weight) correspond to human-relevant doses (2% of their LD 50 ) (3). The neurotoxicity of soman was monitored using the Functional observational battery (FOB) at 24 hours and 7 days following soman poisoning. The evaluated markers of soman-induced neurotoxicity in experimental animals were compared to the parameters obtained from control rats, administered with saline instead of soman and antidotes at the same volume (0.1 ml/100g b. w.).
The FOB consists of 40 measures of sensory, motor and autonomic functions (Tab. 1) (5,14,15,16). First measurements were made while the animal was in the home cage. The observer evaluated each animal posture, palpebral closure, and presence or absence of convulsions. If convulsions were present, they were further categorized. Following observations in the home cage, the animal was removed and briefly held in the hand. Ease of removal and handling, skin and fur abnormalities, lacrimation, salivation and nose secretion were recorded.
Then, the rat was placed on a flat surface, which served as the open field covered with a clean absorbent pad. A timer was started for three minutes during which time the frequency of rearing responses was noted. At the same time, gait characteristics were noted and ranked, and arousal, tremor, convulsions and abnormal postures were evaluated. At the end of the time period (3 min), the number of faecal boluses and urine pools on the absorbent pad were recorded. Reflex testing followed next and consisted of recording each rat's responses to the frontal approach of a blunt object such as a pencil, a touch of an object to the posterior flank, and an auditory click stimulus. Reactivity to a pinch on the tail and the ability of the pupil to constrict in response to light were also assessed. These examinations were followed by a test for the righting reflex, then by measurements of forelimb and hindlimb grip strength, rectal temperature and finally landing foot splay. The entire battery of tests required approximately from six to eight minutes per one rat.
Motor activity data were collected shortly after FOB testing, using an apparatus for testing a spontaneous motor activity of laboratory animals (constructed in Purkyne Military Medical Academy, Hradec Králové, Czech Republic). The animals were placed into the measuring cage for a short time (10 minutes) and their movements (total, horizontal and vertical activity) were recorded.
Data collected with the FOB and motor activity assessment include categorical, ordinal and continuous values. Statistical analyses were performed on a PC with a special interactive programme NTX (5

Results
The results of the evaluation of soman-induced neurotoxicity at 24 hours and 7 days following soman poisoning are summarized in Table 2 and 3. Three non-treated somanpoisoned rats and one soman-poisoned rat treated with HI-6 and atropine died within 24 hours following soman administration. All pretreated soman-poisoned rats survived till the end of experiment (7 days following the intoxication).
The evaluation of soman-induced neurotoxic signs at 24 hours following intoxication proved significant alteration of 27 observed parameters. Soman caused relatively passive behaviour of rats during catching and handling. The animals were hypotonic. Soman also caused an increase in lacrimation, palpebral closure, nose secretion and skin as well as fur abnormalities. The exploratory activity in the open field was significantly decreased (p < 0.05). Abnormal clonic movements appeared, gait and mobility were impaired (p < 0.05). Soman influenced response of animals to approach of the object and pupil response to light too. Grip strength of all limbs as well as the distance between hindpaws after a jump were significantly diminished (p < 0.05), animal's spontaneous horizontal as well as vertical motor activity, food receiving and body temperature were significantly (p < 0.001) decreased (Table 2).
When pharmacological pretreatment (PANPAL or pyridostigmine in combination with biperiden) or antidotal treatment (atropine in combination with HI-6) were administered alone, a few soman-induced neurotoxic signs were only eliminated. On the other hand, if pharmacological pretreatment was combined with antidotal treatment, the soman-poisoned rats were sufficiently protected from acute neurotoxicity of soman. Rats pretreated by pyridostigmine in combination with biperiden and treated by atropine in combination with HI-6 showed the best protection from soman-induced neurotoxicity compared to other groups at 24 hours following soman challenge because this combination was able to eliminate the majority of soman-induced signs of neurotoxicity (excluding abnormal clinic movements, reduced rearing, arousal and rectal body temperature) (Tab. 2).
Passive behaviour of rats during catching and handling, tremor, abnormal clinic movements, impaired gait and mobility score and abnormal righting reflex were only observed in soman-poisoned rats at 7 days following soman administration. While PANPAL pretreatment alone or in combination with antidotal treatment was not able to eliminate soman-induced signs of neurotoxicity, the combination of pyridostigmine with biperiden eliminated all soman-induced neurotoxic signs when it was combined with antidotal treatment (Tab. 3).

Discussion
In the case of a threat of soman exposure, it seems to be very important to have sufficiently effective pretreatment because soman-induced toxic effects are extraordinarily dif-ficult to counteract due to very low reactivating efficacy of currently used oximes (6,7,12,19). Pyridostigmine that is stockpiled by various armed forces including the US army for pretreatment purpose against nerve agent poisoning is not sufficiently effective to increase the resistance of soman-exposed experimental animals (11) because it is only able to protect peripheral AChE from irreversible somaninduced AChE phosphonylation while soman can readily cross the blood-brain barrier and, therefore, exert its deleterious effects through its central toxic effects including centrally mediated seizures (2). The addition of centrally acting anticholinergic drugs to pyridostigmine for pharmacological pretreatment of acute soman exposures seems to be rational because a mixture of pyridostigmine with anticholinergic drugs should be able to increase the resistance of soman-poisoned animals and eliminate side effects of pyridostigmine, especially the effects of accumulated ACh (11).
The prophylactic efficacy of two various combinations of pyridostigmine with anticholinergic drugs were compared in this study. The combination of pyridostigmine with BNZ and THP stockpiled by Czech armed forces as PANPAL and Bulgarian prophylactic mixture consisting of pyridostigmine and biperiden. Both mixtures seem to be useful to increase the resistance of soman-poisoned rats and to increase the neuroprotective effects of common antidotal treatment of acute soman poisonings, nevertheless, the combination of pyridostigmine with biperiden appears to be more efficacious compared to PANPAL to eliminate acute soman-induced signs of neurotoxicity. Thus, biperiden that is characterized by high selectivity to m 1 muscarinic acetycholine receptors in the central nervous system (9) was found to be more efficacious to eliminate soman-induced centrally mediated seizures than BNZ and THP involved in PANPAL. Therefore, it should be considered as means for the currently used pretreatment of the nerve agent poisoning, especially in the case of the threat of exposure to soman.
In conclusion, the combination of prophylactic antidotal mixture containing pyridostigmine and centrally active anticholinergic drugs with common antidotal treatment consisting of anticholinergic drug (mainly atropine) and oxime (mainly obidoxime, pralidoxime or HI-6) seems to be sufficiently effective to counteract acute neurotoxic effects of soman.