A Combination of Pyridostigmine with Anticholinergic Drugs:
Effective Pharmacological Pretreatment of Soman-Poisoned Mice
Jiri Kassa, Josef Vachek, Jiri Bajgar and Josef Fusek
Dept. of Toxicology, Purkyne Military Medical Academy, 500 01 Hradec Kr·lovÈ, Czech Republic.
e-mail: kassa@pmfhk.cz

Introduction
          Despite the entry into force in April 1997 of the Chemical Weapons Convention, which forbids the production, storage and use of chemical warfare agents, the world has seen a rapid proliferation of such agents. Highly toxic organophosphorus compounds (OPs), neurotoxic or nerve agents, are considered the most dangerous of chemical warfare agents. They are also potential terrorist agents for both military and civilian populations, as well as occupational hazards to individuals exposed to OP insecticides. OPs' toxicity results from the irreversible binding to and inactivation of acetylcholinesterase (AChE, EC 3.1.1.7) and subsequent acetylcholine (ACh) accumulation, which leads to severe respiratory distress, prolonged limbic seizures, convulsions and death (1-2). The current standard treatment for poisoning by OPs consists of the combined administration of atropine sulfate and AChE reactivators (oximes). Atropine blocks the effects of overstimulation by accumulated ACh at muscarinic receptor sites, while AChE reactivators (generally nucleophilic compounds with high affinity for phosphorus) repair the biochemical lesion by dephosphonylation of AChE-OP complex, thus restoring AChE's activity (1-3).
           Unfortunately, certain OP compounds are resistant to standard antidotal treatment. One of the most resistant OP compounds is soman (pinacolyl methylphosphonofluoridate). It differs from many OPs in the rate of aging of the phosphonylated AChE; the soman-AChE complexes ages very quickly and this prevents the oxime-induced reactivation of AChE. Therefore the treatment of poisoning with soman is so difficult (4-7). The currently used oximes (obidoxime, pralidoxime), as well as H-oximes including HI-6, in combination with anticholinergic drugs are not considered to be sufficiently effective in decreasing the toxicity of soman (4-8).
           The relatively unsatisfactory treatment available for acute soman poisoning has prompted a study of pretreatment possibilities that allow survival and increase the resistance of organisms exposed to nerve agents. Currently the method of protection used against nerve agent poisoning is the use of pyridostigmine, a reversible carbamate AChE inhibitor (9). The prophylactic effect of pyridostigmine results 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 (10). However, the 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 permits the administration of a dose of pyridostigmine that would otherwise be limited by symptoms caused by elevated concentrations of ACh and results in a higher prophylactic efficacy than that observed for pyridostigmine alone (11-12). One of these mixtures, pyridostigmine in combination with benactyzine (BNZ) and trihexyphenidyle (THP), designated PANPAL, has been developed in our laboratory (13). *
          In the present study, we compared the influence of pyridostigmine alone or in combination with BNZ and THP (PANPAL) on the resistance of soman-exposed mice and on the therapeutic efficacy of antidotal treatment of soman-induced acute poisoning.

Methods

Animals
          Male NMRI mice from Kon·rovice (Czech Republic), weighing 19-22g were kept in an air-conditioned room with light from 07:00 AM to 07:00 PM and were allowed free access to standard chow and tap water. The rats were divided into groups of six animals each. Handling of experimental animals was under the supervision of the Ethics Committee of the Purkyne Military Medical Academy and the Medical Faculty of Charles University (Hradec Kr·lovÈ, Czech Republic).

Chemicals and drugs
          Soman of 98.5% purity was obtained from ZemianskÈ Kostolany (Slovak Republic). The oxime HI-6 of 99% purity was synthesized in the Department of Toxicology of the Purkyne Military Medical Academy. All other chemicals and drugs of analytical grade were obtained commercially and used without further purification.

Animals experiments
            Pyridostigmine (5.82 mg/kg of body weight) alone or in combination with BNZ (70 mg/kg of body weight) and THP (16 mg/kg of body weight) was administered perorally (p.o.) to male NMRI mice. The pyridostigmine or PANPAL was administered as a solution in distilled water (0.2 mL/100g of body weight) 60 or 120 min before the soman challenge. The antidotal treatment (the oxime HI-6 or obidoxime at equi-effective doses - 2% of their LD₅₀ in combination with atropine 8.4 mg/kg of body weight) and diazepam (1 mg/kg of body weight) were administered by intramuscular injection (i.m.) one min following soman administration. The dose of pyridostigmine, used in our experiments, causes 40% inhibition of erythrocyte AChE; the used doses of anticholinergic drugs correspond to common therapeutical doses (5% of their LD₅₀) (10). Soman-induced toxicity was evaluated with the help of LD₅₀ values and 95% confidence limits. The efficacy of tested pretreatment was expressed as protective ratio A (LD₅₀ value of soman in pretreated mice/ LD₅₀ value of soman in non-pretreated mice without antidotal treatment) and protective ratio B (LD₅₀ value of soman in pretreated mice/ LD₅₀ value of soman in non-pretreated mice with antidotal treatment).

Data Analysis
           The LD₅₀ values and their 95% confidence limits were estimated by probit analysis based on 24 h mortality data in at least four groups of six animals each. The differences between LD₅₀ values were considered to be significant when p < 0.05 (14).

Results
          The prophylactic efficacy of pyridostigmine alone and the prophylactic mixture PANPAL is presented in Table 1. While pyridostigmine alone is practically ineffective to decrease the soman-induced acute toxicity regardless of the time of its administration before the poisoning, PANPAL is able to significantly increase the 24h LD₅₀ value of soman in pretreated mice more than three times in comparison with the 24h LD₅₀ value in non-pretreated mice (p < 0.05).
          Similarly, pyridostigmine alone does not influence the efficacy of the antidotal treatment of soman-poisoned mice consisting of the oxime HI-6 and atropine, regardless of the time of pretreatment. On the other hand, the prophylactic mixture PANPAL is able to increase the efficacy of the antidotal mixture approximately two times in comparison with treated soman-poisoned mice without pretreatment (Table 2). PANPAL-induced increase in the effectiveness of the oxime HI-6 in combination with atropine is significant (p < 0.05). For the current antidotal mixture (obidoxime in combination with atropine and diazepam), pyridostigmine is able to increase the effectiveness of antidotal treatment of soman-poisoned mice when it is administered 2 hours before the poisoning, but this increase does not reach the efficacy of PANPAL pretreatment. (Table 3).

Conclusions
          In the case of a threat of soman exposure, it seems to be very important to have sufficiently effective pretreatment because soman-induced deleterious effects are extraordinarily difficult to counteract, due to the rapid aging and the existence of soman reservoir in the poisoned organism (15-16). Pyridostigmine is stockpiled by various armed forces for pretreatment purpose against nerve agent poisoning and has been used by several thousand servicemen during UN operation against Iraq in 1991 (17).
          Unfortunately, our results confirm the limited effectiveness of pyridostigmine to increase the resistance of soman-exposed mice, even with treatment. Pyridostigmine is only able to protect peripheral AChE from irreversible soman-induced AChE phosphonylation, while soman can readily cross the blood-brain barrier. Thus, soman can exert its deleterious effects through its central toxic effects, including centrally mediated seizure activity that can rapidly progress to status epilepticus and contribute to profound brain damage (18).
          In the case of fatal soman poisoning, death is caused by respiratory and subsequent circulatory paralysis, which may be of central origin because soman has been shown to be a potent central respiratory depressant (19). On the other hand, our data show that the pretreatment mixture PANPAL is able to significantly protect soman-poisoned mice, as well as increase the efficacy of antidotal pretreatment, regardless of the type of oxime used. The beneficial effect of this prophylactic mixture, developed in our laboratory, is probably caused not only by the protection of AChE from irreversible soman inhibition but also by a decrease in the cholinergic and stress causing effects of this nerve agent (11). Moreover, PANPAL seems to be very effective in enhancing the neuroprotective efficacy of antidotal treatment in the case of soman poisonings (20)
          The addition of anticholinergic drugs to pyridostigmine is useful, not only in enhancing of the efficacy of pretreatment to increase the resistance of soman-exposed animals, but also in eliminating the side effects of pyridostigmine, especially the effects of accumulated ACh. It is true that pyridostigmine at the commonly used dose (30 mg pyridostigmine tablet three times a day) is thought to be without significant side effects. However, when it was taken by 10 asthmatic solders during Operation Desert Storm, exacerbation of asthma symptoms in seven of the asthmatics was observed (17, 21). In addition, both evidence that some individuals may be more genetically susceptible to pyridostigmine and the potential for pyridostigmine to have synergistic effects with other chemicals have led some researchers and veterans to hypothesize that it may be a cause of postwar morbidity (22-23).
          Based on the presented data, pyridostigmine seems to be sufficiently effective in enhancing the survival of mice poisoned by supralethal doses of soman, when it is combined with anticholinergic drugs. The combination of pyridostigmine with anticholinergic drugs, such as PANPAL, has definite advantages over pyridostigmine alone in the pretreatment of soman poisoning and, therefore, it should be considered as replacement for the currently used pretreatment of the nerve agent poisoning.

Acknowledgement.
The authors express their appreciation to Mrs J Petrov·, E Vod·kov· and J UhlÌrov· for their skill technical assistance and help with the statistical evaluation.

References
1. Marrs TC, Organophosphate poisoning. Pharmacology & Therapeutics 1993; 58: 51 - 66.
2. Taylor P. Anticholinesterase Agents. In: Hardman JG, Limbird LE (eds.), The Pharmacological Basis of Therapeutics. 9th edn. New York: McGraw Hill, 1996.
3. Dawson RM. Review of oximes available for treatment of nerve agent poisoning. Journal of Applied Toxicology 1994; 14: 317 - 331.
4. Shih T-M. Comparison of several oximes on reactivation of soman-induced blood. Brain and tissue cholinesterase activity in rats. Archives of Toxicology 1993; 67: 637 - 646.
5. Koplovitz I, Stewart JR. A comparison of the efficacy of HI-6 and 2-PAM against soman, tabun, sarin and VX in the rabbit. Toxicology Letters 1994; 70: 269 - 279.
6. Kassa J. Comparison of efficacy of two oximes (HI-6 and obidoxime) in soman poisoning in rats. Toxicology 1995; 101: 167-174.
7. Kassa J, Cabal J. A comparison of the efficacy of a new asymmetric bispyridinium oxime BI-6 with currently available oximes and H oximes against soman by in vitro and in vivo methods. Toxicology 1999; 132: 111 - 118.
8. Lallement G, Clarencon D, Brochier G, Baubichon D, Golonnier M, Blanchet G, Mestries J-C., Efficacy of atropine/pralidoxime/diazepam or atropine/HI-6/prodiazepam in primates intoxicated by soman. Pharmacology, Biochemistry and Behavior 1997; 56: 325 - 332.
9. Anderson DR, Harris LW, Woodard CL, Lennox WI. The effect of pyridostigmine pretreatment on oxime efficacy against intoxication by soman or VX in rats. Drug and Chemical Toxicology 1992; 15: 285 - 292.
10. Bajgar J, Fusek J, Vachek J. Treatment and prophylaxis against nerve agent poisoning. ASA Newsletter 1994; 94-4: 10 - 11.
11. Kassa J, Bajgar J. The influence of pharmacological pretreatment on efficacy of HI-6 oxime in combination with benactyzine in soman poisoning in rats. Human & Experimental Toxicology 1996; 15: 383 - 388.
12. Kassa J, Fusek J. The positive influence of a cholinergic-anticholinergic pretreatment and antidotal treatment on rats poisoned with supralethal doses of soman. Toxicology 1998; 128: 1 - 7.
13. Vachek J, Kassa J, Fusek J, Bajgar J. Present possibilities of treatment of organophosphate poisoning (in Czech). SbornÌk v_deck_ch PracÌ VLA JEP Hradec Kr·lovÈ 1993; 116: 67 - 95.
14. Tallarida R, Murray R. Manual of Pharmacological Calculation with Computer Programs. New York: Springer-Verlag, 1987.
15. Bajgar J. Present view on toxidynamics of soman poisoning. Acta Medica (Hradec Kr·lovÈ) 1996; 39: 101 - 105.
16. Kadar T, Raveh L, Cohen G, Oz A, Baraness I, Balan A, Ashani Y, Shapira S. Distribution of 3H-soman in mice. Archives of Toxicology 1985; 58: 45 - 49.
17. Wenger B, Quigley MD, Kokla MA. Seven-day pyridostigmine administration and thermoregulation during rest and exercise in dry heat. Aviation, Space and Environmental Medicine 1993; 64: 905 - 911.
18. Tryphonas L, Clement JG. Histomorphogenesis of soman-induced encephalocardio- myopathy in Sprangue-Dawley rats. Toxicologic Pathology 1995; 23: 393 - 397.
19. Chang EC, Foster RE, Beers ET, Rickett DL, Filbert MG. Neurophysiological concomitants of soman-induced respiratory depression in awake, behaving guinea pigs. Toxicology and Applied Pharmacology 1990; 102: 233 - 238.
20. Koupilov· M, Kassa J. The influence of Panpal pretreatment on the elimination of soman-induced neurotoxicity by antidotes in rats. Homeostasis 1999; 39: 133 - 135.
21. Gouge SF, Daniels DJ, Smith CE. Exacerbation of asthma after pyridostigmine during Operation Desert Storm. Military Medicine 1994; 159: 108 - 111.
22. Loewenstein-Lichtenstein Y, Schwartz, Glick D, Norgaard-PedersenB, Zakut H, Soreq H. Genetic predisposition to adverse consequences of anti-cholinesterases in "atypical" BCHE carriers. Nature Medicine 1995;1: 1082 - 1085.
23. Abou-Donia MB, Wilmarth KR, Jensen KF, Oehme FW, Kurt TL. Neurotoxicity resulting from coexposure to pyridostigmine bromide, DEET, and permethrin: implications of Gulf War chemical exposures. Journal of Toxicology and Environmental Health 1996; 48: 35 - 56.

* Note: Panpal consists of three chemical compounds - pyridostigmine bromide, benactyzine hydrochloride and trihexyphenidyle hydrochloride (3-[[(dimethylamino)-carbonyl]oxy]-1-methyl pyridinium bromide, alpha-hydroxy-alpha-phenylbenzeneacetic acid 2-(diethylamino) ethylester hydrochloride, and alpha-cyclohexyl-alpha-phenyl-1-piperidinepropanol hydrochloride ).

01-3, issue no. 84