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PROPHYLAXIS AGAINST ORGANOPHOSPHORUS POISONING

Jiri Bajgar Purkyne Military Medical Academy, Department of Toxicology, Trebesska 1575, 500 01 Hradec Krlov, Czech Republic Tel.: +420 973 251 507 Fax: +420 495 518 094 E-mail: bajgar@pmfhk.cz ABSTRACT The terms prophylaxis and pre-treatment of organophosphorus (OP) poisoning (including nerve agent poisoning) are discussed. Some treatments to increase the resistance of an organism against the effects of these OP poisons have been developed and are based on the administration of reversible cholinesterase inhibitors. Reversibly inhibited carbamylated cholinesterase enzyme is resistant to subsequent inhibition by OP; it quickly and spontaneously decarbamylates and, thus, serves as a source of normal (uninhibited) enzyme. The administration of therapeutic drugs (antidotes) used for the treatment of OP intoxication is another approach for prophylaxis. Treatments causing a decrease of the level of OP in the blood stream are yet another approach for prophylaxis. Three prophylactics that have been introduced for use by military forces are pyridostigmine bromide alone, pyridostigmine with anticholinergics (PANPAL), and HI-6 for transdermal administration (TRANSANT). Administration of cholinesterase preparations and transdermal administration of physostigmine are new treatment modalities under intensive research. Key words: cholinesterases, inhibitors, organophosphorus, organophosphonates, poisoning, prophylaxis INTRODUCTION Prophylaxis is a medical term used to describe both the prevention of disease and preventive measures, such as immunization or vaccination (against infectious diseases) or mechanical measures (e.g., using condoms to prevent venereal diseases and cleaning teeth as a dental prophylaxis for decay and gum disease). Prophylaxis against chemical poisoning can be understood as preparation of the organism to be resistant or less sensitive to health disturbances/death caused by chemicals. From this point of view, prophylaxis against poisoning could include the use of protective measures (e.g., use of protective mask, etc.). However, the term prophylaxis as used in this article is limited to medical countermeasures applied relatively shortly before exposure of an organism to chemicals. Prophylaxis can be distinguished from pre-treatment. If further medical treatment is not required after the administration of a drug and after an exposure to chemicals, the drug is prophylactic and its administration is prophylaxis. However, if further medical treatment is necessary, despite the administration of a drug and even if the extent of the medical treatment is less than that without the drug, this is called pre-treatment.

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For example, although successful prophylaxis can be achieved for some organophosphorus compounds (OP), the ability of prophylaxis to fully protect the organism without post-exposure treatment is not clear, especially for Soman poisoning. From a practical point of view, it seems obvious that when a drug is administered prior to intoxication (either prophylaxis or pre-treatment) with the aim to protect the organism against a toxic agent, exposure to the agent is expected. For chemical warfare agents, some post-exposure therapy will probably be needed (i.e., pretreatment is the correct term). However, for simplicity, the term prophylaxis is used interchangeably in this article. Similarly, in this article the term toxic agent is limited to the group of organophosphorus chemicals, including nerve agentsthe most dangerous group of these chemicals. They are mainly represented by the organophosphonates Sarin (O-isopropyl methylphosphonofluoridate), Soman (Opinacolyl methylphosphonofluoridate), and VX (O-ethyl S-2-diisopropylaminoethyl methyl phosphonothiolate), though other nerve agents are known (e.g., cyclosarin, Tabun, Russian VX, or GV). GENERAL The basic mechanism of action of OP is known: it is based on irreversible acetylcholinesterase (AChE, EC 3.1.1.7) inhibition at cholinergic synapses. The resulting accumulation of acetylcholine at synaptic junctions overstimulates cholinergic pathways and subsequently desensitizes cholinergic receptor sites. The evidence supporting AChE as the primary site of OP action has been based on a number of observations, such as correlation between AChE inhibition in vivo and in vitro, good therapeutic efficacy of anticholinergics and cholinesterase reactivators, and the possibility of preventing intoxication (and cholinesterase inhibition) using reversible cholinesterase inhibitors (e.g., carbamates and others). However, a variety of data documented that AChE inhibition is not the only important biochemical change during the intoxication. Many other changes were described that accompanied the development of intoxication and might contribute to OP toxicity, including changes in other enzymes or neurotransmitters, immune changes, anaphylactoid reaction, behaviour, etc. The data also indicate prophylactic and therapeutic drugs might have multiple sites of action, similar to those sites of action that were observed during intoxication (Bajgar, 1991, 1992; Bardin et al., 1994; Cowan et al., 1996; Kassa, 1998). Nevertheless, the first reaction of the OP is interaction with cholinesterases, first in the bloodstream (as in first come, first served) (Benschop and de Jong, 2001), and then in the target tissuesthe peripheral and central nervous systems (Bajgar, 1985, 1991; Bardin et al., 1994; Green, 1958; Marrs et al., 1996). From this basic enzyme inhibition mechanism, it appears that the first step for prophylaxis is focused on protecting AChE against the inhibition or decreasing the concentrations of OP. By using enzymes to hydrolyze these agents or enzymes to bind the agents (e.g., to specific proteins or to antibodies), the OP concentrations are reduced, as are the levels of inhibition of cholinesterases, in the organism. The first group of drugs can be considered detoxicants, the second one as scavengers. In general, this approach is described as detoxification. Another approach to prophylaxis is based on the use of current antidotes. The current standard treatment

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for OP combines administration of anticholinergics (preferably atropine) and reactivators. Anticholinergics block the effect of accumulated acetylcholine, while reactivators repair the inhibition of AChE via dephosphorylation and thus regenerate normal enzyme, AChE. For the treatment of convulsions, benzodiazepines (diazepam) are used. Contrary to the treatment of other OPs, treatment of Soman poisoning is very difficult and unsatisfactory. This is the reason for intensive studies using pretreatment/prophylaxis to increase survival and resistance of an organism exposed to Soman. The administration of current antidotes (anticholinergics, reactivators, and other drugs) to prevent effects of OP seems to be described as treatment of the symptoms, not the cause (i.e., administration of the drug to the point that the OP symptoms appear to have been countered). The problem of this approach is achieving sufficient levels of antidotes for a relatively long time. Combinations of these approaches are also possible (table 1). PROTECTION OF AChE AGAINST INHIBITION Keeping AChE active is a basic requirement for effective prophylaxis (i.e., changing the enzyme in a way so that it is resistant to OP). AChE can be kept active using reversible inhibitors, which inhibit the enzyme, thus making it unavailable for reaction with OP but subsequently allowing the recovery of normal AChE activity, ideally after other enzymes have reacted with the OP and hydrolyzed it or otherwise inactivated it. AChE belongs to the group of hydrolases catalyzing hydrolysis of choline esters to choline and relevant acid. They are differentiated into two classes, based on their enzymatic properties and physiological functions: AChE and butyrylcholinesterase (BuChE, EC 3.1.1.8.) (Massouli et al., 1993; Darvesh et al., 2003). AChE splits the neuromediator acetylcholine at the cholinergic synapses. AChE has been observed in erythrocytes but its function there is not yet known. Similarly, the function of BuChE in blood plasma is not yet known, though there is evidence that this enzyme has an important role in cholinergic neurotransmission and could be involved in other nervous system functions and in neurological diseases (Darvesh et al., 2003). Activity of cholinesterases is of fundamental importance for diagnosis of intoxication with cholinesterase inhibitors, including OP and carbamates (Bajgar, 1985; Bardin et al., 1994; Cowan et al., 1996; Marrs et al., 1996). Conversely, the activity depends on many other factors and, therefore, cholinesterase determination is of diagnostic importance in different pathological states (i.e., not only intoxications) (Bajgar, 1985, 1991; Brimijoin and Rakonczay, 1986; Rakonczay, 1988). The activities of these enzymes (AChE and BuChE) are influenced by sex, age, nutrition, hormonal factors, irradiation, etc. (Brown et al., 1981; Darvesh et al., 2003; Herink et al., 1975, 1980; Skau, 1986; Skopec et al., 1981; Whittaker, 1980). The variation of BuChE activity is greater than that of AChE (Bajgar, 1985, 1991; Brown et al., 1981; Kutty, 1980) and is genetically determined (Brown et al., 1981; Whittaker, 1980). As other serine hydrolases, AChE contains a catalytic triad, the so-called esteratic site Ser200-His440-Glu327, at the bottom of a deep and narrow cavity known as the aromatic gorge since more than 50 percent of the gorges lining is composed of the

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rings of 14 conserved aromatic amino acids. Acetylcholine (ACh) must pass down into this gorge and bind to the active site within. Ligands binding to esteratic site are called acylating (OP, carbamates, alkylsulphonates). Non-acylating ligands are bound to sites other than esteratic sites (anionic sites) and comprise a large group of chemicals, including tetralkylammonium, coumarine, tacrine, gallantamine, etc. These anionic sites can be responsible for the allosterism of AChE, along with the complexity of the enzyme structure (Soreq et al., 1992; Sussman et al., 1991). In addition to the subsites of the catalytic center, AChE possesses one or more additional binding sites for ACh and other quaternary ligands. Such peripheral anionic binding sites are at the lip of this gorge. In BuChE, Trp279, an important component of the peripheral binding site in AChE, is missing. This peripheral binding site is believed to be responsible for substrate inhibition (Radic et al., 1991), which is one of the features that distinguish AChE from BuChE. OP inhibits AChE via phosphorylation of the esteratic site. The rate of spontaneous dephosphorylation is very low and can be increased by reactivators (oximes). The efficacy of oxime reactivation is dependent on both oxime and conjugated phosphonate structures (Taylor et al., 1999). Concurrently, the microenvironment of the gorge plays a significant role in determining selectivity of substrate and inhibitors for cholinesterases (Saxena et al., 1999). In the case of some OPs after phosphorylation of the active site, the complex of the enzyme-inhibitor (i.e., the part of the OP-AChE complex that is able to be reactivated by oximes) is changed to an unreactivatable complex, resistant to reactivators. According to the molecular mechanism, the OP-AChE complex splits forming the OP alcohol and an unreactivatable enyzme. This reaction, called aging or dealkylation, is very fast for Soman-inhibited AChE (the half-life is about 10 minutes) and is slower for Sarin (the half-life is about 10 hours) (Bajgar, 1991, 1992; Fleisher and Harris, 1965; Talbot et al., 1988). This is one of the reasons for the difficulty with therapeutic interventions of Soman intoxication, in particular (Bajgar, 1996; Wolthuis et al., 1994). Peripheral site ligands may have selective effects on AChE phosphorylation (Rosenberry et al., 1999). There are many inhibitors of cholinesterases, which can diminish both AChE and BuChE activities to a comparable extent as has been described by Aldridge (1969). Carbamates belong to the group of inhibitors with a large variation in their effectiveness. They are biologically active because of their structural complementarity to the active surface of AChE and their consequent reaction as substrates with very low turnover numbers (Aldridge, 1969). The ability of some carbamates to protect an organism poisoned with OP has been known for many years (Koster, 1946; Koelle, 1946). Physostigmine and neostigmine were used to protect animals against diisopropyl phosphorofluoridate (DFP). The number of OPs studied for protection, as well as the number of carbamates studied, was increased. These studies were performed both in vitro and in vivo. The results are very dependent on experimental conditions; nevertheless, protective effects of physostigmine, aminostigmine, pyridostigmine, and others against AChE inhibition caused by different OPs (mostly soman studies) have been demonstrated (Patocka, 1989; Marrs et al., 1996; Tonkopii, 2003). Important contributions based on

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modelling of different inhibition processes in OP-intoxicated organisms were described by Ellin (1982), Green (1958, 1983), and Maxwell et al. (1987). There have been numerous studies that demonstrated the effectiveness of carbamate pretreatment/prophylaxis against intoxication with OP. From the results, published and unpublished, pyridostigmine appeared to be the most promising prophylactic drug, especially against Soman poisoning (Anderson et al., 1992; Bajgar, 1985, 1991, 2001; Bajgar et al., 1994, 1996; Fusek et al., 2000; Kassa, 1995; Kassa and Fusek, 1998, 2000; Kassa et al., 1997, 2001a, 2001b; Koplovitz et al., 1992; Koupilova and Kassa, 1999; Marrs et al., 1996; Maxwell et al., 1993; Patocka et al., 1991; Tuovinen, 1998; Wolthuis et al., 1994). On the basis of these results, pyridostigmine was introduced into the military forces of several nations as a prophylaxis against nerve agents. Its prophylactic effect (as with the effects of other carbamates) is limited by its dose. In higher doses, higher efficacy was observed but more side effects were expressed. This problem can be solved by adding pyridostigmine antagonizing drugsanticholinergics. Many anticholinergics were tested with respect to their abilities to protect an organism against intoxication with soman (and other nerve agents) and on the basis of this research, a prophylactic combination of pyridostigmine with trihexyphenidyle and benactyzine (PANPAL) was introduced into the Czech Army (Bajgar et al., 1994, 1996; Kassa, 1995; Kassa and Bajgar, 1996; Kassa and Fusek, 1998, 2000; Kassa and Vachek, 2002; Kassa et al., 2001a, 2001b). The presence of two anticholinergics suppressed some of the pyridostigmine side effects and allowed an increase in pyridostigmine dose, which produced an increase in its prophylactic efficacy. This combination (and following therapy) is not limited to Soman, Sarin, and VX poisoning; PANPAL was observed to be highly effective against Tabun (Kassa and Vachek, 2002), GV (Bajgar et al., 1996), and cyclosarin (Kassa and Cabal, 1999) intoxications. These nerve agents are also known to be resistant to common antidotal treatment. PANPAL had no side effects, as was demonstrated on volunteers; no statistically different changes in actual psychic state and no negative changes in dysfunction time were observed. An improvement of results of a tapping test (psychomotoric reaction time) following PANPAL administration was demonstrated. A decrease in heart frequency 60 minutes following PANPAL administration, lasting 480 minutes and returning to normal values within 24 hours, was demonstrated (Fusek et al., 2000). On the basis of tests, which evaluated the prophylactic efficacy of different carbamates, aminostigmine appeared to be the most effective protector of AChE (Tonkopii, 2003). Its ability to protect cholinesterase to a sufficient extent was demonstrated but not sufficiently documented. Aminostigmine may be part of a prophylactic treatment composed of aminostigmine, an antioxidant, diazepam and a cholinolytic. According to personal communications, its efficacy is very high and long-lasting. However, without further information (composition, doses of drugs, form, etc.), aminostigmine by itself or in combination cannot be considered relevant for serious discussions. Other carbamates also have good prophylactic efficacy, especially physostigmine (due to its effect on the central nervous system, in contrast to pyridostigmine) (Kim et al., 2001, 2002; Sket, 1993; Tuovinen and Hanninen, 1999; Wolthuis et al., 1994). Human studies using transdermal physostigmine suggest a serious interest in the prophylactic use of this drug (Walter et al., 1995; Levy et al., 1992). Mobam and decarboxyfuran were also considered as potential candidates for prophylaxis based on

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experiments. Aminophenols were tested for their protective effects against OP poisoning, but their effects were lower than pyridostigmines (Marrs et al., 1996). Inhibitors structurally different from carbamate and OP groups were also studied. From these compounds, those preferably binding to AChE anionic site, tacrine, methoxytacrine, and huperzine A were considered and experiments were performed in vitro and in vivo that studied their abilities with respect to prophylaxis (Ashani et al., 1992; Bajgar et al., 1983; Freeman and Dawson, 1991; Fusek, 1977; Lallement et al., 2002; Lau, 1992; Patocka, 1998; Patocka and Kassa, 1999; Raves et al., 1997). The most interesting results were obtained with huperzine A. It is an inhibitor of rat brain AChE (mixed linear competitive type) (McKinney et al., 1991). Very similar results were obtained with enzymes from other sources (Saxena et al., 1994). Huperzine A was tested as a potential candidate against OP, both for its long-lasting efficacy and relatively low toxicity (Grunwald et al., 1994). The results obtained to date do not support the replacement of pyridostigmine by any of these newer drugs. DETOXIFICATION Detoxification can be accomplished by the administration of enzymes to split the OPAChE complex. Exogenously administered enzyme binds to the OP and thus decreases the OP level in the organism (i.e., the exogenously administered enzyme acts as a scavenger). Enzymes that can hydrolyze OPs are under investigation (Li et al., 1995; Raveh et al., 1992). On the other hand, many studies have been made with cholinesterases as scavengers. BuChE and AChE were observed to be very effective in protection against OP intoxication (Clark et al., 2002; Doctor et al., 1991, 1997, 2002; Marrs et al., 1996; Maxwell et al., 1993, 1998; Moore, 1996; Saxena et al., 1997). The administration of enzymes as scavengers seems to be one very promising avenue of research. The enzyme (either AChE or BuChE) acts at the very beginning of the OPs toxic action, before the OP can interact with target tissues and without side effects (Clark et al., 2002; Doctor et al., 1997, 2002). This was clearly demonstrated not only in rodentsguinea pigs (Allon et al., 1998), mice (Ashani et al., 1991: Raveh et al., 1989) and rats (Brandeis et al., 1993: Genovese and Doctor, 1995)but also in primates (Broomfield et al., 1991; Maxwell et al., 1992; Raveh et al., 1997; Wolfe et al., 1992). All these features are of great interest and research is starting to produce practical results. Moreover, in experiments using BuChE as the pre-treatment, protective effects on AChE inhibition in the brain, following low-level Sarin inhalation exposure, were demonstrated (Sevelova et al., 2003). As we develop better knowledge in bioengineering and biotechnology, a connection between the two enzymes may be possible, with the aim to obtain a modified enzyme, capable of splitting OP and simultaneously reacting with AChE as a scavenger (Broomfield et al., 1997). Antibodies against OP are also possible in future research, but research studies on antibodies are currently focused more on detection of OP.

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SIMULATION OF TREATMENT Therapeutic approaches to OP poisoning using current antidotes are partially effective, and these antidotes may be considered and tested as possible prophylactics. Standard antidotes have been studied in this respect (i.e., anticholinergics, reactivators, anticonvulsants, and others) (Patocka et al., 1991; Samnaliev et al., 1997; Marrs et al., 1996). The problem in their use is the timing, duration and achievement of sufficient concentrations of these antidotes after administration. However, the prophylactic efficacy is good, as has been demonstrated in treatment studies with antidotes, which have been mostly administered within minutes after exposure. Prolonging the duration of the antidote effect by attaining sufficient concentrations in the blood is not possible by oral administration (especially for reactivators) and therefore is excluded. This was the reason we searched for other routes of administration. Transdermal administration of one of the most effective reactivators (HI-6) was shown as the most realistic of these other routes (Dolezal et al., 1988). The final result was the new prophylactic transdermal antidote TRANSANT. This preparation was clinically tested (including dermal sensitivity) without any harmful effects, and field testing was also successful. The final reports were finished and TRANSANT was introduced into the Czech Army. All nerve agents were capable of producing neuropathology. Seizure control was strongly associated with protection against brain pathology and lethality (Shih et al., 2003). Therefore, prophylactic efficacies of anticonvulsants were studied. The benzodiazepines (diazepam, midazolam, alprazolam, triazolam, clonazepam) were studied, but isolated prophylactic administrations have not produced very good effects (Marrs et al., 1996; Herink, 1992; Herink et al., 1990, 1991; Kubova et al., 1990). McDonough and Shih (1997) proposed a three phase model of neuropharmacological processes responsible for the seizures and neuropathology produced by nerve agent intoxication: a cholinergic phase (initiation and earlyexpression of seizures), a transition phase (excitatory amino acid levels increase), and finally a non-cholinergic phase (stimulation of NMDA receptors) leading to the irreversible brain neuropathology. Rapid and aggressive management of each phase is necessary to prevent nerve neuropathology from nerve agent exposure. Six benzodiazepines were studied in guinea pigs to stop seizures produced by exposure to Soman (avizafone, clonazepam, diazepam, loprazolam, lorazepam, and midazolam). Midazolam was the most potent and rapidly acting drug (McDonough et al., 1999). Similar results (i.e., midazolams antiseizure efficacy was better in comparison with diazepam) were obtained in rats (Herink, 1992). Calcium antagonists (nimodipine), neuromuscular blockers (tubocurarine), adamantanes (memantine), and opiate antagonist meptazinol (Galli and Mazri, 1988; Marrs et al., 1996; Karlsson et al., 1998; Stojiljkovic et al., 1998) were also tested with varying results, but these have not been very useful for practical use. Conversely, positive prophylactic effect has been demonstrated with procyclidine (an antimuscarinic, antinicotinic, and anti-NMDA receptor drug) (Myhrer et al., 2003). Special importance can be focused on suramine (protease inhibitor). Administration of procyclidine prior to Soman intoxication (and followed by administration of atropine) showed good prophylactic effect (Cowan et al., 1996). However, all these studies are experimental studies and have not yet reached any practical outputs.

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COMBINATIONS Drug and antidote combinations can be of very different character. They can be used simultaneously (combination of different drugs) or as pre-treatment and following treatment with different antidotes. Administration of pyridostigmine (or other inhibitors) prior to intoxication and treatment with different drugs is a typical example (Anderson et al., 1992; Bajgar et al., 1996; Kassa, 1995; Kassa and Bajgar, 1996; Kim et al., 2001, 2002; Tuovinen and Hanninen, 1999). There are other combinations such as the administration of triesterase (Tuovinen, 1998), procyclidine (Kim et al., 2001, 2002), and clonidine (Loke et al., 2001), and sustained release of physostigmine and scopolamine (Meshulam et al., 2001). The results are very dependent on experimental conditions but this approachadministration of different drugshas produced some good outcomes; however, these combinations have been tried only at the experimental level. The only prophylactic antidote introduced into the Czech armyPANPALis composed of pyridostigmine, benactyzine, and trihexyphenidyle. These and many other studies were performed to improve prophylaxis of OP poisoning. They contribute to elucidation of OP action on the nervous system. This approach could lead to improvement of our knowledge of mechanisms of action of OP and other cholinesterase inhibitors and of the poisoning caused by these chemicals and their possible treament. Simultaneously, it could contribute to better understanding of cholinergic nerve transmission and thus to biochemistry, pharmacology, and neuropharmacology in general. CONCLUSIONS In summary, simple prophylaxis (without post-exposure treatment) against OP is not sufficient; however, prophylaxis with combinations of different drugs is of special importance. Pyridostigmine is important as a prophylactic drug, especially when it is followed by post-exposure antidotal treatment. For further development, it is necessary to search for new drugs for prophylaxis. Preparations of cholinesterases are of special importance for development of more effective prophylactics. New routes of administration of prophylactics are also of interest for further evaluation. A very small number of the prophylactics that have been studied are actually used in the regular equipment of the military medical service of any army.

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REFERENCES Aldridge, W.N. (1969) Organophosphorus compounds and carbamates, and their reactions with esterases. Brit med Bull. 25, 236239. Allon, N., Raveh, L., Gilat, E., Cohen, E., Grunwald, J., and Ashani, Y. (1998) Prophylaxis against soman inhalation toxicity in guinea pigs by pretreatment alone with human serum butyrylcholinesterase. Toxicol Sci. 43, 121128. Ashani, Y., Shapira, S., Levy, D., Wolfe, A.D., Doctor, B.P., and Reveh, L. (1991) Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice. Biochem Pharmacol. 41, 3741. Anderson, D.R., Harris, L.W., Woodard, C.L., and Lennox, W.I. (1992) The effect of pyridostigmine pretreatment on oxime efficacy against intoxication by soman and VX in rats. Drug Chem Toxicol. 15, 285294. Ashani, Y., Peggins, I.I.I., J.O., and Doctor, B.P. (1992) Mechanism of inhibition of cholinesterases by huperzine A. Biochem Biphys Res Commun. 184, 719726. Bajgar, J. (1985) Intoxication with organophosphorus cholinesterase inhibitors. Mechanism of action, diagnosis and treatment (in Czech). Novinky v medicin 34, Avicenum, Prague, 740. Bajgar, J. (1991) The influence of inhibitors and other factors on cholinesterases. Sb Ved Pr Lek Fak UK (Hradec Krlov). 34, 375. Bajgar, J. (1992) Biological monitoring of exposure to nerve agents. Brit J Ind Med. 49, 648653. Bajgar, J. (1996) Present views on toxidynamics of soman poisoning. Acta Med (Hradec Kralove) 39, 101105. Bajgar, J. (2001) Development of new methods and means for diagnosis and prophylaxis of poisoning with nerve agents. (in Czech). Vojenske rozhledy (Military views) 10, 148153. Bajgar, J., Fusek, J., Patoka, J., and Hrdina, V. (1983) Protective effect of 9-amino7-methoxy-1,2,3,4-tetrahydroacridine against O-ethyl S-(2-dimethylaminoethyl) methylphosphonothioate in vivo. Arch Toxicol. 54, 163166. Bajgar, J., Fusek, J., and Vachek, J. (1994) Treatment and prophylaxis against nerve agent poisoning. ASA Newsletter. 94-4, 1011. Bajgar, J., Fusek, J., Vachek, J., Kassa, J., and Patocka, J. (1996) Organophosphate poisoning:imrovement of prophylaxis. Proceedings of the 2nd CBMTS, 712 July 1996, Spiez, Switzerland, pp.201204. Bardin, P.G., van Eeden, S.F., Moolman, J.A., Foden, A.P., and Joubert, J.R. (1994) Organophosphate and carbamate poisoning. Arch Intern Med. 154, 14331441. Benschop, H.P., De Jong, L.P.A.: Toxicokinetics of nerve agents. In: Chemical Warfare Agents: Toxicity at Low Levels (Somani, S.M. and Romano, J.A., Eds.) Chapter 2. CRC Press, Boca Raton, Florida, USA, 2001. Brandeis, R., Raveh, L., Grunwald, J.,Cohen, E., and Ashani, Y. (1993) Prevention of soman-induced cognitive deficits by pretreatment with human butyrylcholinesterase in rats. Pharmacol Biochem Behav. 46, 889896.

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Brimijoin, S. and Rakonczay, Z. (1986)Immunocytology and molecular histology of cholinesterases: current results and prospects. Int Rev Neurobiol. 28, 353410. Broomfield, C.A., Maxwell, D.M., Solana, R.P., Castro, C.A., Finger, A.V., and Lenz, D.E. (1991) Protection by butyrylcholinesterase against organophosphorus poisoning in non human primates. J Pharmacol Exp Ther. 259, 633638. Broomfield C.A., Lockridge, O., Millard C.B., and Lenz, D.E. (1997) Design and construction of butyrylcholinesterase mutants that have organophosphorus acid anhydride hydrolase activity. Mini CB Medical Treatment Symposium, Hradec Kralove, Czech Republic, 2639 May 1997, Abstracts p. 1314 Brown, S.S., Kalow, W., Pilz, W., Whittaker, M., and Woronick, C.L. (1981) The plasma cholinesterases: a new perspectives. Adv Clin Chem. 22, 1123. Clark, M.G., Saxena, A., Anderson, S.M., Sun, W., Bansal, R., Myers, T.M., and Doctor, B.P.(2002) Behavioral toxicity of purified human serum butyrylcholinesterase in mice. The 4th International CBMTS, 28 April3 May 2002, Spiez, Switzerland, Abstract No 19. Cowan, F.M., Shih, T.M., Lenz, D.E., Madsen, J.M., and Broomfield, C.A. (1996) Hypothesis for synergistic toxicity of organophosphorus poisoning-induced cholinergic crisis and anaphylactoid reactions. J. Appl. Toxicol. 16, 2533 Darvesh, S., Hopkins, D.A., Geula, C. (2003) Neurobiology of butyrylcholinesterase. Nature Rev Neurosci. 4, 131138. Doctor, B.P., Raveh, L., Wolfe, A.D., Maxwell, D.M., and Ashani, Y. (1991) Enzymes as pretreatment drugs for organophosphate toxicity. Neurosci Behav Rev. 15, 123128. Doctor, B.P., Maxwell, D.M., and Saxena, A. (1997) Preparation and characterization of bioscavengers for possible use against organophosphate toxicity. M-CB Medical Treatment Symposium, 2630 May 1997, Hradec Kralove, Czech Republic, Abstracts p. 1718. Doctor, B.P., Saxena, A., Clark, M.G., Bansal, R., Luo, C., Rosenberg, Y., Lenz, D., and Ashani, Y. (2002) Scavenger protection against organophosphates by human serum butyrylcholinesterase. The 4th International CBMTS, 28 April3 May 2002, Spiez, Switzerland, Abstract No 24 Dolezal, P., Vachek, J., and Hrabalek, A. (1988) In vitro transdermal permeation of a cholinesterase reactivator HI-6. In: Perspectives in percutaneous penetration (R.K. Brain and K.A. Walters, Eds.). Vol. 6A, STS Publishing, Cardiff, 1988, p. 84. Ellin, R.I. (1982) Anomalies in theories and therapy of intoxication by potent organophosphorus anticholinesterase compounds. Gen Pharmacol. 13, 457456. Fleisher, J.H., and Harris, L.W. (1965) Dealkylation as a mechanism for ageing for cholinesterase after poisoning with pinacolyl methylphosphonofluoridate. Biochem Pharmacol. 14, 641650. Freeman, S.E. and Dawson, R.M. (1991) Tacrine: a pharmacological review. Prog Neurobiol. 36, 257277. Fusek, J. (1977) Tacrin a and its analogues, antidotes against psychotomimetics with anticholinergic effect (in Czech). Voj Zdrav Listy. 46, 2127.

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