Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants (1984)

Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

2 CHOLINESTERASE REACTIVATORS BACKGROUND the nine teenth and tweet ieth mustard gas as a toxic weapon , ~ the warfare potential of chem- ical agents. This led to support for research on lethal nerve agents during and immediately after World War Il. The research was followed by the development of treatment methods, and prominent among these was the use of cholinesterase Deactivators to reverse the lethal ef fec ts of anticholinesterase nerve gases . Rapid advances in chemistry during centuries, coupled with the success of in Wnrl r1 War T attracted attention to The terminal ion of World War II brought to light the highly potent chemical-warfare agents of the organophosphorus ester class that had been synthesized by both sides during the conflict. The properties, pharmacology, and toxicology of these anticholinesterase agents were discussed ire Volume 1 of the National Research Council report . 17 Such agents as diisopropyl fluorophosphate ~ DFP), sarin (GB), soman (GD), and tabun (GA) were a fascination to pharmacolo- gists who conduc ted extensive studies in the early postwar years . It soon became evident that no available antidotes could block the pharmacologic activity of these chemicals, alleviate the signs and symptoms of toxicity, or restore normal bodily functions after exposure. Atropine readily antagonized the muscarinic actions, including those in the central nervous system (CNS), but elicited no reversal of the nicotinic effects. Better forms of therapy were sought, particularly to alleviate the nicotinic effects of anticho- linesterase agents. _ ,. The first suggestion of a practical form of antidotal therapy came in 1949 from Hestrin,9 who found that acetylcholinesterase (ACHE) catalyzed the formation of acetohydroxamic acid when incu- bated with sodium acetate and hydroxylamir~e. Critical in vitro studies in the next decade led to the development of a practical approach to therapy. The crucial concept ire these studies was the recognition that the compound formed when AChE reacted with a phosphorus ester was a covalent phosphoryl-enzyme intermediate similar to that formed in the hydrolysis of acetylcholine.23 Wilson and colleagues, beginning in 1951, demonstrated that AChE inhibited by alkyl phosphate esters (tetraethyl pyrophosphate, TEPP) could be reactivated by water, but that free enzyme formed much more rapidly in the presence of hydroxylamir~e. 20 ~ 21 Similar results —3—

were obtained by Hobbiger.l0 In 1953, Wilson and Meislich26 synthesized nicotinehydroxamic acid and the corresponding meth- iodide; these agents were far more potent Deactivators of Skye phosphate-inhibited AChE than was hydroxylamine. In 1955, Wilson described picolinehydroxamic acid as the best Deactivator among a series of quaternary hydroxamic acids, noting that oximes also were reactiva~cors. In in vitro studies, Davies and Green4 and Childs et al. 3 reported that 2-formyl-N-methy~pyridinium iodide oxide (2-PAM iodide form) was 170 times more active than picoline- hydroxamic acid in reactivating TEPP-inhibited AChE in vitro, 30 times more active in reactivating DFP-inhibited AChE, and 400 times more ac Live ire reactivating GB-inhibited AChE . Similar f indings were reported by Wilson and Gineburg.24,25 The first investigators to show the marked antidotal properties of pral idoxime compounds (Figure 2-~) were Kewitz and Wilson.13 Askew1 noted marked species differences in the rate and effec- tiveness of oximes and hydroxamic acids in reactivating al kyl phosphate-inhibited AChE. Other in viva studies demonstrated that some oximes were effective in antagonizing the blockade of neuro- muscular transmission at ter treatment of animals with TEPP, DFP, or GB. 12 The methyl methanesulfonate derivative of 2-PAM, P2S ~ Figure 2-1), was shown to be very effective by virtue of greater water solubillty. 5 Additional studies demonstrated that the most effective treatment of organophosphonus ester-poisoned animates was to use oximes concomi tantly wi th atropine . The general conclusions were that atropine minimizes or abolishes the toxic actions of AChE- inhibi tiny compounds on mu scarinic and central chorine rgic si tea and the pyridini''m and other quater~ary hydroxamic acids and oxides reverse the toxic ef fects of the organophosphorus esters by reac- t ivating the inhibited AChE. 14 ,19 Success with the pyridinium oximes led to an intensive search for more effective oximes and the discovery of an especially potent derivative, 1, 3-bis(4-formy~pyridinium~propane dibromide biso2rime - (~B-4) (Figure 2-1), which reactivated serum-inhibited AChE in vitro more rapidly than 2-PAb1 or P2S.11,l8 l~lB-4 was an effective chemi cad adjunct to atropine in sarin-poisoned mice, rats, rabbits, cats, and dogs, 2 Further modifications of the bispyridinium structure--insertion of dif ferent groups between the pyridinium rings--led to the synthesis of bis(4-formyl-N-methylpyridinium oxide) ether bichloride known originally as LuH6 and later as toxogonin (Figure 2-1).' Its advantage was that it was less toxic than 1~B-4 and also capable of reactivating AChE inhibited by a variety of organophosphorus esters .6,15 With the demonstration of species variability in response to the various oximes,\ it was noted that results obtained with a parti- cular organophosphate were not obtained with other agents, ever if —4—

H-~-OH O(3 CH3 2-PAM. Pyrldine-2-aldoxime methiodide methyl chloride LOU - H. N - H CH, so.O — 1 CH, P2S. Pyridine-2-aldoxime methyl methanesulfonate CHAT - H CHAT - H (3: e! CH2 - H2-CH2 28r() TMB-4. N,N-trimethylene bis~pyridine-4-aldoxime bromide) HC- - OH HC--N—OH 0~ itch CHINCH: Toxogonin. Bis(4-formyl N-methylpyridini~,m oxime) ether dichloride FIGURE 2-1 Structural formulas of cholinesterase Deactivators tested at Edgewood

they formed the same phosphorylated enzyme. This meant that screening of the reactivators for suitable antidota1 properties necessitated in viva testing with as many organophosphorus esters as possible. It also meant that data from animal studies could not be extrapolated to humans without considerable caution, necessitating testing in humans. The ef fectiveness of oximes in treating poison- ing by organophosphorus eater chemical - carfare agents stimulated the U. S. Army to initiate studies with four compounds--2-PAM, P2S, TMB-4, and tozogonin--in military volunteers from 1958 to 1975, to assess their effectiveness against low doses of suitable militarily impor- tant organosphates. This chapter examines some pertinent features of the pharmacology and toxicology of the pralidoxime Deactivators (Table 2-1~. It presents conclusions of the Panel on Cholinesterase Reactivators as to the potential of these agents to impair chroni- cally the health and well-being of the volunteers who participated in the studies conducted by the Department of the Army. It focuses on the four agents tented at Edgewood. A detailed report of the pharmacology and toxicology of the four oxime Deactivators is in Appendix B. TABLE 2-l Subjects Tested with Cholinesterase Reactivators at Edgewood No . Sub; ec ts Tox. No. EA No. Compound CAS NO. (Approx.) D-1 217 0 2-PAM: pralidoxime 51-15-0 607 chloride ~ or iodide) protopam chloride (or iodide); 2-f ormyl-N-me thylp yrid ini um chloride [or iodide) oxime; pyridine-2-aldoxime methoch'oride (or methiodide) D-2 P2S: methyl methanesulfonate 154-92-2 95 derivative of 2-PAM; pralidoxime me thanesulf onat e; Prot opam methanesulf orate D-3 3475 Toxogonin: LuH6; bier 4-formyl- 114-90-9 41 Name thy~pyridini um o2rime ~ e ther dichloride D-4 1814 TMB-4; 1, 1 ~ -trimethylene- 56-9 7-3 bis ~ 4-formyl-pyridinium ~ bromide (bromide ~ ~ or chloride ~ dioxide; 3613-81-9 trimedoxime; 1,3-bis (chloride) ~ 4-f ormy~pyridinium) propane dibromide (or dichloride) bisoxime —6—

REFERENCES - 1. Aslcaw, B.M. Oximes and hydroxamic acids as antidotes in anticholinesterase poisoning. Br. J. Pharmacol. Chemo the r. 11:417-423, 1956. 2. Bay, E., Krop , S . and Yates , L. F. Chemotherapeutic effec- ti~reness of I,1 '-trimethylene bis (4-formy~pyridinium bromide) dioxime (TMB-4) in experimental anticholinesterase poisoning. Pro c . Soc. Exp . Biol. Med. 98 :107-110 ? 1958. Childs , A. F., Davies, D.R., Green, A.L., and Rutiand, J. P. reacti~ration by oximes and hydroxamic acids of cholinesterase inhibi~ced by organophosphorus compounds. Br. J. Pharmacol. Chemother. 10 :462-465, 1955. Da~ries, D.R., and Green, A.L. General Discussion. Discuss. Faraday Soc. No. 20: 269, 1955. 5. Davies, D.R., Green, A.L., and Willey, G.L. 2-Hydroxy- imiIlomethyt -N~ethy~pyridini ~ me thanesulphonat e and atropine in the treatment of severe organophosphate poisoning. Br. J. Pharmacol. Chemother. 14: 5-8, 1959. 6. Engelhard, N., and Erdmann , W. D. Beziehangen zwischen chemischer Struktur und Cholinesterase reaktivierender Wirk~amkeit bei einen Reihe neuer bis-quartarer Pyridin-4- aldoxime . Arzoem. Forsch. 14: 870-875, 1964. Engelhard , H., and Erdmarm, W. D. Ein neuer Reaktivator fur durch Alky~phosphat gehemmte Acetylcholinesterase. Klin. Wochenschr. 41: 5 25-5 27, 19 63. . Grob, D., and Johns , R. J. Use of oximes in the treatment of intoarication by anticholinesterase compounds in normal subjects Am. J. Med. 24:497-511, 1958. 9. Hestrin, S.J. Acylation reactions mediated by purified acetyl- choline esterase . J. Biol . Chem. lB0: 879-~81, 1949. 10. Hobbiger, F. Inhibition of cholinesterases by irreversible inhibitors in ~ritro and in vivo. Br. J. Pharmacol. Chemother. 6:21-30, 1951. 11. Hobbiger, F., O' Sullivan, D.G., and Sadler, P.W. New potent reactivators of acetocholinesterase inhibited by tetraethyl pyrophosphate. Nature 182: 1498-1499, 1958. —7—

Ho Ime ~ , R., and Robins , E . L. The reversal by oximes of neuro- muscular block produced by anticholinesterases. Br. J. Pharmacol. Chemother. 10: 490-495, 1955. Kewitz, H., and Wilson, I.B. A specific antidote against lethal a~y~phosphate intoxication. Arch. Biochem. Biophys. 60: 261- 263, 1956. 14. Kewitz , H., Wilson, I. B., and Nachmansohn, D. A specific anti- dote against lethal alkyl phosphate intoxication. II. Antidotal properties. Arch. Biochem. Biophys. 64: 456-465, 1956. Milosevic , M., Andjelkovic , D., and Binenfeld, Z . Effete de divers oximes, derives de chlorures de l-phenyle, 1-benzyle et 1-phenacyle pyridine et lea analogues de 4-formyle pyrldin dans 1' intoxication au paragon. Med. Exp. 10: 73-7B, 1964. 16. Namba, T., and Hiraki, K. PAM (pyridine-2-aldoxime methiodide) therapy for alkylphosphate poisoning. J. Am. Med. Assoc. 16 6: 1834-1839, 1958. 17. National Research Council, Committee on Toxicology. Possible Long-Term Health Ef fee ts of Short-Term Exposure to Chemical Agents. Vol. I. Anticholinesterases and Anticholinergica. Washington, D. C.: National Academy Press, 1982. 290 pp. 18. Poziomek, E.J., Hackley, B.E., Jr ., and Steinberg , G.M. Pyridir~ium aidoximes. J. Org. Chem. 23:714-717, 1958. 19. Wills, J. H. Recent studies of organic phosphate poisoning. Fed. Proc., Fed. Am. Soc. Exp. Biol. 18: 1020-1025, 1959. 20. Wilson, I. B. Acety~cholinesterase. XI. Reversibility of tetraethyl pyrophosphate inhibition. J. Blol. Chem. 190:111-~17, 1951. 21. Wilson, I . B. Acety~cholinesterase . XTII. Reactivation of alkyl phosphate-inhibited enzyme. J. Biol.- Chem. 199:~13-120, 1952. 22. Wilson, I. B. Promotion of acetylcho~nesterase activity by the anionic site. Discus s. Faraday Soc. No . 20 :119-125, 1955. 23. Wilson, I.B., Bergman, F., and Nachmansohn, D. Acetyl- cholinesterase. X. Mechanism of the catalysis of acylation reactions. J. Biol. Chem. 186: 781-790, 1950. 24. Wilson, I.B., and Ginsburg, S. A powerful Deactivator of alkyd phosphate-inhibited acetylchol inesterase . Biochim. Biophys. Acta 18 :168-170, 1955. _~_

25. Wilson, I . B. -, and Ginsburg, S . Reactivation of acetyl- cholinestera~e inhibited by aLky~phosphates. Arch. Biochem. 54: 569-5 71, 1955. 26. Wilson, I.B., and Meis~ich, E.K. Reactivation of acetyl- cholinesterase inhibited by alky~phosphates. J. Am. Chem. Soc. 75: 4628-4629, 1953. REVIEW OF ABLE INFO~TION ON l 2-PAM P2S IMB-4 AND TOXOGONIN ~ ~ , CHARACTERISTICS Pralidoxime chloride (EA 2170), [ C7HgN2O]C1 (Figure 2-1), is a white, nonhygroscopic, crystalline powder, with a molecular weight of 172. 63, a melting point of 226-227°C, and a solubility in water of over 1 g/ml. It is a chorine st era se deactivator and was tested alone and with anticholinesterase chemicals on human volun- teers at Edgewood. It is manufactured by Ayerst Laboratories, approved by the F1)A, and wide1 y used in the United States as an antidote to alleviate the acute responses to poisoning by organo- phosphorus insecticides and other anticholinesterase chemicals.4,ll,73 Pralidoxime methanesulfonate (P2S), [C7HgN2O]CH3O3S, consists of hydroscopic crystals with a molecular weight of 232.28 and a melting point of 155°C. 1~117 This is reportedly the preferred oxime in the United Kingdom. 73 Toxogonin (EA 3475), [C14Hl6Cl2N4O3], is a grayish- whi te powder that exists as ache chloride with a molecular weight of 359. 22, a melting point of 229°C, and free volubility in water. A dibromide compound with a melting point of 202°C is also used This oxime is much used in many European countries . 73 4 (EA 48~4~, [Cl5Hl8~4o2]Br2, exists as odorless, light tan crystals with a molecular weight of 446.21 and a melting pa int of 238-2 41°C and is also available as the chloride . 11, 7 3 Russian and Yugoslavian investigators have reported the use of TMB-4 as an antidote in anticholinesterase poisoning.l7~73 All four cholinesterase Deactivator compounds are pralidoximes . The f irst two are monoquaternary oximes; the last two are hi squat ernary oxime ~ . _9_ l

BIOCHEMISTRY The body contains two main classes of cholinesterase: acety~cho- linesterase (EC 3.~.~. 7) and butyry~cholinesterase (EC 3.~.~.~) .27 ^The former, sometimes referred to as true cho1 inesterase, is mainly a tissue enzyme and is found mainly in such tissues as the synapses of the cholinergic system; it is also found in other tissues, such as erythrocytes, where i ts function is uncertain. The latter, referred to as peeudocholinesterase, is a soluble enzyme that is synthesized in the liver and circulates in the plasma. Acetylcholine is the optimal substrate for acetylcholinesterase, whereas butyrY1choline is the optimal substrate for butyrylcholin- esterase. 80~04 Acety~cholinesterase does not hydrolyze butyryl- choline very ef f iciently. Acetylcholinesterase can be extracted from tissues to various extents by saline buf fers, but often a detergent is necessary to solubi lize the larger part of the enzyme . Even a single tie sue usually yields a number of enzyme forms with different sedimentation coefficients. 72 Some of the forms are highly asymmetric; some tend to aggregate, unless the salt concentration is high. Regard- less of the source or the molecular form, the kinetic properties of acety~cholinesterase are similar. Although it contains a number of catal ytic subuni ts, there are no homotropic allosteric ef facts, nor do there appear to be any physiologic regulators of i ts activity . Acetylcholinesterase is sub] ect to substrate inhibition at high concentrations, but Michaelis kinetics are observed at lower concen- trations, because the substrate constant and the Michaelis constant differ by a factor of 100. Turnover numbers run about 2-9 ~ 105 min~1, and Km (Michaelis constant) values are about 0.2 mM. 7616 Whatever the source, the enzyme is sub ject to inhibi- tion by the same reversible and irreversible inhibitors. Most of the kinetic work has been done with the saline-extracted 1iS enzyme from electric eel and the detergent-extracted 6S enzyme from erythro- cytes . The former is a tetramer derived f ram the net ive enzyme by the action of proteases; the latter is a dimer.89 The following diagram illustrates the reaction of acety~cho- linesterase (E) with acetylcholine (S ~ to produce an acetylenzyme intermediate (E' ~ with later hydrolysis of the intermediate and regeneration of the enzyme .113 ~ 114 KM is calculated as shown in the diagram. The catalytic constant, kCat, refers to the overall decomposition of the enzyme intermediate (E.S, E' ). —10—

EOH+S ken E S ken k-1 H2Oi k3 E'+ Choline ELM = k 1 + k~( I + k2 ) E + acetate ~( ) In acetylation, the enzyme acts as a nucleophi'e, and choline as a leaving group; in deacetylation, water is the nucleophile, and the enzyme is the leaving group. The enzymic nucleophile is the hydroxyl group of a specific serine residue. The mechanisms of both acetyl- at ion and deacetylat ion probably involve the formation of tetrahedral intermediates. The active site contains an imidazole group that functions first as a general base and than as a genera' acid in each step and whose ionization determines in part the pH variation of enzyme activity. Details of enzyme-substrate and enzyme-inhibi tor reactions were described in Volume 1 (p. 8~. In summary, organophosphate esters wi th good leaving groups phosphorylate the enzyme by a mechanism similar to acetylation of the enzyme. RO O \11 RG E-OH + ,P-F ~ E-OH (ROXR'O)P(0)F 2~ E_o_p\ + HE OR' These substances are sometimes called irreversible inhibitors, because the hydrolysis of the phosphorylated enzyme by water is slow. Various nucleophiles containing a substituted ~mmorrium group will dephosphorylate the phosphorylated enzyme much more rapidly than water. This was recognized early in the quest for a practical reac- t tvating agent when choline was found to be a Deactivator. Because a hydroxyl group in an alcohol is a weak nucleophile at neutral pH, the capacity of choline to function as a Deactivator must be a con- sequence of its molecular complementarily with the enzyme and the increased acidity of the alcohol. The idea arose of f inding a rigid structure that would include a quaternary ammonium group and an acidic nucleophile that would be complementary with the phospho- rylated enzyme ire such a way that the nucleophilic oxygen would be positioned close to the elec trophilic phosphorus atom. This led t o the pralidoxime compounds ~ PAbls ~ . The syn isomer of 2-PAM (2-PAM-ayn) turned out to be especially active. The reaction of 2-PAM with the phosphorylated enzyme is shown in the following diagram. —11—

E O P/ + :` N-OH +CH 3 H OR 2 PAM EO-P\ KR |k OR E + 0` `_o_p +CH3 H The pseudo-first-order rate constant for reac~ci~ration of ~ iS enzyme is: k (pseudo first-order ) = i;2 I +KR 2 -PAM hen the oxime concentration is smaller than Kp (~2-PAM] <C Key, the reactivation follows second order kinetics, and the rate constant can be described as shown below. k ~ second- order )- K2 (I PAM) R The value for Kp. as determined for eel cholinesterase is about 10~4 M, and that for k:, about 4 min~i, so the second-order race constant, k2/KR, is 4 x to4 M-l mined. Thus, ache biochemical characteristics affecting the reacti- vation of cholinesterase are complex and only partially understood. Knowledge of the kinetics of the various rate-determining processes is essential to the understanding of the inhibitox-reactivation process. MECHANISM; OF ACTION The therapeutic action of oximes resides largely in their capacity to reactivate cholinesterases without contributing markedly toxic ac tions of the ir on at recommended or usual doses . Other actions may contribute to their effectiveness: they may react directly with the antichol1nesterase agent,4~45 block its reaction with cholinesterase,90 modulate the release of acety~chollne, 30~74 block acety~choline's agonistic activity —12—

(cholinolytic effect),l5~35~5l or increase excretion of the anticholinesterase agent.48~86 Some of the other possible mechanisms are discussed later, but the interaction between oxime an free or unbound organophosphorus ester is considered below. The basic reaction believed to occur in the inhibition of acetylcholinesterase by some alkylphosphates is shown in Reaction 1. n |~E + (go)} - ~ ~ (go)} - ~ + ~ (1) active enzyme inhibitor inhibited enzyme The inhibited enzyme is stable in aqueous environments, and lethality results from a blocking of transmission at cortical cholinergic synapses, such as those involved in breathing. The reactivation or the inhibited enzyme can be achieved with selected oximes (such as 2-PAM), as shown in Reaction 2. (lD)2~01! ~ ~ CE-NOB 3] SIDE ~ ;~'d-~_O~P-(08)2 cE3I inhibited enzyme Deactivator active enzyme phosphorylated oxime The reactivation is an equilibria reaction. Under some conditions--e."., when the oxime reacts directly with the organophosphate ester (Reaction 3) or where the equilibria constant is less than 1 (Reaction 21--the phosphorylated oxime can inhibit the enzyme by driving the reaction to the lef t . i+ CII-NOB CE31 reactivator inhibitor ~ (~32P-! ~ ^~ ~ =3 phosphorylated oxime Thus, the phosphorylated oxime can itself be a potent cholin- esterase inhibitor. Wilson and Ginsburg advanced this concept when they noticed incomplete react ivation of acety~cholinesterase by 2-PAM and suggested that the phosphorylated oxide reinhibited the enzyme (according to Reaction 2~. —13— (3)

Studies of the in vitro reaction between the alkaline oximes and sarin (isopropyl methy~phosphonofluoridate, GB) revealed that 2-PAM reacted rapidly with the organophosphorus ester in solution at physi- ologic pH and temperature (Figure 2-2~.43 CH, 1° CH—tJOH q CH3-P-F OCH(CH3 ): 2-~= G8 CH3 1~ O 1 11 N CH ~ N~P'H3 H(CH3)2 WHO FIGURE 2-2 Suggested interac Lion be tween 2-PAM and GB ~ isopropyl methylphosphonofluoridate) . Reprinted with permission from Hackley et al.45 Many of the oximes reacted in two distinct acid-producing steps: an initial phosphonylation of the oxime and then a breakdown into secondary product9.44 Although the phosphonylated intermediate of 2-PAM could not be prepared, the phosphonylated derivative of 4-PAM was synthesize d and found to be a potent inhibitor of eel acetylcho- linesterase and to be toxic to mice, having an intravenous LDso of 0.2 mg/kg of body weight.44 Green and Saville found that MINA (monoisonitrosoacetone or 1,2-propanedione 1-oxime) underwent a stoichiometric reaction with sarin.4i Additional studies con- firmed that 2-PAM, 4-PAM, and probably TMB-4 were converted to potent inhibitora of AChE when they reacted with sarin in vitro.45 That TMB-4 was converted to a potent anticholinest erase agent when it reacted with satin in vitro was confirmed in studies that suggested that this bis-quaternary oxime could exist either as a mono~hos- phonylated or as a diphosphonylated compound (Figure 2_3~.6 Zech et al. demonstrated that 2-PAM, 4-PAM, and TMB-4 were more potent inhibitors of cholinesterases after incubation with dimethoate (O,O-dimethyl S-methylcarbamoylmethyl phosphorodithioate) or deriva- tives.ll9 Later studies, in which sarin was incubated with P2S and to~ogonin, demonstrated that unstable phosphonylated oximes were formed, but that these intermediates were potent inhibitors of cholinesterase.26 - Although it appears that these phosphonylated oxides rapidly degrade in aqueous solutions, it has been suggested that they can have a harmful effect on the oxide therapy of poisoning by some organophosphates if they are formed in vivo, react with excess -14-

Br- Br- + + ON ~ CH' CH2 CH2 N CH=NOH CH=NOH THE- 4 GB B- Br- o + CH3 -P- F OCH(C.H3 )2 CH2 CH2 CH2 ~ CH=NO`tI CH=NOH P-CH3 OCIl((~H3 )2 FIGURE 2-3 Interaction between TMB-4 and GB to form the monophosphonylated oxime. Reaction with a further molecule of GB would form the diphosphonylated compound. ~ "free" ~ circulating ester in cases of severe poisoning, and create a new inhibitor as potent as or more potent than the original anti- cho ~ nesterase . The inhibitory potency of phosphonylated oxides has been estimated from their effects on the course of the reactivation of phosphon;lated AChE when they were formed during the reaction. 2, ~ Thus, it appears that the potential for the phos- phorylated oximes to inhibit cholinesterases in viva is substantial, but depends entirely on the stability of these intermediates in the body. A prac tical limi tat ion on the usefulness of reac tivators comes about because cholinesterase inhibited by organophosphates undergoes a secondary reac tion termed "aging . " This phenomenon is discussed in Volume ~ of this report series. In brief an alkyd group is cleaved from the phosphorylated enzyme, leaving an "aged" enzyme that is resistant to reactivation by the oxime. The rate at which a phos- phorylated enzyme "ages" is determined by the alkyl groups on the organophosphate, as described in Volume ~ . PHARMAC0KINETICS The pharmacokinetic features of these drugs , based on the evidence produced and the conclusions drawn by various investigators, are described in Appendix B. This section touches on the highlights and restates the material from another viewpoint. Two features of the cho1 inesterase reactivating agents are critical for their pharmacokinetica: —~ 5—

Oxime deactivators (R-CH=NOH) are weak acids that partly ionize at biologic pH. This property allows them to function as nucleo- philes and displace organophosphate moieties from inhibited acetyl- cholinesterase. It also makes them vulnerable to decomposition by other mechanisms in the body. The oximes contain a quaternary ammonium group that contributes to their acidity and their strong binding to the inhibited enzyme. This appears to be a key struc tura1 element in known Deactivators, but it tends to make them poorly soluble in lipids . Practically, this means that the drugs are slowly absorbed from the gastrointes- tinal tract, have dif ficulty entering the brain, do not easily enter hepatic cells to be biotransformed, and are not reabsorbed from the renal tubular urine. 2-PAM has been studied extensively by pharmacokinetic means, and data are available on its absorption, tissue distribution, bl cod concentration, and elimination by humans and ant orals. Although it has been given as various salts, the particular form admirli`3tered is of no consequence once the material is absorbed and dissociated into the cation by body fluids . 61 ,100,106 2-PAM is poorly and variably absorbed from the human gastro- intestinal tract; only 10-50% of an oral dose is recovered in urine unchanged or as metabolites . 53, 61, 99, 100 ,106 The absorption occurs, at least in dogs, in a segment of the ileal-JeJunal area68 either by passage through the small membrane pores in this section or by some more active absorption process that carries a variety of quaternary ammonium compounds into the blood. The overall absorp- tion rate is lower than the elimination rate ; that is, absorption is the rate-limiting step. Hence, blood concentrations are always low after oral administration, arid substantial blood concentrations are dif ficult to produce or maintain when the drug is given by this route. 28~99 Marked gastrointestinal distress is produced by oral 1 y given oximes, particularly if therapy is continued.99 2-PAM may ef fective 1 y be given either intravenously or intramuscularly, although the latter route is more erratic and sl ower in producing high blood concentrations and may in~rol~re some local pain. 96 Like most quaternary ammonium compounds, 2-PAM is distributed mainly to extracellular water, and some tissue sequestration occurs . 100 2-PAM is only weakly bound to plasma proteins . 34, 100 2-PAM cro sees the blood-brain barrier wi th dif ficulty. 2-PAM ire rat brain, 10 min after injection, is only about 5-12% of that in plasma; higher percentages are in the more heavi 1 y vascularized areas, such as cerebral and cerebellar cortex and inferior colliculi.34 This low brain-to-blood ratio persists, but over the next 6 h the brain and blood come closer to equilibrium as the blood —16—

concentration decreases sharply.34 The low brain concentration is puzzling, inasmuch as the oximes depress anticholinesterase-induced seizures and void the part of the weakness of respiratory muscle con- trac tions that is thought to be due in part to the ac tion of organo- pho sphate in the brain by suppers sing ant icholinesterase inact ivation of central respiratory drive. 4 Hypoxia might cause disruption of the blood-brain barrier, thereby allowing the oxime to enter the brain. Some anticholinesterases may also facilitate the entry of oximes into the central nervous system. 34 Regardless of mechanism involved, oxides can cause remarkable improvement in ENS signs and symptoms in some patients. There is some evidence that peripheral actions, such as might occur a70n~e~romuscu~ ar junctions, could con- tribute to muscle weakness.60, ~ 2-PAM is eliminated rapidly by man and animals. In humans, biologic half-life is about ~ -2 h.100 This short half-life is due in part to metabolism, but more to the fact that renal clearance approaches 700 mI/min,l°° i.e., almost that of ~aminohippuric acid (PAH). Investigators have therefore suggested that 2-PAM might be secreted into the urine . 14, 50, i0O, 108 Interpretation of data on renal elimination is complicated by the presence of a quaternary group on one end of the oxime molecule and a weak acid at the other ~ Figure 2-l) . They imply dif ferent mecha- nisms of renal handling. Swartz and Sidell 108 showed that in human beings both chemical groups are important. The more important of the two actions is the act ive secretion of the compound by the organic-base-secreting system of the kidney. This process can be slowed by one-third by coadministering high doses of thiamine.57 Das Gupta _ al.25 claimed that the inhibiting effect of thiamine occurs in female, but not in male, rats. The net clearance of 2-PA approaches that of PAH, 100 but is not as great; hence there may be some simultaneous active reabsorption of the material. SO Like most cations, 2-PAM ire the urine does not readily dif fuse back from renal tubule to blood. The process here is complicated, in that the oxime has a pKa of 7. 8-~. 108 It is more ionized at lower pH, and the renal loss is inverse, y proportional to pH. 14 Although the pH/pKa relation and active absorption contribute to the resulting or bio- logic half-life, active secretion into the urine is the predominant feature, and the total half-life is correspondingly short. Because of this, it is dif f icult to accumulate and maintain a substantial concentration of material. The rapid renal elimination does not leave much time for metabolism of 2-PAM about 90% of injected material is recovered unchanged in urine . i00 Nonetheless, 2-PAM is metabolized by liver, primarily by removal of the oxime moiety, and a number of metabolites have been identified,32~78~1] ,\12 some of them in rat brain, plasma, and urine.34 —17—

The blood concentrations, time-courses, and half-li~res (~.2-~.4 h) of 2-PAM and tozogonin are similar in humans,l°° but toxogonin has a smaller volume of distribution than 2-PAM (174 vs. 795 mI/kg) and a smaller renal elimination (clearance, 133 vs. 717 mI/min). The smaller volume of distribution and slower clearance imply dif ferent distribution and handllog of the two oximes. They also imply that, if toxogonin and 2-PAM were given in equimolar concentrations, the to~ogonin plasma concentration would be greater than that of 2-PAM. T!IB-4 is unstable in fecal matter in vitro; only about 3% of an oral dose is found in urine and 2: in feces. 61 ADVERSE EFFECTS The adverse effects of various oximes ~ including diacetyl- monoxime, which was not used at Edgewood) in humans are listed in Tables 2-2 through 2-7 . These tables list only signif icant f indings . Although Hopff and Waser54 have listed mechanisms of action whereby reactivators of inhibited cholinesterase could be harmful, the real cause of most of the observed adverse ef fects is obscure. One exception is the slight inhibition of cholinesterase caused by single dose of TMB-4 (dibromide form) .61 Regardless of the underlying mechanism, the observed adverse effects are usually mild and brief. Many last only a few minutes; almost all disappear within a few hours after a single dose. A num- ber of investigations, especially those concerning metabolism or pharmacokinetics revealed no adverse effects, even though they were looked for. 31~10{ ,107 Gastrointestinal disturbance after oral admini Stratton of oxime s, e specially P2S and TMB-4 chloride, may be severe enough to require discontinuation. 20 But doses of P2S as high as ~ g caused no symptoms (including gastritis) when giver as tablets coated with dimethylaminoethyl methacrylate, each containing 400 mg of the drug and 71 mg of excipierlt.~°° Local pain and increased creative phosphokinase in the blood follow intramuscular injection. Using 2-PAM chloride and saline, Sidell_ al.95 showed that the degree of tissue in Jury was directly related to the osmolarity of the injected solutions when the volume was kept constant and directly related to the volume when the osmolarity was constant . However, under identical conditions, 2-PAM chloride was somewhat more injurious than saline. Hepatic injury has been recorded in connection with 2-PAtI (chloride), TMB-4 (chloride) and toxogonin. In the case of 2-PAM chloride, hepatic injury was manifest by transient increases in SGPT -18-

TABLE 2-2 Adverse Effects Observed in Humans After Administration of 2-PAM loaf ide Ef fects Dose and Route References Taste (bitter, metallic, salty, or mushy) Iodi sm Local irritation He adache Dizziness and nausea, possibly progressing to vomiting Blurred vision, impaired accommodat ion Mus cu 1 ar weakne s s and/or malaise, fatigue, etc. Gastrointestinal distress Paresthesia and/or anesthesia Rash Bleeding Hypertension (moderate systolic and diastolic with increased pulse pressure and tachycardia) Initial hypertension followed by hypotension ECG changes Icterus and/or clinical test implicating liver Other biochemical changes Loss of consciousness Convulsions b ~5 g, pa &1 a, iv, NEG ~1 g, iv ~1 g, iv '1 g, iv b b b 61 56 56 56 ~6 b b '10 g, pa, NEG 61 &1 g, iv, NEG S6 b b ~1 g, iv, NEG 56 b b . . . a pa = oral; iv ~ intravenous; NEG = administration fa i 1 ed to produce e f fee t ind ice ted . b Effect looked for, but not observed at any dose or by any route. —19—

TABLE 2-3 Adveree Effects Observed in Humans After Administration of 2-PAM Cl,loride Ef fect~ Dose and Routea References Taste (bitter, metallic, b s a 1 ty, or mushy ~ Todism b Local irritation 0.6 g, im 101 ~0.175 g, im 101 Headache 32 g, iv 20 Dizziness and nausea, b pos s ib ly progres ~ ing to vomi t ing Blurred vision, ~2 g, iv 20 impaired acconmodation 0.25 g, iv 108 Muscular weakness and/or b malaise, fatigue, etc . Gastrointestinal J8 g, po 99 diseress ~2 g, po qid, 2 d 99 2.1 g, iv 20 2 g, po, b id, 180 d NEG 20 Pares tines ia and /or b ane s the s ia Rash b Bleeding b Hypertension (moderate .3.7 g, po, NEG 61 sys tol ic and d ias tol ic ^4 g, po, NEG 20 with increased pulse 2 g, iv 20 pressure and tachycardia) 2 g, iv 20 1 g, iv, NEG 20 ^2 g, po, qid, 3 d, NEG 20 2 g, po, bid, 180 d, NEG 20 0.35 g, iv, NEG 108 Ini t ial hypertens ion b followed by hypotension ECG changes 3 g, iv 20 2 g, po, bid, 180 d Icterus and/or clinical 2 g, po, bid, 180 d 20 ' tes t impl icat ing ~ iver Other biochemical .3 g, iv, NEG 20 changes '2 g, po, q id, 3 d, NEG 20 Loss of consciousness b Convul ~ ions b a in = intramuscular; iv = intravenous; po = oral; q id ~ 4 t imes a day; bid = 2 times a day; NEG s administration failed to produce effect indicated. b Ef feet looked for, but not observed at any dose or by anY route . —20—

TABLE 2-4 Adverse Effects Observed in Humans After Admini~tration of P2S Ef fec ts Taste (bitter, meeallic sa 1 ty ~ or mushy ) Iodism Local irritation Headache Dizziness and nau~ea, possibly progressing to vomiting B1urred vis ion, impaired acco~mnodat ion Muscular weakness and/or malaise, fatigue, etc. Gastroin~cestinal distress &2. 5 Pare~ thesia and/or anesthes ia Ra sh Bleed ing Hypertens ion (moderate systolic and diastolic with increased pulse pressure and tachycardia, Initial hypertension followed by hypotension ECG changes Icterus and/or clinical test implicating liver Other biochemical changes Loss of consciousnese Convulsions Dose and Routea References &0. 7 g, im 0.5 g, im b b 0.5 g, im, NEG 1 g, po, qid + 0.5 g, im b 6 52 g, po, bid, 180 d 20 b ~5 b b g, po, NEG 3 g, po, NEG 3g, iv b 3 g, 3 g, 2.5 g, po, bid, 77 d b po, NEG iV b b b a im ~ intramuscular; po ~ oral; iv ~ intravenous; qid s 4 times a day; bid ~ 2 times a day; NEG ~ administration failed to produce ef fec t ind icated . b Ef feet looked for, but not obeerved at any dose or by any route . —21— 61 20 20 20 20 20

TABLE 2-5 Adverse Effects Observed in Humans After Administration of TMB-4 Dich lor ide Effects ~ . . _ Do~e and Routea References . Tast (bitter, metallic, b salty ~ or mushy) Iodism b Local irritation b Headache b Dizziness and nausea, possibly &1 g, po, 70 d 20 progres s ing to vomi t ing Blurred vis ion, impaired b acco~nmodat ion Muscular weakness and/or d! g, po, 70 d 20 malaise, fatigue, etc. Gastrointe~tinal distress ,! g, po, 70 d 20 Pares tines ia and /or anes tines ia &1 g, po, 70 d 20 Rash &1 g, po, 70 d 20 Bleeding *t g, po, 70 d 20 Hypertens ion (moderate ~ys eol ic b and d ias tol ic wi th increased pulse pressure and tachycard ia ~ Initial hypertension 4 8, P° 20 fol lowed by hypotens ion 2 g, iv ECG changes 4 g, po, NEG 20 2 g, iv Icterus and/or clinical ~1 g, po, 70 d 20 tes impl icating liver Other biochemical changes b Loss of consciousness b Convulsions b a po = oral; iv s intravenou produce e f feet indicated . s; NEG ~ administration failet to b Ef feet tooked for, but not obeerved at any dose or by any route . —22—

TABLE 2-6 Adverse E f fec t ~ Ob served in Human ~ Af ter Admin is trat ion o f Toxogon in Ch 1 or ide Ef f~cts Dose and Routea Re ferences . Taste (bitter, metallic 0.175 g, im 9f salty, or mushy) &3 g, po 98 ~1.84 g, po 102 Iodism b Local irritation ~0.175 g, im 97 0.25 g, im 33 Headache ~2.76 g, po 102 Dizziness and nausea, po~sibly 7 g, po 98 progres ~ ing to vomi t ing Blurred vis ion, impaired b accononodat ion Muscular weakness and/or malaise, fatigue, etc. Gastrointestinal distress Pares tines ia and /or anes tines ia '2.76 g, po b 0.175 g, im &3 g, po 0.25 g, im &2.76 g, po b b Ra sh B1 eed ing Hypertension (moderate systolic 0.175 g, im and diastol ic with increased &4 .6 g, po, NEG pulse pressure and tachycard ia ~ Init ial hypereension followed by hypotens ion ECG changes Icterus and/or cl~nical tes t impl icating 1 iver Other b iochemical changes Loss of consciousness Convul ~ ions a im ~ intramuscular; po ~ ore to prociuce ef feet indicated. 1; 102 97 98 33 102 b 97 102 b 0.25 8, im, 2 d, NEG 16 ^4.6 g, po, NEG 102 b b b NEG ~ administration failed b Ef feet looked for, but not observed at any dose or by any rou te . —23—

TABLE 2- 7 Adverse Ef fects Observed in Humans Af ter Atministrat ion of Diacetylmonoxime Ef fee t ~ Dose and Routea . . . Taste (bitter, metallic, salty, or mushy) lad ism Local irritation Headache Dizziness and nausea, possibly progress ing to vomi t ing Blurred vis ion, impaired acco~ranodat ion Muscular weakness and/or malaise, fatigue, eec. Gastrointestinal distress Paresthesia and/or anesthesia Rash Bleeding Hypertension (moderate systolic and diastolic with increased pulse pressure and tachycardia) Initial hypertension followed by hypotension ECG changes Icterus and/or clinical test impl ice t ing 1 iver Other biochemica 1 change s Loss of consciousness Convulsions a iv ~ intravenous. &1 g, iv b ~1 g, iv ~0.5 g, iv b ~1 g, iv ^0.5 g, iv 41 g, iv b b ~1 g, iv ~0.5 g, iv b b b b b b b &t 8, iv &1 g, iv b Ef feet looked for, but not observed at any dose or by any route . —24— Re ferences 56 56 42 56 42 56 56 42 56 56

and SCOT in 4 of 29 subjects, positive thymol flocculation test in 4 sub Recta, and positive urobilinogen test in Il sub jects. This occurred when substantial oral 608e8 (2 g twice a day) were a~in- istered for 6 mot Ire the case of TMB-4 chloride, hepatic enzyme changes were accompanied by icterus, petechial bleeding, and pro- thrombin times of 40: and 50: of normal, respectively, in two of Il subjects. All adverse effects were marked by the time ~B-4 chloride had been administered for 6 wk orally at increasing rates of 0.~-2.3 g. All symptoms except icterus subsided completely in 1 or 2 wk after administration was stopped; clearing of ic~cerus required 3-6 wk. The investigators considered the icterus similar to that pro- duced by chlorpromazine. 20 GASTROINTESTINAL EFFECTS Direct ef fects of oximes on ache gastrointestinal tract are related entirely to the route of admir~istration--they are seen only after oral administration. Ingestion of tablets of 2-PAM or related salts is often accompanied by transient diarrhea and cramps. The full explanation is not known. In the case of P2S, it has been sug- gested that a dose of 20 mg/kg inhibits ATPase and therefore pro- duces excess intestinal fluid and diarrhea. The ef feet depends on concentration and is reversible. 2-PAM has no effect on cyclic AMP (cAMP), adenylate cyclase, or phosphodiesterase.66 Repeated oral add nistration of 2-PAM or TMB-4 in dogs may lead to areas of acute necrosis and to deposition of fibrous tissue in the stomach. ~ Whether those effects are due to a specific action of the oxime or to chronic irritation related to repeated adminis- tration is not known. Nor are the long-term ef fects of such chronic administration and consequent fibrosis known. However, single doses, even oral' y, have not beer reported to produce tissue changes. No change was found in the gastrointestinal tracts of rats given P2S (200 mg/kg) orally each day for 50 d.23 Large doses of 2-PAM or to~ogollin (at 7100 mg/kg) cause death that may be associated with acute en~ceritis,37 but small doses do not seem to produce tissue changes. 23 With small doses, enteritis is not suf ficiently severe to cause death. CARDIOVASCULAR EFFECTS The adverse ef fects of cholinest~erase reactivating chemicals on the human cardiovascular system are shown in Tables 2-2 through 2-7. These ef fects may be classified as hypertension characterized by moderate increased systolic and diastolic pressure with hypertension fold owed by hypotension, and ECG changes. 20,6i,97,:02~07 The —25—

occurrence of adverse cardiovascu;Lar effects depends or dosage and route of administration. In general, these effects were seen when large doses of the cholinesterase Deactivators were given intraven- ously or intramuscularly or af ter repetitive oral doses. Although therapeutic doses of to~ogonin produced no effects on heart rate or blood pressure in humans, 2-PAM caused a signif icant increase in car- diac output in dogs. 2\ 2-PAM increases blood pressure in experi- mental animals and humans . l8 ~ 2o, 38 ~ 6o, 6 7 ~ lo 3 ~ llB It causes ala increase in mycocardial contractility in experimental animals85 and cardiac arrythmias when used in humans to treat organophosphorus insecticide poisoning . 18, 24 The mechanisms by which 2-P~ exerts its cardiac effects have been studied in experimental animals. At least three classes of action have been attributed to the effects of altered calcium meta- bolism on autonomic ganglia. A sympathomimetic action of 2-P~ was postulated to explain the increase in blood pressure and the augmen- ted myocardial contractility by one or more of the following mecha- nisms: 2-PAM may not be ock the release of the endogenous compounds, but may prevent the uptake of catecholamine; ~ O it may stimul ate the release of norepinephrine;l8 it increases m;}ocardial contrac- tility by directly stimulating beta receptors ;3 and it increases blood pressure by directly stimulating alpha receptors.85 2-PAM increased the contracti~ e force of stimulated rabbit atria that did not result from an increase in the frequency response. 2t Similar results were obtained in rabbits pretreated with reserpine, a catecholamine~epleting agent. These findings are in agreement with previous reports that the cardiovascular ef fects of 2-PAM on anes- thetized dons were not due to a release of tissue catechol- amir~e . 38 ~ i03 Other studies, however, have shown that 2-PAM affects blood pressure in viva by changing the release or metabolism of endogenous norepirlephrine . 10 ~ 18 Most at tent ion has been focused on adrenergic receptors ~ Recent study of the mechanism of the effect of 2-PAl1 on cardiac action in rabbit atria indicated that 2-PAl1 exerts a direct action on vascular smooth muscle that is not mediated through stimulation of the alpha receptors. 2\ These results have led to the conclusion that the effect of 2-PAM on the cardiovascular system cannot be explained ent irely by its sympathon~imetic ef feet. This conclusion was suppor- ted by the finding that 2-PAlI caused an increase in the contractile for`,e of isolated aortic strips from rabbits and the finding that phentolamine, an alpha-adrenergic blocking drug, did not affect this response of the aorta. These results contradict the previous find- ing that phenoxybenzamine, an adrenergic blocking drug, decreased the press or effect of 2-PAM. 118 The latter finding may be explained by the inhibiting ef feet of phenoxybenzam'4e on the drug- illduced calcium movement in vascular smooth muscle. —26—

Conflicting results have been reported in regard to the involve- ment of beta receptors in the mechanism of the isotropic effect of 2-PAM. It was concluded that the direct stimulation of the myocar- dium by 2-PAM that could be blocked by dichioroisoproterenol (DCI) was responsible for the locrease in blood pressure . 38 A recent report, however, has indicated that the increase in blood pressure caused in the dog by 2-PAM was not blocked by propranolol.;L03 Similar results were obtained with rabbit atria in vitro2i in a study that al so showed that 2-PAM augments muscle contractility and alters calcium movement. The studies reported above imp' icated calcium movement in the mechanism.21 It was postulated that 2-PAM may increase the rate of calcium movement either by interfering with calcium binding to its binding site or by directly stimulating the release of - bound Cal cium. 21 It was suggested that 2-PAl1 may not have increased cell permeability or af fected the rate of re-entry of calcium into the cell, or 2-PAM may have increased calcium distribution in the vicinity of myofilaments. Studies of the efflux of 45Ca by stimulated rabbit atria have characterized three calcium pools. Phase I may represent extra- cellular washout of the 45Ca that binds to the surface of muscle membrane arid is characterized by a high rate constant. Phase II may represent loosely bound calcium present in cell membrane and calcium red eased at the sarcoplasmic reticulum. Calcium in this pool is directly related to contractility.65~84~93 Phase III may repre- sent the t ightly bound calcium that exchanges very slowly and does not play a role ire maintaining calcium cor~cerltrations. Recent study has shown that the storage or release of calcium at the sarcoplasmic reticulum and other loosely bound calcium sites (cell membrane) that are involved in muscle contractility can be directly affected by 2-PAM.21 These results indicate that 2-PAM increases the rate of release of Phase II calcium. It has been postulated that 2-PAM exerts its cardiac action in rabbit atria through its alteration of calcium metabolism. The relaxation phase of skeletal muscle contraction seems to be directly affected by the sarcoplasmic reticulum because of its ability to sequester calcium actively.29~46 A similar role has been su,- gested for the sarcoplasmic reticulum in cardiac muscle. 46, ~ The onset of muscle contraction takes place when calcium reaches a crit- cal concentration. This contraction is later reduced by the increased calcium-sequestering activity of the sarcoplasmic reti- culum. Thus, 2-PAM can affect this process by decreasing the rate of calcium uptake by the sarcoplasmic reticulum, which results in increasing the time required to reduce the calcium concentration enough to allow relaxation to take place. This was demonstrated by the increase in the relaxation phase. It was suggested that this —27—

delay in binding of calcium to the sarcoplasmic reticulum may increase the active state that increased the contractile force. 2-PAM either activates or blocks autonomic ganglia, depending on dose and route and speed of administration. The changes in gang- lionic function induced by 2-PAM will be reflected in sympathetic and parasympathetic activity, which may cause changes in cardiovascular functions . These are di scussed further in the next section. NEUROPHARMACOLOGY The four oximes have, in addition to reactivation of phosphory- lated AChE, a variety of actions that can be demons bated in the absence of previous exposure to organosphosphorus compounds. Experi- meets that involved only short-term exposure to high concentrations of 2-PAM showed the following short-lived and reversible actions of oximes in nervous tissues: inhibition AChE, agonist-antagor~ist ef feet on cholinergic receptors, gangs: blockade, and presynap- tic actions, such as modification of the Please of acetylcholine (ACh) from the nerve terminal. ~ - Effects of Oximes on AChE Activity ACh contrac Lion of frog rectus abdominis muscle was potent fat ed by 2-PAM, and progressive AChE inhibition was seen with increasing concentrations of 2-PAM. 2-PAM (at l.5 ~ 10~3 M) produced a 66X inhibition. 36 This is far higher than the concentration that woul d be produced in viva by admini Stratton of ordinary doses . Similar effects were seen on isolated rabbit intestine and atriumi5 and on chicken muscle.22 In phrenic nerve diaphragm preparations of rat, 2-PAM increased the neuromuscular block induced by depolarizing blocking agents and antagonized the nondepolarizing block induced by curare. 6 Recep tar Int erac tions Receptor interac tions were seen at nicotinic and muscarinic receptors. No correlations were found, however, between atropine- like or curare-like actions of these compounds and their protective effects against organophosphate poisoning. Nicotinic Receptor Interactions. Af ter producing an initial . ~ brief augmentation of the twitch response and fasciculations ~ ,~ 2-PAM and TMB-4 at 1-20 mg/kg abolished the indirectly stimulated twit ch response of skeletal muscle ~ but did not alter the response to direct stimulation. Neostigmine and edrophonium antagonized, whereas —28—

d-tubocurarine increased these ef fects, which are due to competition for nicotir~ic acetylcholir~e receptor sites.9 Both agents anta- gonized the response to decamethonium and reduced the effects of carbamoylcholine .36 Muscarinic Receptor Interactions. Excitatory muscarinic effects, such as temporary stimulation of salivation and stimulation of intes- tinal peristalsis, were seen with 2-PAM. Atropine-like actions were seen at high concentrations (15-20 mg/kg or more), and, when injected rapidly, 2-PAM caused temporary diplopia (nicotinic block) and 108s of accommodation in the eye.56 Both TMB-4 and 2-PAM blocked brady- cardia induced by vagal stimulation. At low concentrations, neither compound affected normal intestinal peristalsis, but they did block peristalsis caused by lucreased vagal stimulation. TMB-4, 2-PAM, and to~ogonin antagonized the effect of acetylcholine, acetyl- -methyl choline , and other agonists on isolated guinea pig ileum. 62 Ga~ylionic Blocking Action Doses of 2-PAM larger than 40 mg/kg, as well as TMB-4 and tozo- gonin, produced a t emporary block of the cardiac response to vagal stimulation and of the nictitating membrane response to pregang- lionic, but not postganglionic, stimulation. There was transient hypotension due to block of ganglionic tranamission.9~63~7 Presynaptic Effects of Oximes Concentration-dependent presynaptic effects of 2-PAM on the release of acetylcholine from terminals of nerves innervating the rat diaphragm muscle were seen; at concentrations of 10-4-10-3 M 2-PAM stimulated the release of acetylcholine; higher concentrations led to a total block of the evoked release of acetylcholine .30 ,40 MUTAGENIC, REPRODUCTIVE, AND CARCINOGENIC EFFECTS No information on mutagenic, carcinogenic, reproduct ive , or tera- togenic effects of any of the compounds in question is available. None of the compounds or their in viva intermediates is likely to bind covalently with DNA and other macromolecules. Other ways of binding with DNA, such as intercalation, cannot be ruled out. There is some membrane transport, albeit limited. It is not possible, therefore, to conclude that they are not mutagens, teratogens, or carcinogens . —29—

tTHERAPEUTIC USE Little information is avail able on the treatment of humeri exposed to military agents. However, pralidoxime is an FDA-approved marketed drug in the U.S. and there is substantial experience with therrapeutic use on civilians exposed to agricultural organophos- phorus products. That poisoning by diethoxy compounds can be treatedO'effectively by oximes is nearly universally agreed.55~59~82~87~92~i There is general agreement that the therapeutic effects of oxides are less spectacular in ~ isonin' by dimethoxy compounds as a group,87 and several reports ,8~53~7 ,1 5 indicate lack of efficacy. However, oxime treatment of patients poisoned by clime thoxy compounds has been beneficial in many cases.3~1 ,13~79~87 Thus, 2-PAM in particular and oximes in general are recommended for therapeutic use in severe poisoning by either diethoxy or clime thoxy phosphorus compounds.47 Two factors contribute to the safety of offices in the treatment of acute poisoning by organophosphorus compounds: recommended doses are small, compared with doses likely to cause even mild toxic effects in coral subjects; and adverse effects of o~cimes are reduced in the presence of poisoning by an organophosphorus compound. More reports deal with 2-PAM than with other oximes in the treat- ment of poisoned patients. High dosages have been used in patients who survived without sequelae. Milthers et al.77 reported the administration of 21 g. Gitelson et al. 3 - administered total doses . _ _ . as high as 24 g in 6 d. Namba ~ reported the use of 40.5 g in 7 d, with 26 g ir1 the f irst 54 h . Hiraki et al .49 administered 65 g in 16 d. Warriner_ al.~° gave 92 g in 23 d; this case is espe- cially instructive, because the patient suf fered a relapse on the tenth day of illness af ter his dosage was reduced, but responded favorably to a temporary return to full dosage ( 12 g/d) . In ore case of successful treatment with to~ogonin, 16 doses of 250 mg each (total, 4 g) were administered during the first day of illness and somewhat small er doses later. 5 DELAYED AND LONG-TERM EFFECTS Appendix B reviews some important animal studies of cholin- esterase Deactivator chemicals.- The extensive literature reviewed offers little definitive information with which to project possible long-term effects or delayed sequelae in hen subjects tested at Edgewoode These compounds have a short biologic half-life of 1 to 3 h. However, no chronic studies were found. Consequently, the carcinogenic potential of cholinesterase Deactivators remains —30—

up gown. 61, 96, 98, 99 Acute ef fects have been reported that may be rev ersible, but these require additional study. Reports in the Polish it terature (see Appendix B) that single exposure to obidoxime (to~ogonin ~ may af feet the integrity of renal tubules need explora- tion and confirmation. These findings certainly raise questions about the long-term use of these compounds in humans. Another find- ing that needs further investigation, substantiation, and extension is ache apparent production of muscle necrosis from intramuscular in j action of 2-PAMi, 9 5 and P2S .1 It is not known to what extent and under what circumstances this effect occurs in humans. Oral administration of cholinest erase reactlvators for up to 17 wk pro- duced erosion of the mucosa along ridges of the rugae in the fundus of the stomach and fibrosis in vicinities of the cardiac and pyloric sphincters . ~ The finding that an azotemic sub ject had markedly decreased clearance from blood56 is of interest, although probably not pertinent to the Edgewood studies on hen subjects, because the volunteers were in good physical condition when tested. These find- ings require further analysis to determine dose-frequer~cy and dose- response relationships and their potential significance with regard to hewn experiments conducted at Edgewood. EFFECTS ON VOLIJNTF.FRS The medical records of the volunt eers who received cholin- esterase-reactivating chemicals consisted of the test protocol, physicians' orders , nursing notes ~ including clinical observations), a checklist of symptoms, and laboratory and performance test results. The reports of physicians' examinations and physical findings were generally not included. Volunteers were identified by number. The Committee on Toxicology's assessment was based on records and sum- maries provided by the Department of the Army and NRC staff. The procedures were described fully in Volume 1. In most cases, the analysis was based on summaries of drug admini s "rations prepared by a consultant to the Panel. The number of subjects tested with each compound, the number of records examined, routes of administration, and doses are shown in Tables 2-8 through 2-~. In some instances, the compounds were given before or after anticholinesterase compounds or in conjunction with other drugs; thus, some of the results shown here were also presented in Volume 1. The data on TMB-4 (Table 2-11) are difficult to interpret, because, in the instances on which clinical data are available, other potent drugs were given as well. The manifestations experienced by the subjects in these tests (Tables 2-8 through 2-11) were the moderate clinical effects that —31—

TABLE 2-8 Manifestations after Administration of 1-7 Doses (on Different Days) of 2-PAM (Chloride Form) to 79 Subjectsa Manifestationab None Dry mouthb Diz z ines s Diplopia Eye discomfort Blurred vi sion Mood e' evasion Voiding dif ficulty Nausea Vomi tiny Uric acid increase Faintness Claustrophobia Abdominal cramps Diarrhea Muscle pain Tachycard lab Grand mal seiz urea Intravenous, 5 mg/kg, to total of 500 ma, N = 29 o 2 23 8 12 9 1 2 6 l 1 1 1 __ Oral, 5-7 g total, N - 13 6 1 Intramuscular, 2.5 mg/kg, to total of 600 ma, N ~ 37c o __ __ 37 1 1 a Task plan: absorption efficacy of 2-PAN (chloride form) as function of pH (intramuscular studies). Studies conducted: plasma and urine content, renal clearance, CNS ef fects, blood pressure, and in some cases, pupil size and number facility. Test conditions: heat and exercise in some tests. Clinical evaluat ion tests: uric acid, creatinine, BUN, SOOT, and CPK in serum. Records on 79 subjects selected, on basis of high dose or high frequency of administration, from records on 607 subjects tested. b Dry mouth and increased heart rate were associated with atropine administrated to some subjects. c Several sub ~ ec ts received atropine in addition to 2-PAM. d Sub] ect 6849 ~ see text) . —32—

TABLE 2-9 Manifestations after Administration of To~ogonin to 41 Subjectea Intravenous, b 0. 5-~. 0 ,ug/kg, 60-128 me infused, Manifestations N ~ 11 None Hot, cold, numb, tingling sensation Eye discomfort Dizzinen ~ Dry mouth Local pain Peculiar taste Drowsines ~ Blurred vision Headache Nausea Vomiting o 10 1 l Oral, 1-9 g total, N ~ 20 3 9 2 2 10 51 3 1 a Records on all 41 subjects tested were summarized. b Five sub jects, single injection; six sub jects, infusion as total dose in combination with aminohippuric acid (PAH). —33— Intramuscular, 2.5-1 0 mg/kg, N ~ 10 o 10 1 2 ll /

TABLE 2-10 Manifestations after Administration of P2S to 75 SubJectsa Oral Intramuscular Before After Nerve Nerve Intra- Oral, Agent, Agent, venous, 2-9 g 1-3 g 1-2 g 5 mg/kg, total, total, topical, Manifestations N = 3 N - 49 N ~ 12 N -~1 None Headache ~ s light ~ Voiding difficulty Diarrhea St omach ache Nausea Ventricular extra syst oles Anxiety, agitation, vomi tiny 38 2 4 4 2 1 10 1 . . . a Clinical tests included CBC, blood oximes, creat ine, and urinalysis. Results were in normal range after transitory manifestations. Nerve agent resulted in decrease in RBC cholinesterase content. Records on 75 of 95 subjects tested were summarized; each subject was tested once with P2S. b Subject 2307 (see text). —34— \

TABLE 2-11 Manifestations after Administration of TMB-4 to 24 Subjectsa Protocol TtIB-4 alone Wi th intravenous soman With intravenous soman and P2S With percutaneous VX and atropine With percutaneous V.X, atropine, and 2-PAM (chloride) The TAB and heat stres ~ TAB and physostigmi ne Manifestationsf None Dry mouth Blurred vision Lethargy, sluggishness Dizziness Dreaming (hallucinations) Nausea Restlessness Muscl e twitching Heat, flush Headache Abdominal discomf art Anxiety, agitation, vomit ing No . Sub] ec ts 6b 1 c,d 2 1 ad 2d 4 No. Sub jects __ 9 6 6 4 3 2 2 2 1 1 c ) a Records on 24 of 34 subjects tested were summarized; clinical data available on 11, only laboratory data on eight, and no data (except RBC cholinest erase content) on five. b Clinical data afraid able on one c Sub] ect 2 307; see test. Clinical data available e TAB - TMB-4, atropine, and benzactyzine . f Among 11 with clinical data . —35—

have been reported in the literature ~ and they are discussed in Appendix B. In all but two instances (described below), moderate effects disappeared within 24 h. One volunteer (2307) received 2 g of P2S and ~ g of TMB-4 oral ly 68 mire before being given 1.5,ug of soman by intravenous infusion. He had minor symp tome, but in 12 h became anxious, restless, and agitated. He was transferred to Walter Reed Hospital. His Multi- phasic Multiple Personali ty Inventory (MMPI) and psychiatric inter- view before exposure were described as "doubtful in regard to suit- abi ~ ity, but riot grossly pathological . " Postexposure physical and necrologic examinations showed no organic signs of neurologic disease. The discharge diagnosis from Walter Reed Hospital in 1961 specified acute, moderate anxiety reaction manifested by restless- ness, anxiety, agitation, and hysterical reaction. He has experi- enced further problems requiring inpatient and outpatient psychi- atric care. The Veterans' Administration diagnoses between 1966 and 1980 noted infantile personality with strong paranoid trends, organic brain syndrome, and severe a ~ iety neurosis with depression. This subject was also noted in Volume 1 (p. 30~. A second sub ject ~ 6849) experienced a grand Sal seizure 3 h af ter receiving 300 mg of 2-PAM (chloride form) intramuscularly. He regained consciousness within 5 mini he had bitten his tongue. He was initially lethargic, but felt well 10 h later. He was trans- ferred to Walter Reed Hospital, but followup records are not avail- able. He had received 300 mg of 2-PA}1 intramuscularly 5 and 8 ~ before the episode. The only symptom on those occasions was local pain at the injection site. Although 2-PAM is an FDA-approved ding, data submitted to the FDA to obtain approval are proprietary and were not released when requests for them were made to the FDA and to the manufacturer. A report of the FDA Adverse Drug Experiences Monitoring Program ~ October 30, 1983) contained one adverse reaction (hypotension fol1 owing 1 g by intravenous injection). In seminary, with the possible exception of those two cases, the records contained no evidence of delayed or persistent effects after administration of the chorine at erase Deactivators. Such data cannot, however, address the issue of long-term effects or delayed sequelae. REFERENCES 1. Albanus, L., Jarplid, B., and Sundwall, A. The toxicity of some cholinesterase reactivating oximes. Br. J. Exp. Pathol. 45: 120-127, 1964. —36—

2. Aldridge, W.N., and Reiner, E. Enzyme Inhibitors as Substrates. Frontiers of Biology, Volume 26. New York: American Elsevier Publishing Co., Inc. 1972. p. 75-77. 3 . Amo ~ , W. C., Jr ., and Hal ~ , A. Malathion poisoning treated with protopam. Ann. Intern. lied. 62 :1013-1016, 1965. Ayerst Laboratories, Division of American Home Products Corp., New York, N.Y. Protopam(R) chloride (Pralidoxime chloride). Professional brochure . 197 3. 39 p . 5. Barckow, D., Nenhaus, G., aid Erdmann, W.D. Zur Behandlung der schweren Parathion-(E 605(R)~-Vergiftung mit dem Cholin- esterase-Reaktivator Obidoxim (To~ogonin(R)~. Arch. Toxikol. 24:133-146, 1969. (English Summary) 6. Barkman, R., Edgren, B., and Sundwall, A Self-administration of pralidoxime in nerve gas poisoning with a note on the stability of the drug. J. Pharm. Pharmacol. 15:671-677, 1963. Barr, A.M. Further experience in the treatment of severe organic phosphate poisoning. tied. J. Aust. 1:490-492, 1966. 8. Barr, A. M. Poisoning by antic ho1 inesterase organic phosphates: Its significance in anaesthesia. Med. J. Aust. 1: 792-796, 1964. ^. Bay ~ E. PharmacodyDamice of 1,1'-trimethylene bis(4-formyl- pyridirM um bromide) dioxime (TMB-4~. Fed. Proc. Fed. Ame SOC. Exp. Biol. I8: 366, abet . no. 1445, 19 59. 10. Bay, E. lathe action of 2-formyl-1 Methyl pyridinium chloride oxime ~ 2-PAM Cl) on catechlolamine induced response in rabbits. Pharmacologist 4: 149, 1962. 11. Beilsteins Handbuch der Organischen Chemie, edited by Reiner Luckenbach. Supplementary Series III/IV, Vol. 21, part 4, pp . 3514-3515, 3542-3543. Heidelberg: Springer-Veriag Berlin, 1978. 12. Bell, A., Barnes, R., and Simpe on, G.R. Cases of absorption and poisoning by the pesticide "Phosdrin. " Med. J. Aust . 1: 178-180 , 1968. 13. Bennett, B.G ., and Best , J. Successful use of an antidote in phosphorus insecticide poisoning . Med. J. Aust . 2:150, 1962. 14 . Berglund , F., Elwin, C. E., and Sundwall , A. Studies on the renal elimination of N~ethylpyridinium-2-aldo2rime. Biochem. Pharmacol. 11: 383-388, 1962. —37—

15. Bethe, K., Erdmann, W.D., Lendle, L., and Schmidt, G. Spezifische Antidot-Behandlung bei protrahierterVergiftung mit Alky~phosphaten (Parao~on, Parathlon, DFP) und Eserin an Meerechweinchen. Naunyr~-Schmiedebergs Arch. Exp. Pathol. Pharmako ~ . 2 31: 3-2 2, 195 7 . 16 . Boelcke , G., Creutzfeldt, W., ErdmaDn , W. D., Gaaz , J.W., and Jacob, G. Untersuchungen zur Frage der Lebertoxizitat von Obido~cim (Toxogonin (R3) am Menechen. Dtech. Med. Wochenechr. 95:1175-117S, 1970. 17. Boskovic, B. The treatment of Soman poisoning and its perspec- tives. Eundam. Appl. Toxicol. 1:203-213, 1981. 18. Brachfeld, J., and Zavon, M. R. Organic phosphate ~ Phos~rin) intoxication: Report of a case and the results of treatment with 2-PAM. Arch. Environ. Health I] :859-862, 1965. 19. Brown, R.V., Kunkel, A.M., Somers , L.M., and Wills , J. FI. Pyridine-2-aldoxime me thiodide in the treatment of sarin and tabun poisoning, with notes on its pharmacology. J. Pharmacol. Exp. Ther. 120: 276-284, 1957. 20. Calesnick, B., Christensen, J.A., and Richter, M. Human toxicity of various oximes. Arch. Eaviron. Health ~ 5: 599-60B, 1967. 21. Carrier, G. O., Peters, T., and Bishop , V. S . Alteration in calci''m metabolism as a mechanism for pyridine aldoxime metho- chloride ~ 2-PAM) cardiac action in rabbit atria. J. Pharmacol. Exp. Ther. 193: 218-231, 1975. 22. Clement, J.G. Pharmacological actions of MS-6, an oxime, on the neuromuscular junction. Eur . J . Pharmacol. 53 :135-141, 1979 . 23. Coleman, I.W., Little, P.E., and Grant, G.A. Oral prophylaxis for anticholinesterase poisoning. Can. J. Biochem. Physiol. 39:351-363, 1961. 24. Crowley, W. J., Jr ., and Johns , ~ . R. Accidental malathion poisoning. Arch. Neurol. 14: 611-616, 1966. 25. Das Gupta, S., Moorthy, M.V., Chowdhri, B.L. and Ghosh, A.K. Effect of thiamine hydrochloride on the blood level of 2-formyl 1-me tiny! pyridir~ium oxime chloride ~ 2-PAM. C1) in rats. Experientia 35: 249-250, 1979. 26. DeJong , L. P.A., and Ceulen, D. I. Arlticholinesterase acti~rity and rate of decomposition of some phosphylated oximea. Biochem. Pharmacol. 27: 857-863, 1978. —38—

27. Dixon, M. and Webb, E. Enzymes, 3rd ed. New York: Academic Press. lll6 p. ~ 980. 28. Duke , E.E., and DeCandole , C .A. Absorption of injected oximes . J. Biochem. Physiol . 41: 2473-247B, 1963. ~ IM 5 :N-340, 1 964 ~ . 29. Ebashi , S ., and Endo, M. Calcium ion and muscle contraction. Prog. Biophys . Mol. Biol. 18 :125-183, 1968. 30. Edwards , C., and Ikeda , K. Ef fects of 2-PAbI and succiny~choline on neuromuscular transmission in the frog. J. Pharmacol. Exp. Ther. 138:322-327, 1962. I'd . Enander, I ., Sundwall, A., and Sorbo, B. Metabolic studies on N-methy] pyridinium-2-aldoxime. II. The conversion to N~ethyl- pyridinium-2-nitrile. Biochem. Pharmacol. 7: 232-236, ~ 961. 3 2. Enander , I ., Sundwall , A., and Sorbo , B . Metabolic studies on N-methyl pyridinium-2-aidoxime. III. Experiments with the 14C-labell ed compound. Biochem. Pharmacol. 11:377-382, 1962. 33. Erdmann, W. D., Bosse, I., and Franke , P. Zur Resorption und Ausscheidung von Toxogonin nach intramuskularer Injektion am Menschen. Dtsch. Med. Wochenschr. 90: 1436-1433, 1965. 34. Firemark H., Barlow, C. F., and Roth, L. J. The penetration of 2-PAM-C~ ~ into brain and the effect of cholinesterase inhi bitors on i ts transport . J . Pharmacol . Exp . The r . 145:252-265, 1964. 35. Fleisher, J.H., Howard, J.W., and Corrigan, J.P. Effect of pyridine aldoximes on response of frog rectus muscle to acetyl- choline. Br. J. Pharmacol. Chemother. 13: 288-290, 1958. 36. Fleisher, J.H., Moen, T.H., and Ellingson, N.R. Effects of 2-PAM and TMB-4 or~ neuromuscular transmission. J. Pharmacol. Exp . Ther. 149: 311-319, 196 5. Georgiev, B., and Khristosko~ra, E. Toxicity of some oximes and their ef feet on the activity of acetylcholine esterase in the blood and myoneural synapses in animals. Vet. Med. Nauki 15:108-115, 1978. (Abstract from To~back 74) 38. Gilmore, J.P., and Skinner, N.S., Jr. Studies on the systemic effects of 2-PAMCf. Arch. Int. Pharmacodyn. Ther. 151:503-509, 1964. 39. Gitelson, S., Alad jemoff, L., Ben-Hador, S., and Katzoelson, R. Poisonir~g by a mal athion-xylene mixture. J. Am. Med. Assoc. 197: 819-821, 1966. —39—

40. Goyer, R . G. The ef fee ts of 2-PAM on the release of acetyl- choUne from the isolated diaphragm of the rat. J. Pharm. Pharmaco l . 2 2 :4 20 5, 197 0 . 41 . Green, A.L., and Saville , B. The reaction of oximes with isopropyl methy~phosphonofl uoridate ~ Sarin) . J. Chem. Soc., Part I II: 3887-3892, 19 56. 42. Grob, D., arid Johns, R. J. Use of oxides in the treatment of intoxication by anticholinesterase compounds in normal subjects. Am. ]. Med. 24: 497-511, 1958. 43. Hackley, B . E., Jr. The kinetics of reaction of isopropyl methylphosphonofluoridate ~ Sarin) and diisopropyl phosphoro- fluoridate (DFP) with oximes . PhD., University of Delaware, 1957. 88p. 44. Hackley, B . E ., Jr ., and Owens , O. . O . Preparation of 0- ( is opropylme thylpho sphono ) -4-f ormyl-l~e thy~pyridinium iodide oxime . J . Org . Chem. 24: 1120, 19 59. 45. Hackley, B . E., Jr., Steinberg , Gaff., and Lamb, J. C. Formation of po tent inhibitors of AChE by reaction of pyridinaidoximes with isopropyl methylphosphonofluoridate (GB). Arch. Biochem. 80:21 1-21 4, 1959. 46. Hasselbach, W., and Makinose, M. fiber den Mechanismus des Calciumtransportes durch die Membranen des sarkoplasmatischen Reticulums. Biochem. Z. 339: 94-111, 1963. 47. Hayes, W.J., Jr. Pesticides Studied in Man. Baltimore: Williams and Wilkins, 1982. 672 p. 48. Heilbronn, E., Appel gren, I.E., arid Sundwall, A. The fate of tabun in atropine and atropine-oxime treated rats and mice. Biochem. Pharmacol. 13 :1189-1i9 5, 1964. 49. Hiraki, K., Iwasaki , I ., and Namba, M. Electroencepha' ograms pesticide poisoning cases. Rinsho No ha (Clint Electro- encephalog.) 14:333-340, 1972. 5 0 ~ Hobbiger, F. . React ivation of pho sphorylated acetylcholin- esterase . In Koelle, G . B. ., ed. Chol inesterases and Anticholinesterase Agents . Handb. E;xp. Pharmakol. 15: 921-988, ~ 963. Berlin: Springer-Verlag. 51. Hobblger, F., and Sadler, P.W . Protection against lethal organosphosphate poisoning by quaternary pyridine aidoximes. Br. J. Pharmacol. Chemother. 14:192-201, ~ 959. —40—

52. Holland, P., and Parke s, D. C. Plasma concentrations of the oxime Pralidoxime Me sylate ~ P2S ~ af ter repeated oral and intr~muscular administration. Br. J. Ind. Med . 33: 43-46, 1976 . 53. Holmes, J.H., Starr, H.G., Jr., Hanisch, R.C., and vonRaulla, K.N. Short-term toxicity of mevinphos in man. Arch. Eaviron. Health 29 :84-89, 1974. 54. Hopff, W.H., and Waser, P.G. Warum konnen Reaktivatoren schadlich sein? Abgehandelt am Beispie' der Reakti~rierung der blockierten Acety~cholinesterase. Pharmaceut. Acta Helv. 45:414-423, 1970. 55. Imo, K. Behandiung einer E605-Vergif tuna mit Atropin und PAM. Medizinische No. 44: 2114-2115, 1959. 56. Jager, B.V., and Stagg, G.N. Toxicity of diacetyl monoxime and of pyridine-2-aldoxime methiodide in man. Bull. Johns Hopkins Hosp. 102: 203-211, 1958. 57. Josselson, J., and Sidel I, F.R. Effect of intravenous thiamine on pra] idoxime kinetics. Clin. Pharmacol. Ther. 24:95-100, 1978. 58. Kal ser, S.C. Metabolism of 2-formyl-1-methyl pyridinium iodide oxime ~ 2-PAM) . Chemical \]arfare Laboratories Technical Report CWLR 2347. U. S. Army Chemical Center, Md., 1959. 12 p. 59. Karlog , 0 ., Nimb, M., and Poulsen , E . Parathion ~ bladan) forgif tning behandlet med 2-PAM (pyridyl-~2~-aidoxim-N- methyljodid). Ugeskr. Laeg. 120:177-183, 1958. 60. Kewitz, H., Wilson, I.B., and Nachmansohn, D. A specific antidote against lethal alkyl pho sphate into~cication: I I . Antidotal properties. Arch. Biochem. Biophys. 64:456-465, 1956. 61. Kondritzer , A.A., Zvirblis , P., Goodman, A., and Paplanus , S .H. Blood plasma level ~ and el ~ mination of salts of 2-PAbI in man after oral admir~istration. J. Pharm. Sci. 57 :1142-1146, 1968. 62. Kuhnen-Clausen, D. Investigations on the parasympatholytic ef feet of tozogonin on the guinea-pig isolated ileum. Eur. J. Pharmacol. 9: 85-92, 1970. 6 3. Kunke l, A. M., Dikemus, A. H ., Wills , J . H ., and O ' Leary , J . F . The pharmacology of 2-formyl-l~ethylpyridinium iodide oxime ~ 2-PAM) . Fed. Proc . Fed . Am. Soc . Exp . Biol . 18 : 412 , abst . no . 16 29 , 19 59 . 64. Lamb , J. C., Steinberg , G.M., and Hackley, B . E., Jr . Isopropyl methylphosphonylated bisquaternary oximes; powerful inhibitors of cholinesterase . Biochim. Biophys . Acta 89: 174-176, 1964 . —41—

65. Langer, G.A. Ion fluxes in cardiac excitation arid contraction aM their relation to myocardial contractility. Physiol. Rev. 48: 708-757, 1968. 66. Laufer, J., Karmell, F., Rachmilewitz, D., and Scapa, E. Effect of N~ethylpyridinium-2-aldoxime methane suiphonate (P2S ~ on rat intestinal, (Na-K)-ATPase and adenyl cyclase activities. Eur. J. Pharmacol. 74: 61-66, 1981. 67. Lehman, R.A., and Nicholls, M.E. Antagonism of phospholine (echothiophate) iodide by certain quaternary oximes. Proc. Soc. Exp. Biol. Med. 104: 550-554, 1960. 68. Lindsey , D., Pfautz , J. H., Dill, D. B., and Ward , D.M . Oral toxicity of EA 2071 ~ 2-PAM lactate) . CWL111 20-17. U. S. Army Chemical Warfare Laboratories, Army Chemical Center, Md., 1959. 1 p. 69. Loomis, T.A. Reversal of a soman-induced effect on neuro- muscular function by oximes . Life Sci. 5 :1255-1261, 1966. 70. Loomis, T.A., and Johnson, D.D. Reversal of a soman-induced effect on neuromuscular function without reactivation of cholinesterase. Toxicol. Appl. Pharmacol. B: 528-532, 1966. 71. Lundy, P.M., and -Tremb' ay, K. P. Ganglion blocking properties of some hi spyridinium soman antagonists. Eur. J . Pharmacol . 60:47-53, 1979. 7 2. Massoulie, J ., and Rieger , F. . L' acetylcholinesterase des organes el ectriques de Poissons (torpille et gymnote); complexes membranaires. Eur. J. Biochem. 11: 441-455, 1969. 73. McNamara, B.P. Oxides as antidotes in poisoning by anti- cholinesterase compounds. Edgewood Arsenal Special Publication EB-SP-76004. U. S . Dept. of the Army, Edgewood Arsenal, Aberdeen Proving Ground, Md . 21010 . 19 76. 122 p . 74 . fleeter, E. The ef feet of diacety' monoxide ~ DAM) on the neuro- muscu' ar junction. Acta Physiol. Pharmacol. Neerl. 10: 286-287, 1962. 75. Merck, E., A-G. Pyridinium salts. Brit. Pat . 930, 040, July 3 , 1963. 76. Michel, H. O ., arid Krop , S . The reaction of cholinesterase with diisopropy} fluorophosphate. J. Biol. Chem. 190 :~19-125, 1951. —42—

77. Milthers, E., Clemmesen, C., and Nimb, M. Poisoning with phosphostigaines treated with atropine, pralidoxime methiodide and diacety] monoxime. Dan. Med. Bull . 10: ~ 22-1 29, 1963. 78. Miranda, P.M.S., and Way, J.L. lathe metabolism of C14 2-PAM in the isolated perfused rat liver. IV. I-Methyl -2-pyridone. Mol. Pharmacol. 2:~17-124, ~ 966. 79. Molphy, R., and Rathus, E . M. Organic phosphorus poisoning and therapy. Med . J. Aust . 2: 337-340, 1964. 80. Nachma~ohn, D., and Rothenberg , M.A. Studies on cholin- esterase. I. On the specificity of the enzyme in nerve tissue. J. Biol. Chem. 158:653-666, 1945. 81. Namba, T. Cholinesterase inhibition by organophosphorus compounds arid its clinical ef fects. Bu' 1. WHO 44: 289-307, 1971. 82. Namba, T., and Hiraki, K. PAM (pyridine-2-aldoxime methiodide) therapy for alkylphosphate poisoning. J. Am. Med. Assoc. 16 6: 1834-1839, 1958. 83. Nayler, W. G. Calcium exchange in cardiac muscle: A basic mechanism of drug action. Am. Heart J. 73:379-394, 1967. 84. Niedergerke, R. The rate of action of calcium ions on the contraction of the heart . J. Physiol. (London) 138: 506-515, 19 57. 85. O 'Leery , J. F. ., Kunke l , A. M., Murtha , E. F. ., and Somers , L. M. Sympathomimetic actions of 2-formyl-l~e thylpyridinium chloride oxime ~ 2-PAMC1) . Fed. Proc ., Fed. Am. Soc . Fop. Biol. 21 :112 (abstract section), 1962. 86. Polak, R.L., and Cohen, E.M. The influence of oximes on the distribution of 32p in the body of the rat at ter in jection of 32P-sarin. Biochem. Pharmacot . 19: 865-876, 1970. 87. Quiaby, G. E. Further therapeutic experience with pralidoximes in organic phosphorus poisoning. J. Am. Med. Assoc. 187: 202-206, 1964. 88. Raines, A., Helke, C.J., Iadarola, M.J., Britton, L.W., and Anderson, R. J. Blockade of the tonic hindlimb extensor component of maximal electroshock and pentylene~cetrazol-induced seizures by drugs acting on muscle and muscle spindle systems: - perspecti~re on method. Epilepsia 17:395-402, 1976. —43—

89. Romer-Luthi , C.R., Ha~du, J ., and Brodbeck, U. Molecular f orms of purified hl~mAn erythrocyte membrane acetylcholinesterase investigated by crosslinking with diimidates. Hoppe-Seyler's Z. Physiol. Chem. 360: 929-934, 1979. 90. Scaife, J.F. Protection of human red cell cholinesterase against inhibition by tabun and O,O - iethyl-S-2 - iethyl- aminoethyl phosphorothiolate. Can. J. Biochem. Physiol. 38:301-303, 1960. 91. Schoene, K. Reaktivierung ~ron O,O-diathy~phosphoryl- acetylcholine st erase . Reakt i~rierunga-repho s phorylierunga- gleichgewicht. Biochem. Pharmacol. 21:163-170, 1972. 92. Shankar, P. S . Pralidoxime chloride in diazinon poisoning. A report of six cases. J. Indian Med. Assoc. 46:263-264, 1966. 93 . Shelburne , J . C., Serena , S . D ., and Langer ? G . A. Rate-tension staircase in rabbi t ventricular muscle: Relat ion to ionic exchange. Am. J. Physiol . 213 :1115-1124, 1967. 94. Shibata, S., and Carrier, 0., Jr. Antagonizing action of chlorpromazines dibenamine, and phenoxybenz amine on potassium- induced contraction. Can. J. Physiol. Pharmacol. 45: 587-596, 196 7 . 95. Sidell, F.R., Cul~rer, D.L., and Kaminskis, A. Serum creatine pho sphokinase act ivity af t er intramuscular in jection. The ef f ec t of dose, concentrat ion, and ~rolume . J . Am. Med . Assoc . 229:1894-1897, 1974. 96. Sidell, F.R., and Groff , W.A. Intramuscular and intravenous administration of small doses of 2-pyridinium aidoxime metho- chloride to man. J. Pharm. Sci. 60: 1224-122B, 1971. 97. Sidell, F.R., and Groff, W.A. Tozogonin: Blood levels and side ef fects af ter intramuscular administration in man. J. Pharm. Sci . 59: 793-79 7, 197 0. 98. Sidell, F.R., and Grof f , W.A. Tozogonin : Oral administration to man. J. Pharm. Sci . 60: 860-863, 1971. 99. Sidell, F . R., Grof f , W.A., and Ellin, R. I . Blood levele of oxime and symptoms in hu nans after sing' e and mutlip] e oral doses of 2-pyridine aidoxime methochloridee J. Pharm. Sci. 58: ~ 093-109B, 1969. —44—

100. Sidell,. F.~., Groff, W.A., and Kaminakis, A. Pralidoxime methanesulfona'ce: plasma levele and pharmacokinetics af ter oral administration to man. J. Pharm. Sci. 61:~136-~140, 1972. 101. Sidell, F.R., Magmess, J.S., and Bollen, T.E. bIodification of the effects of atropine on h''m~n heart rate by pralidoxime. Clin. Pharmacol. Ther. 11: 68-76, 1970. 102. Simon, G.A., Tirosh, M.S., and Edery, H. Adminis~cration of obidoxime tablets to man. Plasma levels and side reactions. Arch. Toxicol. 36:83-~S, 1976. 103. Stavinoha, W.B., Hinshaw, L.B., Smith, P.W., and Rieger, J.A., Jr. Cardiovascu:lar effects of 2-pyridine aldoxime methyl- chloride (pralidoxime and b~ ood pressure) . Arch. Int. Pharmacodyn. Ther. ~ 87 :52-65, 1970. 104. Stedmall, E., Stedman, E., and Easson, L. H. Choline-esterase . An enzyme present in blood serum of the horse. Biochem. J. 26: 2056-2066, 1932. 105. Stoeckel, H., and Meinecke, K.H. Uber eir~e gewerbliche Vergiftung durch Mevinphos. Arch. Toxikol. 21: 284-28B, 1966. 106. Sur~dwall, A. P~ a ;ma concentration curves of N-methyI- pyridinium-2-aldoxime methane sulphonate (P2S ~ af ter intra- venous, intramuscular, and oral administration in man. Biochem. Pharmacol. 5: 225-230, 1960. 107. Swartz, R.D., and Sidell, F.R. Effects of heat and exercise on the elimination of pralidoxime in- man. Clin. Pharmacol. Ther. 14: 83-89, 1973. 108. Swartz, R.1)., and Sidell, F.R. Renal tubular secretion of pralidoxime in man. Proc. Soc. Exp. Biol. Med. 146:419-424, 1974. 109. Wadia, R. S., Karnik, V.M., and Ihaporia, R.N. Cholinesterase leYels in diazillon poisoning (a study of the ef feet of PAfI in treatment) . J. Assoc. Physicians India 19 :185-191, 1971. 110. Warriner, R.A., III, Nies, A.S., and Hayes, W.J., Jr. Severe organophosphate poisoning complicated by alcohol and turpentine ingestion. Arch. Environ. Health 32: 203-205, 1977 111. Way, J. L. The metabolism of C14 2-PAM in the isolated perfused rat liver. II. 2-0-conjugate pyridinium ion. Pharmacol. Exp. Ther. 138 :264-26B, 1962. 112. Way, J.I.., Masterson, P. B., and Beres, J.A. The metabolism of C 2-~iM in the isolated perfu~;ed rat liver. III. 1-Methyl- 2`yanopyridinium ion. J. Pharmacol . Exp . Ther. 140 :117-124, 1963. —45—

Il3. Wilson, I.B., Bergmann, F., and Nachmansohn, D. Acetyl- cholinesterase. X. Mechanism of the catalysis of acylation reactions . J. Biol. Chem. IB6: 781-790, 1950. 114. Wilson, I. B., and Cabib, E. Acety~cholinesterase: Enthalpies and entropies of activation. J. Am. Chem. Soc. 78: 202-207, 1956. 115. Wi1son, I.B., and Ginaburg, S. A powerful reactivator of alky~pho sphat e-inhibited acet ylcholinesterase . Biochim. Biophys . Acta. 18 :168-170, 1955. 116. Wilson, I.B., and Harrison, M.A. Turnover number of acetyl- cholines~cerase. J. Biol. Chem. 236: 2292-2295, 1961. 117. Windho~z, M., ed. The Merck Index, 9th ed. Rahway, N.J l~lerck and Co ., 19 76. 1313 p . · ~ 118. Zarro , V. J., and DiPalma, J. R. The symathomimetic effects of 2-pyridine aldoxime methy~ch, oride ~ 2-PAMCl) . J. Pharmacol. Exp . Ther. 147 :153-160, 1965. 119 . Zech, R., Engelhard , H ., and Erd~un, W-1) . Reaktionen von pyridinium oximen mit dem alky~pho sphat dimethoat und seinen derivaten. Wirkung auf cholinesterasen. Biochim. Biophys. Acta 128: 36 3-371, 1966. CONCLUSIONS 0n the basis of an examination of toxicologic litera~cure, case reports from Edgewood volunteers, and a review of mortality data (reported in Volume 1), the Committee found no evidence of chronic disease ir~ animals or humans associated with single or repeated doses of the cholinesterase reactivators (oximes). The paucity of chronic-exposure data from aMmals and the lack of follownp data on volunteers prevent certainty in predicting occurrence or absence of delayed ef fects. The compounds are eliminated very rapidly from the body, but they produce a variety of acute effects that are short- lived a~ re~rersible, such as gastrointestinal distress following oral administration, pain at ar~ injection site, dizzir~ese, headache, a~ ocular discomfort. The Committee found no data on the basis of which to deter~ine or rule out carcinogenicity, mutagenicity, teratogenici~cy, or reproductive ef fects of the four oximes and therefore did not reach a conclusion in this area. —46—