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It has been demonstrated that cholinesterases (ChEs) are an effective mode of pretreatment to prevent organophosphate (OP) toxicity in mice and rhesus monkeys. The efficacy of ChE as a bioscavenger of OP can be enhanced by combining enzyme pretreatment with oxime reactivation, since the scavenging capacity extends beyond a stoichiometric ratio of ChE to OP. Aging has proven to be a major barrier to achieving oxime reactivation of acetylcholinesterase (AChE) inhibited by the more potent OPs. To further increase the stoichiometry of OP to ChE required, we have sought AChE mutants that are more easily reactivated than wild-type enzyme. Substitution of glutamine for glutamate (E199) located at the amino-terminal to the active-site serine (S200) in Torpedo AChE generated an enzyme largely resistant to aging. Here we report the effect of the corresponding mutation on the rate of inhibition, reactivation by 1-(2-hydroxyiminomethyl-1-pyridinium)-1(4-carboxyaminopyridinium)- dimethyl ether hydrochloride (HI-6), and aging of mouse AChE inhibited by C(+)P(-)- and C(-)P(-)-epimers of soman. The E202 to Q mutation decreased the affinity of soman for AChE, slowed the reactivation of soman-inhibited AChE by HI-6, and decreased the aging of mutant AChE. These effects were more pronounced with C(-)P(-)-soman than with C(+)P(-)-soman. In vitro detoxification of soman and sarin by wild-type and E202Q AChE in the presence of 2 mM HI-6 showed that, E202Q AChE was 2-3 times more effective in detoxifying soman and sarin than wild-type AChE. These studies show that these recombinant DNA-derived AChEs are a great improvement over wild-type AChE as bioscavengers. They can be used to develop effective methods for the safe disposal of stored OP nerve agents and potential candidates for pre- or post-exposure treatment for OP toxicity.

Fetal bovine serum actylcholinesterase (FBS-AChE, EC 3.1.1.7) was titrated, both in vitro and in vivo , with a highly toxic anti-ChE organophosphate, 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodie (MEPQ). Approximately 1:1 stoichiometry was obtained for the sequestration of MEPQ by FBS-AChE in mice. A quantitative, linear correlation was demonstrated between blood-AChE levels and the protection afforded by exogenously administered AChE in mice when challenged with anti-ChE MEPQ. The results presented in this report demonstrate that such prophylactic measures are indeed sufficient to protect animals against poisoning by as high as an 8 × ld 50 dose of organophosphate without the administration of any supportive drug. Despite the relatively large toxic dose, most of the mice that survived the challenge did not show any classical clinical signs of severe anti-ChE poisoning. MEPQ may be considered a suitable model compound for studying the quantitative aspects of the scavenger prophylactic approach described here.

The protection exerted by carbamate anticholinesterases against the lethal effects of organophosphate anticholinesterases is interpreted kinetically. The protective action is shown to depend on the provision (by carbamylation) of a pool of sequestered cholinesterase resistant to organophosphates but which furnishes (by decarbamylation) sufficient active enzyme to essential cholinergic synapses to ensure survival until all free organophosphate is cleared from the tissues. The two main factors governing the extent of protection are the minimum cholinesterase activity compatible with survival and the “enzyme conservation index” defined essentially as the ratio of the rate of enzyme decarbamylation to the rate of enzyme phosphorylation; the lower the “minimum essential cholinesterase” and the higher the “enzyme conservation index”, the greater the protection. The theory is shown to be in excellent qualitative agreement with experiment.

Soman inhibits the enzyme acetylcholinesterase, essentially irreversibly, producing an accumulation of acetylcholine (ACh) which is responsible for many of its toxic effects. Current approaches to treatment include: (1) atropine, a muscarinic receptor blocker; (2) pyridine-2-aldoxime methylchloride (2-PAM), an enzyme reactivator; and (3) carbamate protection of the enzyme. However, no fully satisfactory regimen has been found, primarily because of the rapid aging process. In this study, compounds known to inhibit ACh synthesis in vitro were evaluated in combination with atropine and 2-PAM so as to assess their potential utility in protection against soman toxicity in rats. Acetylsecohemicholinium (100 micrograms/kg, i.c.v.t., 30 min prior to soman), an inhibitor of high affinity choline uptake (HAChU) and cholineacetyltransferase (ChAT) activity in vitro, enhanced the protective effects of atropine and 2-PAM, reducing the mortality within the first 2 hr following soman. N-Hydroxyethylnaphthylvinylpyridine (NHENVP), a quaternary ChAT inhibitor (1.7 mumol/kg, i.m.), significantly reduced the overall percent mortality due to soman from 80% to 20%. The compound was most effective when administered 2-3 min prior to soman and was effective only by the intramuscular route. N-Allyl-3-quinuclidinol, a potent HAChU inhibitor (1 mumol/kg, i.m.) was the most effective quinuclidine analog evaluated, also reducing the percent mortality for a 24-hr period. Unlike NHENVP, it was most effective when given 30-60 min prior to soman. It is suggested from the data that compounds that disrupt presynaptic ACh synthesis in vitro may prove effective in treating organophosphate poisoning. The results demonstrate interesting differences among the compounds studied and provide insight for the design of protectants against soman toxicity. These findings further underscore the need to examine the structure activity and pharmacokinetic properties of these compounds, i.e. comparison of routes of administration, dose-response relationships, and time to effect.

Intravenous injection of32P-isopropyl methylphosphonofluoridate (Sarin) into rats resulted in inhibition of brain acetylcholinesterase (AChE) activity and phosphonylation of the enzyme. Spontaneous recovery from inhibition occurred between 0.5 and 48 hr in the Sarin-intoxicated animals in correlation with comparable dephosphorylation of Sarin-derived phosphorus bound to the enzyme. Injection of 44 mg/kg of monoisonitrosoacetone (MINA) into the Sarin-intoxicated rats resulted in significant reactivation of inhibited brain AChE over and above that occurring spontaneously, accompanied by additional dephosphorylation of enzymatically bound phosphorus. Loss of phosphorus in vivo took place exclusively as 32P-isopropyl methylphosphonic acid (32P-IMPA). No significant loss of Sarin-derived32P bound to AChE as 32P-methylphosphonate was apparent under these conditions.

The biochemical lesion in organophosphate poisoning is inhibition of cholinesterase which results in auto-intoxication by accumulated acetylcholine. Possible therapeutic processes are antagonism of acetylcholine, direct detoxication of the organophosphate, or reactivation of the inhibited enzyme. In this paper a simple mathematical model of the physiology of poisoning and treatment has been constructed by kinetic analysis of the basic biochemical reactions involved in these processes, with the added assumption that an animal will die once the fraction of cholinesterase in its body has fallen below an arbitrary minimum. Formulae have been derived from the model which enable the therapeutic efficiencies of the different forms of treatment, either alone or in combination, to be calculated. Qualitatively, the experimental and calculated relative efficiencies of the different therapeutic processes are in good agreement.

Some of the factors contributing to the failure of TMB-4 (1,3-di(4-hydroxy-iminomethylpyridinium) propane dichloride) and atropine to treat rats poisoned with MBPF (3-methylbutyl-2 methylphosphonofluoridate) have been investigated. The largest doses of oxime that the animal could tolerate produced in the diaphragm concentrations which, according to studies in vitro , would be expected to produce barely enough reactivation of the acetylcholinesterase to restore normal function. Greater reactivation in vitro could be achieved by using much higher concentrations of oxime. The “ageing” of inhibited diaphragm acetylcholinesterase was found to be relatively slow in vivo . The importance of ageing in the therapy of organophosphate poisoning is discussed, with particular reference to poisoning by Soman (3,3-dimethylbutyl-2 methylphosphonofluoridate).

The effects of pralidoxime (N-methylpyridinium-2-aldoxime) and TMB-4 (N,N'-trimethylene bis(pyridinium-4-aldoxime)) on blood cholinesterases (ChE) in Tabun poisoned dogs have been studied. When given prophylactically TMB-4 dibromide (7 mg/kg intravenously) partly prevented the decrease of the ChE activity caused by 1 LD50 of Tabun. When given after the organophosphorus compound an initial enzyme inhibition was first observed followed by a rapid reactivation. Pralidoxime methane sulphonate (20 mg/kg intravenously) had no significant reactivating effect, and it made no difference whether the oxime was given before or after Tabun. The lowest plasma concentration of TMB-4 dibromide that produced within 2 hr a significant reactivation of the erythrocyte ChE was 4–7 × 10−6 M. This concentration produced a 60 per cent reactivation within 48 hr (dose of Tabun 10 LD50). Ageing of phosphorylated dog erythrocyte ChE was found to be slower in vivo than in vitro ( t 1 2 about 48–72 and 30 hr respectively).

A comparative study of the reactivation by two oximes of acetylcholinesterase inhibited by several organophosphates has been made, with particular reference to the dependence of the degree of reactivation produced by an oxime (reactivating potency) upon the concentration of inhibited enzyme. In the case of one inhibitor it is demonstrated that the relative reactivating potency of the two oximes can be reversed by a change in experimental conditions. It is concluded that the measurement of the reactivation produced by two or more oximes, particularly when carried out under standardized conditions, is of little value in determining their relative reactivating potencies, and of negligible value in predicting their likely therapeutic effectiveness against organophosphate poisoning.

HI-6, ([[[(4-aminocarbonyl)pyridino]methoxy]methyl]-2-[(hydroxyimino)methyl]-pyridinium dichloride), is an oxime which, when combined with atropine, is an extremely effective therapy against organophosphate poisoning. It was found that, following soman (287 μg/kg) poisoning, HI-6 reactivated acetylcholinesterase in the diaphragm and intercostal muscles but not in the brain. At a lower dose of soman (110 μg/kg), HI-6 reactivated acetylcholinesterase in the brain. HI-6 reactivated sarin-inhibited acetylcholinesterase in the brain and in the respiratory musculature but did not reactivate tabun-inhibited acetylcholinesterase. It was also found that soman produced a differential inhibition of diaphragm and intercostal muscle acetylcholinesterase in vivo , whereas the in vitro I 50 for soman was the same in both areas. HI-6 was capable of reactivating soman-inhibited acetylcholinesterase when administered up to 30 min post-soman, indicating that the rate of aging of the soman-acetylcholinesterase complex is slower than previously reported. The above results suggest that, in severe soman poisoning, the primary lesion occurs in peripheral acetylcholinesterase in the respiratory musculature (specifically the diaphragm).

Fetal bovine serum acetylcholinesterase (FBS-AChE, EC 3.1.1.7) was titrated, both in vitro and in vivo, with a highly toxic anti-ChE organophosphate, 7-(methylethoxyphosphinyloxy)-1-methyl-quinolinium iodie (MEPQ). Approximately 1:1 stoichiometry was obtained for the sequestration of MEPQ by FBS-AChE in mice. A quantitative, linear correlation was demonstrated between blood-AChE levels and the protection afforded by exogenously administered AChE in mice when challenged with anti-ChE MEPQ. The results presented in this report demonstrate that such prophylactic measures are indeed sufficient to protect animals against poisoning by as high as an 8 x LD50 dose of organophosphate without the administration of any supportive drug. Despite the relatively large toxic dose, most of the mice that survived the challenge did not show any classical clinical signs of severe anti-ChE poisoning. MEPQ may be considered a suitable model compound for studying the quantitative aspects of the scavenger prophylactic approach described here.

Brief treatments with paraoxon ( O , O -diethyl- p -nitrophenyl phosphate) irreversibly inhibited the acetylcholinesterase (AChE, acetylcholine hydrolase, EC 3.1.1.7) activity of cultured chick embryo muscle. Enzyme activity recovered as long as protein synthesis occurred, and was most rapid during the first 4 hr after paraoxon treatment. The initial recovery rate was related to the extent of initial inhibition of AChE activity: the more activity inhibited the more rapid the recovery. Differences noted between paraoxon-treated and untreated cultures during recovery included a 192 per cent increase in net AChE activity and an increase of 200 per cent in cell protein levels. AChE activity first appeared around the nuclei after paraoxon treatment, spread through the rest of the cell, and was released into the medium. The results suggest the presence of feedback control of the rapid recovery of AChE activity after organophosphate poisoning.

A study has been made of the relationship between the inhibition of plasma or brain cholinesterase of the chicken and the paralytic syndromes caused by DFP, TOCP, malathion, ronnel, EPN, and Trithion. DDVP and Dipterex served as non-paralytic controls. TOCP was the only compound tested that was a specific inhibitor of brain cholinesterase. All compounds except EPN inhibited plasma cholinesterase. All compounds tested inhibited brain true and/or pseudocholinesterase. All paralytic compounds tested except DFP produced a prolonged inhibition of brain true and/or pseudocholinesterase which lasted for 5 to 17 days. It was concluded that no causative relationship exists between the prolonged inhibition of brain cholinesterase and the paralytic syndromes. The regeneration of brain cholinesterase after inhibition with DFP, Dipterex, or DDVP is probably due to protein synthesis de novo ; the prolonged inhibition of these enzymes after the administration of the other compounds may be due in part to the persistence of the inhibitor in the body.

The half-time for the conversion of the reversibly inhibited form of the dimethyl phosphorylated cholinesterase to the irreversibly inhibited form of this enzyme in the brains of chickens given DDVP or malathion has been found to be 2 hr. The same half-time is observed in vitro in brain homogenates inhibited with DDVP or malaoxon, the active form of malathion.

The following types of phosphorus compounds were found to be active in potentiating the toxicity of malathion to mice: triphenyl phosphates and phosphonates containing o- and p-, methyl and ethyl substitutents; certain di-(substituted-phenyl) phenylphosphonates and N-methylphosphoramidates; S,S,S-trialkyl phosphorotrithioites and phosphorotrithioates; and certain saligenin cyclic phosphorus esters. Some compounds in the latter two groups also produced ataxia in hens. Certain of the saligenin cyclic phosphorus esters were as potent in effecting ataxia as the dialkyl phosphorofluoridates, but required much larger doses to produce parasympathomimetic effects. Also considered are the activity of the 112 phosphorus esters investigated for inhibition in vitro of mouse plasma esterases hydrolyzing malathion and propionylcholine, and the stability of the saligenin cyclic phosphorus esters to enzymatic and nonenzymatic hydrolysis.

An enzyme hydrolysing the acetyl, acetyl-β-methyl and butyryl esters of choline has been demonstrated in homogenates of the parasitic nematode, Haemonchus contortus . It is concluded that there is no evidence for the existence of more than one cholinesterase in this species. The effects of temperature, pH, substrate concentration and inhibitors on the cholinesterase of H. contortus have been described. The reactions of Di-(2-chloroethyl) (3-chloro-4-methylcoumarin-7-yl) phosphate (Haloxon) with the cholinesterases of H. contortus and sheep erythrocytes have been studied. The results are discussed with regard to the fact that Haloxon is toxic to H. contortus but not to the sheep host. The cholinesterase of another helminth parasite, Trichuris ovis , is much less susceptible to inhibition by Haloxon than that of H. contortus . The inability of Haloxon to remove T. ovis from infected sheep is attributed to this reduction in cholinesterase inhibition.

Doses of physostigmine which produce no clinical signs will, when given to guinea pigs together with atropine before poisoning, raise the ld 50 of Soman (1,2,2-trimethylpropyl methylphosphonofluoridate) seven to eight times. Prostigmine is equally effective, but other carbamates are less so and simple reversible diquaternary inhibitors are almost ineffective. If physostigmine and atropine are given after Soman, no protection occurs beyond that given by atropine alone; the toxicity of Soman is slightly decreased. Prophylaxis with physostigmine and atropine varies in effectiveness in different species in the order guinea pig > dog > rabbit > mouse = chicken > rat. Physostigmine plus atropine gave rather more protection to guinea pigs against Sarin (isopropyl methyl-phosphonofluoridate) but not so much as was given by P2S (2-hydroxyiminomethyl-N-methylpyridinium methanesulphonate) and atropine. The protective action of P2S and atropine was not affected by the addition of physostigmine to the mixture.

The antiesterase activity of tri-o-cresyl phosphate (TOCP) and its cyclic phosphate metabolite (SM-1) was examined. Administration of TOCP and SM-1 to hens in ataxia-inducing doses resulted in selective rather than prolonged inhibition of the nerve esterases hydrolyzing butyryl esters of choline, glycerol, and phenols. Nerve esterase inhibition was more persistent with TOCP than with SM-1. Selective inhibition of esterases hydrolyzing butyryl esters was also evident in studies in vitro with spinal cord homogenates and SM-1 or diisopropyl fluorophosphate (DFP). TOCP and SM-1 did not affect succinate, α-ketoglutarate, or pyruvate oxidation by preparations of nerve and liver from treated birds, nor was the response of the pyruvate oxidation by brain homogenates to thiamine pyrophosphate affected by TOCP. Many compounds tested as possible antidotes for the neurotoxicity induced by TOCP, SM-1, and DFP proved ineffective. Cortisone acetate gave limited improvement in the condition of the birds, thiamine and oxythiamine appeared to be detrimental, and many potential cholinesterase reactivators and other materials were without effect on the neurotoxicity.