Toxicodynamic modeling of highly toxic organophosphorus compounds.

Although the in vitro effect of organophosphorus (OP) compounds on acetylcholine-esterase (AChE) has been studied extensively, the hypothesis that OP inhibition of AChE is the primary mechanism of acute in vivo OP toxicity has been controversial. For example, a recent review (Pope and Liu, 2004) suggested that OP compounds have direct toxic effects on other enzymes, ACh receptors, and receptor/ channel complexes that are independent of AChE inhibition. The purpose of this report is to examine the hypothesis that AChE inhibition is the mechanism of acute toxicity of OP compounds by mathematically modeling the in vivo lethal effects of highly toxic OP compounds and determining the amount of variation in OP toxicity that is explained by AChE inhibition.

In vitro and in vivo characterization of recombinant human butyrylcholinesterase (Protexia) as a potential nerve agent bioscavenger.

Previous studies in rodents and nonhuman primates have demonstrated that pretreatment with cholinesterases can provide significant protection against behavioral and lethal effects of nerve agent intoxication. Human butyrylcholinesterase (HuBuChE) purified from plasma has been shown to protect against up to 5 x LD50s of nerve agents in guinea pigs and non-human primates, and is currently being explored as a bioscavenger pretreatment for human use. A recombinant form of HuBuChE has been expressed in the milk of transgenic goats as a product called Protexia. Protexia was supplied by Nexia Biotechnologies (Que., Canada) as a purified solution with a specific activity of 600 U/mg. Initial in vitro studies using radiolabeled 3H-soman or 3H-DFP (diisopropyl fluorophosphate) demonstrated that these inhibitors specifically bind to Protexia. When Protexia was mixed with soman, sarin, tabun or VX using varying molar ratios of enzyme to nerve agent (8:1, 4:1, 1:1 and 1:4, respectively), the data indicated that 50% inhibition of enzyme activity occurs around the 1:1 molar ratio for each of the nerve agents. Protexia was further characterized for its interaction with pyridostigmine bromide and six unique carbamate inhibitors of cholinesterase. IC50 and Ki values for Protexia were determined to be very similar to those of HuBuChE purified from human plasma. These data suggest that Protexia has biochemical properties very similar to those HuBuChE when compared in vitro. Together these data the continued development of the goat milk-derived recombinant HuBuChE Protexia as a potential bioscavenger of organophosphorus nerve agents.

Central neuro-inflammatory gene response following soman exposure in the rat.

Effective treatments to improve survivability following exposure to the nerve agent soman have been established and are currently available. Unfortunately, electrographic brain seizures, neuroinflammation and brain cell death are still a potential problem even with treatment. In the present study we have characterized the time course of the central neuro-inflammatory gene response using quantitative real time-PCR (TaqMan). Male Sprague-Dawley rats were pre-treated with HI-6 (1-2-hydroxy-iminomethyl-1-pyridino-3-(4-carbamoyl-1-pyridino-2-oxapropane dichloride); 125 mg/kg, i.p.) and exposed 30 min later to 1.6 x LD(50) of soman (pinacolyl methyl-phosphonofluoridate, 180 microg/kg, s.c.) followed at 1 min by atropine methyl nitrate (4 mg/kg, i.m.). Initially, a significant and dramatic upregulation of tumor necrosis factor-alpha and vascular cell adhesion molecule-1 mRNA levels was measured 2 h post-exposure followed at 6 h by upregulation of interleukin-1beta, interleukin-6, E-selectin, and intercellular adhesion molecule-1 with eventual resolution by 24-48 h. In conclusion, an acute and transient upregulation of the inflammatory gene response is activated following soman exposure that may be involved in the soman-induced brain injury process.

Combination anticonvulsant treatment of soman-induced seizures.

These studies investigated the effectiveness of combination treatment with a benzodiazepine and an anticholinergic drug against soman-induced seizures. The anticholinergic drugs considered were biperiden, scopolamine, trihexaphenidyl, and procyclidine; the benzodiazepines were diazepam and midazolam. Male guinea pigs were implanted surgically with cortical screw electrodes. Electrocorticograms were displayed continually and recorded on a computerized electroencephalographic system. Pyridostigmine (0.026 mg x kg(-1), i.m.) was injected as a pretreatment to inhibit red blood cell acetylcholinesterase by 30-40%. Thirty minutes after pyridostigmine, 2 x LD50 (56 microg x kg(-1)) of soman was injected s.c., followed 1 min later by i.m. treatment with atropine (2 mg x kg(-1)) + 2-PAM (25 mg x kg(-1)). Electrographic seizures occurred in all animals. Anticonvulsant treatment combinations were administered i.m. at 5 or 40 min after seizure onset. Treatment consisted of diazepam or midazolam plus one of the above-mentioned anticholinergic drugs. All doses of the treatment compounds exhibited little or no antiseizure efficacy when given individually. The combination of a benzodiazepine and an anticholinergic drug was effective in terminating soman-induced seizure, whether given 5 or 40 min after seizure onset. The results suggest a strong synergistic effect of combining benzodiazepines with centrally active anticholinergic drugs and support the concept of using an adjunct to supplement diazepam for the treatment of nerve-agent-induced seizures.

Anticonvulsants for soman-induced seizure activity.

This report describes studies of anticonvulsants for the organophosphorus (OP) nerve agent soman: a basic research effort to understand how different pharmacological classes of compounds influence the expression of seizure produced by soman in rats, and a drug screening effort to determine whether clinically useful antiepileptics can modulate soman-induced seizures in rats. Electroencephalographic (EEG) recordings were used in these studies. Basic studies were conducted in rats pretreated with HI-6 and challenged with 1.6 x LD50 soman. Antimuscarinic compounds were extremely effective in blocking (pretreatment) or terminating soman seizures when given 5 min after seizure onset. However, significantly higher doses were required when treatment was delayed for more than 10 min, and some antimuscarinic compounds lost anticonvulsant efficacy when treatment was delayed for more than 40 min. Diazepam blocked seizure onset, yet seizures could recur after an initial period of anticonvulsant effect at doses

Electrocorticographic changes during generalized convulsive status epilepticus in soman intoxicated rats.

Generalized convulsive status epilepticus (GCSE) is the most common and potentially most damaging form of status epilepticus (SE). It has been previously reported, in both human GCSE and animal models of GCSE, that the electroencephalographs (EEGs) and electrocorticographs (ECoGs) recorded during GCSE contain an ordered sequence of five identifiable patterns: discrete seizures (phase 1), waxing and waning ictal discharges (phase 2), continuous ictal discharges (phase 3), continuous activity with flat periods (phase 4), and periodic epileptiform discharge on a flat background (phase 5). In this paper, we report the same pattern of ECoG changes in 15 rats exposed to soman, an acetylcholinesterase (AChE) inhibitor. Phase 1 was observed in 12 of 15 animals, but phases 2-5 were recorded in all the animals. Taken together, these findings suggest that the sequence of EEG changes is independent of the initiating cause, represent a common electrical response to GCSE, and reflect a common underlying neurochemical mechanism.

Dose-response effects of atropine and HI-6 treatment of organophosphorus poisoning in guinea pigs.

HI-6 (1-2-hydroxyiminomethyl-1-pyridino-3-(4-carbamoyl-1-pyridino -2- oxapropane dichloride) has been evaluated as an oxime alternative to pralidoxime, and toxogonin in the treatment of organophosphorus (OP) poisoning. The dose response effects of atropine (ATR) and HI-6 were investigated to more fully explore the interaction of these compounds in the treatment of OP poisoning. ATR, HI-6 and various combinations of the two drugs were evaluated against lethal poisoning by soman (GD) and tabun (GA) in guinea pigs. The effect of adjunctive diazepam treatment on the efficacy of atropine and HI-6 against soman was also investigated. Animals of either sex were challenged s.c. with OP and treated i.m. 1 min later with ATR and/or HI-6. When used, diazepam was injected immediately after ATR+HI6. LD50s of each treatment were calculated from probit models based on 24-hour survival against 5 levels of nerve agent and 6 animals per challenge level. A protective index (PI) was calculated by dividing the nerve agent LD50 in the presence of treatment by the LD50 in the absence of treatment. Treatment with HI6 alone had little effect on the toxicity of either OP. Treatment with ATR alone was more effective than HI-6 alone and was significantly more effective against soman than against tabun. When used in combination atropine and HI-6 had a strong synergistic effect against both agents. The dose of atropine used with HI-6 was critical in determining the efficacy of HI-6 against either agent. The slopes of the dose-lethality curves were minimally affected by the dose of ATR or HI-6. Adjunctive treatment with diazepam enhanced the efficacy of HI-6 and atropine against soman. It is concluded that 1) ATR has a large effect on the efficacy of HI-6 against OP poisoning, 2) the dose of ATR must be carefully selected in studies investigating the efficacy of HI-6 against OP poisoning, 3) the effective dose of ATR in the guinea pig is approximately 16 mg/kg, and 4) diazepam is a useful adjunct to atropine and HI-6.

Acute toxicity of cyclohexylmethylphosphonofluoridate (CMPF) in rhesus monkeys: serum biochemical and hematologic changes.

Changes in serum biochemical and hematological parameters were studied in 20 male rhesus monkeys following acute poisoning by the organophosphate nerve agent cyclohexylmethylphosphonofluoridate (CMPF or GF). Animals were challenged with 5 x LD50 GF (233 micrograms/kg, IM) following pretreatment with pyridostigmine (0.3-0.7 mg/kg per 24 h) and treated with atropine (0.4 mg/kg, IM) and either 2-PAM (25.7 mg/kg, IM) or H16 (37.8 mg/kg, IM) at the onset of clinical signs or at 1 min after exposure. Muscle fasciculations, tremors, or convulsions occurred in 19 of 20 animals. Serum biochemical and hematologic parameters were analyzed 2 days and 7 days after exposure and compared to pre-exposure baseline values. Significant increases in creatine kinase (CK), lactate dehydrogenase (LD), aspartate transaminase (AST), alanine transaminase (ALT) and potassium ion (K+), associated with damage to striated muscle and metabolic acidosis, occurred in both oxime-treated groups 2 days after exposure. Total protein, albumin, red blood cell (RBC) count, hemoglobin concentration (Hb) and hematocrit (Hct), were decreased in both oxime-treated groups at 7 days. The results demonstrate that animals exposed to a single high dose of GF and treated with standard therapy exhibit changes in serum biochemical and hematological indices directly and indirectly associated with their clinical presentations.

A comparison of the efficacy of HI6 and 2-PAM against soman, tabun, sarin, and VX in the rabbit.

This study compared the efficacy of HI6 and 2-PAM against nerve agent (soman, tabun, sarin, and VX)-induced lethality in the atropinesterase-free rabbits pretreated with vehicle (controls) or pyridostigmine. Treatment was administered at signs or 2 min after agent challenge and consisted of oxime (100 mumol/kg) + atropine (13 mg/kg) (alone or together with diazepam). Twenty-four-h LD50 values were calculated for soman- and tabun-intoxicated animals, whereas 24-h survival was noted in animals given 10 LD50s of sarin or VX. In pyridostigmine and control rabbits intoxicated with soman and treated with oxime + atropine (alone or together with diazepam), HI6 was 3-5 times more effective than 2-PAM. In contrast, HI6 was less effective than 2-PAM against tabun poisoning. In pyridostigmine-pretreated animals exposed to tabun, efficacy was increased more than 3-fold when compare to tabun-challenged animals treated with atropine + HI6 alone. Both oximes were highly effective against sarin and VX. These findings suggest that HI6 could replace 2-PAM as therapy for nerve agent poisoning, because it is superior to 2-PAM against soman, and when used in pyridostigmine-pretreated animals, it affords excellent protection against all four nerve agents when used in combination with atropine (alone or together with diazepam) therapy.

Efficacy of tacrine as a nerve agent pretreatment.

Tacrine (THA) was evaluated in vitro and in vivo as a pretreatment for nerve agent intoxication. In vitro experiments showed that the primary effect of THA was direct inhibition of purified fetal bovine serum acetylcholinesterase (AChE) with a slight effect on slowing the aging rate of nerve agent-inhibited AChE. THA produced significant behavioral effects at doses above 1.7 mg/kg, i.m., in the mouse and 3.4 mg/kg, i.m., in the guinea pig. At the no observable effect level (NOEL) for mice (1.7 mg/kg), THA was effective (P < or = 0.05) in reducing tabun- and soman-, but not sarin-induced lethality in mice. Experiments in the guinea pig showed that at the NOEL (3.4 mg/kg, i.m.) THA was not effective in decreasing lethality due to soman exposure. Since there was significant overlap between pharmacologically effective doses of THA and those which produce behavioral toxicity, THA was not considered a suitable pretreatment for nerve agent intoxication.

Efficacy of oxime plus atropine treatment against soman poisoning in the atropinesterase-free rabbit.

The oximes pralidoxime chloride (2-PAM), MMB4, and HI-6 were evaluated in combination with atropine as treatments against soman poisoning in atropinesterase-free rabbits. Animals were challenged i.m. with 2 x LD50 soman and treated at the onset of toxic signs with 50 mumols/kg of oxime and 5 or 13 mg/kg atropine. Survival and time to death were compared at 48 hours post-soman challenge. Survival rates in MMB4 and HI-6 treated animals were higher than in 2-PAM-treated animals. The increase in survival was significant at the 13 mg/kg dose of atropine. MMB4 and HI-6 also significantly delayed time to death after soman compared to 2-PAM. The results suggest that MMB4 and HI-6 have potential as useful oximes for treating soman poisoning.

Reduction by pyridostigmine pretreatment of the efficacy of atropine and 2-PAM treatment of sarin and VX poisoning in rodents.

This study concerned the effect of pyridostigmine pretreatment on (a) the antidotal efficacy of atropine and 2-PAM in sarin, tabun, and VX poisoning in mice and guinea pigs and on (b) the oxime-induced reactivation of VX-inhibited whole blood acetylcholinesterase (AChE) of guinea pigs. One hour prior to organophosphate (OP) challenge with sarin, tabun, or VX, animals were given oral doses of pyridostigmine to induce approximately 30 and 60% inhibition of whole blood AChE; controls received vehicle. Mice were challenged im and guinea pigs sc with the OP compounds. Treatment with atropine (11.2 mg/kg to mice; 32 mg/kg to guinea pigs) plus 2-PAM (25 mg/kg) was given im at 10 sec postchallenge in mice and 1 min postchallenge in guinea pigs. In the reactivation experiments, pyridostigmine or saline was given im to guinea pigs 30 min prior to VX (8.24 micrograms/kg, sc), atropine (16 mg/kg) was given im at 1 min, and 2-PAM (25 mg/kg) at 16 min postchallenge. Pyridostigmine significantly enhanced the efficacy of atropine and 2-PAM against tabun in both species. In contrast, pyridostigmine reduced or did not increase the efficacy of atropine and 2-PAM against sarin or VX in both species. Recovery of VX-inhibited AChE by 2-PAM was decreased significantly in pyridostigmine pretreated animals. The results suggest that pyridostigmine pretreatment may adversely effect the efficacy of atropine and 2-PAM as antidotes for VX and sarin intoxication.

Evaluation of the toxicity, pathology, and treatment of cyclohexylmethylphosphonofluoridate (CMPF) poisoning in rhesus monkeys.

Cyclohexylmethylphosphonofluoridate (CMPF) is an organophosphate cholinesterase inhibitor with military significance. The purpose of these studies was 1) to determine the acute toxicity of CMPF in the male rhesus monkey, 2) to evaluate the efficacy of pyridostigmine (PYR) pretreatment plus atropine and oxime (2-PAM or H16) treatment, and 3) to evaluate the pathological consequences of acute poisoning. An i.m. LD50 of CMPF was estimated using an up-and-down dose selection procedure and 12 animals. The 48-h and 7-day LD50 was 46.6 micrograms/kg, i.m. In the protection experiments, pyridostigmine (0.3-0.7 mg/kg/24 h) was administered by surgically implanted osmotic minipumps for 3-12 days resulting in 21-65% inhibition of erythrocyte acetylcholinesterase activity. Animals were challenged with 5 x L50 CMPF (233 micrograms/kg) and treated with atropine (0.4 mg/kg) and either 2-PAM (25.7 mg/kg) or HI6 (37.8 mg/kg) at the onset of signs or 1 min after challenge. Osmotic pumps were removed within 30 min after agent challenge. Pyridostigmine, atropine, and either 2-PAM or H16 were completely effective against CMPF, saving ten of ten animals in each group. In comparison, three of five animals challenged with 5 x LD50 of soman and treated with atropine and 2-PAM survived 7 days. The primary histologic lesions in the acute toxicity group were neuronal degeneration/necrosis and spinal cord hemorrhage. The CMPF treated groups (total of 20 animals) had minimal nervous system changes with no significant lesion difference resulting from the different oxime therapies. The primary non-neural lesions were degenerative cardiomyopathy and skeletal muscle degeneration which occasionally progressed to necrosis and mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)

Quaternary salts of 2-[(hydroxyimino)methyl]imidazole. 5. Structure-activity relationships for side-chain nitro-, sulfone-, amino-, and aminosulfonyl-substituted analogues for therapy against anticholinesterase intoxication.

Several quaternary imidazolium oxime derivatives incorporating side chains bearing nitro, sulfone, amino, and aminosulfonyl substituents were prepared and evaluated as treatment therapeutics for anti-AChE intoxication. In vivo test results in the mouse revealed that many of these compounds are highly effective in providing life-saving protection against the extremely toxic cholinesterase inhibitors soman and tabun. Several structure-activity relationships were noted that were characteristic of the side-chain substituent. In vivo test results for additional selected derivatives of some of the more therapeutically active compounds indicated that the quaternary heteroaryl nucleus is essential for activity whereas a nucleophilic moiety (i.e., oxime) is not. In support of previous suspicions, these results afforded additional evidence suggesting that reactivation is not the main mode of antidotal action by the imidazolium oximes. An alternative antidotal mechanism is postulated that is consistent with all data and that involves enzyme protection by the compounds.

Quaternary salts of 2-[(hydroxyimino)methyl]imidazole. 4. Effect of various side-chain substituents on therapeutic activity against anticholinesterase intoxication.

A series of quaternary salt derivatives of 2-[(hydroxyimino)methyl]-1-methylimidazole incorporating various side chains bearing ether, silyl, nitrile, ester, halogen, nitro, sulfone, amino, or aminosulfonyl substituents was prepared and evaluated in vivo for the treatment of anticholinesterase intoxication. Test results in the mouse revealed that the type and location of the side-chain substituent both have a significant influence on the toxicity and antidotal effectiveness of the compounds. Some of the more active examples represent the most potent therapeutics to date against intoxication by the powerful cholinesterase inhibitors soman and tabun. Significantly, the antidotal effectiveness of the compounds was not dependent on the inhibiting agent nor was there any correlation between in vivo efficacy and in vitro reactivation of ethyl (4-nitrophenyl)methylphosphonate inhibited human acetylcholinesterase. These observation suggested that the main mode of antidotal protection by the compounds is something other than enzyme reactivation.

Effect of endogenous carboxylesterase on HI-6 protection against soman toxicity.

Species variation in oxime protection against soman was examined in mice and guinea pigs with the bis-pyridinium oxime HI-6. HI-6, an effective reactivator of soman-inhibited acetylcholinesterase, produced greater maximal protection against soman in mice than in guinea pigs, although there was no species difference in acetylcholinesterase reactivation. In mice the maximal therapeutic dose of HI-6 increased the LD50 of soman from 113 micrograms/kg in unprotected animals to 992 micrograms/kg in animals receiving 76.6 mg/kg of HI-6 and 11.2 mg/kg of atropine. In guinea pigs the maximal therapeutic dose of HI-6 increased the LD50 of soman from 28.2 micrograms/kg in unprotected animals to 179 micrograms/kg in animals receiving 136 mg/kg of HI-6 and 16 mg/kg of atropine. In animals whose carboxylesterase had been inhibited with 2 mg/kg of cresylbenzodioxaphosphorin oxide, the soman LD50 values in unprotected mice and guinea pigs were similar (10.2 vs. 12.2 micrograms/kg), as were the soman LD50 values in mice and guinea pigs protected with HI-6 and atropine (159 vs. 151 micrograms/kg). In cresylbenzodioxaphosphorin oxide-treated mice and guinea pigs the achievement of equal HI-6 protection against soman correlated with the ability of HI-6 to produce equal levels of reactivation of soman-inhibited acetylcholinesterase in both species. In animals whose carboxylesterase levels were different, the animals with higher carboxylesterase levels (i.e., mice) achieved higher levels of HI-6 protection against soman than did animals with lower levels of carboxylesterase (i.e, guinea pigs). This observation suggested that, in addition to its reactivation of soman-inhibited acetylcholinesterase, HI-6 produced an effect on carboxylesterase that increased its therapeutic effect in mice.

Assessment of motor performance decrement following soman poisoning in mice.

A simple motor performance test, the inverted screen test, was used to assess the incapacitating effects of soman in mice and to evaluate the effectiveness of carbamate pretreatment and/or treatment with atropine plus 2-PAM on soman-induced debilitation. The test requires minimal equipment and personnel training, and can be done rapidly on untrained animals. Mice were placed individually on wire mesh screens which were horizontally mounted on a metal rod. The rod was rotated 180 degrees so that the mice were oriented upside down on the bottom of the screen. The animals were observed for their ability to climb to the top of the screen within one minute. Mice exhibited significant disruption of performance on the screen test 24 hours after soman doses of 0.71 LD50 or more. Treatment with atropine and 2-PAM i.m. 10 sec post-soman (1.1 x LD50) did not improve screen test performance when measured at 24 hours. Pretreatment with pyridostigmine or physostigmine in combination with post-soman therapy with atropine and 2-PAM significantly improved screen test performance as early as 2 hours after soman. The results suggest that the inverted screen test may be useful as a first-line assessment of the efficacy of pretreatment and treatment compounds against nerve agent-induced incapacitation.

Quaternary salts of 2-[(hydroxyimino)methyl]imidazole. 3. Synthesis and evaluation of (alkenyloxy)-, (alkynyloxy)-, and (aralkyloxy)methyl quaternarized 2-[(hydroxyimino)methyl]-1-alkylimidazolium halides as reactivators and therapy for soman intoxication.

A series of structurally related monosubstituted 1-[(alkenyloxy)methyl]-, 1-[(alkynyloxy)methyl]-, and 1-[(aralkyloxy)methyl]-2-[(hydroxyimino)methyl]-3-methyli midazolium halides were prepared and evaluated. All new compounds were characterized with respect to (hydroxyimino)methyl acid dissociation constant, nucleophilicity, and octanol-buffer partition coefficient. The alkynyloxy-substituted compounds were also evaluated in vitro with respect to reversible inhibition of human erythrocyte (RBC) acetylcholinesterase (AChE) and kinetics of reactivation of human AChE inhibited by ethyl p-nitrophenyl methylphosphonate (EPMP). In vivo evaluation in mice revealed that coadministration of alkynyloxy-substituted imidazolium compounds with atropine sulfate provided significant protection against a 2 x LD50 challenge of GD. For the alkynyloxy-substituted imidazolium drugs there is a direct relationship between in vitro and in vivo activity: the most potent in vivo compounds against GD proved to be potent in vitro reactivators against EPMP-inhibited human AChE. These results differ from the observations made on the sterically hindered imidazolium compounds (see previous article) and suggest that several antidotal mechanisms of protective action may be applicable for the imidazolium aldoxime family of therapeutics. The ability of the alkynyloxy substituents to provide life-saving protection against GD intoxication was not transferable to the pyridinium or triazolium heteroaromatic ring systems.

Acetylcholinesterase prophylaxis against organophosphate toxicity.

Fetal bovine serum acetylcholinesterase (FBS-AChE) protected mice from multiple LD50 doses of organophosphorus (OP) nerve agents. Mice were injected intraperitoneally (ip) with up to 3.3 mg (11,000 U) of FBS-AChE which exhibited a relatively long serum half-life and appeared well tolerated. The enzyme protected mice from the OP ethyl-S-2-diisopropylamino-ethylmethylphosphonothiolate (VX) with a stoichiometry equal to approximately 2 moles of enzyme active site per mole of VX. FBS-AChE, at a lower enzyme OP ratio, protected mice from 2 LD50s of the nerve agent methylphosphonofluoridic acid 1,2,2,-trimethylpropyl ester (soman) when used in conjunction with atropine and 2[(hydroxyimino)methyl]-1-methylpyridinium chloride. It is concluded that sequestration of highly toxic OPs by administration of AChE occurs in mice and suggests a new approach to treatment of OP intoxication.