Reactivator Report for: Trimedoxime

Organophosphorus (OP) compounds are used as both chemical weapons and pesticides. However, these agents are very dangerous and toxic to humans, animals, and the environment. Thus, investigations with reactivators have been deeply developed in order to design new antidotes with better efficiency, as well as a greater spectrum of action in the acetylcholinesterase (AChE) reactivation process. With that in mind, in this work, we investigated the behavior of trimedoxime toward the Mus musculus acetylcholinesterase (MmAChE) inhibited by a range of nerve agents, such as chemical weapons. From experimental assays, reactivation percentages were obtained for the reactivation of different AChE-OP complexes. On the other hand, theoretical calculations were performed to assess the differences in interaction modes and the reactivity of trimedoxime within the AChE active site. Comparing theoretical and experimental data, it is possible to notice that the oxime, in most cases, showed better reactivation percentages at higher concentrations, with the best result for the reactivation of the AChE-VX adduct. From this work, it was revealed that the mechanistic process contributes most to the oxime efficiency than the interaction in the site. In this way, this study is important to better understand the reactivation process through trimedoxime, contributing to the proposal of novel antidotes.

The aim of the study was to examine antidotal potency of trimedoxime in mice poisoned with three direct dimethoxy-substituted organophosphorus inhibitors. In order to assess the protective efficacy of trimedoxime against dichlorvos, heptenophos or monocrotophos, median effective doses and efficacy half-times were calculated. Trimedoxime (24 mg/kg intravenously) was injected 5 min. before 1.3 LD50 intravenously of poisons. Activities of brain, diaphragmal and erythrocyte acetylcholinesterase, as well as of plasma carboxylesterases were determined at different time intervals (10, 40 and 60 min.) after administration of the antidotes. Protective effect of trimedoxime decreased according to the following order: monocrotophos > heptenophos > dichlorvos. Administration of the oxime produced a significant reactivation of central and peripheral acetylcholinesterase inhibited with dichlorvos and heptenophos, with the exception of erythrocyte acetylcholinesterase inhibited by heptenophos. Surprisingly, trimedoxime did not induce reactivation of monocrotophos-inhibited acetylcholinesterase in any of the tissues tested. These organophosphorus compounds produced a significant inhibition of plasma carboxylesterase activity, while administration of trimedoxime led to regeneration of the enzyme activity. The same dose of trimedoxime assured survival of experimental animals poisoned by all three organophosphorus compounds, although the biochemical findings were quite different.

Tabun (O-ethyl-N,N-dimethyl phosphoramidocyanidate) belongs to highly toxic organophosphorus compounds misused as chemical warfare agents for military as well as terroristic purposes. It differs from other highly toxic organophosphates by its chemical structure and by the fact that tabun-inhibited acetylcholinesterase is extraordinarily difficult to reactivate. The potency of trimedoxime and other commonly used oximes (pralidoxime, obidoxime, the oxime HI-6) to reactivate tabun-inhibited acetylcholinesterase and to eliminate tabun-induced acute effects was evaluated using in vitro and in vivo methods. In vitro calculated kinetic parameters of reactivation of tabun-inhibited acetylcholinesterase from rat brain homogenate and in vivo determined percentage of reactivation of tabun-inhibited blood and tissue acetylcholinesterase in poisoned rats show that trimedoxime seems to be the most efficacious reactivator in the case of tabun poisonings. Trimedoxime was also found to be the most efficacious oxime in the elimination of acute lethal toxic effects in tabun-poisoned rats and mice. The oxime HI-6, so efficacious against soman, does not seem to be sufficiently effective oxime to reactivate tabun-inhibited acetylcholinesterase and to counteract acute lethal effects of tabun.

Organophosphorus (OP) compounds are used as both chemical weapons and pesticides. However, these agents are very dangerous and toxic to humans, animals, and the environment. Thus, investigations with reactivators have been deeply developed in order to design new antidotes with better efficiency, as well as a greater spectrum of action in the acetylcholinesterase (AChE) reactivation process. With that in mind, in this work, we investigated the behavior of trimedoxime toward the Mus musculus acetylcholinesterase (MmAChE) inhibited by a range of nerve agents, such as chemical weapons. From experimental assays, reactivation percentages were obtained for the reactivation of different AChE-OP complexes. On the other hand, theoretical calculations were performed to assess the differences in interaction modes and the reactivity of trimedoxime within the AChE active site. Comparing theoretical and experimental data, it is possible to notice that the oxime, in most cases, showed better reactivation percentages at higher concentrations, with the best result for the reactivation of the AChE-VX adduct. From this work, it was revealed that the mechanistic process contributes most to the oxime efficiency than the interaction in the site. In this way, this study is important to better understand the reactivation process through trimedoxime, contributing to the proposal of novel antidotes.

The ability of two newly developed oximes (K305, K307) to protect tabun-poisoned rats from tabun-induced inhibition of brain acetylcholinesterase, acute neurotoxic signs and symptoms and brain damage was compared with that of the oxime K203 and trimedoxime. The reactivating and neuroprotective effects of the oximes studied combined with atropine on rats poisoned with tabun at a sublethal dose were evaluated. The reactivating efficacy of a newly developed oxime K305 is lower compared to the reactivating efficacy of the oxime K203 and trimedoxime while the ability of the oxime K307 to reactivate tabun-inhibited acetylcholinesterase (AChE) in the brain roughly corresponds to the reactivating efficacy of the oxime K203 and it is slightly lower compared to trimedoxime. In addition, only one newly developed oxime (K307) combined with atropine was able to markedly decrease tabun-induced neurotoxicity although it did not eliminate all tabun-induced acute neurotoxic signs and symptoms. These results correspond to the histopathological evaluation of tabun-induced brain damage. Therefore, the newly developed oximes are not suitable for the replacement of commonly used oximes (especially trimedoxime) in the treatment of acute tabun poisonings.

The ability of two newly developed bispyridinium oximes (K920, K923) to reduce tabun-induced acute neurotoxic signs and symptoms was compared with the oxime K203 and trimedoxime using a functional observational battery. The neuroprotective effects of the oximes studied combined with atropine on rats poisoned with tabun at a sublethal dose (130 mug/kg i.m.; 80% of LD50 value) were evaluated. Tabun-induced neurotoxicity was monitored by functional observational battery at 2 hours after tabun administration. The results indicate that all tested oximes combined with atropine enable tabun-poisoned rats to survive till the end of experiment while one non-treated tabun-poisoned rat died within 2 hours. Both newly developed oximes (K920, K923) combined with atropine were able to markedly decrease tabun-induced neurotoxicity in the case of sublethal poisoning although they did not eliminate all tabun-induced acute neurotoxic signs and symptoms. Their ability to decrease tabun-induced acute neurotoxicity did not prevail the neuroprotective efficacy of trimedoxime and the oxime K203. Therefore, the newly developed oximes are not suitable for the replacement of currently available oximes (especially trimedoxime) in the treatment of acute tabun poisonings.

The reactivating and therapeutic efficacy of three original bispyridinium oximes (K727, K733 and K203) and one currently available oxime (trimedoxime) was evaluated in tabun-poisoned rats and mice. The oxime-induced reactivation of tabun-inhibited acetylcholinesterase was measured in diaphragm and brain of tabun-poisoned rats. The results showed that the reactivating efficacy of two recently developed oximes (K727 and K733) does not achieve the level of the reactivation of tabun-inhibited acetylcholinesterase induced by oxime K203 and trimedoxime. While all oximes studied were able to increase the activity of tabun-inhibited acetylcholinesterase in diaphragm, oxime K733 was not able to reactivate tabun-inhibited acetylcholinesterase in the brain. The therapeutic efficacy of all oximes studied roughly corresponds to their reactivating efficacy. While both recently developed oximes were able to reduce acute toxicity of tabun less than 1.5-fold, another original oxime K203 and commonly used trimedoxime reduced the acute toxicity of tabun almost two times. In conclusion, the reactivating and therapeutic potency of both newly developed oximes does not prevail the effectiveness of oxime K203 and trimedoxime, and therefore, they are not suitable for their replacement of commonly used oximes for the antidotal treatment of acute tabun poisoning.

Abstract The potency of two newly developed oximes (K361 and K378) to reactivate tabun-inhibited cholinesterase and to reduce acute toxicity of tabun was compared with the oxime K203 and trimedoxime using in vivo methods. The study determining percentage of reactivation of tabun-inhibited diaphragm cholinesterase in poisoned rats showed that the reactivating efficacy of the oxime K378 is slightly lower than the reactivating potency of the oxime K203 and trimedoxime while the ability of the oxime K361 to reactivate tabun-inhibited cholinesterase is markedly lower compared with the oxime K203 and trimedoxime. In the brain, the potency of both newly developed oximes to reactivate tabun-inhibited cholinesterase was negligible. The therapeutic efficacy of both newly developed oximes roughly corresponds to their weak reactivating efficacy. Their potency to reduce acute toxicity of tabun was significantly lower compared with the oxime K203 as well as trimedoxime. In conclusion, the reactivating and therapeutic potency of both newly developed oximes does not prevail the effectiveness of the oxime K203 and trimedoxime and, therefore, they are not suitable for their replacement of commonly used oximes for the treatment of acute tabun poisoning.

K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-propane dibromide] is a promising new reactivator of organophosphate- or organophosphonate-inhibited acetylcholinesterase (AChE) with low acute toxicity and broad spectrum efficacy. The aim of the present study was to compare the pharmacokinetics of both compounds. Male Wistar rats (body weight = 320 +/- 10 g) were administered a single intramuscular dose of K027 (22.07 mg kg(-1)) and an equimolar dose of trimedoxime. Blood was collected at various time intervals until 180 min. Plasma samples were analyzed by reversed-phase HPLC with ultraviolet (UV) detection. The recovery of both oximes from the plasma was approximately 90% and a linear relationship (R(2) > 0.998) was observed between the peak areas and concentrations of calibrated standards in the range 1-100 microg ml(-1). Near-identical plasma profiles were obtained for both compounds. No differences were found in the mean +/- SD values of C(max) (18.6 +/- 2.5 vs 20.0 +/- 6.3 microg ml(-1), P = 0.72) and AUC(0-180min) (2290 +/- 304 vs 2269 +/- 197 min microg ml(-1), P = 0.84). However, the percentage coefficient of variation of the first-order rate constant of absorption (k(a)) was 3-fold higher (P < 0.01) providing evidence for more erratic absorption of intramuscular trimedoxime as compared with K027. In conclusion, oxime K027 might have superior pK properties that may be translated in its faster absorption and subsequent tissue distribution.

The potency of three newly developed bispyridinium compounds (K454, K456, K458) to reactivate tabun-inhibited acetylcholinesterase and reduce tabun-induced lethal toxic effects was compared with the oxime K203 and trimedoxime using in vivo methods. The study determining percentage of reactivation of tabun-inhibited diaphragm and brain acetylcholinesterase in poisoned rats showed that the reactivating efficacy of all newly developed oximes is comparable with K203 but lower than the reactivating potency of trimedoxime in diaphragm. In the brain, their potency to reactivate tabun-inhibited acetylcholinesterase is lower compared with trimedoxime and the oxime K203. All three newly developed oximes were also found to be relatively effective in reducing lethal toxic effects in tabun-poisoned mice. Their therapeutic efficacy is consistent with the therapeutic potency of the oxime K203. On the other hand, their potency to reduce acute toxicity of tabun is significantly lower compared with trimedoxime. In conclusion, the reactivating and therapeutic potency of all three newly developed oximes does not prevail the effectiveness of the oxime K203 and trimedoxime and, therefore, they are not suitable for their replacement of commonly used oximes for the treatment of acute tabun poisoning.

Multiple oximes have been synthesized and evaluated for use as countermeasures against chemical warfare nerve agents. The current U.S. military and civilian oxime countermeasure, 2-[(hydroxyimino)methyl]-1-methylpyridin-1-ium chloride (2-PAM), is under consideration for replacement with a more effective acetylcholinesterase reactivator, 1,1'-methylenebis{4-hydroxyiminomethyl}pyridinium dimethanesulfonate (MMB-4). Kinetic data in the scientific literature for MMB-4 are limited; therefore, a physiologically based pharmacokinetic (PBPK) model was developed for a structurally related oxime, 1,1'-trimethylenebis{4-hydroximinomethyl}pyridinium dibromide. Based on a previous model structure for the organophosphate diisopropylfluorophosphate, the model includes key sites of acetylcholinesterase inhibition (brain and diaphragm), as well as fat, kidney, liver, rapidly perfused tissues and slowly perfused tissues. All tissue compartments are diffusion limited. Model parameters were collected from the literature, predicted using quantitative structure-property relationships or, when necessary, fit to available pharmacokinetic data from the literature. The model was parameterized using rat plasma, tissue and urine time course data from intramuscular administration, as well as human blood and urine data from intravenous and intramuscular administration; sensitivity analyses were performed. The PBPK model successfully simulates rat and human data sets and has been evaluated by predicting intravenous mouse and intramuscular human data not used in the development of the model. Monte Carlo analyses were performed to quantify human population kinetic variability in the human evaluation data set. The model identifies potential pharmacokinetic differences between rodents and humans, indicated by differences in model parameters between species. The PBPK model can be used to optimize the dosing regimen to improve oxime therapeutic efficacy in a human population.

The penetration of acetylcholinesterase reactivators (oximes) into the central nervous system is typically restricted by the blood-brain barrier. Although oximes are highly hydrophilic compounds, some contradictory results confirming permeation into the brain exist. The aim of this study is to verify the penetration of oximes through the blood-brain barrier and to detect their levels achieved in different brain regions 60 min after the administration. It was confirmed that oximes are able to penetrate into the brain after injection of therapeutic doses corresponding with 5% of LD(50). The level in whole brain was 0.58% for trimedoxime and 0.85% for the experimental drug oxime K074 as the percentage of their plasma concentration. The highest concentration was found in frontal cortex (trimedoxime 2.27%; oxime K074 0.95%) and lowest in basal ganglia (trimedoxime 0.86%; oxime K074 0.42%). Entry of oximes into the brain is minimal, but some low reactivation effect should be expected. The reactivation potency of oximes might be higher or lower, depending on the real oxime concentration in a given area.

OBJECTIVE: The present experiment is based on biochemical assessment of nerve agent soman intoxication and atropine, respectively atropine and HI-6, trimedoxime or K203 treatment in rats. BACKGROUND: Nerve agents are toxic substances irreversibly inhibiting enzyme acetylcholinesterase (AChE). Treatment is typically based on application of atropine and oxime reactivator. Atropine is able to protect overstimulation of muscarinic acetylcholine receptors. Application of oxime reactivator enable return of AChE activity and full suppression of intoxication. METHODS: In a total, fifteen biochemical markers were assayed in plasma or blood of intoxicated animals. 42 rats were divided into 7 groups each 6 individuals. The first group was exposed to atropine; the second group was exposed to one LD50 of soman and atropine. The groups 3-5 were exposed in a same way as the second group and were treated with oxime reactivators: HI-6 (group 3), trimedoxime (4) and K203 (5). The sixth group was control treated with saline solution only. The last (seventh) group was intoxicated with soman only. RESULTS: The most striking shifts were found for blood acetylcholinesterase and plasma creatinine, glucose, inorganic phosphate as well as uric acid. Lactate dehydrogenase and aspartate aminotransferase assays were useless due to soman interference. CONCLUSION: It was demonstrated that treatment was able to protect poisoned animals from metabolic disorder represented by hyperglycemia and nephropathy represented by hyperuricemia and elevated creatinine. Soman exposure and treatment with the oxime reactivators and/or atropine contains quite complex and still not well understood side mechanisms (Tab. 2, Fig. 1, Ref. 25).

Up to now, intensive attempts to synthesize a universal reactivator able to reactivate cholinesterases inhibited by all types of nerve agents/organophosphates were not successful. Therefore, another approach using a combination of two reactivators differently reactivating enzyme was used: in rats poisoned with tabun and treated with combination of atropine (fixed dose) and different doses of trimedoxime and HI-6, changes of acetylcholinesterase activities (blood, diaphragm and different parts of the brain) were studied. An increase of AChE activity was observed following trimedoxime treatment depending on its dose; HI-6 had very low effect. Combination of both oximes showed potentiation of their reactivation efficacy; this potentiation was expressed for peripheral AChE (blood, diaphragm) and some parts of the brain (pontomedullar area, frontal cortex); AChE in the basal ganglia was relatively resistant. These observations suggest that the action of combination of oximes in vivo is different from that observed in vitro.

The potency of newly developed bispyridinium compound K203 and its fluorinated analog KR-22836 in reactivating tabun-inhibited acetylcholinesterase and reducing tabun-induced lethal toxic effects was compared with commonly used oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies determining the percentage of reactivation of tabun-inhibited blood and tissue acetylcholinesterase in rats showed that the reactivating efficacy of K203 is higher than the reactivating efficacy of its fluorinated analog KR-22836 as well as currently available oximes studied. The therapeutic efficacy of the oxime K203 and its fluorinated analog corresponds to their potency to reactivate tabun-inhibited acetylcholinesterase. According to the results, the oxime K203 is more suitable than KR-22836 for the replacement of commonly used oximes for the antidotal treatment of acute tabun poisoning due to its relatively high potency to counteract the acute toxicity of tabun.

Introduction. The new K-oximes, K-27 [1-(4-hydroxyimino-methylpyridinium)-4-(4-carbamoylpyridinium) propane dibromide] and K-48 [1-(4-hydroxyimino-methylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide], show good in vitro efficacy in protecting acetylcholinesterase from inhibition by different organophosphorus compounds (OPCs), including nerve agents. To assess their efficacy in vivo, the extent of oxime-conferred protection from mortality induced by diisopropylfluorophosphate (DFP) was quantified and compared with that of five established oximes. Materials and Methods. Rats received DFP intraperitoneally in a dosage of 6, 8, or 10 micromol/rat and immediately thereafter intraperitoneal injections of K-27, K-48, pralidoxime, obidoxime, trimedoxime, methoxime, or HI-6. The relative risk (RR) of death over time (48 h) was estimated by Cox survival analysis, comparing results with the no-treatment group. Results. Best protection was observed when K-27 was used, reducing the RR of death to 19% of control RR (p < or = 0.005), whereas obidoxime (RR = 26%, p < or = 0.01), K-48 (RR = 29%, p < or = 0.005) and methoxime (RR = 26%, p < or = 0.005) were comparable. The RR of death was reduced only to about 35% of control by HI-6, to 45% by trimedoxime, and to 59% by 2-PAM (p < or = 0.005). Whereas the differences between the best oximes (K-27, obidoxime, methoxime, and K-48) were not statistically significant; these four oximes were significantly more effective than 2-PAM (p < or = 0.05). The efficacy of K-27 was also significantly higher than that of HI-6, trimedoxime, and 2-PAM (p < or = 0.05). Conclusion. Our data provide further evidence that K-27 is a very promising candidate for the treatment of intoxication with a broad spectrum of OPCs.

The potency of newly developed bispyridinium compounds (K117, K127) to reactivate tabun-inhibited acetylcholinesterase and reduce tabun-induced lethal toxic effects was compared with currently available oximes (obidoxime, trimedoxime, oxime HI-6) by using in vivo methods. A study that determined the percentage of reactivation of tabun-inhibited blood and tissue acetylcholinesterase in poisoned rats showed that the reactivating efficacy of newly developed oxime K127 is comparable with obidoxime and trimedoxime in blood but lower than the reactivating potency of trimedoxime and obidoxime in the diaphragm and brain. The potency of another newly developed K117 to reactivate tabun-inhibited acetylcholinesterase is comparable with obidoxime or trimedoxime in the diaphragm, but it is significantly lower than the reactivating potency of trimedoxime and obidoxime in the blood and brain. The oxime, K127, was also found to be relatively effective in reducing lethal toxic effects in tabun-poisoned mice. Its therapeutic efficacy is consistent with the therapeutic potency of obidoxime. On the other hand, the potency of the oxime, K117, to reduce acute toxicity of tabun is significantly lower compared to trimedoxime and obidoxime. The therapeutic efficacy of K117 and K127 corresponds to their potency to reactivate tabun-inhibited acetylcholinesterase, especially in the diaphragm and brain. Contrary to obidoxime and trimedoxime, the oxime, HI-6, is not an effective oxime in the reactivation of tabun-inhibited acetycholinesterase and in reducing the lethal effects of tabun. The reactivating and therapeutic potency of both newly developed oximes does not prevail over the effectiveness of currently available obidoxime and trimedoxime and, therefore, they are not suitable for their replacement of commonly used oximes for the treatment of acute tabun poisoning.

The potency of newly developed monoxime bispyridinium compounds (K156, K203) in reactivating tabun-inhibited acetylcholinesterase and reducing tabun-induced lethal toxic effects was compared with commonly used oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies determining percentage of reactivation of tabun-inhibited blood and tissue acetylcholinesterase in poisoned rats showed that the reactivating efficacy of newly developed oxime K203 is comparable with obidoxime and trimedoxime in blood and higher than the reactivating potency of trimedoxime and obidoxime in diaphragm and brain, where the difference in reactivating efficacy of obidoxime, trimedoxime and K203 is significant. On the other hand, the potency of newly developed K156 to reactivate tabun-inhibited acetylcholinesterase is comparable with obidoxime or trimedoxime in diaphragm and brain. It is significantly lower than the reactivating efficacy of trimedoxime and obidoxime in blood. Moreover, both newly developed oximes were found to be relatively efficacious in the reduction of lethal toxic effects in tabun-poisoned mice. Especially, the oxime K203 is able to decrease the acute toxicity of tabun nearly two times. The therapeutic efficacy of K156 and K203 corresponds to their potency to reactivate tabun-inhibited acetylcholinesterase, especially in diaphragm and brain. In contrast to obidoxime and trimedoxime, the oxime HI-6 is not effective in reactivation of tabun-inhibited acetycholinesterase and in reducing tabun lethality. While the oxime K156 does not improve the reactivating and therapeutic effectiveness of currently available obidoxime and trimedoxime, the newly developed oxime K203 is markedly more effective in reactivation of tabun-inhibited acetylcholinesterase in rats, especially in brain, and in reducing lethal toxic effects of tabun in mice and, therefore, it is suitable for the replacement of commonly used oximes for the antidotal treatment of acute tabun poisoning.

Nerve agents and pesticides belong to the group of organophosphates. They are able to inhibit irreversibly the enzyme acetylcholinesterase (AChE). Acetylcholinesterase reactivators were designed for the treatment of nerve agent intoxications. Their potency to reactivate pesticide-inhibited AChE was many times evaluated. In this study, five commonly used AChE reactivators (pralidoxime, methoxime, HI-6, obidoxime, trimedoxime) for the reactivation of AChE inhibited by two pesticides (chlorpyrifos and methylchlorpyrifos) were used. Russian VX (nerve agent) as a member of nerve agents' family was taken for comparison. Obtained results show that oximes developed against nerve agent intoxication are less effective for intoxication with organophosphorus pesticides. Especially, methylchlorpyrifos-inhibited AChE was found to be poorly reactivated by the compounds used.

There is a clear need for broad-spectrum cholinesterase reactivators (active against a multitude of organophosphorus ester enzyme inhibitors) with a higher efficacy than pralidoxime. The purpose of the study was to quantify in vivo the extent of oxime-conferred protection, using methyl-paraoxon [dimethyl p-nitrophenyl phosphate; (methyl-POX)] as a cholinesterase inhibitor. There were seven groups of six rats in each cycle of the experiment. Group 1 (G1) received 2 micromol methyl-POX ( approximately LD(50)), the other groups (G2-7) received 2 micromol methyl-POX + one of the six reactivators. The animals were monitored for 48 h and the time of mortality was recorded. The procedure was repeated six times. All substances were applied i.p. The experiments were repeated using 3 and 5 micromol methyl-POX. Mortality data were compared and hazards ratios (relative risks) ranked using the Cox proportional hazards model with methyl-POX dose and group (reactivator) as time-independent covariables. The relative risk of death estimated by Cox analysis (95% CI) in oxime-treated animals when compared with untreated animals, adjusted for methyl-POX dose (high/low) was K-27, 0.58 (0.42-0.80); K-48, 0.60 (0.43-0.83); trimedoxime, 0.76 (0.55-1.04); pralidoxime, 0.88 (0.65-1.20); obidoxime, 0.93 (0.68-1.26); HI-6, 0.96 (0.71-1.31). Only K-27 and K-48 provided statistically significant protection in rats exposed to methyl-POX. Despite the lower inhibitory potency (higher IC(50)) of methyl-POX compared with POX (ratio 4:1), the ability of oxime reactivators to protect from methyl-POX induced mortality was reduced compared with protection from POX (ethyl-analog).

The hydrolysis of acetylthiocholine iodide (ATCh) by pralidoxime chloride (2-PAM Cl), trimedoxime (TMB(4)) and obidoxime chlpride (LUH(6)) was studied at pH 5.8-8.0 and incubation temperature from 5 to 40 degrees C in vitro. Significant ATCh hydrolysis by 2-PAM Cl, TMB(4) and LUH(6) was found, with the exceptions of those at pH 7.0, 6.2 and 5.8 at 5 degrees C and those at pH 6.2 and 5.8 at 15 degrees C. The hydrolysis by TMB(4) and LUH(6) was significantly stronger than that by 2-PAM Cl. The hydrolysis increased with increasing pH, incubation temperature and three oxime or ATCh concentration. Significant hydrolysis of ATCh by the three oximes could be found when the terminal concentration of oxime was higher than 0.01 mM at pH 7.0 and 7.4 at 30 and 37 degrees C. However, no hydrolysis of natural substrate (acetylcholine iodide) by the three oximes was found when very high terminal concentrations of oximes were used. In addition, the three oximes displayed an extraordinary efficiency in the reactivation of phoxim-inhibited acetylcholinesterase (AChE) from fish (Carassius auratus) or rabbit (Oryctolagus cuniculus domestic) brain in vitro. Parallel to the level of ATCh hydrolysis by the oximes, TMB(4) and LUH(6) displayed significantly higher reactivation efficiency than 2-PAM Cl to phoxim-inhibited AChE. And, the extent of reactivation by 2-PAM Cl was also lower than the other two. Plausible antidotal actions of the oximes against organophosphate poisoning AChE and erroneously high estimation of AChE activity by the Ellman method were discussed.

The efficacy of H oximes (HI-6, HLo-7), the oxime BI-6, and currently used oximes (pralidoxime, obidoxime, trimedoxime) to reactivate acetylcholinesterase inhibited by two nerve agents (tabun, VX agent) was tested in vitro. Both H oximes (HI-6, HLo-7) and the oxime BI-6 were found to be more efficacious reactivators of VX-inhibited acetylcholinesterase than pralidoxime and obidoxime. On the other hand, their potency to reactivate tabun-inhibited acetylcholinesterase was low and did not reach the reactivating efficacy of trimedoxime and obidoxime. Thus, none of these compounds can be considered to be a broad-spectrum reactivator of nerve agent-inhibited acetylcholinesterase in spite of high potency to reactivate acetylcholinesterase inhibited by some nerve agents. More than one oxime may be necessary for the antidotal treatment of nerve agent-exposed individuals.

The aim of the study was to examine antidotal potency of trimedoxime in mice poisoned with three direct dimethoxy-substituted organophosphorus inhibitors. In order to assess the protective efficacy of trimedoxime against dichlorvos, heptenophos or monocrotophos, median effective doses and efficacy half-times were calculated. Trimedoxime (24 mg/kg intravenously) was injected 5 min. before 1.3 LD50 intravenously of poisons. Activities of brain, diaphragmal and erythrocyte acetylcholinesterase, as well as of plasma carboxylesterases were determined at different time intervals (10, 40 and 60 min.) after administration of the antidotes. Protective effect of trimedoxime decreased according to the following order: monocrotophos > heptenophos > dichlorvos. Administration of the oxime produced a significant reactivation of central and peripheral acetylcholinesterase inhibited with dichlorvos and heptenophos, with the exception of erythrocyte acetylcholinesterase inhibited by heptenophos. Surprisingly, trimedoxime did not induce reactivation of monocrotophos-inhibited acetylcholinesterase in any of the tissues tested. These organophosphorus compounds produced a significant inhibition of plasma carboxylesterase activity, while administration of trimedoxime led to regeneration of the enzyme activity. The same dose of trimedoxime assured survival of experimental animals poisoned by all three organophosphorus compounds, although the biochemical findings were quite different.

Tabun (O-ethyl-N,N-dimethyl phosphoramidocyanidate) belongs to highly toxic organophosphorus compounds misused as chemical warfare agents for military as well as terroristic purposes. It differs from other highly toxic organophosphates by its chemical structure and by the fact that tabun-inhibited acetylcholinesterase is extraordinarily difficult to reactivate. The potency of trimedoxime and other commonly used oximes (pralidoxime, obidoxime, the oxime HI-6) to reactivate tabun-inhibited acetylcholinesterase and to eliminate tabun-induced acute effects was evaluated using in vitro and in vivo methods. In vitro calculated kinetic parameters of reactivation of tabun-inhibited acetylcholinesterase from rat brain homogenate and in vivo determined percentage of reactivation of tabun-inhibited blood and tissue acetylcholinesterase in poisoned rats show that trimedoxime seems to be the most efficacious reactivator in the case of tabun poisonings. Trimedoxime was also found to be the most efficacious oxime in the elimination of acute lethal toxic effects in tabun-poisoned rats and mice. The oxime HI-6, so efficacious against soman, does not seem to be sufficiently effective oxime to reactivate tabun-inhibited acetylcholinesterase and to counteract acute lethal effects of tabun.

Mono- and bisbenzyloxime ethers of the bispyridinium derivative TMB-4(Trimedoxime) (UNO, DUO) are potent allosteric modulators of the muscarinic receptor attracting clinical interest in case of organophosphate poisoning. In order to work out the stability of these compounds oximes, different oxime ethers and potential degradation products were synthesized and UV- and NMR-spectroscopically characterized. The process of degradation of all compounds was observed under stress conditions at varying pH-values and different temperatures by means of time-dependent NMR- und UV-measurements. The pyridinium aldoxime turned out to be rather stable, whereas the oxime ether and cyano derivatives convert to the pyridone at high pH-values and high temperature. The mechanism of degradation is discussed.

The reaction of human erythrocyte acetylcholinesterase (AChE) with a set of structurally related phosphoramidates was studied in order to investigate the properties of phosphorylated enzyme and the effects of 4 oximes PAM-2, TMB-4(Trimedoxime), HI-6 and BDB-106 on the reactivation of inhibited AChE. Second-order rate constant of the phosphorylation reaction of the compounds towards the active site of AChE range between 5.0 x 10(2) and 4.9 x 10(6) M-1min-1 and their inhibitory power (I50) was from 7.3 x 10(-5) to 5.7 x 10(-9) M for 20 min incubation at 37 degrees C. The oximes used were weak reactivators of inhibited AChE except for (C4H9O)(NH2)P(O)DCP (DCP, -O-2,5-dichlorphenyl group) and (C6H13O)(NH2)P(O)SCH3 where we have obtained good reactivation. Imidazole oxime BDB-106 proved to be a potent reactivator of tabun-inhibited AChE.

The effects of the oximes 2-pyridine aldoxime methiodide (PAM), HI-6, HS-6, toxogonin and TMB-4(Trimedoxime) on the rate of carbamylation of membrane-bound bovine erythrocyte acetylcholinesterase were studied. The second-order rate constant of carbamylation (ki) and the first-order rate constant of decarbamylation (k3) were calculated from the proportion of free acetylcholinesterase at equilibrium and the rate of approach to equilibrium. Twenty insecticidal carbamates plus physostigmine and pyridostigmine were studied. The oximes increased ki for several carbamates, with HI-6 causing an increase in the most number of cases (12) and PAM the least (3). HI-6 was also a potent accelerator of decarbamylation (increase in k3) in all cases, whereas PAM caused a significant decrease in k3 in 15 cases and a nonsignificant decrease in the other 7. Toxogonin and TMB-4(Trimedoxime) increased k3 or had no significant effect. The results were generally consistent with a proposal in the literature that there is a correlation between increased ki and increased toxicity of the carbamate in the presence of an oxime.

1. The dispositions of the acetylcholinesterase reactivators: 2PAM-I, TMB4 and R665, labelled with 14C on the oxime group, have been studied in normal rats and rats poisoned by the organophosphates Soman and A4. 2. For all three compounds, radioactivity was eliminated mostly in the urine (60-90% dose in 24 h). Faecal elimination was low (5.8-17.2% in 72 h). 3. All three compounds concentrated in kidney, but only 2PAM-I and R665 concentrated in liver. TMB4 and R665 concentrated in mucopolysaccharide-containing tissues such as cartilage and intervertebral disc. Other tissues were weakly and uniformly labelled. Soman poisoning does not modify the kinetic parameters of both compounds, but A4 poisoning increases 2PAM-I tissue concentration. 4. Chromatography of urine and plasma showed only unchanged 2PAM-I, TMB4 and R665 in both healthy and poisoned animals. Despite the high concentration of 2PAM-I and R665 in liver, these oximes are not metabolized.

The therapeutic and reactivating activities of a new antidote alloxime were studied as compared with dipiroxime (TMB-4(Trimedoxime)) during oral intoxication of animals by chlorophos and carbophos (LD50). When administered intramuscularly in a dose of 10 mg/kg, alloxime exhibited more pronounced therapeutic and reactivating effects (particularly in the central nervous system) as compared to dipiroxime.

Relative stability studies of three organophosphate-inhibited acetylcholinesterase reactivators, 1-(2-hydroximinomethyl-1-pyridinium)-3-(4-carbamoyl-1-pyridinium)- 2-oxapropane dichloride (HI-6), 1,1'-methylenebis(4-hydroximinomethylpyridinium) dichloride (MMB-4), and 1,1'-trimethylenebis(4-hydroximinomethylpyridinium) dibromide (TMB-4(Trimedoxime)) were carried out by semiquantitative TLC and NMR methods. TMB-4(Trimedoxime) appears to be the most, and HI-6 the least stable of the three compounds. The extent of hydrolysis of HI-6, MMB-4, and TMB-4 in 0.05 M, pH 7 phosphate buffer was approximately 50, 25, and less than 1%, respectively, after 20 d at room temperature. The hydrolysis products of HI-6 were identified by NMR and MS (electron impact) as 2-pyridinealdoxime, picolinamide, and isonicotinamide, whereas that of MMB-4 was identified as 4-pyridinealdoxime. The stability of these reactivators decreases with increasing pH. TMB-4 was stable under both neutral and basic conditions at room temperature. Deuterium exchange of the methylene protons of MMB-4 in D2O and of the protons at the 2- and 6-positions of the pyridinium ring of TMB-4 in NaOD/D2O were observed.

After passing toxicity and experimental therapeutic tests, four oxime cholinesterase reactivators [PAM (pyridine aldoxime methiodide), PAC (pralidoxime, pyridine aldoxime methylchloride), TMB4 (trimedoxime), and DMO4 (obidoxime, Toxogonin, LH6)] were compared in clinical trials. All of them proved capable of restoring erythrocyte cholinesterase activity and relieving symptoms and signs of organophosphate insecticide poisoning. Mildly and moderately poisoned patients can be treated by several injections of any one of these drugs alone, but severe cases need the synergistic action of atropine, as well as treatments for two to three consecutive days. Although response to treatment is stronger with TMB4 and DMO4, they are not recommended for routine treatment because of their dangerous adverse side effects.

HI-6 and TMB-4(Trimedoxime) were the most effective and safe of 7 cholinesterase reactivators tested as agents for the prophylaxis of proserine poisoning of male mice. The reactivator HI-6 strongly potentiated the prophylactic efficacy of a mixture of atropine and arpenal administered in the doses sufficient for the blockade of both the m- and h-cholinoreactive systems of mice. As demonstrated by experiments in vitro, HI-6 and TMB-4(Trimedoxime) did not reacivate proserine-inhibited cholinesterase. The natural anticholinesterase activity of HI-6 was negligible. Based on the correlation of the data obtained to the reported data indicating that HI-6 has a low ganglioblocking activity it is inferred that the direct effect on the receptor is of no importance for the potentiating effect. It is assumed that HI-6 modulates the cholinoreactive systems, which leads to a dramatic increase of the efficacy of cholinolytics.

The authors found that the phosphororganic pesticide neguvon in a dose of 200 mg/kg of body weight diminished the content of dopamine in the striatum of white rats. This effect was increased after daily administration of the same dose for a period of 5 days. The reactivator of cholinesterase TMB-4(Trimedoxime) and H-choline blocker tropacine prevented this effect. They discussed the connection between the changes in the content of dopamine and modulating role of presynaptic choline receptors of H- and M- types in the dopaminergic terminals of the striatum.

Sheep were studied for the possibility of treatment after parenteral (intramuscular) intoxication with EDMM (methylthiophosphorous acid O-ethyl-S-2-dimethylamino-ethylester) and with EDIM (methylthiophosphorous acid O-ethyl-S-2-diisopropyl-aminoethylester). In both cases of intoxication, the therapy was based on a system of an anticholinergic and cholinesterase reactivator administered singly at a time of the maximum development of the clinical signs of poisoning and maximum inhibition of both erythrocytic (AChE, E.C.3.1.1.7.) and plasma (BChE, E.C.3.1.1.8.) cholinesterase. The optimum therapeutic system requires the administration of 20.0 mg atropine s. c. pro toto and 10.0 mg trimedoxim per kg 1. w. i. v. In both cases of poisoning with doses = LD50 in i. m. administration, the mentioned system was actually positive. In a single administration irrespective of the doses of the used drugs, the system does not guarantee survival after ingestion of anticholinesterasic doses above LD50.

The authors found that the contractile activity of isolated myofibers as-well as superprecipitation of myosin B were reduced after intoxication of white rats with 1/2 of dose of LD50 of neguvon. The reaktivator of cholinesterase TMB-4(Trimedoxime) (20 mg/kg) recovered the contractile capability of myofibres even on the third day after treatment, but superprecipitation of myosin B-after the twentieth day.

A single application of a mixture of cholinolytic and reactivator of cholinesterase (TMB-4(Trimedoxime) compos. SPOFA) administered intravenously in the dose of 10.0 mg of trimedoxim per kg of live weight to sheep for 60 minutes after an intramuscular intoxication with O-ethyl S-(2-dimethylaminoethyl) methyl phosphonothioate (EDMM) in the dose of 0.00835 mg per kg of live weight (i.m. LD50, 2h) produces an immediate clinical effect. The reactivation of the erythrocytary acetyl cholinesterase (AChE, E.C.3.1.1.7.) examined in 15 minutes after the administration of the antidotal mixture is almost 100 p.c., the reactivation of the plasmatic butyryl cholin esterase (ChE, E.C.3.1.1.8.) approx. from 70 to 80 p. c. Restitution ad integrum occurred not later than in 14 days after the intoxication.

Per os intoxication of sheep with O-ethyl S-(2-dimethylaminoethyl)methyl phosphonium thioate (EDMM) in the dose of 0.209 mg per kg of live weight, that means p.o. LD50 (2 h), can be therapeutically mastered by means of an antidotal mixture of the reactivator of cholinesterase (trimedoxim) and of the cholinolytic (atropine), which is commercially available in the SPOFA preparation TMB-4(Trimedoxime) compos. for human purposes, if the effective dose of the reactivator was 10.0 mg per kg of live weight and if it was applied intravenously, and if the dose of the cholinolytic consisted, per 1 kg of live weight, of 0.08 mm. The antidotal mixture was applied, when the degree of the muscarinic and nicotinic symptoms and the corresponding degree of the inhibition of erythrocytary and plasmatic cholinesterase were on such a level which, according to the classification criteria, we consider a severe grade of intoxication with a dubious diagnosis.

The authors examined the changes in the nerve-muscular conductivit occuring after acute poisoning with phosdrine and treatment with arpenal and oximic reacytivators of HE-toxogonine and TMB-4(Trimedoxime) on 20 cats, anesthethised with urethane and atropine protection under the conditions of artificinal respiration. They found that there was a total nerve-muscular block cessation of the mediation in the myoneural synaptic zones, characterized by complete cessation also of the induced electrical responses after single and serial stimuli, during acute intoxications with phosdrine, used in lethal doses of 300 mg/kg intramuscularly. After treatment with H-cholinolitics (arpenal) there was a quick recovery of the muscle contractions as well as of the evoked myopotentials after stimulation with single electric stimuli. In contrast to the oximnic reactivators of He (toxogonine, TMB-4(Trimedoxime)) during serial electric stimuli (10,20 and 30 per second after treatment with arpenal there was a quick contraction only after the first stimulus, after which a progresive weakening of the induced electric myopotentials followed till the appearence of a total nerve-muscular block. This phenomenon was explained by the lack of reactivating properties of H-cholinolitics to HE, inhibited by FOV.

Studied was the activity of low concentrations --- 1-10-12, and 1.10-18 -- of the specific reactivators of the enzyme cholinesterase (ChE): 2-PAM, TMB-4(Trimedoxime) Toxogonin, HS-3, and HS-6, in a chromodacryorrhea test (ChDT) with rats, daily (in the course of 30 days -- 1 ml/100 g), twice (at an interval of 10 days -- 1 ml/100 g) and once (5 mg/kg) at subcutaneous application. Described is an activating affect (observed for the first time in vivo) on ChE preduced by low and very low concentrations of reactivators of the group of oximes. HS-3 produced best activating effects. The low concentrations of the specific oxime ractivators of ChE, followed up by ChDT, can be used as sensitive quantitative indicators of the activity of ChE. They can also serve to specify the mode of action power and term of effectiveness of these reactivators.