Tabun-poisoned Mice Research Articles

In vivo evaluation of cholinesterase activity, oxidative stress markers, cyto- and genotoxicity of K048 oxime–a promising antidote against organophosphate poisoning.

K048 is a member of K-oximes, a new oxime class that has recently been confirmed effective against poisoning by the nerve agent tabun and several pesticides. The toxicity profile of the K048 oxime has not been fully characterized and its optimal therapeutic dose has not yet been established. Earlier studies report excellent results with K048 in reactivating tabun-phosphorylated AChE and in the therapy of tabun-poisoned mice. It possesses a low acute toxicity and exerts an acceptable toxicity profile on isolated human peripheral blood lymphocytes in vitro. Intraperitoneal administration of K048 in rats resulted in an LD50 of 238.3 mg/kg. In this in vivo study, we investigated cholinesterase (ChE) activity and oxidative stress marker levels (lipid peroxidation and superoxide dismutase activity) in the plasma of exposed rats after administering the compound at 25% of its LD50. Lymphocyte viability was evaluated using an acridine orange/ethidium bromide in situ fluorescent assay. The levels of primary DNA damage in rat white blood cells were measured using the alkaline comet assay. The compound applied at 25% of its LD50 did not significantly affect ChE activity and lipid peroxidation and did not cause significant changes in the SOD activity in plasma. The cytotoxicity profile of K048 in the tested dose was also acceptable, and it did not possess significant DNA-damaging potential. The obtained results are promising for further evaluations of the K048 oxime, which should include tests on a broader concentration range and longer incubation times.

The evaluation of the reactivating and therapeutic efficacy of three novel bispyridinium oximes (K454, K456, K458) in comparison with the oxime K203 and trimedoxime in tabun-poisoned rats and mice

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.

A comparison of the reactivating and therapeutic efficacy of newly developed oximes (K347, K628) with commonly used oximes (obidoxime, HI-6) against tabun in rats and mice

The potency of newly developed reactivators of nerve agent–inhibited acetylcholinesterase (K347, K628) in reactivating tabun-inhibited acetylcholinesterase and reducing tabun-induced lethal toxic effects was compared with currently available oximes (obidoxime, the oxime HI-6), using in vivo methods. Studies that determined the percentage of reactivation of tabun-inhibited blood and tissue acetycholinesterase in poisoned rats showed that the reactivating efficacy of both newly developed oximes is comparable with the oxime HI-6, but it is significantly lower than the reactivating effects of obidoxime. The monopyridinium oxime, K347, was also found to be able to reduce lethal toxic effects in tabun-poisoned mice, while the therapeutic efficacy of another newly developed bispyridinium oxime, K628, was negligible. The therapeutic efficacy of K347 was higher than the potency of the oxime, HI-6, but it was lower than the therapeutic effects of obidoxime. Thus, the reactivating and therapeutic potency of both newly developed oximes (K347, K628) was not more effective then currently available oximes, and therefore, they are not suitable for the replacement of commonly used oximes (especially obidoxime) for the treatment of acute tabun poisoning.

In vivo experimental approach to treatment against tabun poisoning

Organophosphorus compounds pose a potential threat to both military and civilian populations. Since post-exposure therapy has its limitations, our research was focused on the possibility of improving pretreatment in order to limit the toxic effects of tabun. We determined the protective index of various combinations of atropine, oximes (K074, K048, and TMB-4), and pyridostigmine given to mice before tabun intoxication. Although the tested oximes showed very good therapeutic efficacy in tabun-poisoned mice, the given pretreatments improved therapy against tabun poisoning. These regimens ensured survival of all animals up to 25.2 LD50 of tabun. Our results indicate that even pretreatment with atropine alone is sufficiently effective in enhancing the survival of mice poisoned by multiple doses of tabun, if oxime therapy follows. K048 is our oxime of choice for future research, as it shows better protective and reactivating potency.

The antidotal efficacy of the bispyridinium oximes K027 and TMB-4 against tabun poisoning in mice

A toxic effect of highly toxic nervous agents is irreversible inhibition of vitally important enzyme acethylcholinesterase (AChE). Inhibition of AChE results in accumulation of acetylcholine (ACh) at the synaptic cleft of the cholinergic neurons, leading to overstimulation of cholinergic receptors. The highly toxic nature of tabun has been known for many years, but there are still serious limitations to the antidotal therapy. In this paper a bispyridinium compound K027 [1-(4-hydroxyiminomethylpyridinium)-3-(-4-carbamoylpyridinium) propane dibromide] was tested as potential antidote in tabun poisoned mice. Oxime TMB-4 was included for comparison. The therapeutic efficacy of applied antidotal regimens was tested as pretreatment given 15 min before tabun poisoning and/or as therapy given 1 min after tabun poisoning. Using oxime K027 (25% of its LD 50) plus atropine as both, pretreatment and therapy, we showed that this combination can protect mice 8 times better than the therapy alone. Under these experimental conditions we confirmed good antidotal efficacy of K027. Moreover, its low acute toxicity is as much as beneficial effect in contrast to high toxicity of currently used TMB-4.

The influence of combinations of oximes on the reactivating and therapeutic efficacy of antidotal treatment of tabun poisoning in rats and mice

The influence of the combination of oximes on the reactivating and therapeutic efficacy of antidotal treament of acute tabun poisoning was evaluated. The ability of two combinations of oximes (HI-6 + obidoxime and HI-6 + K203) to reactivate tabun-inhibited acetylcholinesterase and reduce acute toxicity of tabun was compared with the reactivating and therapeutic efficacy of antidotal treatment involving single oxime (HI-6, obidoxime, K203) 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 both combinations of oximes is higher than the reactivating efficacy of the most effective individual oxime in blood and diaphragm and comparable with the reactivating effects of the most effective individual oxime in brain. Moreover, both combinations of oximes were found to be slightly more efficacious in the reduction of acute lethal toxic effects in tabun-poisoned mice than the antidotal treatment involving individual oxime. A comparison of reactivating and therapeutic efficacy of individual oximes showed that the newly developed oxime K203 is slightly more effective than commonly used obidoxime and both of them are markedly more effective than the oxime HI-6. Based on the obtained data, we can conclude that the antidotal treatment involving chosen combinations of oximes brings beneficial effects for the potency of antidotal treatment to reactivate tabun-inhibited acetylcholinesterase in rats and to reduce acute toxicity of tabun in mice.

A comparison of the reactivating and therapeutic efficacy of newly developed bispyridinium oximes (K250, K251) with commonly used oximes against tabun in rats and mice

The potency of newly developed bispyridinium compounds (K250, K251) in reactivating tabun-inhibited acetylcholinesterase and reducing tabun-induced lethal toxic effects was compared with currently available oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies determined percentage of reactivation of tabun-inhibited blood and tissue AChE in poisoned rats and showed that the reactivating efficacy of both newly developed oximes is comparable with the oxime HI-6 but it is significantly lower than the reactivating effects of obidoxime and trimedoxime, especially in diaphragm and brain. Both newly developed oximes were also found to be able to slightly reduce lethal toxic effects in tabun-poisoned mice. Their therapeutic efficacy is higher than the potency of the oxime HI-6 but it is lower than the therapeutic effects of trimedoxime and obidoxime. Thus, the reactivating and therapeutic potency of both newly developed oximes (K250, K251) does not prevail over the effectiveness of currently available oximes and, therefore, they are not suitable for their replacement for the treatment of acute tabun poisoning.

Evaluation of Oxime K203 as Antidote in Tabun Poisoning

We studied bispyridinium oxime K203 [(E)-1-(4-carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide] with tabun-inhibited human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in vitro, and its antidotal effect on tabun-poisoned mice and rats in vivo. We compared it with oximes K048 and TMB-4, which have proven the most efficient oxime antidotes in tabun poisoning by now. Tabun-inhibited AChE was completely reactivated by K203, with the overall reactivation rate constant of 1806 L mol(-1) min(-1). This means that K203 is a very potent reactivator of tabun-inhibited AChE. In addition, K203 reversibly inhibited AChE (Ki = 0.090 mmol L(-1)) and BChE (K(i) = 0.91 mmol L(-1)), and exhibited its protective effect against phosphorylation of AChE by tabun in vitro. In vivo, a quarter of the LD50 K203 dose insured survival of all mice after the application of as many as 8 LD50 doses of tabun, which is the highest dosage obtained compared to K048 and TMB-4. Moreover, K203 showed high therapeutic potency in tabun-poisoned rats, preserving cholinesterase activity in rat plasma up to 60 min after poisoning. This therapeutic improvement obtained by K203 in tabun-poisoning places this oxime in the spotlight for further development.

New bispyridinium oximes: In vitro and in vivo evaluation of their biological efficiency in soman and tabun poisoning

Improving the efficacy of antidotal treatment of poisonings with nerve agents is still a challenge for the scientific community. This study investigated the interactions of four bispyridinium oximes with human erythrocyte acetylcholinesterase (AChE) and their effects on soman- and tabun-poisoned mice. Oximes HI-6 and TMB-4 were used for comparison. These oximes inhibited AchE with inhibitory potency (IC 50) ranging from 0.02 to 1.0 mM. The best reactivating potency (%R) was obtained with K074, when AChE was inhibited by tabun. The protective potency (P 50) of all oximes in human erythrocyte AChE inhibited by soman and tabun could not be determined. In tabun-poisoned mice very good antidotal efficacy was obtained with K027, K048, and K074, which makes them interesting for future investigation. The combination of HI-6 and atropine is the therapy of choice for soman poisoning.

Oximes: Reactivators of phosphorylated acetylcholinesterase and antidotes in therapy against tabun poisoning

One of the therapeutic approaches to organophosphate poisoning is to reactivate AChE with site-directed nucleophiles such as oximes. However, pyridinium oximes 2-PAM, HI-6, TMB-4 and obidoxime, found as the most effective reactivators, have limiting reactivating potency in tabun poisoning. We tested oximes varying in the type of ring (pyridinium and/or imidazolium), the length and type of the linker between rings, and in the position of the oxime group on the ring to find more effective oximes to reactivate tabun-inhibited human erythrocyte AChE. Three of our tested pyridinium oximes K027, K048, K074, along with TMB-4, were the most promising for AChE reactivation. Promising oximes were further tested in vivo on tabun poisoned mice not only as antidotes in combination with atropine but also as pretreatment drug. Herein, we showed that a promising treatment in tabun poisoning by selected oximes and atropine could be improved if oximes are also used in pretreatment. Since the reactivating efficacy of the oximes in vitro corresponded to their therapeutic efficacy in vivo, it seems that pharmacological effect of these oximes is indeed primarily related to the reactivation of tabun-phosphorylated AChE.

A Comparison of the Therapeutic and Reactivating Efficacy of Newly Developed Oximes (K117, K127) and Currently Available Oximes (Obidoxime, Trimedoxime, HI-6) in Tabun-Poisoned Rats and Mice

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.

A comparison of the therapeutic and reactivating efficacy of newly developed bispyridinium compounds (K206, K269) with currently available oximes against tabun in rats and mice

The potency of newly developed bispyridinium compounds (K206, K269) in reactivating tabun-inhibited acetylcholinesterase and eliminating tabun-induced lethal toxic effects was compared with commonly used oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies which determined percentage of reactivation of tabun-inhibited blood and tissue AChE in poisoned rats showed that the reactivating efficacy of both newly developed oximes is comparable with obidoxime and trimedoxime in blood but lower than the reactivating potency of trimedoxime and obidoxime in the diaphragm and brain. Nevertheless, the differences in reactivating efficacy of obidoxime, trimedoxime and K206 was not significant while the potency of K269 to reactivate tabun-inhibited acetylcholinesterase was significantly lower. Both newly developed oximes were also found to be relatively efficacious in elimination of the lethal toxic effects in tabun-poisoned mice. Their therapeutic efficacy corresponds to the therapeutic potency of obidoxime. The oxime HI-6, relatively efficacious against soman, did not seem to be an adequately effective oxime in reactivation of tabun-inhibited AChE and to counteract lethal effects of tabun. Both newly developed oximes (K206, K269) are significantly more efficacious in reactivating tabun-inhibited AChE in rats and to eliminate lethal toxic effects of tabun in mice than the oxime HI-6 but their reactivating and therapeutic potency 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.

An evaluation of therapeutic and reactivating effects of newly developed oximes (K156, K203) and commonly used oximes (obidoxime, trimedoxime, HI-6) in tabun-poisoned rats and mice

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.

Pretreatment with pyridinium oximes improves antidotal therapy against tabun poisoning

Oximes K033 [1,4-bis(2-hydroxyiminomethylpyridinium) butane dibromide] and K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide] were tested as pretreatment drugs in tabun-poisoned mice followed by treatment with atropine plus K033, K048, K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], TMB-4 [1,3-bis(4-hydroxyiminomethylpyridinium) propane dibromide] and HI-6 [(1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-oxapropane dichloride)]. Oxime doses of 25% or 5% of its LD 50 were used for pretreatment 15 min before tabun-poisoning and for treatment 1 min after tabun administration to mice. The best therapeutic effect was obtained when oxime K048 (25% of its LD 50) was used in both pretreatment and treatment with atropine. This regiment insured survival of all tested animals after the application of 10 LD 50 of tabun. In addition, since butyrylcholinesterase (BChE; EC 3.1.1.8) is considered an endogenous bioscavenger of anticholinesterase compounds and its interactions with oximes could be masked by AChE interactions, we evaluated kinetic parameters for interactions of tested oximes with native and tabun-inhibited human plasma BChE and compared them with results obtained previously for human erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7). Progressive inhibition of BChE by tabun was slightly faster than that of AChE. The reactivation of tabun-inhibited BChE by oximes was very slow, and BChE binding affinity for oximes was lower than AChE's. Therefore, BChE could scavenge tabun prior to AChE inhibition, but fast oxime-assisted reactivation of tabun-inhibited AChE or protection of AChE by oxime against inhibition with tabun would not be obstructed by interaction between BChE and oximes.

Effect of Methoxime Combined with Anticholinergic, Anticonvulsant or Anti-HCN Drugs in Tabun-Poisoned Mice

The effect of methoxime combined with a) atropine, b) benactyzine, c) atropine and natrium thiosulphate, d) atropine and diazepam on antidotal treatment effectiveness was studied in tabun-poisoned mice. In addition, the influence of pretreatment consisiting of pyridostigmine, benactyzine and trihexyphenidyle (PANPAL) administered 2 hours before tabun intoxication on the treatment effectivity of methoxime combined with e) atropine or f) benactyzine was tested. The most efficacious therapeutic mixture in non-pretreated mice was methoxime, atropine and diazepam. Natrium thiosulphate did not significantly increase neither decrease the antidotal treatment efficacy in comparison with methoxime and atropine alone. Pretreatment with PANPAL significantly decreased tabun toxicity (nearly 4 times in methoxime and benactyzine combination and more than 4 times in atropine and methoxime mixture). The present study demonstrates that the tabun toxicity in mice is more effectively reduced when PANPAL prophylactically is administered than in case of treatment with methoxime and cholinergic drug alone. We established that anticholinergic drug option in the therapeutic mixture of methoxime and anticholinergic drug did not cause the difference in the antidotal treatment effectivities.

P2H232 - Dietary treatment with N-propyl gallate (nPG) enhances the survival of mice exposed to phosgene

Improving the efficacy of antidotal treatment of poisonings with nerve agents is still a challenge for the scientific community. This study investigated the interactions of four bispyridinium oximes with human erythrocyte acetylcholinesterase (AChE) and their effects on soman- and tabun-poisoned mice. Oximes HI-6 and TMB-4 were used for comparison. These oximes inhibited AchE with inhibitory potency (IC50) ranging from 0.02 to 1.0 mM. The best reactivating potency (%R) was obtained with K074, when AChE was inhibited by tabun. The protective potency (P50) of all oximes in human erythrocyte AChE inhibited by soman and tabun could not be determined. In tabun-poisoned mice very good antidotal efficacy was obtained with K027, K048, and K074, which makes them interesting for future investigation. The combination of HI-6 and atropine is the therapy of choice for soman poisoning.