Loading and Sustained Release of Pralidoxime Chloride from Swellable MIL-88B(Fe) and Its Therapeutic Performance on Mice Poisoned by Neurotoxic Agents.

Maintaining a long-term continuous and stable reactivator blood concentration to treat organophosphorus nerve agent poisoning using acetylcholinesterase (AChE) reactivator pralidoxime chloride (2-PAM) is very important yet difficult. Because the flexible framework of MIL-88B(Fe) nanoparticles (NPs) can swell in polar solvents, pralidoxime chloride (2-PAM) was loaded in MIL-88B(Fe) NPs (size: ca. 500 nm) by stirring and incubation in deionized water to obtain 2-PAM@MIL-88B(Fe), which had a maximum drug loading capacity of 12.6 wt %. The as-prepared composite was characterized by IR, powder X-ray diffraction (P-XRD), scanning electron microscopy (SEM), ζ-potential, Brunauer-Emmett-Teller (BET), and thermogravimetry/differential thermal analysis (TG/DTA). The results showed that under constant conditions, the maximum drug release rates of 2-PAM@MIL-88B(Fe) in absolute ethanol, phosphate-buffered saline (PBS) solution (pH = 7.4), and PBS solution (pH = 4) at 150 h were 51.7, 80.6, and 67.1%, respectively. This was because the composite showed different swelling behaviors in different solvents. In PBS solution with pH = 2, the 2-PAM@MIL-88B(Fe) framework collapsed after 53 h and released 100% of 2-PAM. For mice after intragastric poisoning with sarin (a neurotoxic agent), an atropine-assisted 2-PAM@MIL-88B(Fe) treatment experiment revealed that 2-PAM@MIL-88B(Fe) continuously released 2-PAM for more than 72 h so that poisoned AChE was continuously and steadily reactivated. The reactivation rate of AChE was 56.7% after 72 h. This composite is expected to provide a prolonged, stable therapeutic drug for the mid- and late-stage treatment of neurotoxic agent poisoning.

Comparative physiology and efficacy of atropine and scopolamine in sarin nerve agent poisoning.

Anticholinergic treatment is key for effective medical treatment of nerve agent exposure. Atropine is included at a 2 mg intramuscular dose in so-called autoinjectors designed for self- and buddy-aid. As patient cohorts are not available, predicting and evaluating the efficacy of medical countermeasures relies on animal models. The use of atropine as a muscarinic antagonist is based on efficacy achieved in studies in a variety of species. The dose of atropine administered varies considerably across these studies. This is a complicating factor in the prediction of efficacy in the human situation, largely because atropine dosing also influences therapeutic efficacy of oximes and anticonvulsants generally part of the treatment administered. To improve translation of efficacy of dosing regimens, including pharmacokinetics and physiology provide a promising approach. In the current study, pharmacokinetics and physiological parameters obtained using EEG and ECG were assessed in naïve rats and in sarin-exposed rats for two anticholinergic drugs, atropine and scopolamine. The aim was to find a predictive parameter for therapeutic efficacy. Scopolamine and atropine showed a similar bioavailability, but brain levels reached were much higher for scopolamine. Scopolamine exhibited a dose-dependent loss of beta power in naïve animals, whereas atropine did not show any such central effect. This effect was correlated with an enhanced anticonvulsant effect of scopolamine compared to atropine. These findings show that an approach including pharmacokinetics and physiology could contribute to improved dose scaling across species and assessing the therapeutic potential of similar anticholinergic and anticonvulsant drugs against nerve agent poisoning.

Efficacy of atropine sulfate/obidoxime chloride co-formulation against sarin exposure in guinea pigs.

The efficacy and pharmacokinetics of the aqueous co-formulation contents of the Trobigard™ (atropine sulfate, obidoxime chloride) auto-injector were evaluated in a sarin exposed guinea pig model. Two subcutaneous (sc) sarin challenge doses were evaluated in guinea pigs instrumented with brain and heart electrodes for electroencephalogram (EEG) and electrocardiogram (ECG). Sarin challenge doses were chosen to reflect exposure subclasses with sublethal (moderate to severe clinical signs) and lethal consequences. The level of protection of intramuscular human equivalent doses of the co-formulation was defined by (1) the mitigation of signs and symptoms at a sublethal level and (2) the increase of survival time at the supralethal sarin dose levels. Pharmacokinetics of both atropine sulfate and obidoxime were proportional at 1 and 3 human equivalent doses, and only a small increase in heart rate was observed briefly as a side effect. At both sarin challenge doses, 54 µg/kg and 84 µg/kg, the co-formulation treatment was effective against sarin-induced effects. Survival rates were improved at both sarin challenge levels, whereas clinical signs and changes in EEG activity could not in all cases be effectively mitigated, in particular at the supralethal sarin challenge dose level. Reactivation of sarin inhibited cholinesterase was observed in blood, and higher brain cholinesterase activity levels were associated with a better clinical condition of the co-formulation treated animals. Although the results cannot be directly extrapolated to the human situation, pharmacokinetics and the effects over time related to plasma levels of therapeutics in a freely moving guinea pig could aid translational models and possibly improve prediction of efficacy in humans.

The estimation of oxime efficiency is affected by the experimental design of phosphylated acetylcholinesterase reactivation.

Reactivation of acetylcholinesterase (AChE), an essential enzyme in neurotransmission, is a key point in the treatment of acute poisoning by nerve agents and pesticides, which structurally belong to organophosphorus compounds (OP). Due to the high diversity of substituents on the phosphorous atom, there is a variety of OP-AChE conjugates deriving from AChE inhibition, and therefore not only is there no universal reactivator efficient enough for the most toxic OPs, but for some nerve agents there is still a lack of any reactivator at all. The endeavor of many chemists to find more efficient reactivators resulted in thousands of newly-designed and synthesized oximes-potential reactivators of AChE. For an evaluation of the oximés reactivation efficiency, many research groups employ a simple spectrophotometric Ellman method. Since parameters that describe reactivator efficiency are often incomparable among laboratories, we tried to emphasize the critical steps in the determination of reactivation parameters as well as in the experimental design of a reactivation assay. We highlighted the important points in evaluation of reactivation kinetic parameters with an aim to achieve better agreement and comparability between the results obtained by different laboratories and overall, a more efficient evaluation of in vitro reactivation potency.

Is it possible to reverse aged acetylcholinesterase inhibited by organophosphorus compounds? Insight from the theoretical study.

The main treatment for organophosphorus (OP) compound poisoning in clinics is to restore the activity of acetylcholinesterase (AChE) through oxime-induced reactivation of the phosphorylated OP-AChE adduct. It suffers from a competitive and irreversible aging reaction of the phosphorylated OP-AChE adduct, resulting in permanent inactivity of AChE. However, it was recently reported that N-methyl-2-methoxypyridinium species can act as methylating agents to methylate the methyl methane-phosphonate monoanion, in which the reaction mimics the reverse of the aging reaction of the phosphorylated OP-AChE adduct. If the aging reaction could be really reversed, the efficiency for the OP detoxification should be significantly improved, bringing up the possibility to develop an agent to reverse the aging process of the phosphorylated OP-AChE adduct. However, such a reaction with the N-methyl-2-methoxypyridinium species in the enzyme is still not reported so far. It is of great interest to know whether or not this reaction is observable in the enzyme, and more importantly, if it turns out to be not observable in the enzyme, why such a reaction proceeds quickly in aqueous solution but not in the enzyme. In the present study, we performed DFT calculations and quantum mechanical/molecular mechanical (QM/MM) calculations to reveal the fundamental mechanism for the methylation of both the methyl methane-phosphonate monoanion and the aged sarin-AChE adduct by N-methyl-2-methoxypyridinium species, respectively. The obtained results support the SN2 reaction mechanism, not the stepwise mechanism, for the methylation of the methyl methane-phosphonate monoanion by 9 reported N-methyl-2-methoxypyridinium compounds. The calculated free energy barriers are in good agreement with the experimental data. The methylation of the aged sarin-AChE adduct by one N-methyl-2-methoxypyridinium compound (labeled as compound 2) also employs the SN2 reaction mechanism with an extremely high free energy barrier of 30.4 ± 3.5 (or 26.6) kcal mol(-1), implying that this reaction in the enzyme hardly occurs. Our results clearly show that compound 2 forms a strong π-π stacking interaction with the aromatic ring of the W86 residue of AChE, making itself unable to approach sarin for the reverse of the aging process. On the basis of the structure and mechanism, several possible strategies have been suggested for designing methylating agents with higher activity against the aged sarin-AChE adduct.

A comparison of the reactivating and therapeutic efficacy of two novel bispyridinium oximes (K727, K733) with the oxime HI-6 and obidoxime in sarin-poisoned rats and mice.

The ability of two novel bispyridinium oximes K727 and K733 and currently available oximes (HI-6, obidoxime) to reactivate sarin-inhibited acetylcholinesterase and to reduce acute toxicity of sarin was evaluated. To investigate the reactivating efficacy of the oximes, the rats were administered intramuscularly with atropine and oximes in equitoxic doses corresponding to 5% of their LD50 values at 1 min after the intramuscular administration of sarin at a dose of 24 µg/kg (LD50). The activity of acetylcholinesterase was measured at 60 min after sarin poisoning. The LD50 value of sarin in non-treated and treated mice was assessed using probit-logarithmical analysis of death occurring within 24 h after intramuscular administration of sarin at five different doses. In vivo determined percentage of reactivation of sarin-inhibited rat blood, diaphragm and brain acetylcholinesterase showed that the potency of both novel oximes K727 and K733 to reactivate sarin-inhibited acetylcholinesterase roughly corresponds to the reactivating efficacy of obidoxime. On the other hand, the oxime HI-6 was found to be the most efficient reactivator of sarin-inhibited acetylcholinesterase. While the oxime HI-6 was able to reduce the acute toxicity of sarin >3 times, both novel oximes and obidoxime decreased the acute toxicity of sarin <2 times. Based on the results, we can conclude that the reactivating and therapeutic efficacy of both novel oximes K727 and K733 is significantly lower compared to the oxime HI-6 and, therefore, they are not suitable for the replacement of the oxime HI-6 for the antidotal treatment of acute sarin poisoning.

Exploring the physicochemical properties of oxime-reactivation therapeutics for cyclosarin, sarin, tabun, and VX inactivated acetylcholinesterase.

The inactivation of acetylcholinesterase (AChE) by organophosphorus agent (OP) compounds is a serious problem regardless of how the individual was exposed. The reactivation of OP-inactivated AChE is dependent on the OP conjugate, and commonly a specific oxime is better at reactivating a specific OP conjugate than several diverse OP conjugates. The presented research explores the physicochemical properties needed for the reactivation of OP-inactivated AChE. Four different OPs, cyclosarin, sarin, tabun, and VX, were analyzed using the same set of oxime reactivators. A trial descriptor pool of semiempirical, traditional, and molecular interaction field descriptors was used to construct an ensemble of QSAR models for each OP-conjugate pair. Based on the molecular information and the cross-validation ability, individual QSAR models were selected to be part of an OP-conjugate consensus model. The OP-conjugate specific models provide important insight into the physicochemical properties required to reactivate the OP conjugates of interest. The reactivation of AChE inactivated with either cyclosarin or tabun requires the oxime therapeutic to possess an overall polar-positive surface area. Oxime therapeutics for the reactivation of sarin-inactivated AChE are conformationally dependent while oxime reverse therapeutics for VX require a compact region with a highly hydrophilic region and two positively charged pyridine rings.

A comparison of the potency of a novel bispyridinium oxime K203 and currently available oximes (obidoxime, HI-6) to counteract the acute neurotoxicity of sarin in rats.

The neuroprotective effects of a newly developed oxime K203 and currently available oximes (obidoxime, HI-6) in combination with atropine in rats poisoned with sarin were studied. The sarin-induced neurotoxicity was monitored using a functional observatory battery at 2 hr after sarin challenge. The results indicate that the potency of a novel bispyridinium oxime K203 to counteract sarin-induced neurotoxicity is relatively low and roughly corresponds to the neuroprotective efficacy of obidoxime. Among tested oximes, the oxime HI-6 seems to be significanlty more efficacious to counteract acute neurotoxicity of sarin than commonly used obidoxime and a newly developed oxime K203. Thus, the oxime K203 does not provide any beneficial effect for the antidotal treatment of acute poisoning with sarin in comparison with the oxime HI-6 that should be considered to be the best oxime for antidotal treatment of acute sarin poisonings.

Reactivation kinetics of a homologous series of bispyridinium bis-oximes with nerve agent-inhibited human acetylcholinesterase.

The reactivation of organophosphorus compound (OP)-inhibited acetylcholinesterase (AChE) by oximes is inadequate in case of different OP nerve agents. This fact led to the synthesis of numerous novel oximes by different research groups in order to identify more effective reactivators. In the present study, we investigated the reactivation kinetics of a homologous series of bispyridinium bis-oximes bearing a (E)-but-2-ene linker with tabun-, sarin-, and cyclosarin-inhibited human AChE. In part, marked differences in affinity and reactivity of the investigated oximes toward OP-inhibited human AChE were recorded. These properties depended on the position of the oxime groups and the inhibitor. None of the tested oximes was equally effective against all used OPs. In addition, the data indicate that a (E)-but-2-ene linker decreased in most cases the reactivating potency in comparison to oximes bearing an oxybismethylene linker, e.g., obidoxime and HI-6. The results of this study give further insight into structural requirements for oxime reactivators, underline the necessity to investigate the kinetic interactions of oximes and AChE with structurally different OP inhibitors, and point to the difficulty to develop an oxime reactivator which is efficient against a broad spectrum of OPs.

Comparative protective effects of HI-6 and MMB-4 against organophosphorous nerve agent poisoning.

The oximes pralidoxime (2-PAM), its dimethanesulphonate salt derivative P2S, and obidoxime (toxogonin) are currently licensed and fielded for the treatment of chemical warfare (CW) organophosphorous (OP) nerve agent poisoning. While they are effective against several of the identified threat CW OP agents, they have little efficacy against others such as soman (GD) and cyclosarin (CF). In addition, they are also significantly less effective than other investigational oximes against the nerve agent known as Russian VX (RVX). Among the oximes currently being investigated, two in particular, HI-6 (asoxime) and MMB-4 (ICD-039, methoxime) have been proposed as replacement therapies for the currently licensed oximes. HI-6 has been safely used in individuals to treat OP insecticide poisoning, as well as in human volunteers, although its efficacy against OP nerve agent poisoning in humans cannot be demonstrated due to ethical considerations. It is currently available for use in defined military settings in Canada, Sweden and the Czech Republic, and is also under development in a number of other countries. The oxime MMB-4 has not yet been studied clinically, but is fielded by the Czech Republic, and is being developed by the United States armed services as a replacement for the currently fielded 2-PAM. This review compares the effectiveness of HI-6 and MMB-4 against nerve agent threats where comparisons can be made. HI-6 has been demonstrated to be generally a superior reactivator of nerve agent inhibited enzyme, particularly with human and non-human primate derived enzyme, and has also shown better protective effects against the lethality of most OP agents in a variety of species. Both compounds appear to be clearly superior to the available oximes, obidoxime and 2-PAM.

In vitro reactivation of sarin-inhibited human acetylcholinesterase (AChE) by bis-pyridinium oximes connected by xylene linkers.

A series of bis-pyridinium oximes connected by xylene linkers were synthesized and their in vitro reactivation potential was evaluated against human acetylcholinesterase (hAChE) inhibited by nerve agent sarin and the data were compared with 2-PAM and obidoxime. Among the synthesized compounds, N,N'-p-xylene-bis-[(2,2'-hydroxyiminomethyl)pyridinium] dibromide (3c) was found to be the most potent reactivator for hAChE inhibited by sarin. The oxime 3c exhibited 45% regeneration of inhibited hAChE, in comparison to 34% and 24% regeneration by 2-PAM and obidoxime, respectively, at a concentration of 10(-3) M within 10 min. The higher reactivation efficacies of these oximes were attributed to their acid dissociation constants (pKa). The pKa values of all the oximes were determined spectrophotometrically and correlated with their observed reactivation potential. This method involving the in vitro reactivation of inhibited hAChE may be useful for the screening of new oximes as reactivators.

Molecular and cellular actions of galantamine: clinical implications for treatment of organophosphorus poisoning.

There have been continued efforts to develop effective antidotal therapies against poisoning with organophosphorus (OP) compounds, including nerve agents and pesticides. We reported recently that galantamine, a drug used to treat Alzheimer's disease, administered before (up to 3 h) or soon after (up to 5 min) an exposure of guinea pigs to 1.5-2 x LD50 soman or sarin effectively counteracted the acute toxicity and lethality of the nerve agents provided that the animals were also post-treated with atropine. Here, we demonstrate that administered to guinea pigs at 30 min before or up to 15 min after an acute challenge with 1 x LD50 soman, galantamine (8 mg/kg, intramuscular) alone is sufficient to counteract the lethality and acute toxicity of the nerve agent. Evidence is also provided that 100% survival can be attained when the association of appropriate doses of galantamine and atropine is administered 30-45 min after the challenge of the guinea pigs with 1 x LD50 soman. Galantamine counteracts the neurodegeneration and the changes in the nicotinic cholinergic system that result from an acute exposure of guinea pigs to 1 x LD50 soman. The results presented herein corroborate that galantamine is an effective antidote against OP poisoning.

Targeting cholinesterase inhibitor poisoning with a novel blocker against both nicotinic and muscarinic receptors.

Clinicians have been treating poisoning by acetylcholinesterase inhibitors (ChEI) for more than half a century. However, the current atropine-centered therapy still cannot protect completely against all ChEIs, and poisoning by ChEIs is fatal in more than 20% of cases. Various solutions that try to enhance atropine's antimuscarinic effects have been used, but these fail to increase the antidotal effect, and their too potent muscarinic antagonism may produce incapacitating side effects. We hypothesized that, in the treatment of ChEI poisoning, the high death rate may not be attributed to the insufficient muscarinic antagonism but to the lack of nicotinic antagonism. To test this hypothesis, we designed and synthesized benthiactzine, a drug that blocks both muscarinic acetylcholine receptors (mAChRs) and nicotinic acetylcholine receptors (nAChRs). A specific [(3)H]quinuclidinyl benzilate-binding assay showed that benthiactzine was much weaker than atropine in binding to five different mAChR subtypes or to mAChRs expressed in 14 different tissues. Electrophysiological measures were used to identify and characterize benthiactzine's antinicotinic effect on three typical neuronal nAChRs subtypes, alpha4beta2, alpha4beta4, and alpha7, which are expressed heterogenously in SH-EP1 cells. Finally, benthiactzine afforded better protection than atropine against the most lethal ChEI, VX or sarin, in a mouse model. These results indicate that the antidotal effect may not be directly related to the antidote's antimuscarinic effect and that the antinicotinic effect may provide additional protection against ChEI poisoning. This new drug may benefit future antidote discovery.

[Immunostimulant properties of polyoxidon on the model of acute poisoning with anticholinesterase chemicals in rats].

Experiments on noninbred rats showed that, upon acute poisoning with toxic chemicals (sarin, VX agent; 1.0 LD50) and the treatment with atropine (10 mg/kg), the administration of polyoxidon in a daily dose of 100 mg/kg over 4 days partly reduces the degree of immune system suppression and the level of lipid peroxidation induced by toxic chemicals.

A comparison of the potency of newly developed oximes (K005, K027, K033, K048) and currently used oximes (pralidoxime, obidoxime, HI-6) to reactivate sarin-inhibited rat brain acetylcholinesterase by in vitro methods.

The potency of newly developed and currently used oximes to reactivate sarin-inhibited acetylcholinesterase was evaluated using in vitro methods. A rat brain homogenate was used as a source of acetylcholinesterase. Significant differences in reactivation potency among all tested oximes were observed. Although the ability of newly developed oximes to reactivate sarin-inhibited acetylcholinesterase does not reach the reactivating potency of the oxime HI-6, the oxime K033 seems to be a more efficacious reactivator of sarin-inhibited acetylcholinesterase than other currently available oximes (pralidoxime, obidoxime) at concentrations (10(-5)-10(-4)M) corresponding to recommended doses in vivo. The results of our study also confirm that the reactivation potency of the tested reactivators depends on many factors, such as (1) the number of pyridinium rings, (2) the number of oxime groups and their position, and (3) the length and the shape of the linkage bridge between pyridinium rings.

Partial adenosine A(1) receptor agonists inhibit sarin-induced epileptiform activity in the hippocampal slice.

Organophosphate poisoning can result in seizures and subsequent neuropathology. One possible therapeutic approach would be to employ adenosine A(1) receptor agonists, which have already been shown to have protective effects against organophosphate poisoning. Using an in vitro model of organophosphate-induced seizures, we have investigated the ability of several adenosine A(1) receptor agonists to inhibit epileptiform activity induced by the organophosphate sarin, in the CA1 stratum pyramidale of the guinea pig hippocampal slice. Application of the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) or the partial adenosine A(1) receptor agonists 2-deoxy-N(6)-cyclopentyladenosine (2-deoxy-CPA) and 8-butylamino-N(6)-cyclopentyladenosine (8-butylamino-CPA) abolished epileptiform activity in a concentration-related manner. The rank order of potency was CPA (IC(50) 4-5 nM) >2-deoxy-CPA (IC(50) 113-119 nM)=8-butylamino-CPA (IC(50) 90-115 nM). These data suggest that partial adenosine A(1) receptor agonists, which have fewer cardiovascular effects, should be further evaluated in vivo as potential treatments for organophosphate poisoning.

Comparative efficacy of diazepam and avizafone against sarin-induced neuropathology and respiratory failure in guinea pigs: influence of atropine dose.

This investigation compared the efficacy of diazepam and the water-soluble prodiazepam-avizafone-in sarin poisoning therapy. Guinea pigs, pretreated with pyridostigmine 0.1 mg/kg, were intoxicated with 4LD(50) of sarin (s.c. route) and 1 min after intoxication treated by intramuscular injection of atropine (3 or 33.8 mg/kg), pralidoxime (32 mg/kg) and either diazepam (2 mg/kg) or avizafone (3.5 mg/kg). EEG and pneumo-physiological parameters were simultaneously recorded. When atropine was administered at a dose of 3 mg/kg, seizures were observed in 87.5% of the cases; if an anticonvulsant was added (diazepam (2 mg/kg) or avizafone (3.5 mg/kg)), seizure was prevented but respiratory disorders were observed. At 33.8 mg/kg, atropine markedly increased the seizure threshold and prevented early respiratory distress induced by sarin. When diazepam was administered together with atropine, seizures were not observed but 62.5% of the animals displayed respiratory difficulties. These symptoms were not observed when using avizafone. The pharmacokinetic data showed marked variation of the plasma levels of atropine and diazepam in different antidote combination groups, where groups receiving diazepam exhibited the lowest concentration of atropine in plasma. Taken together, the results indicate that avizafone is suitable in therapy against sarin when an anticonvulsant is judged necessary.

Assessment of a combination of physostigmine and scopolamine as pretreatment against the behavioural effects of organophosphates in the common marmoset (Callithrix jacchus).

RATIONALE: There is a requirement to ensure that UK armed forces are provided with the best possible medical countermeasures to prevent or mitigate the effects of exposure to nerve agents. When pretreatments are under consideration, it is of particular importance to ensure that they do not in themselves give rise to adverse effects and do not exacerbate the effects of agent exposure. OBJECTIVES: The present study was designed to address these considerations for a combination of physostigmine and scopolamine as a potential pretreatment regimen. METHODS: Common marmosets were trained to perform a two-choice discrimination serial reversal task, and baseline data were collected. Subjects received a dose of either soman or sarin after 2 weeks of pretreatment with either saline or physostigmine and scopolamine via miniosmotic pump. RESULTS: No effects of physostigmine and scopolamine were seen on task accuracy or response rates. Neither accuracy of reversal performance nor number of responses made were significantly changed by administration of either soman or sarin subsequent to pretreatment with physostigmine/scopolamine. In the groups pretreated with saline, performance of the behavioural task, in terms of responses made, was virtually abolished on the day the OP was administered, but a significant increase in accuracy of performance was seen over the 2- to 14-day period following administration. CONCLUSIONS: A combination of physostigmine and scopolamine, which is known to protect against nerve-agent lethality, offers protection against the effects of soman and sarin on behavioural performance, as measured by a discrimination reversal task. The improved performance observed following nerve agent requires further investigation.

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.