Oxime-Mediated Reactivation of Organophosphate-Inhibited Acetylcholinesterase with Emphasis on Centrally-Active Oximes

Abstract

This review provides an overview of the global research leading to the large number of compounds developed as reactivators of acetylcholinesterase inhibited by a variety of organophosphate compounds, most of which are nerve agents but also some insecticides. A number of these organophosphates are highly toxic and effective therapy by reactivators contributes to saving lives. Two major challenges for more effective therapy with reactivators are identification of a broad spectrum reactivator efficacious against a variety of organophosphate structures, and a reactivator that can cross the blood-brain barrier to protect the brain. The most effective of the reactivators developed are the nucleophilic pyridinium oximes, which bear a permanent positive charge from the quaternary nitrogen in the pyridinium ring. The permanent positive charge retards the oximes from crossing the blood-brain barrier and therefore restoration of normal cholinergic function in the brain is unlikely. A number of laboratories have developed nucleophiles, mostly oximes, that are theorized to cross the blood-brain barrier by several strategies. At the present time, no reactivator is optimally broad spectrum across the wide group of organophosphate chemistries. Some oximes, including the substituted phenoxyalkyl pyridinium oximes invented by our laboratories, have the potential to provide neuroprotection in the brain and show evidence of efficacy against both nerve agent and insecticidal chemistries, so these novel oximes have promise for future development.

1. INTRODUCTION

The most toxic of the organophosphorus compounds (OPs) are potent and persistent anticholinesterases and inhibit the neural target enzyme acetylcholinesterase (AChE) at very low concentrations. Cholinergic pathways are widely distributed in both the peripheral and the central nervous systems in vertebrates, and they serve many functions that are essential for life or normal physiological function. AChE is of critical importance in nervous system function of both vertebrates and invertebrates because of its high turnover rate, thereby allowing only a short time of action (i.e., msec) for the neurotransmitter acetylcholine (Taylor, 2002). The OP anticholinesterases were developed initially as insecticides and subsequently during World War II as chemical warfare agents (Hersh, 1968; Tucker, 2006). The inhibition of AChE allows hypercholinergic activity to occur throughout the nervous system, with subsequent derangement of many normal physiological functions, some of which derangements can translate into life-threatening signs of poisoning. Numerous toxic responses occur in the cholinergic syndrome such as tremors, nausea, salivation, lacrimation, urination, emesis, defecation, contraction of the iris, convulsions, respiratory distress and ultimately respiratory shut-down in the case of a fatal poisoning. Respiratory failure occurs largely from peripheral nervous system hypercholinergic activity: bronchiolar constriction, excess bronchiolar secretion and spasm of the respiratory muscles, but also contributing is inhibition of the respiratory rhythm set by the brain’s respiratory control center (Watson et al., 2009).

In addition to the effects on respiratory rhythm, hypercholinergic activity in the brain also leads to over-stimulation of postsynaptic nicotinic and muscarinic receptors and seizures which, if not stopped, elicit excitatory amino acid release followed by excessive calcium influx and stimulation of N-methyl-D-aspartate (NMDA) receptors resulting in neuropathology (Shih et al., 2003; McDonough and Shih, 1997).

Examinations of OP victims, who avoided respiratory failure, suggest that this brain damage can result in long-term behavioral and cognitive deficits. For example, sarin manufactured and released by a Japanese cult in 1994 and 1995 resulted in 19 deaths and 6,100 exposures. Up to five years later, EEG abnormalities were still present and many of the victims experienced post-traumatic stress disorder (PTSD) (Yanagisawa et al., 2006). Although they received a lower dose than the victims, first responders at the incident were found to have poorer memory function than nonexposed first responders when tested 2 to 4 years later (Nishiwaki et al., 2001).

At the end of the first Gulf War in March 1991, an ammunitions dump was exploded in Khamisiyah, Iraq. In 1996 the dump was found to have contained the OPs sarin and cyclosarin. Cognitive (Proctor et al., 2006) and structural brain changes (Heaton et al., 2007) were found in veterans with predicted exposure to the Khamisiyah plume compared to unexposed veterans. Using structural magnetic resonance imaging Chao et al. (2010) found that veterans exposed to the Khamisiyah chemicals had reduced grey matter, white matter and hippocampal volumes and poorer performance on neurobehavioral tests compared to matched, nonexposed veterans. Later work showed predicted Khamisiyah exposure to be associated with hippocampal structural changes including volume changes in the CA2, CA3, and dentate gyrus (Chao et al., 2014, 2017).

OPs, used as nerve agents or some insecticides, are among the most toxic synthetic chemicals in military applications and in commercial agricultural use. The OPs certainly have terrorist implications as well, evidenced by the poisoning with sarin in the Japanese subway system in 1995 (Yanagisawa et al., 2006), the assassination with VX of Kim Jun Un in a Kuala Lampur airport (Swenson, 2017; Chai et al., 2017) and an attempted assassination of a former British spy and his daughter with a Novichok agent (Hay, 2018). The use of sarin against citizens in Syria resulted in many deaths including children (Loveluck, 2017; van der Schans et al., 2018). It has been estimated that about 300,000 poisonings occur annually from OP agents, largely from suicides in developing countries (Pope and Brimijoin, 2018; Eddleston, 2019). Therefore effective therapy certainly should be made available to warfighters, first responders, remediation personnel and the overall civilian population.

The OPs of concern for high potency acute neurotoxicity are pentavalent phosphorus esters that phosphylate the serine in the active site of AChE while releasing one of the moieties bound to the phosphorus, i.e., the “leaving group”, thus inhibiting AChE’s activity; this inhibition is usually persistent (Chambers, H.W., 1992). The current therapy for OP anticholinesterase poisoning consists of the muscarinic receptor antagonist atropine to suppress hypercholinergic activity and maintain respiratory function as well as other autonomic functions at a life-sustaining level. An oxime reactivator is also provided to restore AChE activity (Eddleston and Chowdhury, 2015). Oximes are strong nucleophiles that are able to remove the phosphylating OP moiety through a transesterification reaction, thus restoring the AChE active site for its normal hydrolytic activity on acetylcholine and thus can dampen or eliminate the hypercholinergic activity. However, the phosphylated oxime resulting from the transesterification reaction is frequently a potent AChE inhibitor, which limits some of the oxime structures that could be considered as therapy (de Jong and Ceulen, 1978). This reinhibition is also called the recapture phenomenon (Wei et al., 2016).

Because of the possibility of seizures, an antiepileptic benzodiazepine, currently in the USA diazepam and with midazolam under consideration, is administered. However, as demonstrated in animal models, the seizures can become recurrent and become refractory to benzodiazepine suppression (Aroniadou-Anderjaska et al., 2016), so there remains a need for improved therapy to provide long-term protection of brain structure and function. The most effective therapeutic strategy to insure both survival and preservation of brain function would be one which attenuates or reverses the hypercholinergic activity induced by AChE inhibition (the initial toxicological lesion) in both the central and the peripheral nervous systems. Attenuation or stopping the initial lesion (i.e., restoring AChE activity) should dampen or prevent any of the down-stream effects of the hypercholinergic activity. Much of the research on therapies for OP poisoning, particularly with respect to nerve agents, is conducted with surrogates because few laboratories throughout the world are able to handle actual nerve agents; nevertheless, research with many of these surrogates yield important insights on the likely efficacy of potential therapeutics (Cavalcante et al., 2019). The remainder of this review will summarize research on reactivators.

2. ACETYLCHOLINESTERASE REACTIVATORS

The first reactivator recognized for OP-inhibited AChE was hydroxylamine in 1957 (reviewed in Karczmar, 1970). However the pyridinium oximes were found to be far more effective reactivators than hydroxylamine because of the presence of the charged quaternary nitrogen in the pyridine ring (Wilson and Ginsburg, 1955; Bismuth et al., 1992). These effective pyridinium oximes are sometimes mono-pyridinium and sometimes bis-pyridinium compounds. The positive charge of the quaternary nitrogen in the pyridinium ring assists with positioning the oxime in the active site. Those oximes that are currently approved or under consideration for approval, such as 2-PAM (pralidoxime), HI-6, MMB-4 or obidoxime, all contain one or two pyridinium rings. Many oximes have been synthesized and tested experimentally by a number of laboratories with a goal of finding an oxime reactivator that is effective against many OP structures. A partial list of papers describing this variety of oximes and their efficacy includes: Poziomek et al., 1958; Wilson and Ginsburg, 1959; Hobbiger et al., 1960; Hagedorn et al., 1969; Schoene and Oldiges, 1972; Lüttringhaus et al., 1964; Clement, 1982, 1992; Cetkovic et al., 1984; Lundy et al., 1992; Koplovitz and Stewart, 1994; Kassa, 2002; Worek et al., 2002, 2007, 2016; Aas, 2003; Kuca and Patocka, 2004; Marrs et al., 2006a; Kassa et al., 2012; Gorecki et al., 2016; Chambers et al., 2013. However, no single oxime has emerged as an effective reactivator against all nerve agent and insecticidal adducts of AChE despite the massive amount of research effort (Worek et al., 2016).

Additionally, the pyridinium oximes, which possess one or two permanent positive charges, are essentially unable to cross the blood-brain barrier (BBB) since the BBB restricts passage of charged molecules (Ballabh et al., 2004). The activity of OP-inhibited AChE can be restored in the peripheral nervous system at muscles or glands by these oximes, but the activity of OP-inhibited AChE in the brain cannot be restored. Thus the majority of these oximes have not been able to suppress seizures and brain damage, although they are effective in maintaining respiratory function and contribute to preserving life.

The oximes have not proven to be effective antidotes used independently but must be used in conjunction with the muscarinic cholinergic receptor antagonist atropine, the latter frequently requiring multiple administrations to maintain suppression of life-threatening hypercholinergic activity. The oximes are strong nucleophiles, which contributes to their ability to reactivate AChE, but it also makes some of them relatively toxic and therefore multiple applications, like atropine, may be less feasible, although there has been a recommendation that oxime be maintained for as long as atropine is required (Marrs and Vale, 2006b).

In addition, oxime therapy is only effective early in the OP toxidrome before the process of “aging” occurs, a non-enzymatically-mediated loss of an alkyl group from the phosphylated AChE, rendering the inhibited AChE charged and refractory to either spontaneous or oxime-mediated reactivation (Smith and Usdin, 1966). Some OPs, such as soman, age so quickly (minutes) as to make oxime therapy almost an impractical option. In addition to rapid aging following inhibition by many of the OP’s, conformational changes within the inhibited active site may also occur, reducing the effectiveness of oximes as reactivators (Stojiljkovic et al., 2001; Bajgar et al., 2007; Artursson et al., 2013).

Much research has occurred in the search for improved AChE reactivators with a major goal of a broad spectrum reactivator effective as therapy for some of the more recalcitrant OPs. Because the pyridinium group imparts great efficacy as a reactivator, many pyridinium oximes have been synthesized for preventing lethality with knowledge that these oximes will not reactivate brain AChE. Some research has involved a search for brain-penetrating reactivators that have the potential to restore normal cholinergic activity in the brain that might prevent neuropathology and brain damage; both uncharged and charged reactivators have emerged in these studies. This paper has provided a broad overview of reactivator research, and now presents some of the main studies in the search for brain-penetrating reactivators, with a more detailed presentation of the results our laboratory has obtained on a very promising platform of centrally active oxime reactivators.

3. SEARCH FOR BRAIN-PENETRATING REACTIVATORS.

While, as indicated above, the pyridinium oximes are the most effective reactivators, the presence of one or two permanent positive charges in the pyridinium ring(s) has prevented appreciable entry of the typical pyridinium oximes into the brain. Typical mono- and bis-pyridinium oximes only penetrate into the brain about 10% and 6%, respectively (Sakurada et al., 2003; Lorke et al., 2008; Kalasz et al., 2015). Therefore several strategies have been attempted to create an oxime that can provide therapy within the brain to not only restore normal cholinergic pathway activity, but also to dampen glutamatergic system-induced seizures and the resultant brain damage.

Pro-PAM:

2-PAM (pralidoxime) was the first effective pyridinium oxime characterized and developed as a drug. It is a mono-pyridinium oxime and is approved in the United States and other countries. Some of the AChE reactivator research has tested compounds which are uncharged with the anticipation that these reactivators would be able to cross the blood-brain barrier. Of these lines of investigation, one of the earliest was the use of pro-PAM, a dihydropyridine pro-drug of 2-PAM that can enter the brain and then theoretically be oxidized within the brain to 2-PAM which is the active reactivator (Bodor et al., 1976; Khan et al., 2011); this possibility would potentially be a more effective strategy for remediating some of the central toxicity with a well-known effective oxime. However, less than 10% of 2-PAM entered the brain in in vivo tests (Shek et al., 1976), and, unfortunately, Pro-Pam did not prove sufficiently effective in in vivo tests in laboratory animals (Clement, 1979; Shih et al., 2011) to warrant further development.

Monoisonitrosoacetone (MINA):

Tertiary oximes that are uncharged have also been considered as potential brain-penetrating reactivators. Monoisonitrosoacetone (MINA) has been investigated as a potential centrally acting reactivator, and it was shown to reactivate brain AChE and enhance survival in a dose-related manner in guinea pigs challenged with three nerve agents (VX, sarin and cyclosarin) (Skovira et al., 2010). MINA was not as effective as other reactivators in preventing lethality in a larger study in guinea pigs involving a number of OP compounds (Wilhelm et al., 2014).

Amidine oximes:

Another chemistry for uncharged oximes is a platform of amidine oximes which demonstrated efficacy in vitro with AChE and butyrylcholinesterase (BChE) reactivation (Kalisiak et al., 2011, 2012). In addition, very good survival efficacy was observed in mice challenged with a surrogate of soman, but the oximes were administered either prophylactically at 30 min prior to challenge or therapeutically at 5 min after challenge. Therefore it cannot be concluded that the survival efficacy resulted entirely or in part from oxime penetration into the brain or from reactivation of brain AChE. These amidine oximes are zwitterionic and can exist in an uncharged form, so their potential for brain penetrability was ascribed to this property and the amidine was considered to provide enhanced nucleophilicity to the reactivator; these two properties were concluded to contribute to the survival efficacy observed (Okolotowicz et al., 2014).

Sugar-oximes:

Significant work has been done on sugar-oximes (conjugates of oximes and glucose), using the premise that the oxime in this conjugate could be carried into the brain through the glucose transporter and subsequently could reactivate OP-inhibited AChE. Brain AChE inhibited by tetraethylpyrophosphate (TEPP) was reactivated by sugar-oximes, and these sugar oximes did promote survival from TEPP acute toxicity in mice (Rachaman et al., 1979). Further support for the brain penetration of sugar-oximes came from in vivo studies in which body temperature reductions following OP exposure was attenuated by the sugar-oximes (Heldman et al., 1986). Garcia et al. (2010) produced a platform of sugar-oximes with a linker chain between the pyridinium oxime and the sugar which was theorized to fit well into the AChE gorge. Human blood AChE and BChE could be reactivated by some of these sugar-oximes following inhibition by paraoxon (the active anticholinesterase metabolite of the insecticide parathion) or the model compound diisopropylfluorophosphate (DFP), with efficacy in some cases approaching 2-PAM’s efficacy. Further some of the sugar-oximes showed reactivation kinetics similar to the parent (non-conjugated) oximes with human AChE inhibited by sarin or VX (Bhonsle et al., 2013).

Non-quaternary ortho-hydroxylbenzaldoximes:

The ortho-hydroxylbenzaldoxime platform was synthesized with the rationale that attaching a ligand for the peripheral site of AChE would assist in preventing the recapture phenomenon (Wei et al., 2016). These were developed to reactivate AChE following soman inhibition. Very few oximes are effective against soman, and even though less effective than HI-6 against soman, the fact that they are non-quaternary and therefore uncharged may give them the ability to enter the brain.

Imidazole and imidazolium oximes:

A series of imidazole and imidazolium oximes was synthesized linked to a peripheral site ligand, and they were assessed by molecular docking modeling to human AChE inhibited by sarin, cyclosarin, tabun and VX (de Koning et al., 2017). In vitro efficacy was studied with human erythrocyte AChE. In vivo efficacy was observed in rats challenged with a 1.8 LD50 dosage of sarin with respect to attenuating seizures and convulsions, providing suggestions of central nervous system therapy.

3-Hydroxy pyridine aldoximes:

A non-quaternary uncharged pyridine oxime can be achieved by placing a hydroxyl group in the 3 position of the pyridine ring, so such oximes have the potential to cross the blood-brain barrier (Mercey et al., 2011, 2012; Renou et al., 2013, 2014). This platform of compounds showed very good in vitro efficacy with human AChE. In in vivo studies with mice challenged with VX, two of the members of this platform showed comparable protection to 2-PAM, but less protection than HI-6 (Calas et al., 2017; Zorbaz et al., 2018).

Zwitterionic hydroxyiminoacetamido alkylamines:

This platform, which includes primarily N-substituted 2-hydroxyiminoacetamides and imidazole aldoximes, relies on the equilibrium that exists between the charged and uncharged forms of the molecules whereby the uncharged forms would have the ability to cross the blood-brain barrier (Radic et al., 2012). The lead compound in the series, RS194B, showed good efficacy in in vitro tests with human AChE (Sit et al., 2011), and these results were verified by molecular dynamics simulations. There was good survival efficacy in mice challenged with VX, although relatively poor survival efficacy in guinea pigs challenged with several OP’s (Wilhelm et al., 2014). RS194B provided a rapid attenuation of signs of poisoning from sarin in macaques; some of the macaques had been pretreated with aerosolized human recombinant butyrylcholinesterase before receiving the sarin challenge (Rosenberg et al., 2018). Studies were also conducted with paraoxon with similar efficacy against signs of poisoning (Rosenberg et al., 2017).

Substituted phenoxyalkyl pyridinium oximes:

Our laboratories have invented a platform of substituted phenoxyalkyl pyridinium oximes that was designed to include a more lipophilic moiety (the phenoxyalkyl group) while still retaining the pyridinium group for greater reactivation efficacy. The greater lipophilicity was theorized to provide a greater likelihood for crossing the BBB. In vitro tests were conducted with highly relevant surrogates of sarin (nitrophenyl isopropyl methylphosphonate, NIMP) and VX (nitrophenyl ethyl methylphosphonate, NEMP) that leave AChE phosphylated with the same chemical moiety as their respective nerve agents (Meek et al., 2012). These initial in vitro tests identified several novel oximes that were effective as AChE reactivators using rat brain, although none were as effective as 2-PAM (Chambers et al., 2013).

An in vivo paradigm was developed in male rats to deliver test oximes (in DMSO, intraperitoneal) at the time of peak brain AChE inhibition following high sublethal level nerve agent surrogate challenge. The rationale behind this paradigm was that the OP was being cleared at that time and little to none would be available to reinhibit reactivated peripheral AChE, since reinhibition would lower circulating levels of OP available to enter the brain and could erroneously give the appearance of brain entry compared to non-oxime-treated animals. Therefore this paradigm would provide convincing evidence of brain penetration if a reduction in brain AChE inhibition was observed. Indeed, a reduction of brain AChE inhibition of up to 35% was observed for several of our novel oximes but not for 2-PAM, supporting the concept of brain entry by the novel oximes (Chambers et al., 2013).

Following these initial efficacy tests, male rats were challenged with an LD99 level of sarin or VX surrogates or paraoxon (in Multisol vehicle, subcutaneous) and survival to 24 hrs and signs of toxicity during the initial 8 hrs were observed with a comparison of therapy with atropine (in saline at the human equivalent dosage, intramuscular) and either 2-PAM or one of our novel oximes used at the same molar equivalent as the FDA-approved level of 2-PAM (Multisol vehicle, intramuscular); atropine and oxime were delivered at time of onset of signs of OP toxicity, about 30 minutes post OP challenge (Chambers et al., 2016). This experiment yielded a reduction of time to cessation of seizure-like behavior by several of our novel oximes, but not by 2-PAM (i.e., 6–7 hours for our most effective novel oxime, Oxime 20) compared to the 8+ hours for 2-PAM), providing behavioral support for the concept that our novel oximes can enter the brain, despite the presence of the charged quaternary nitrogen in the pyridine ring. The efficacy observed for both brain AChE reactivation and for seizure-like behavior cessation was not proportional to novel oxime lipophilicity.

Subsequent in vitro tests with a model transporter system indicated that the in vivo efficacy was only partially explained by the novel oximes’ ability to serve as P-glycoprotein (Pgp) substrates; however it appeared that some of the more efficacious oximes in vivo were the poorest Pgp substrates and some of the least efficacious oximes in vivo were the best Pgp substrates, suggesting that the most efficacious oximes might enter the brain and not be efficiently transported back out (Dail et al., 2018). This characteristic is an additional advantage to our oximes and would allow them to remain in the brain without the addition of a Pgp inhibitor to suppress export.

A subsequent immunohistochemical analysis of rat brain slices was performed in both the piriform cortex and the dentate gyrus of the hippocampus of rats challenged with high sublethal dosages (not requiring atropine for survival) of the sarin surrogate NIMP or the VX surrogate NEMP (DMSO vehicle delivered intraperitoneally) followed by 2-PAM or one of our novel oximes (Oxime 20) (DMSO vehicle delivered intramuscularly). Glial fibrillary acidic protein (GFAP) did not accumulate with Oxime 20 but did with 2-PAM similar to the non-oxime controls (Pringle et al., 2018). Since GFAP accumulation is a marker of gliosis and damage, this study provided functional evidence of brain penetration by one of our novel oximes.

Also providing the most convincing functional evidence of neuroprotection in the brain by two of our three most promising oximes (Oximes 15, 20 and 55; Figure 1) was a study in rats with lethal dosages of NIMP or paraoxon (to assure survival antidoted with atropine in saline, intramuscular at time of development of signs of toxicity, in addition to oximes in Multisol intramuscular) which showed protection of CA1 region hippocampal neurons, using the histological marker NeuN, by Oximes 20 and 55, but not Oxime 15 (Figure 2) (Dail et al., 2019). A pharmacokinetic analysis showed longer plasma half-lives for Oximes 20 and 55 (5.2 and 13.8 hours, respectively) than for Oxime 15 (2.5 hours), suggesting that a longer residence time in the blood might be needed to provide neuroprotection. Oxime 15 is the least lipophilic of the three oximes, Oxime 20 intermediate and Oxime 55 the most. The octanol:water partition coefficients were 0.056, 0.352 and 1.461, respectively, and these were proportional to the plasma half-lives (Chambers and Meek, 2020). This study also demonstrated that our oximes show some broad-spectrum efficacy in that they displayed neuroprotection against both a nerve agent chemistry (sarin surrogate) and an insecticidal chemistry (paraoxon). Another advantage of these novel oximes is their slower clearance compared to the rapid clearance of 2-PAM (34 min; Green et al., 1986) for such agents as VX that are non-volatile making both slower contact and slower dermal absorption more likely. Also the longer clearance time of these oximes might allow greater efficacy for a single administration compared to 2-PAM, thereby reducing the likelihood of adverse drug reactions. Altogether, we believe that the experimental paradigms we have employed and the functional efficacy that we have observed provide convincing evidence that our oximes can enter the brain and provide therapy within the brain. In particular, the in vivo neuroprotection data obtained in glial and neuronal cells from our novel oximes, but not 2-PAM at the same molar equivalent, are unique among oxime studies, and these protective results from our oximes would not have occurred from strictly peripheral AChE reactivation.

Figure 1:

Structures of three of the substituted phenoxyalkyl pyridinium oximes.

Figure 2:

Relative neuropathology damage scores observed in sequential NeuN stained sections from the hippocampal CA1 region of rats (n = 7 to 14) challenged subcutaneously with organophosphates (OP) [either nitrophenyl isopropyl methyl phosphonate (NIMP) at 0.6 mg/kg or paraoxon (PXN) at 0.8 mg/kg) in Multisol]. Therapy was administered at 30 minutes, consisting of 0.65 mg/kg atropine sulfate in saline +/− 0.146mmol/kg oxime (2-PAM, Oxime 15, Oxime 20, or Oxime 55) in Multisol intramuscularly. Brains were sampled at 4 days post-treatment. The # symbol indicates a significant statistical difference between the median of this group and that of the control; bars reflect SD. (Figure modified from Dail et al., 2019).

Therefore there seems to be exceptional promise for the development of these substituted phenoxyalkyl pyridinium oximes as potential therapeutics with efficacy within the brain against both nerve agent and insecticidal chemistries, and pharmacokinetics that favor efficacy in the brain and protection against slowly absorbed OPs. Experiments are on-going to provide additional information on our oximes.

4. CONCLUSION

Much research from a number of laboratories world-wide has occurred to create and identify oximes that could provide effective therapy for the highly toxic organophosphate anticholinesterases. A few of these oximes, for example, 2-PAM and HI-6, have shown a great deal of efficacy against lethality and are approved for therapeutic use in some countries, but they still are not maximally broad-spectrum for all nerve agent chemistries as well as insecticidal chemistries. The currently approved oximes do not appreciably cross the blood-brain barrier and therefore do not provide central neuroprotection. A number of laboratories, including ours, have developed reactivators theorized to enter the brain, but relatively few have provided convincing functional evidence of brain entry because of the nature of the experimental paradigms employed. However, some oxime reactivators, including ours, are currently under investigation for neuroprotection in the brain and the goal is that one or more of these promising oximes will soon arise as an additional approved therapeutic for the brain damage induced by high level nerve agent and insecticidal poisoning.

Highlights.

• Acetylcholinesterase reactivators are therapeutic for OP poisoning.

• Most reactivators are nucleophilic pyridinium oximes with positive charges.

• Several labs are trying to develop brain-penetrating reactivators.

• Substituted phenoxyalkyl pyridinium oximes show survival efficacy.

• These oximes show neuroprotection from OP-induced brain damage.