Mechanism-based novel antidotes for organophosphate neurotoxicity

Abstract

This article describes current pursuits for developing novel antidotes for organophosphate (OP) intoxication. Recent mechanistic studies of benzodiazepine-resistant seizures have key consequences for victims of OP pesticide and nerve agent attacks. We uncovered why current therapies are not able to stop the OP-induced seizures and brain cell death and what type of drug might be better. OP exposure down regulates critical inhibitory GABA-A receptors, kills neurons, and causes massive neuroinflammation that will cause more neuronal death, which causes the problem of too few benzodiazepine receptors. The loss of inhibitory interneurons creates a self-sustaining seizure circuit and refractory status epilepticus. Thus, there is an urgent need for mechanism-based, new antidotes for OP intoxication. We have discovered neurosteroids as next-generation anticonvulsants superior to midazolam for the treatment of OP poisoning. Neurosteroids that activate both extrasynaptic and synaptic GABA-A receptors have the potential to stop seizures more effectively and safely than benzodiazepines. In addition, neurosteroids confers robust neuroprotection by reducing neuronal injury and neuroinflammation. The synthetic neurosteroid ganaxolone is being considered for advanced development as a future anticonvulsant for nerve agents. Experimental studies shows striking efficacy of ganaxolone and its analogs in OP exposure models. They are also effective in attenuating long-term neuropsychiatric deficits caused by OP exposure. Overall, neurosteroids represent rational anticonvulsants for OP intoxication, even when given late after exposure.

1. Introduction

Organophosphate (OP) pesticides and nerve agents produce severe, fast-acting effects on both the body and the brain. Nerve agents are among the most toxic of known chemical agents; agents such as tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), and VX are deployed as chemical warfare weapons in combat or as bioterror agents against civilians. Chemical attack is becoming a real threat worldwide. In 2013, about 1,400 civilians died from the nerve gas sarin attacks in Syria [1]. Over a decade ago, Tokyo civilians were exposed to sarin gas on the subway [2]. These compounds are known to be hazards in both liquid and gas form, killing individuals within minutes of exposure. Many countries have the technology to weaponize these deadly agents. Compounds known as Novichok are considered among the deadliest chemical weapons ever made and are difficult to identify due to their poorly defined composition. The substance Novichok, which means “newcomer” in Russian, is a more dangerous and sophisticated nerve agent than sarin which has been used in chemical weapons attacks in Syria in 2013, or VX, which was used to assassinate Kim Jong Nam at an airport in Malaysia in 2017. Novichok exposure incident in Salisbury in England was widely reported in the media. Although the substance was undetectable by standard detection equipment, the patient’s response to certain antidotes confirmed that it resembles OPs. In addition, thousands of OP pesticide poisonings occur annually due to suicides or agriculture accidents worldwide [3]. OP pesticides such as monocrotophos, parathion, chlorpyrifos, paraoxon, and diisopropylfluorophosphate (DFP) are considered credible threat agents. This article describes a concise overview of current pursuits of mechanistic toxicology and mechanism-based therapies for effective treatment of OP neurotoxicity.

OP pesticides and nerve agents produce lethal neurotoxicity via common mechanisms (Fig. 1), primarily causing neurotoxicity by irreversibly (permanently) inhibiting acetylcholinesterase (AChE); this, in turn, leads to an excessive accumulation of acetylcholine (ACh) in the synaptic cleft in peripheral and central nervous systems (CNS). These compounds interfere with brain chemicals that turn neurons and muscles “on” and “off.” They inhibit AChE in plasma, red blood cells, tissues, and brain [4–6]. In a normal, healthy person, ACh is released at the junction between neurons and muscles, acting as an “on” switch by allowing the brain to contract the muscles. So, every time someone wants to walk—or breathe—ACh is released, causing certain muscles to contract and facilitating movement. When the body needs to stop contracting its muscles, AChE acts as the “off” switch. AChE essentially cuts up the ACh into choline and acetate so that the muscles stop contracting. When an individual is exposed to OPs, it blocks AChE. As a result, ACh builds up in massive quantities in the brain and causes widespread nerve excitation and muscle contraction. Without an “off” switch, the brain is overly excited, and the muscles in the body begin to continuously contract, lacking the ability to relax (cholinergic crisis). This results in muscle spasms, convulsions, continuous seizures, respiratory arrest, and eventually death [7,8]. If a person is able to survive OP attack, they will likely have serious brain damage due to severe secondary neuronal damage (Fig. 1). Besides brain damage, it also effects the peripheral nervous and other systems.

Fig. 1. Schematic illustration of potential cholinergic and non-cholinergic mechanisms of organophosphate (OP) neurotoxicity.

OP pesticides and nerve agents produce acute and long-term neurotoxicity. The primary mechanism of action of both classes is irreversible inhibition of acetylcholinesterase (AChE) resulting in accumulation of toxic levels of acetylcholine (ACh) at the synaptic junctions which induces muscarinic and nicotinic receptor stimulation, and host of other pathways. Muscle fasciculation and cardiac and respiratory distress occur very rapidly after exposure and can ultimately lead to death if left untreated or treated too late. The autonomic nervous system is heavily affected by OPs since the transmission from pre-ganglionic to post-ganglionic neurons is dependent on ACh. The levels of AChE remain low for several days after OP exposure, but the range of autonomic changes is also dependent on the exposure route. If exposure to OPs is via inhalation, the vapour comes into contact with the eyes and respiratory tract, causing miosis (pupil constriction) and difficulty breathing, respectively. Further, OPs can rapidly cross the blood brain barrier (BBB) and induce severe seizures, initially through overstimulation of cholinergic pathways. Seizures can reversibly open the BBB with permeability and trigger a massive inflammation response in the brain. As status epilepticus (SE) progresses, glutamatergic networks are recruited and several other neurochemical changes may occur. Once this hyperexcitability is initiated, such seizures are difficult to reverse. The secondary events of SE and non-seizure activity, such as neuronal necrosis, cell death, and axonal degeneration can potentially result in severe brain damage. Thus, survivors of OP exposure suffer from long-term neurological problems including cognitive deficits, anxiety, depression and epileptic seizures.

Since OP chemicals are cholinesterase inhibitors, measuring levels of AChE in the blood provides a reliable biomarker of their exposure. The extent of AChE activity also reflects the exposure severity and timeline. OP pesticides and nerve agents present major differences in both duration of neurotoxicity and response to therapy. The initial effects of exposure to these compounds depend on the dose and route of exposure [7,8]. Inhalation (rapid absorption), oral (medium absorption), and dermal (slow absorption) routes are most common routes of OP exposure. Miosis is an indicative sign of exposure to OP agents. However, the major effect of OPs is on skeletal muscle, producing muscular fasciculation and twitching. In contrast, exposure to nerve agent (vapor) is initially indicated by rhinorrhea. Such compounds cause bronchoconstriction and increased gland secretions in the airways, often times producing tightness in the chest. Cessation of respiration occurs later after the onset of toxic signs. For both classes of compounds, the severe CNS signs of exposure are loss of consciousness, seizure activity, and apnea. Status epilepticus (SE) occurs within minutes after exposure and may persist for up to 30 min or longer. SE is a life-threatening emergency in which an individual, without regaining consciousness, experiences a prolonged, continuous state of convulsions. If not controlled immediately, this medical emergency could result in tragic consequences, resulting in widespread brain damage or death. Thus, there is an urgent need for a better understanding of the molecular neurotoxicity to design effective countermeasures for OP attacks.

2. The Current Situation

Managing an individual that is intoxicated with OP consists of decontamination, ventilation, and administration of antidotes. Decontamination includes the application of reactive skin decontamination lotion, soap and water, and 5% hypochlorate solution. Three drugs-atropine sulfate, pralidoxime chloride (2-PAM), and diazepam– are used to treat OP intoxication [9,10]. This regimen is distributed as CHEMPACKs with auto-injectors for use in case of a chemical attack or accident. Pyridostigmine bromide is used as protective pretreatment for military personnel at risk for a nerve agent exposure. After exposure, atropine, a muscarinic receptor antagonist, is extremely effective at blocking the effects of excess ACh at peripheral sites. Atropine produces life-saving effects by decreasing hypersecretions and relieving bronchoconstriction, allowing for easier breathing. The nicotinic effects of OPs, such as spasms and fasciculations, will not be improved by atropine. Atropine has a limited effect on CNS due to its poor entry into the brain. Pralidoxime chloride (2-PAM) is an AChE reactivator that can break the agent-enzyme bond to release the free AChE enzyme. Like atropine, 2-PAM has poor penetration of the brain. Hence, despite atropine and 2-PAM therapy, excess ACh remains uncontrolled in the brain resulting in cholinergic crisis including seizures and SE (Fig. 1).

Currently, there are two drugs used to control OP neurotoxicity. If given promptly after exposure, diazepam and midazolam are benzodiazepine anticonvulsants that can work to prevent OP-induced seizures and brain damage. Both drugs are effective anticonvulsants when given within 30 minutes of OP exposure but do not have much effect beyond an hour or two after exposure. In the context of chemical warfare and unexpected civilian bioterrorism, this is not a realistic timeline. Thus, the neurotoxic signs of OP exposure, including convulsive seizures and SE, cause profound permanent brain damage that will result in neuronal damage or death (Fig. 1). Brain damage can occur not only by seizure-related excitotoxicity but also via mechanisms independent of seizures such as the activation of microglia, astrocytes, and cellular inflammation [11,12]. The effects of OP intoxication are long lasting and pose a greater risk of long-term neurologic and cognitive deficits [4, 13,14].

Although soldiers often carry antidote kits for personal use in case of a nerve agent attack, civilians have less convenient access to anticonvulsant medications; for many civilians, a trip to the hospital is required to receive the needed medication. The process of getting to the hospital and being administered the drugs would most likely take at least 40 minutes in the majority scenarios [7,21]. This represents the critical time period, meaning any anticonvulsant antidote for these OP chemical seizures has to work after 40 minutes following exposure. This is the critical goal of the antidote therapy for OP intoxication. However, this timeline is often not practical in many emergencies. Hence, OP-induced neurotoxicity can lead to long-term brain injury and devastating neuropsychiatric dysfunction in survivors of chemical attacks. Five years after the attacks with the nerve agent sarin in Matsumoto and Tokyo, individuals exposed to sarin reported devastating neurological and psychiatric disorders [15–18]. Likewise, thousands of survivors in Syria following sarin exposure may live with the aftereffects for the rest of their lives.

3. Mechanisms of benzodiazepine refractory seizures

Benzodiazepines are the primary drugs for treatment of SE, but there is strong evidence of refractoriness to diazepam and midazolam [19–21]. We have investigated the mechanistic basis of benzodiazepine refractory seizures after OP intoxication [22, 23]. After DFP exposure in animal models, we examined two major points for diazepam and midazolam, which are most widely used anticonvulsants in OP intoxication. First, we examined how efficiently each drug suppressed seizures and SE. Second, we looked at how efficiently each drug protected against brain damage. The results of both studies showed that diazepam and midazolam were very effective at controlling seizures, neurodegeneration, and neuroinflammation when given 10 minutes after exposure. However, both medications were completely ineffective when administered at 60 minutes or 120 minutes after exposure. Delayed therapy (40 min), a simulation of the practical therapeutic window for first responders or hospital admission, was associated with reduced seizure protection and neuroprotection [22,23]. These results strongly reaffirm the notion that OP-induced seizures and brain damage are progressively resistant to delayed treatment with diazepam or midazolam. This condition is referred to as the benzodiazepine refractory SE caused by OP exposure [12,23]. Since benzodiazepines are positive allosteric agonists of synaptic GABA-A receptors [24], multiple mechanisms may contribute to their reduced efficacy, including pharmacodynamic (loss or internalization of receptors) rather than a pharmacokinetic mechanism [21,25–27]. It is concluded that the benzodiazepines don’t control seizures at later time points (after 40 min), but it’s not because they aren’t reaching enough quantities in the brain. Instead, it is considered more likely to the loss of target receptors, the loss of neurons, and damage-induced inflammation. It is likely that OP exposure somehow affects the receptor targets, causing diazepam and midazolam to be unable to find their receptors and diminish the seizure circuits.

Benzodiazepine receptors disappear in more than 50 percent of neurons within 10 to 20 minutes of OP-induced seizure onset [26,26]. When benzodiazepines were administered at 40 min, it meant that 50% of the benzodiazepine receptors had already vanished or not functional at the neuronal membrane targets. The administered benzodiazepines bound to the remaining 50% of receptors, but the maximum effect they could produce depended on the number of receptors available, regardless how large of a dose was given. This is why repeated doses of diazepam are needed for partial control of seizures, resulting in sedation, respiratory depression, and tolerance in victims.

Moreover, OP poisoning also kills neurons, which worsens the problem of too few benzodiazepine receptors. Massive brain cell death further exacerbates the problem of a lack of receptors, as demonstrated by massive neurodegeneration of principal and interneurons [22,23]. The cell must be alive for the drug to bind to its targets receptors. The benzodiazepine receptors are on the main neurons. However, so many of these neurons are dead that it further reduces the number of available receptors. The loss of inhibitory interneurons, which apply strong breaks on excessive neuronal excitation and synchronization to manifest into seizures, creates a self-sustaining seizure circuit. Finally, OP poisoning causes persistent inflammation in the brain, as evident by astrogliosis and microgliosis [22,23], which will cause more cell death and a loss of more receptors. This forms the mechanistic reason for why benzodiazepines are failing when they are administered later in a field setting.

These studies have provided deeper insights for developing next-generation anticonvulsants that are better than benzodiazepines. Benzodiazepine receptors are exclusively present in post-synaptic junctions. However, a new type of GABA-A receptors are present at perisynaptic and extrasynaptic sites (Fig. 2) [33]. While OP molecules can destroy the “synaptic” forms comprising benzodiazepine receptors, they do not affect the “extrasynaptic” forms of GABA-A receptors. These GABA-A receptors should be targets for new drugs because they will not disappear in 10 to 20 minutes after OP exposure. However, it is still a reverberating circuit because the neuronal loss leads to neuroinflammation and loss of receptors. By targeting extrasynaptic receptors, it’s more likely to control the seizures and stop neuronal loss. Essentially, it is like breaking the circuit.

Fig. 2. Mechanisms of ganaxolone (GX) at extrasynaptic and synaptic GABA-A receptors in the brain.

(A) Like other neurosteroids, ganaxolone enhances the function of extrasynaptic and synaptic GABA-A receptors by binding to “neurosteroid binding” sites, which are distinct from sites for GABA, benzodiazepines, and barbiturates. There are two subtypes of GABA-A receptors: (i) synaptic receptors composed of 2α2βγ subunits, mediate the phasic inhibition in response to action potential-dependent vesicular release of high levels of GABA; and (ii) extrasynaptic receptors composed of 2α2βδ subunits, primarily contribute to tonic inhibition when exposed to low, ambient levels of GABA. GX can bind to both subtypes and enhance the phasic and tonic currents. (B,C) Panels B and C depicts GX potentiation of phasic current and tonic current in hippocampal granule cells. Panel 2B shows sample traces, frequency, amplitude, and decay time of mIPSCs with or without GX. Panel 2C shows sample traces, current shift, and current density of tonic currents before and after GX. Overall, GX and other neurosteroids can significantly enhance the phasic inhibition and tonic inhibition, and thereby promote maximal inhibition. This contributes to their robust anticonvulsant actions, including controlling organophosphate-induced seizures and brain damage. In contrast, benzodiazepines (e.g. midazolam) bind specifically to γ2-containing synaptic receptors and augment phasic inhibition. Benzodiazepines do not bind to extrasynaptic δ-containing extrasynaptic receptor and do not affect tonic inhibition in the brain.

4. Neurosteroids as Novel Anticonvulsant Antidotes for OP Intoxication

Neurosteroids with a unique mechanism of action have the potential to stop seizures more effectively and safely than benzodiazepines. In addition, neurosteroids may confer neuroprotection as well. This is achieved by shunting the excessive excitability and its exacerbating impact on neuronal injury and neuroinflammation, which are typically associated with OP poisoning. These products are being developed for approval by the FDA, which could be revolutionary for both military members and civilian victims of nerve agent attacks [28–30]. The term neurosteroid refers to steroids that are synthesized within the brain which rapidly alter neuronal excitability through interactions with non-genomic, membrane receptors, and lack conventional hormonal effects. A variety of neurosteroids are present in the brain, including allopregnanolone (brexanolone), pregnanolone, androstanediol, and allotetrahydrodeoxycorticosterone (THDOC) [31]. They play critical roles in modulating neuronal excitability and neuroplasticity [31].

Neurosteroids act as positive allosteric modulators and direct activators of GABA-A receptors (Fig. 2). They bind to “neurosteroid binding sites” on the receptor channel, which are distinct from the benzodiazepines and GABA sites [32]. Neurosteroids act on all GABA-A receptor isoforms in the brain. They activate receptor channels primarily via allosteric potentiation of GABA at nanomolar concentrations and through direct activation of the channel at micromolar concentrations [33]. There are two distinct categories of GABA-A receptors that are stratified into synaptic and extrasynaptic receptors (Fig. 2). They exhibit different characteristics in their affinity and efficacy to GABA, desensitization rate, and drug sensitivity [32,33]. Synaptic (γ-containing) receptors produce rapid and transient phasic currents in response to the presynaptic release of GABA (at μM). However, extrasynaptic (δ-containing) receptors generate persistent, non-desensitizing tonic currents by ambient GABA (at M). Tonic currents contribute to the overall basal tone and shunting inhibition via continuous channel conductance, thereby regulating network excitability and seizure susceptibility. Neurosteroids have a high potency for synaptic receptors that mediate phasic inhibition, whereas they exhibit greater efficacy or sensitivity for extrasynaptic receptors that promote tonic inhibition [32,33]. The net output is maximal inhibitory tone that can effectively shunt hyperexcitability and focal discharges in the brain. Hence, neurosteroids have broad-spectrum anticonvulsant activity and promising clinical potential for treating seizure disorders [34].

Natural neurosteroids such as allopregnanolone (brexanolone) have limited therapeutic utility because after oral administration these are inactive due to first-pass metabolism; they exhibit short half-lives and hormonal side effects via metabolism into C3-keto steroids, which can bind to steroid hormone receptors such as the progesterone receptor [31]. Synthetic neurosteroids are designed to overcome these limitations [35–37]. For example, the 3β-methyl substitution in ganaxolone minimizes the metabolic conversion to hormonally-active C3-keto forms, renders it orally-active, and has a longer half-life (4–6 fold) than endogenous neurosteroids. Several synthetic compounds are prepared using structure-activity designs (Fig. 3). In addition, the molecular modelling of neurosteroid potentiation of GABA-A receptors has created new opportunities for creating novel neurosteroid analogs for general treatment of seizure conditions, including OP-induced refractory SE [34].

Fig. 3. Chemical structures of neurosteroids tested in OP intoxication models.

Natural neurosteroids such as allopregnanolone, also called brexanolone (3α-hydroxy-5α-pregnan-20-one), tetrahydrodeoxycorticosterone (THDOC, 5α-pregnan-3α,21-diol-20-one), AD (5α-androstan-3α,17β-diol) and synthetic neurosteroids ganaxolone (3α-hydroxy-3β-methyl-5α-pregnan-20-one), SAGE-516 (3α-hydroxy-3β-methyl-21-(1′,2′,4′-triazol-1′-yl)-19-nor-5β-pregnan-20-one), UCI-50027 (3-[3α-hydroxy-3β-methyl-5α-androstan-17β-yl]-5-(hydroxymethyl)-isoxazole) and alfaxolone (3α-hydroxy-5β-pregnan-11,20-dione) are screened in specific OP exposure models.

Refractory SE is a hallmark of OP intoxication. Mechanistically, a rapid decline in synaptic GABA-A receptors and consequent reduction in phasic inhibition occurs in the hippocampus during SE [25–27]. These changes may account for the resistance to benzodiazepines in SE [19–23]. To overcome this, we have proposed neurosteroids as novel anticonvulsants against OP-induced SE [28]. Our “neurosteroid therapy” is based on the premise that extrasynaptic δGABA-A receptors which generate tonic inhibition do not internalize during SE, so that neurosteroids, which activate both extrasynaptic and synaptic receptors, are more effective treatments for SE. In experimental paradigms, SE causes a reduction in synaptic phasic inhibition (due to internalization or downregulation of synaptic receptors) but not in extrasynaptic tonic inhibition [25]. Since neurosteroids can enhance extrasynaptic inhibition to a greater extent than benzodiazepines, they offer a rational treatment strategy for OP exposure as they can maximally enhance inhibition for counteracting the sustained seizure activity. We are among the first to identify the anticonvulsant potential of neurosteroids for SE therapy [38–40]. Consequently, allopregnanolone, THDOC and ganaxolone have been tested in rodent models of cholinergic SE induced by pilocarpine [41–43], the OP pesticide DFP [30], and the nerve agent soman [30,44]. Recently, we further characterized the efficacy of natural neurosteroids (see Fig. 3), synthetic analogs (alfaxolone, ganaxolone), and super analogs (ganaxolone analogs) in OP exposure models including DFP, soman, and VX [30]. Neurosteroids were effective when given 40-min or later after OP exposure in rat models; they produced rapid and effective control of SE and neuronal damage [30]. Overall, neurosteroids are more effective as anticonvulsants and neuroprotectants than midazolam for OP intoxication at doses that are lower than LOAEL as evident from multiple preclinical toxicokinetic studies [30].

5. Ganaxolone and its analogs as future anticonvulsants for refractory SE

Ganaxolone, the 3β-methylated synthetic analog of allopregnanolone, has been tested extensively in OP exposure models. Ganaxolone potentiates GABA currents by allosteric potentiation and direct activation of synaptic receptors as well as extrasynaptic GABA-A receptors [45] (Fig. 2A). It potentiates the GABA-gated phasic currents by significantly increasing the amplitude and prolongation of mIPSC decay without altering the frequency of mIPSC in the hippocampus (Fig. 2B) and other brain regions. The unique mechanism of ganaxolone on GABA-gated tonic currents is characterized in a hippocampal slice preparation, in which synapses and dendritic connections remain functional [36]. It strikingly enhances the tonic current for the entire duration of its application with little rundown (Fig. 2C) as evident from the persistent tonic current measured as the shift in mean conductance before and after application of the GABA-A receptor antagonist gabazine [36].

Ganaxolone has a unique advantage over midazolam in that tolerance does not appear to occur with extended use. In preclinical models, ganaxolone causes mild side effects such as sedation and hypoactivity, which are comparable to that of the benzodiazepine midazolam [31–36]. Among the synthetic neurosteroids, ganaxolone has been well-studied; the mechanism of action, anticonvulsant profile, pharmacokinetics, and safety profile have been well documented [46,47]. Hence, ganaxolone makes an excellent practical option for development as a medical countermeasure for OP intoxication.

We have developed an intramuscular (IM) formulation of ganaxolone [30]. The product has demonstrated desirable features of efficient absorption and rapid distribution to the brain. Plasma and brain levels of ganaxolone increased proportionately with increasing dosage. The bioavailability of ganaxolone is >95% after IM administration with a C_max of 0.167 hr and t_½ of 2.4 hr. We tested the efficacy of IM ganaxolone in rodent models of nerve agent exposure using a delayed post-exposure protocol in rats [30]. Ganaxolone produced a dose-dependent protection against DFP- and soman-induced seizures. In addition, it produced protection against SE even when administered 40–120 min after agent exposure (Table 1). It displayed strong neuroprotectant activity even with delayed treatment (40 min) after soman exposure. Ganaxolone therapy significantly prevented cell deaths of principal neurons and markedly decreased the loss of inhibitory interneurons. In the same setting, midazolam alone failed to protect against soman-induced SE and neuronal damage. Multiple combination regimens of ganaxolone and midazolam have been tested in the DFP and soman models. A combination regimen of ganaxolone+midazolam produced a superior efficacy for controlling SE than midazolam alone. This combination produced a greater neuroprotectant efficacy, indicating a strong synergistic protective potential of the combination regimen.

Table 1.

A summary of experimental efficacy studies of ganaxolone in OP intoxication models.

Study Type and protocol	Species	Overall Outcomes
		DFP Model	Soman Model
(a) Anticonvulsant efficacy: Drug given @ 40, 60 or 120 min after OP	Rats	Stopped electrographic SE Stopped behavioral SE Significantly decreased seizure activity Significantly decreased duration of SE 100% survival rate	Stopped electrographic SE Stopped behavioral SE Significantly decreased seizure activity Significantly decreased duration of SE 100% survival rate
(b) Acute neuroprotective efficacy: Drug given @ 40, 60 or 120 min after OP	Rats	Significantly reduced neuronal cell injury Significantly prevented cell death of principal neurons Significantly decreased cell death of interneurons	Significantly reduced neuronal cell injury Significantly prevented cell death of principal neurons Significantly decreased cell death of interneurons
(c) Chronic neuroprotective efficacy: Animals tested 3 months after DFP exposure. Drug given @ 40, 60 or 120 min after OP	Rats	Significant reduction in neurodegeneration of principal cells and interneurons	Significant reduction in neurodegeneration of principal cells and interneurons.
(d) Chronic neuroprotective efficacy: Animals tested 3 months after DFP exposure. Drug given @ 40 or 60 min after OP		Significant decrease in frequency and severity of epileptic seizure development. Attenuation or reduction in behavioral memory deficits	Significant reduction in incidence of epilepsy development. Attenuation or reduction in behavioral memory deficits
(e) Combination anticonvulsant efficacy: ± Midazolam Drug given @ 40 min after OP	Rats	Combination regimen was more effective anticonvulsant than midazolam alone Neuroprotection was much better than midazolam alone	Combination regimen was more effective anticonvulsant than midazolam alone Neuroprotection was moderately better than midazolam alone
(f) Combination neuroprotectant efficacy: ± Midazolam Drug given @ 40 min after OP	Rats	Significantly reduced neuronal cell injury Combination regimen was more effective neuroprotectant than midazolam alone	Significantly reduced neuronal cell injury Combination regimen was more effective neuroprotectant than midazolam alone

Many analogs around ganaxolone structure are synthesized and tested for anticonvulsant efficacy [36], including several water-soluble analogs of ganaxolone (an essential feature for intravenous formulation). Ganaxolone analogs are designed to exhibit greater potency and efficacy than ganaxolone on extrasynaptic GABA-A receptor-mediated tonic inhibition (Fig. 3). The ganaxolone analogs at C-21 position (e.g. SGE–516) displayed a stronger potentiation of GABA currents, possibly conveying a stronger antiseizure effect than ganaxolone. There is strong evidence that ganaxolone and its analogs are preferential allosteric modulators and direct activators of extrasynaptic δGABA-A receptors, regulating network inhibition and seizures. Hence, these findings provide a mechanistic rationale for the clinical use of ganaxolone or its analog for OP intoxication [34]. Neurosteroid therapy with ganaxolone partly meet or exceed the expectations of a practical anticonvulsant antidote for OP intoxication in military and civilian persons. It offers many advantages over the current benzodiazepines, including its broad-spectrum efficacy, lack of tolerance upon repeated use, rapid onset and intermediate duration of action, well-characterized mechanism of action, safety profile from clinical trials, and amenability for autoinjector formulations for rapid use by first responders [48–50]. Future studies will ascertain if this drug might be valuable for the treatment of refractory SE, especially OP-induced SE.

6. Conclusion and future research needs

Current treatment for OP intoxication includes a specialized drug combination containing atropine sulfate, 2-PAM and diazepam. However, benzodiazepine anticonvulsants have significant limitations. Recent mechanistic studies with diazepam and midazolam in OP intoxication models support this conclusion. Currently, there are no FDA-approved post-exposure medical countermeasures available to mitigate the effects of OP intoxication [51]. Available pretreatments (pyridostigmine bromide) and post-exposure countermeasures (atropine, 2-PAM, and diazepam) do not effectively prevent or mitigate all symptoms of nerve agent intoxication. Overall, there are urgent unmet medical needs for novel and innovative antidotes to protect the civilians and soldiers against adverse effects of OP nerve agents.

Recently, neurosteroids have been proposed as effective anticonvulsant antidotes than benzodiazepines for OP poisonings. Dual acting neurosteroids at extrasynaptic and synaptic GABA-A receptors appears to be more effective than benzodiazepines to control seizures, even when administered very late after OP exposure. Neurosteroids are able to mitigate the chronic neurological effects caused by OP neurotoxicity. Neurosteroids can produce synergistic protection in combination with midazolam, making them practical medical countermeasures for OP attacks. Ganaxolone is being considered for advanced development and FDA approval for treatment of OP poisonings. Future studies will ascertain the utility of neurosteroid-benzodiazepine combination and its biological variability for treating OP seizures and SE [52].

Highlights.

• Organophosphate (OP) agents are among the most toxic of known chemical agents.

• Current antidotes are ineffective for delayed postexposure treatment of OP neurotoxicity.

• Loss of inhibitory interneurons and neuroinflammation may underlie drug resistance.

• Neurosteroids are proposed as next-generation anticonvulsants superior to current therapies.

• Ganaxolone appears suitable for development as a future anticonvulsant for OP agents.