Enaminone Modulators of Extrasynaptic α4β3δ γ-Aminobutyric AcidA Receptors Reverse Electrographic Status Epilepticus in the Rat After Acute Organophosphorus Poisoning.

Timothy B C Johnstone,F Edward Dudek,Kelvin W Gee,John H Mcdonough,Hilary S Mccarren,Jay Spampanato,Derk Hogenkamp

doi:10.3389/fphar.2019.00560

Timothy B C Johnstone, F Edward Dudek + Show 5 more

Open Access

https://doi.org/10.3389/fphar.2019.00560

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Abstract

Seizures induced by organophosphorus nerve agent exposure become refractory to treatment with benzodiazepines because these drugs engage synaptic γ-aminobutyric acid-A receptors (GABAARs) that rapidly internalize during status epilepticus (SE). Extrasynaptic GABAARs, such as those containing α4β3δ subunits, are a putative pharmacological target to comprehensively manage nerve agent-induced seizures since they do not internalize during SE and are continuously available for activation. Neurosteroids related to allopregnanolone have been tested as a possible replacement for benzodiazepines because they target both synaptic and extrasynaptic GABAARs receptors. A longer effective treatment window, extended treatment efficacy, and enhanced neuroprotection represent significant advantages of neurosteroids over benzodiazepines. However, neurosteroid use is limited by poor physicochemical properties arising from the intrinsic requirement of the pregnane steroid core structure for efficacy rendering drug formulation problematic. We tested a non-steroidal enaminone GABAAR modulator that interacts with both synaptic and extrasynaptic GABAARs on a binding site distinct from neurosteroids or benzodiazepines for efficacy to control electrographic SE induced by diisopropyl fluorophosphate or soman intoxication in rats. Animals were treated with standard antidotes, and experimental therapeutic treatment was given following 1 h (diisopropyl fluorophosphate model) or 20 min (soman model) after SE onset. We found that the enaminone 2-261 had an extended duration of seizure termination (>10 h) in the diisopropyl fluorophosphate intoxication model in the presence or absence of midazolam (MDZ). 2-261 also moderately potentiated MDZ in the soman-induced seizure model but had limited efficacy as a stand-alone anticonvulsant treatment due to slow onset of action. 2-261 significantly reduced neuronal death in brain areas associated with either diisopropyl fluorophosphate- or soman-induced SE. 2-261 represents an alternate chemical template from neurosteroids for enhancing extrasynaptic α4β3δ GABAAR activity to reverse SE from organophosphorous intoxication.

Highlights

Acute organophosphorus poisoning is a military and civilian threat that causes a complex cascade of lethal central and peripheral nervous system toxicities
2-261 was ineffective on EEG measures when tested at 60 mg/kg intraperitoneal alone or in combination with MDZ after diisopropyl fluorophosphate intoxication. 2-261 alone significantly reduced neuronal death as measured by FluoroJade B staining relative to MDZ alone in piriform cortex, thalamus, entorhinal cortex, and multiple areas of the hippocampus, consistent with the long duration of anticonvulsant effect on the EEG (Figure 2A). 2-261 did not cause reductions in FluoroJade B staining in the parietal cortex or amygdala and slightly enhanced FluoroJadeB staining in the hilus (Figre 2A)
We tested whether a non-sedating enaminone, 2-261 (Gee et al, 2010; Yoshimura et al, 2017), could be an alternative to other extrasynaptic GABAARs modulators to reverse organophosphosphorus nerve agent-induced SE

Summary

Introduction

Acute organophosphorus poisoning is a military and civilian threat that causes a complex cascade of lethal central and peripheral nervous system toxicities. Organophosphorus nerve agents cause levels of acetylcholine to accumulate and remain at synapses, leading to overstimulation of peripheral and central muscarinic/nicotinic receptors (McDonough and Shih, 1997; Watson et al, 2009). In vitro studies with soman in hippocampal slices suggest that a 30% reduction of surface expression of GABAARs renders hippocampal neurons unresponsive to diazepam (Wang et al, 2011). These remaining GABAARs may be extrasynaptic receptors responsible for setting the background tone of global neuronal inhibition (Farrant and Nusser, 2005; Chuang and Reddy, 2018). Extrasynaptic GABAARs could be a critical pharmacological target to control soman-induced SE since benzodiazepines are almost inactive at GABAAR subtypes containing δ-subunits (Reddy, 2016; Reddy and Estes, 2016; Chuang and Reddy, 2018) and depend on the presence of synaptic γ-subunits for GABAAR receptor binding and modulation (Pritchett et al, 1989)

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