The main goal of this CounterACT application is to develop a novel class of therapeutics that will mitigate the morbidity and mortality caused by acute exposure to diisopropylfluorophosphate (DFP), a lethal organophosphate insecticide that is considered a credible chemical threat. Organophosphates (OP) are irreversible cholinesterase inhibitors and OP intoxication following exposure to DFP and related military nerve agents results in persistent seizures, status epilepticus (SE) and permanent brain injury. Current medical countermeasures for acute OP intoxication do not sufficiently protect the brain from SE, a prolonged seizure activity lasting 30 min or longer with significant neuronal injury and mortality. We propose that neurosteroids that promote tonic inhibition produce rapid and effective protection against persistent SE, prevent irreversible brain injury, and extend the therapeutic window. This novel therapeutic strategy is based on the emerging molecular mechanisms of neurosteroids and also cellular changes involved in SE, a common toxicity by OP-like agents. Neurosteroids are steroids synthesized locally within the brain that control inhibition. Neurosteroids are the most powerful anticonvulsants against seizures induced by cholinergic agents. Since OP nerve agents cause persistent seizures and brain damage by cholinergic hyperactivation, it is proposed that neurosteroids are an effective antidote for OP intoxication. Neurosteroids act principally at GABA-A receptors, which mediate phasic and tonic inhibition, that play a critical role in controlling seizures by """"""""setting"""""""" the baseline excitabilit. Recent work has shown that SE cause a significant decrease in synaptic inhibition with minimal changes in extrasynaptic (neurosteroid-sensitive) tonic inhibition. Therefore, enhanced sensitivity at extrasynaptic GABA-A receptors and maximally stimulating efficacy at synaptic receptors makes neurosteroids ideal new drugs for controlling SE and neuronal damage following OP intoxication. This novel treatment strategy has not been tested previously. Our preliminary studies in the DFP model of OP intoxication demonstrate the feasibility and promising efficacy of this rationale therapy. We hypothesize that neurosteroids and selective drugs that enhance phasic and extrasynaptic tonic inhibition effectively control OP intoxication-induced SE and neuronal damage. To test this hypothesis, we will address two specific aims:
(Aim 1) : Determine the efficacy of simultaneous augmentation of synaptic and tonic inhibition by ganaxolone in DFP intoxication;
and (Aim 2) : Determine the efficacy of selective augmentation of extrasynaptic tonic inhibition by gaboxadol in DFP intoxication. Test drugs will be given 40 min, 1 hr or 2 hr after exposure to DFP. Behavioral and EEG seizures will be recorded for assessment of drug efficacy, and neuroprotection will be assessed by counting neuron density by immunohistochemical techniques. The proposed studies will provide """"""""proof-of-efficacy"""""""" of the new therapy. Synthetic neurosteroids such as ganaxolone are undergoing clinical trials and have shown an excellent safety record. Therefore, the outcome of this research will set the stage for rapid development of neurosteroids as effective countermeasures against chemical threats.
The insecticide DFP and related OP compounds are high priority chemical threat agents. Current medical countermeasures for acute OP intoxication do not sufficiently protect the brain from seizures and SE, which is characterized by a prolonged seizure activity with significant neuronal injury and mortality. Control of persistent seizure actiity is critical for neuroprotection and survival following OP intoxication. The main therapeutic challenges are the short therapeutic window of current antiseizure drugs and the lack of effective drugs to prevent subsequent brain injury. This CounterACT project is designed to develop neurosteroids as specific treatment for OP intoxication, persistent seizure activity, and brain damage. Synthetic neurosteroids are undergoing clinical trials for epilepsy and are readily available for rapid development for clinical use in treating OP intoxication.
