Protective Activity of Novel Hydrophilic Synthetic Neurosteroids on Organophosphate Status Epilepticus-induced Chronic Epileptic Seizures, Non-Convulsive Discharges, High-Frequency Oscillations, and Electrographic Ictal Biomarkers

Abstract

Nerve agents and organophosphates (OP) are neurotoxic chemicals that induce acute seizures, status epilepticus (SE), and mortality. Long-term neurologic and neurodegenerative effects manifest months to years after OP exposure. Current benzodiazepine anticonvulsants are ineffective in preventing such long-term neurobehavioral and neuropathological changes. New and effective anticonvulsants are needed for OP intoxication, especially for mitigating the long-term sequelae after acute exposure. We developed neurosteroids as novel anticonvulsants and neuroprotectants in OP exposure models. In this study, we evaluated the long-term efficacy of novel synthetic neurosteroids in preventing the development of chronic epilepsy and hyperexcitable ictal events in a rat OP model of SE. Rats were exposed to the OP nerve agent surrogate diisopropylfluorophosphate (DFP), and the experimental groups were treated with the synthetic neurosteroid valaxanolone (VX) or lysaxanolone (LX) 40 minutes post-exposure in conjunction with midazolam. Video-electroencephalography was monitored for two months to assess spontaneous recurrent seizures (SRS), epileptiform discharges, interictal spikes, and high-frequency oscillations (HFOs). Within 60 days of DFP exposure, rats developed chronic epilepsy characterized by frequent SRS, epileptiform discharges, and HFOs. LX treatment was associated with a dose-dependent reduction of epilepsy occurrence and overall seizure burden with a significant decrease in SRS and epileptiform discharges. It also significantly reduced the occurrence of epileptic biomarkers of HFOs and interictal spikes, indicating potential disease-modifying activity. Similarly, the neurosteroid analog VX also significantly attenuated SRS, discharges, HFOs, and ictal events. These results demonstrate the long-term protective effects of synthetic neurosteroids in the OP-exposed post-SE model, indicating their disease-modifying potential to prevent epilepsy and ictal abnormalities. SIGNIFICANCE STATEMENT The effects of nerve agents and organophosphate (OP) exposure are persistent, and survivors suffer from a number of devastating, chronic neurological dysfunctions. Currently, there is no specific therapy for preventing this disastrous impact of OP exposure. We propose synthetic neurosteroids that activate tonic inhibition provide viable options for preventing the long-term neurological effects of OP intoxication. The results from this study reveal the disease-modifying potential of two novel synthetic neurosteroids in preventing epileptogenesis and chronic epileptic seizures after OP-induced SE.

Neuroprotectant Activity of Novel Water-Soluble Synthetic Neurosteroids on Organophosphate Intoxication and Status Epilepticus-Induced Long-Term Neurological Dysfunction, Neurodegeneration, and Neuroinflammation

“…The observed improvements in neuropathological defects align with the improvement of neurologic functional deficits. As projected, VX and LX elicited protection against OP-induced SE, chronic epileptic seizures, and ictal biomarkers in the DFP model ( Ramakrishnan et al, 2024 ). Together, these findings establish VX and LX as potent and efficacious synthetic neurosteroids with drug-like properties suitable for counteracting the long-term neuropsychiatric sequel following OP exposure.…”

“…Neurosteroid analogs decreased degeneration of neurons, specifically inhibitory interneurons in the hippocampus and other regions. Moreover, they exhibit striking disease-modifying effects on DFP-induced long-term epileptic seizures and discharges ( Ramakrishnan et al, 2024 ). These mechanistic and neuropathological improvements likely led to the protective effects observed in seizure outcomes.…”

“…VX and LX were designed to improve hydrophilic properties for better bioavailability and tissue distribution than ganaxolone. In addition, they decreased the occurrence of chronic epileptic seizures and ictal biomarkers in the DFP-induced refractory SE model ( Ramakrishnan et al, 2024 ). Collectively, these findings demonstrate the application of these synthetic neurosteroids for ameliorating the long-term neurologic consequences after OP intoxication with drug-like properties for hospital and field-ready injectable dosing.…”

Neurosteroids as Novel Anticonvulsants for Refractory Status Epilepticus and Medical Countermeasures for Nerve Agents: A 15-Year Journey to Bring Ganaxolone from Bench to Clinic

“…When tested in animal models of OPNA-induced RSE, these lead analogs effectively blocked RSE when given intramuscularly 40 minutes after DFP administration, indicating their efficacy in the DFP model. The lead analogs significantly reduced neuronal injury, neurodegeneration, and inflammation in the hippocampus and other regions ( Ramakrishnan et al, 2024 ; Reddy et al, 2024 ), suggesting their neuroprotectant potential to mitigate OPNA-induced brain damage. The most promising drug-like compounds are in advanced development as anticonvulsants and neuroprotectants for SE indications.…”

Progress and Challenges in Developing Medical Countermeasures for Chemical, Biological, Radiological, and Nuclear Threat Agents

“…Reddy and team have developed novel hydrophilic neurosteroids, such as valaxanolone and lysaxanolone, showing promising results in animal studies. In an antiseizure study, Reddy’s team reported valaxanolone and lysaxanolone effectively protected against DFP-induced epileptic seizures and electrographic biomarkers of ictal activity ( Ramakrishnan et al, 2024 ). In a neuroprotection study, Reddy and the team found that valaxanolone and lysaxanolone significantly improved behavioral and memory deficits, correlated with their ability to reduce neurodegeneration and neuroinflammation ( Reddy et al, 2024 ).…”