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

Abstract

Organophosphates (OPs) and nerve agents are potent neurotoxic compounds that cause seizures, status epilepticus (SE), brain injury, or death. There are persistent long-term neurologic and neurodegenerative effects that manifest months to years after the initial exposure. Current antidotes are ineffective in preventing these long-term neurobehavioral and neuropathological changes. Additionally, there are few effective neuroprotectants for mitigating the long-term effects of acute OP intoxication. We have pioneered neurosteroids as novel anticonvulsants and neuroprotectants for OP intoxication and seizures. In this study, we evaluated the efficacy of two novel synthetic, water-soluble neurosteroids, valaxanolone (VX) and lysaxanolone (LX), in combating the long-term behavioral and neuropathological impairments caused by acute OP intoxication and SE. Animals were exposed to the OP nerve agent surrogate diisopropylfluorophosphate (DFP) and were treated with VX or LX in addition to midazolam at 40 minutes postexposure. The extent of neurodegeneration, along with various behavioral and memory deficits, were assessed at 3 months postexposure. VX significantly reduced deficits of aggressive behavior, anxiety, memory, and depressive-like traits in control (DFP-exposed, midazolam-treated) animals; VX also significantly prevented the DFP-induced chronic loss of NeuN(+) principal neurons and PV(+) inhibitory neurons in the hippocampus and other regions. Additionally, VX-treated animals exhibited a reduced inflammatory response with decreased GFAP(+) astrogliosis and IBA1(+) microgliosis in the hippocampus, amygdala, and other regions. Similarly, LX showed significant improvement in behavioral and memory deficits, and reduced neurodegeneration and cellular neuroinflammation. Together, these results demonstrate the neuroprotectant effects of the novel synthetic neurosteroids in mitigating the long-term neurologic dysfunction and neurodegeneration associated with OP exposure. SIGNIFICANCE STATEMENT Survivors of nerve agents and organophosphate (OP) exposures suffer from long-term neurological deficits. Currently, there is no specific drug therapy for mitigating the impact of OP exposure. However, novel synthetic neurosteroids that activate tonic inhibition provide a viable option for treating OP intoxication. The data from this study indicates the neuroprotective effects of synthetic, water-soluble neurosteroids for attenuation of long-term neurological deficits after OP intoxication. These findings establish valaxanolone and lysaxanolone as potent and efficacious neuroprotectants suitable for injectable dosing.:

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

“…In contrast to midazolam, neurosteroid treatment reduces DFP-induced SRS, epileptiform discharges, and HFOs, along with reduced interictal events, possibly explaining their disease-modifying features. These effects are consistent with neuroprotectant outcomes in neuropathological and behavior tests ( Reddy et al, 2024 ), indicating a robust protective activity of neurosteroid treatments. Further studies are needed to advance the potential of synthetic neurosteroids in mitigating the long-term neurologic effects following OP intoxication.…”

“…Despite its superior protective effects, GX has certain limitations such as poor bioavailability, lack of aqueous solubility, short half-life, and rapid hepatic inactivation ( Reddy, 2023 ; 2024 ). We have developed novel analogs to overcome these limitations ( Reddy et al, 2024 ). Synthetic neurosteroids with greater water solubility and bioavailability offer a superior therapeutic profile for clinical use.…”

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.…”

“…Many analogs of brexanolone and ganaxolone structure have been designed and tested for anticonvulsant efficacy ( Reddy and Kulkarni, 2000 ; Hogenkamp et al, 2014 ; Qian et al, 2014 ; Blanco et al, 2018 ; Zorumski et al, 2019 ; Reddy, 2023 ). Several water-soluble analogs were synthesized (superganaxolones) with improved hydrophilicity, an essential feature for intravenous and injectable formulations ( Reddy, 2023 ; Reddy et al, 2024 ). These novel analogs are designed to exhibit subunit selectivity and preferential interaction with extrasynaptic GABA-ARs to achieve greater therapeutic outcomes than GX ( Fig.…”

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

“…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 ). These findings provide insights into the potential of hydrophilic neurosteroid analogs to overcome the limitations of current neurosteroid products and provide new options for developing effective treatments for nerve agent-induced seizures and neurodegeneration.…”