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

This Commentary delves into the current progress and challenges on ongoing research on medical countermeasures (MCs) for chemical, biologic, radiologic, and nuclear (CBRN) threats. CBRN agents pose a serious risk to human health and safety, with the potential for mass casualties in both military and civilian settings. Chemical threats are toxic compounds that could be used in a terrorist attack, an accidental release, or chemical warfare. They include nerve agents, organophosphates, pulmonary agents, metabolic/cellular agents, vesicants, ocular toxicants, and opioid agents. Developing effective MCs is crucial for mitigating the acute and chronic effects of exposure to CBRN agents. The papers in this special issue of JPET highlights the latest advancements in MC research, showcasing insightful outcomes on experimental models, mechanisms, and translational research on MCs for CBRN threats. They portray several notable contributions, including the development of neurosteroid and combination anticonvulsant therapies for nerve agent poisoning, the exploration of chronic impacts and diagnostic tracers for OP neurotoxicity, the establishment of innovative pediatric OP models, the identification of novel molecules for ocular, pulmonary and vesicant injuries, and the repurposing of existing drugs for the treatment of botulism, cyanide, and OP poisoning. These crucial outcomes underscore the breadth of current research covering a variety of chemical threats. Overall, this collection of articles highlights the importance of ongoing research and development in the field of MCs, emphasizing the potential of these countermeasures to effectively treat and mitigate the effects of toxicant exposures and thereby enhance our preparedness for mass casualty incidents. SIGNIFICANCE STATEMENT: CBRN agents pose a significant threat to public health. Effective MCs exist for certain chemical threats, but there is a need for new and improved MCs for many others. The research presented in this special issue of JPET highlights the latest advancements in MCs for CBRN threats. This research has the potential to lead to the development of new and repurposed MCs that are more effective, broad-spectrum, and easier to administer to mitigate acute and long-term consequences of chemical exposures.

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.

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.

A Pediatric Rat Model of Organophosphate-Induced Refractory Status Epilepticus: Characterization of Long-Term Epileptic Seizure Activity, Neurologic Dysfunction and Neurodegeneration.

Children are highly vulnerable to the neurotoxic effects of organophosphates (OPs), which can cause neuronal developmental defects, including intellectual disability, autism, epilepsy, and related comorbidities. Unfortunately, no specific pediatric OP neurotoxicity model currently exists. In this study, we developed and characterized a pediatric rat model of status epilepticus (SE) induced by the OP diisopropylfluorophosphate (DFP) and examined its impact on long-term neurological outcomes. Postnatal day 21 rats were exposed to a DFP regimen with standard antidotes. Progressive behavioral deteriorations were assessed over a three-month period. Development of epileptic seizures, ictal discharges, high-frequency oscillations (HFOs), and interictal spikes were monitored by video-electroencephalography recordings. Histology-stereology analysis was performed to assess neurodegeneration, neuroinflammation, and morphologic abnormalities. DFP-exposed, post-SE animals exhibited significantly elevated levels of anxiety and depression than age-matched controls at 1, 2, and 3 months post-exposure. DFP-exposed animals displayed aggressive behavior and a marked decline in object recognition memory, as well as prominent impairment in spatial learning and memory. DFP-exposed animals had striking electrographic abnormalities with the occurrence of displayed epileptic seizures, ictal discharges, HFOs, and interictal spikes, suggesting chronic epilepsy. Neuropathological analysis showed substantially fewer principal neurons and inhibitory interneurons with a marked increase in reactive microglia and neuroinflammation in the hippocampus and other brain regions. DFP-exposed animals also exhibited mossy fiber sprouting indicating impaired network formations. Long-term epileptic seizures and neuropsychiatric functional deficits induced by DFP were consistent with neuropathological defects. Collectively, this pediatric model displays many hallmarks of chronic sequelae reminiscent of children exposed to OPs, suggesting that it will be a valuable tool for investigating pathologic mechanisms and potential treatment strategies to attenuate long-term OP neurotoxicity. SIGNIFICANCE STATEMENT: Millions of children are exposed to organophosphates (OPs) used in agriculture or chemical incidents. This study investigated the long-term impact of neonatal exposure to the OP chemical diisopropylfluorophosphate (DFP) on neurobehavioral and neurodevelopmental outcomes in adulthood. DFP exposure caused long-lasting behavioral abnormalities, epileptic seizures, and bilateral brain defects with an array of neurological sequelae seen in children's OP neurotoxicity. Thus, this model provides a novel tool to explore therapeutic interventions that mitigate long-term neurotoxic effects of children exposed to OP-induced seizures and status epilepticus.

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.

This article describes recent advances in the use of neurosteroids as novel anticonvulsants for refractory status epilepticus (RSE) and as medical countermeasures (MCs) for organophosphates and chemical nerve agents (OPNAs). We highlight a comprehensive 15-year journey to bring the synthetic neurosteroid ganaxolone (GX) from bench to clinic. RSE, including when caused by nerve agents, is associated with devastating morbidity and permanent long-term neurologic dysfunction. Although recent approval of benzodiazepines such as intranasal midazolam and intranasal midazolam offers improved control of acute seizures, novel anticonvulsants are needed to suppress RSE and improve neurologic function outcomes. Currently, few anticonvulsant MCs exist for victims of OPNA exposure and RSE. Standard-of-care MCs for postexposure treatment include benzodiazepines, which do not effectively prevent or mitigate seizures resulting from nerve agent intoxication, leaving an urgent unmet medical need for new anticonvulsants for RSE. Recently, we pioneered neurosteroids as next-generation anticonvulsants that are superior to benzodiazepines for treatment of OPNA intoxication and RSE. Because GX and related neurosteroids that activate extrasynaptic GABA-A receptors rapidly control seizures and offer robust neuroprotection by reducing neuronal damage and neuroinflammation, they effectively improve neurologic outcomes after acute OPNA exposure and RSE. GX has been selected for advanced, Biomedical Advanced Research and Development Authority-supported phase 3 trials of RSE and nerve agent seizures. In addition, in mechanistic studies of neurosteroids at extrasynaptic receptors, we identified novel synthetic analogs with features that are superior to GX for current medical needs. Development of new MCs for RSE is complex, tedious, and uncertain due to scientific and regulatory challenges. Thus, further research will be critical to fill key gaps in evaluating RSE and anticonvulsants in vulnerable (pediatric and geriatric) populations and military persons. SIGNIFICANCE STATEMENT: Following organophosphate and nerve agent intoxication, refractory status epilepticus (RSE) occurs despite benzodiazepine treatment. RSE occurs in 40% of status epilepticus patients, with a 35% mortality rate and significant neurological morbidity in survivors. To treat RSE, neurosteroids are better anticonvulsants than benzodiazepines. Our pioneering use of neurosteroids for RSE and nerve agents led us to develop ganaxolone as a novel anticonvulsant and neuroprotectant with significantly improved neurological outcomes. This article describes the bench-to-bedside journey of bringing neurosteroid therapy to patients, with ganaxolone leading the way.

Long-Term Neuropsychiatric Developmental Defects after Neonatal Organophosphate Exposure: Mitigation by Synthetic Neurosteroids.

Children are much more susceptible to the neurotoxic effects of organophosphate (OP) pesticides and nerve agents than adults. OP poisoning in children leads to acute seizures and neuropsychiatric sequela, including the development of long-term disabilities and cognitive impairments. Despite these risks, there are few chronic rodent models that use pediatric OP exposure for studying neurodevelopmental consequences and interventions. Here, we investigated the protective effect of the neurosteroid ganaxolone (GX) on the long-term developmental impact of neonatal exposure to the OP compound, diisopropyl-fluorophosphate (DFP). Pediatric postnatal day-28 rats were acutely exposed to DFP, and at 3 and 10 months after exposure, they were evaluated using a series of cognitive and behavioral tests with or without the postexposure treatment of GX. Analysis of the neuropathology was performed after 10 months. DFP-exposed animals displayed significant long-term deficits in mood, anxiety, depression, and aggressive traits. In spatial and nonspatial cognitive tests, they displayed striking impairments in learning and memory. Analysis of brain sections showed significant loss of neuronal nuclei antigen(+) principal neurons, parvalbumin(+) inhibitory interneurons, and neurogenesis, along with increased astrogliosis, microglial neuroinflammation, and mossy fiber sprouting. These detrimental neuropathological changes are consistent with behavioral dysfunctions. In the neurosteroid GX-treated cohort, behavioral and cognitive deficits were significantly reduced and were associated with strong protection against long-term neuroinflammation and neurodegeneration. In conclusion, this pediatric model replicates the salient features of children exposed to OPs, and the protective outcomes from neurosteroid intervention support the viability of developing this strategy for mitigating the long-term effects of acute OP exposure in children. SIGNIFICANCE STATEMENT: An estimated 3 million organophosphate exposures occur annually worldwide, with children comprising over 30% of all victims. Our understanding of the neurodevelopmental consequences in children exposed to organophosphates is limited. Here, we investigated the long-term impact of neonatal exposure to diisopropyl-fluorophosphate in pediatric rats. Neurosteroid treatment protected against major deficits in behavior and memory and was well correlated with neuropathological changes. Overall, this pediatric model is helpful to screen novel therapies to mitigate long-term developmental deficits of organophosphate exposure.

Sex Differences in Organophosphate Model of Benzodiazepine-Refractory Status Epilepticus and Neuronal Damage.

Sex differences are common in human epilepsy. Although men are more susceptible to seizure than women, the mechanisms underlying sex-specific vulnerabilities to seizure are unclear. The organophosphate (OP) diisopropylfluorophosphate (DFP) is known to cause neurotoxicity and status epilepticus (SE), a serious neurologic condition that causes prolonged seizures and brain damage. Current therapies for OP poisoning and SE do not consider neuronal variations between male and female brains. Therefore, we investigated sex-dependent differences in electrographic seizure activity and neuronal injury using the DFP model of refractory SE in rats. Electroencephalogram recordings were used to monitor DFP-induced SE, and the extent of brain injury was determined using fluoro-jade-B staining to detect cellular necrosis. After DFP exposure, we observed striking sex-dependent differences in SE and seizure activity patterns as well as protective responses to midazolam treatment. Following acute DFP exposure, male animals displayed more severe SE with intense epileptiform spiking and greater mortality than females. In contrast, we observed significantly more injured cells and cellular necrosis in the hippocampus and other brain regions in females than in males. We also observed extensive neuronal injury in the somatosensory cortex of males. The anticonvulsant effect of midazolam against SE was limited in this model and found to be similar in males and females. However, unlike males, females exhibited substantially more protection against neuronal damage after midazolam treatment. Overall, these results demonstrate significant sex-dependent differences in DFP-induced refractory SE and neuronal damage patterns, suggesting that it may be possible to develop sex-specific neuroprotective strategies for OP intoxication and refractory SE. SIGNIFICANCE STATEMENT: Sex-dependent differences in neurotoxicity and status epilepticus (SE) are key biological variables after organophosphate (OP) exposure. Here, we investigated sex-dependent differences in SE and brain injury after acute diisopropylfluorophosphate exposure. Male rats had more severe SE and less survival than females, while females had more neuronal damage. Females had more neuroprotection to midazolam than males, while both sexes had similar but partial anticonvulsant effects. These findings suggest that a sex-specific therapeutic approach may prevent neurological complications of 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.

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.

Neuroendocrine insights into neurosteroid therapy for postpartum depression.

Postpartum depression (PPD) is associated with a decline in progesterone-derived anxiolytic-antidepressant neurosteroids after delivery. Neurosteroid replacement therapy (NRT) with GABA-A receptor-modulating allopregnanolone (brexanolone) shows promise as the first drug treatment for PPD. Here we describe the molecular insights of the neurosteroid approach for rapid relief of PPD symptoms compared with traditional antidepressants.

Preclinical and clinical pharmacology of brexanolone (allopregnanolone) for postpartum depression: a landmark journey from concept to clinic in neurosteroid replacement therapy.

This article describes the critical role of neurosteroids in postpartum depression (PPD) and outlines the landmark pharmacological journey of brexanolone as a first-in-class neurosteroid antidepressant with significant advantages over traditional antidepressants. PPD is a neuroendocrine disorder that affects about 20% of mothers after childbirth and is characterized by symptoms including persistent sadness, fatigue, dysphoria, as well as disturbances in cognition, emotion, appetite, and sleep. The main pathology behind PPD is the postpartum reduction of neurosteroids, referred to as neurosteroid withdrawal, a concept pioneered by our preclinical studies. We developed neurosteroid replacement therapy (NRT) as a rational approach for treating PPD and other conditions related to neurosteroid deficiency, unveiling the power of neurosteroids as novel anxiolytic-antidepressants. The neurosteroid, brexanolone (BX), is a progesterone-derived allopregnanolone that rapidly relieves anxiety and mood deficits by activating GABA-A receptors, making it a transformational treatment for PPD. In 2019, the FDA approved BX, an intravenous formulation of allopregnanolone, as an NRT to treat PPD. In clinical studies, BX significantly improved PPD symptoms within hours of administration, with tolerable side effects including headache, dizziness, and somnolence. We identified the molecular mechanism of BX in a neuronal PPD-like milieu. The mechanism of BX involves activation of both synaptic and extrasynaptic GABA-A receptors, which promote tonic inhibition and serve as a key target for PPD and related conditions. Neurosteroids offer several advantages over traditional antidepressants, including rapid onset, unique mechanism, and lack of tolerance upon repeated use. Some limitations of BX therapy include lack of aqueous solubility, limited accessibility, hospitalization for treatment, lack of oral product, and serious adverse events at high doses. However, the unmet need for synthetic neurosteroids to address this critical condition supersedes these limitations. Recently, we developed novel hydrophilic neurosteroids with a superior profile and improved drug delivery. Overall, approval of BX is a major milestone in the field of neurotherapeutics, paving the way for the development of novel synthetic neurosteroids to treat depression, epilepsy, and status epilepticus.

Experimental Models of Gulf War Illness, a Chronic Neuropsychiatric Disorder in Veterans.

Gulf War illness (GWI) is a chronic multifaceted condition with debilitating pain and fatigue, as well as sleep, behavioral, and cognitive impairments in war veterans. Currently, there is no effective treatment or cure for GWI; therefore, there is a critical need to develop experimental models to help better understand its mechanisms and interventions related to GWI-associated neuropsychiatric disorders. Chemical neurotoxicity appears to be one cause of GWI, and its symptoms manifest as disruptions in neuronal function. However, the mechanisms underlying such incapacitating neurologic and psychiatric symptoms are poorly understood. The etiology of GWI is complex, and many factors including chemical exposure, psychological trauma, and environmental stressors have been associated with its development. Attempts have been made to create GWI-like symptomatic models, including through chronic induction in mice and rats. Here, we present a brief protocol of GWI in rats and mice, which exhibit robust neuropsychiatric signs and neuropathologic changes reminiscent of GWI. This article provides a guide to working protocols, application of therapeutic drugs, outcomes, troubleshooting, and data analysis. Our broad profiling of GWI-like symptoms in rodents reveals features of progressive morphologic and long-lasting neuropsychiatric features. Together, the GWI model in rodents shows striking consistency in recapitulating major hallmark features of GWI in veterans. These models help identify mechanisms and interventions for GWI. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Experimental induction of Gulf War illness in rats Support Protocol 1: Monitoring of Gulf War illness signs and neuroimaging analysis in rats Basic Protocol 2: Experimental induction of Gulf War illness in mice Support Protocol 2: Monitoring of Gulf War illness signs and neuropathology analysis in mice.

Pathophysiological basis and promise of experimental therapies for Gulf War Illness, a chronic neuropsychiatric syndrome in veterans.

This article describes the pathophysiology and potential treatments for Gulf War Illness (GWI), which is a chronic neuropsychiatric illness linked to a combination of chemical exposures experienced by service personnel during the first Gulf War in 1991. However, there is currently no effective treatment for veterans with GWI. The article focuses on the current status and efficacy of existing therapeutic interventions in preclinical models of GWI, as well as potential perspectives of promising therapies. GWI stems from changes in brain and peripheral systems in veterans, leading to neurocognitive deficits, as well as physiological and psychological effects resulting from multifaceted changes such as neuroinflammation, oxidative stress, and neuronal damage. Aging not only renders veterans more susceptible to GWI symptoms, but also attenuates their immune capabilities and response to therapies. A variety of experimental models are being used to investigate the pathophysiology and develop therapies that have the ability to alleviate devastating symptoms. Over two dozen therapeutic interventions targeting neuroinflammation, mitochondrial dysfunction, neuronal injury, and neurogenesis are being tested, including agents such as curcumin, curcumin nanoparticles, monosodium luminol, melatonin, resveratrol, fluoxetine, rolipram, oleoylethanolamide, ketamine, levetiracetam, nicotinamide riboside, minocycline, pyridazine derivatives, and neurosteroids. Preclinical outcomes show that some agents have promise, including curcumin, resveratrol, and ketamine, which are being tested in clinical trials in GWI veterans. Neuroprotectants and other compounds such as monosodium luminol, melatonin, levetiracetam, oleoylethanolamide, and nicotinamide riboside appear promising for future clinical trials. Neurosteroids have been shown to have neuroprotective and disease-modifying properties, which makes them a promising medicine for GWI. Therefore, accelerated clinical studies are urgently needed to evaluate and launch an effective therapy for veterans displaying GWI.

Clinical efficacy and safety of cannabidiol for pediatric refractory epilepsy indications: A systematic review and meta-analysis.

Antiseizure medications (ASMs) are the mainstay for the treatment of seizure disorders. However, about one-third of people with epilepsy remain refractory to current ASMs. Cannabidiol (CBD) has recently been approved as ASM for three refractory epilepsy syndrome indications in children and adults. In this study, we evaluated the overall clinical potential of an oral CBD to treat refractory epilepsy in patients with Dravet syndrome (DS), Lennox-Gastaut syndrome (LGS), and tuberous sclerosis complex (TSC) through a systematic review and meta-analysis. A comprehensive search of databases was conducted, including randomized controlled trials (RCTs) assessing the effect of CBD in epilepsy patients. The review was conducted as per the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The review focused on RCTs involving patients receiving highly purified oral CBD (Epidiolex, 10 to 50 mg/kg/day) for up to 16 weeks. A subgroup analysis by syndrome and CBD with or without concomitant clobazam was conducted. The key outcomes were reduction in seizure frequency, differences in 50% responder rates, adverse events, and interactions with clobazam as co-therapy. Odds ratio (OR) with 95% confidence interval (CI) were estimated. Of 1183 articles screened, we included 6 RCTs meeting our eligibility criteria. All studies were considered to have a low risk of bias. In the pooled analysis, CBD treatment was found to be more efficacious compared to placebo (OR = 2.45, 95% CI =1.81-3.32, p < 0.01). Subgroup analysis by syndrome demonstrated the odds of ≥50% reduction in seizures with CBD treatment in patients with DS (OR = 2.26, 95% CI:1.38-3.70), LGS (OR = 2.98, 95% CI:1.83-4.85) and TSC (OR = 1.99, 95% CI = 1.06-3.76). Compared with placebo, CBD was associated with increased adverse events (OR = 1.81, 95% CI = 1.33-2.46) such as diarrhea, somnolence, and sedation, and any serious adverse events (OR = 2.86, 95% CI = 1.63-5.05). Other factors, including dosage and clobazam co-therapy, were significantly associated with a greater effect on seizure control and side effects of CBD. In conclusion, the study shows that CBD is highly efficacious both as standalone and adjunct therapy with clobazam for controlling seizures in DS, LGS, and TSC conditions while limiting side effects. Further pharmacodynamic investigation of CBD actions, drug interaction assessments, and therapeutic management guidelines are warranted.

Therapeutic and clinical foundations of cannabidiol therapy for difficult-to-treat seizures in children and adults with refractory epilepsies.

Novel and effective antiseizure medications are needed to treat refractory and rare forms of epilepsy. Cannabinoids, which are obtained from the cannabis plant, have a long history of medical use, including for neurologic conditions. In 2018, the US Food and Drug Administration approved the first phytocannabinoid, cannabidiol (CBD, Epidiolex), which is now indicated for severe seizures associated with three rare forms of developmental and epileptic encephalopathy: Dravet syndrome, Lennox-Gastaut syndrome, and tuberous sclerosis complex. Compelling evidence supports the efficacy of CBD in experimental models and patients with epilepsy. In randomized clinical trials, highly-purified CBD has demonstrated efficacy with an acceptable safety profile in children and adults with difficult-to-treat seizures. Although the underlying antiseizure mechanisms of CBD in humans have not yet been elucidated, the identification of novel antiseizure targets of CBD preclinically indicates multimodal mechanisms that include non-cannabinoid pathways. In addition to antiseizure effects, CBD possesses strong anti-inflammatory and neuroprotective activities, which might contribute to protective effects in epilepsy and other conditions. This article provides a succinct overview of therapeutic approaches and clinical foundations of CBD, emphasizing the clinical utility of CBD for the treatment of seizures associated with refractory and rare epilepsies. CBD has shown to be a safe and effective antiseizure medicine, demonstrating a broad spectrum of efficacy across multiple seizure types, including those associated with severe epilepsies with childhood onset. Despite such promise, there are many perils with CBD that hampers its widespread use, including limited understanding of pharmacodynamics, limited exposure-response relationship, limited information for seizure freedom with continued use, complex pharmacokinetics with drug interactions, risk of adverse effects, and lack of expert therapeutic guidelines. These scientific issues need to be resolved by further investigations, which would decide the unique role of CBD in the management of refractory epilepsy.

Efficacy of the FDA-approved cannabidiol on the development and persistence of temporal lobe epilepsy and complex focal onset seizures.

Presently there is no drug therapy for curing epilepsy. Despite many advancements in epilepsy research, nearly 30% of people with epilepsy remain refractory to current antiseizure medications (ASM). Cannabidiol (CBD) has recently been approved as an ASM for pediatric refractory seizures, but it has not been widely tested for adult epileptogenesis and focal onset seizures. In this study, we investigated the efficacy of the FDA-approved CBD in controlling epileptogenesis and complex focal onset seizures using the mouse kindling model of human temporal lobe epilepsy. We also tested combination regimens of CBD with other ASMs. The two primary outcome measures were disease modification and suppression of generalized seizures. In the epileptogenesis study, CBD had a striking effect in attenuating kindling development, with a dose-dependent decrease in behavioral and electrographic seizure activity. In the retention study, mice previously treated with CBD had significantly reduced overall seizure burden, suggesting disease modification. In a fully-kindled seizure study, CBD produced rapid and atypical U-shaped dose-dependent protection against generalized seizures (ED50, 52 mg/kg, i.p.). In a time-course study, CBD showed a maximal protective effect within 1 h of injection, and it declined within 4 h with a biphasic response. In the combination study, CBD produced synergistic/ additive protection when given with midazolam and ganaxolone but not with tiagabine, indicating its strong potential as an adjunct ASM. Finally, the protective effects of CBD were not associated with motor and functional impairments. These preclinical findings demonstrate the potential of adjunct CBD for controlling adult complex focal onset seizure conditions.

Isobolographic analysis of adjunct antiseizure activity of the FDA-approved cannabidiol with neurosteroids and benzodiazepines in adult refractory focal onset epilepsy.

Epilepsy is a serious neurological disorder associated with recurrent and unpredictable seizures and extensive neuropsychiatric comorbidities. There is no cure for epilepsy, and over one third of epileptic patients have been diagnosed with drug-refractory epilepsy, indicating the critical need for novel antiseizure medications (ASMs). Cannabidiol (CBD) has been shown to decrease seizures in pediatric epilepsies, such as Dravet and Lennox-Gastaut syndromes; however, it has not been rigorously tested for adult seizures or in models of refractory focal epilepsy. Although the exact mechanism is unknown, it is likely to act in a way that is unique to certain GABA-A receptor-modulating drugs, such as neurosteroids and benzodiazepines. In this study, we sought to determine the adjunct antiseizure activity of a clinical CBD product in an adult 6-Hz model of focal refractory epilepsy. CBD was evaluated alone in both a dose-response and time-course manner and in an adjunct combination with two ASMs ganaxolone (neurosteroid) and midazolam (benzodiazepine) against 6-Hz-induced refractory focal onset, generalized seizures. In pharmacological studies, CBD produced dose-dependent protection against seizures (ED50, 53 mg/kg, i.p.) without any side effects. CBD significantly reduced both electrographic activity and behavioral ictal responses with no apparent sex differences. CBD was evaluated in an isobologram design in conjunction with ganaxolone or midazolam at three standard ratios (1:1, 1:3, 3:1). Isobolographic analysis shows the combination regimens of CBD + ganaxolone and CBD + midazolam exerted combination index of 0.313 and 0.164, indicating strong synergism for seizure protection, with little to no toxicity. Together, these results demonstrate the therapeutic potential of CBD monotherapy and as an adjunct therapy for adult focal refractory epilepsy in combination with GABAergic ASMs.

Experimental and Clinical Biomarkers for Progressive Evaluation of Neuropathology and Therapeutic Interventions for Acute and Chronic Neurological Disorders.

This article describes commonly used experimental and clinical biomarkers of neuronal injury and neurodegeneration for the evaluation of neuropathology and monitoring of therapeutic interventions. Biomarkers are vital for diagnostics of brain disease and therapeutic monitoring. A biomarker can be objectively measured and evaluated as a proxy indicator for the pathophysiological process or response to therapeutic interventions. There are complex hurdles in understanding the molecular pathophysiology of neurological disorders and the ability to diagnose them at initial stages. Novel biomarkers for neurological diseases may surpass these issues, especially for early identification of disease risk. Validated biomarkers can measure the severity and progression of both acute neuronal injury and chronic neurological diseases such as epilepsy, migraine, Alzheimer's disease, Parkinson's disease, Huntington's disease, traumatic brain injury, amyotrophic lateral sclerosis, multiple sclerosis, and other brain diseases. Biomarkers are deployed to study progression and response to treatment, including noninvasive imaging tools for both acute and chronic brain conditions. Neuronal biomarkers are classified into four core subtypes: blood-based, immunohistochemical-based, neuroimaging-based, and electrophysiological biomarkers. Neuronal conditions have progressive stages, such as acute injury, inflammation, neurodegeneration, and neurogenesis, which can serve as indices of pathological status. Biomarkers are critical for the targeted identification of specific molecules, cells, tissues, or proteins that dramatically alter throughout the progression of brain conditions. There has been tremendous progress with biomarkers in acute conditions and chronic diseases affecting the central nervous system.

A Comprehensive and Advanced Mouse Model of Post-Traumatic Epilepsy with Robust Spontaneous Recurrent Seizures.

Traumatic brain injury (TBI) is a leading cause of epilepsy in military persons and civilians. Spontaneous recurrent seizures (SRSs) occur in the months or years following the injury, which is commonly referred to as post-traumatic epilepsy (PTE). Currently, there is no effective treatment or cure for PTE; therefore, there is a critical need to develop animal models to help further understand and assess mechanisms and interventions related to TBI-induced epilepsy. Despite many attempts to induce PTE in animals, success has been limited due to a lack of consistent SRSs after TBI. We present a comprehensive protocol to induce PTE after contusion brain injury in mice, which exhibit robust SRSs along with neurodegeneration and neuroinflammation. This article provides a complete set of protocols for injury, outcomes, troubleshooting, and data analysis. Our broad profiling of a TBI mouse reveals features of progressive, long-lasting epileptic activity, hippocampal sclerosis, and comorbid mood and memory deficits. Overall, the PTE mouse shows striking consistency in recapitulating major hallmark features of human PTE. This mouse model will be helpful in assessing mechanisms of and interventions for TBI-induced epileptogenesis, epilepsy, and neuropsychiatric dysfunction. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Inducing controlled cortical impact injuries Support Protocol: Creating the custom domed camp Basic Protocol 2: Recording long-term video-EEG signals Basic Protocol 3: Analyzing video-EEG recordings.

Post-Traumatic Epilepsy and Comorbidities: Advanced Models, Molecular Mechanisms, Biomarkers, and Novel Therapeutic Interventions.

Post-traumatic epilepsy (PTE) is one of the most devastating long-term, network consequences of traumatic brain injury (TBI). There is currently no approved treatment that can prevent onset of spontaneous seizures associated with brain injury, and many cases of PTE are refractory to antiseizure medications. Post-traumatic epileptogenesis is an enduring process by which a normal brain exhibits hypersynchronous excitability after a head injury incident. Understanding the neural networks and molecular pathologies involved in epileptogenesis are key to preventing its development or modifying disease progression. In this article, we describe a critical appraisal of the current state of PTE research with an emphasis on experimental models, molecular mechanisms of post-traumatic epileptogenesis, potential biomarkers, and the burden of PTE-associated comorbidities. The goal of epilepsy research is to identify new therapeutic strategies that can prevent PTE development or interrupt the epileptogenic process and relieve associated neuropsychiatric comorbidities. Therefore, we also describe current preclinical and clinical data on the treatment of PTE sequelae. Differences in injury patterns, latency period, and biomarkers are outlined in the context of animal model validation, pathophysiology, seizure frequency, and behavior. Improving TBI recovery and preventing seizure onset are complex and challenging tasks; however, much progress has been made within this decade demonstrating disease modifying, anti-inflammatory, and neuroprotective strategies, suggesting this goal is pragmatic. Our understanding of PTE is continuously evolving, and improved preclinical models allow for accelerated testing of critically needed novel therapeutic interventions in military and civilian persons at high risk for PTE and its devastating comorbidities. SIGNIFICANCE STATEMENT: Post-traumatic epilepsy is a chronic seizure condition after brain injury. With few models and limited understanding of the underlying progression of epileptogenesis, progress is extremely slow to find a preventative treatment for PTE. This study reviews the current state of modeling, pathology, biomarkers, and potential interventions for PTE and comorbidities. There's new optimism in finding a drug therapy for preventing PTE in people at risk, such as after traumatic brain injury, concussion, and serious brain injuries, especially in military persons.