TRPV1 channel in the pathophysiology of epilepsy and its potential as a molecular target for the development of new antiseizure drug candidates.

Identification of transient receptor potential cation channel, subfamily V member 1 (TRPV1), also known as capsaicin receptor, in 1997 was a milestone achievement in the research on temperature sensation and pain signalling. Very soon after it became evident that TRPV1 is implicated in a wide array of physiological processes in different peripheral tissues, as well as in the central nervous system, and thereby could be involved in the pathophysiology of numerous diseases. Increasing evidence suggests that modulation of TRPV1 may also affect seizure susceptibility and epilepsy. This channel is localized in brain regions associated with seizures and epilepsy, and its overexpression was found both in animal models of seizures and in brain samples from epileptic patients. Moreover, modulation of TRPV1 on non-neuronal cells (microglia, astrocytes, and/or peripheral immune cells) may have an impact on the neuroinflammatory processes that play a role in epilepsy and epileptogenesis. In this paper, we provide a comprehensive and critical overview of currently available data on TRPV1 as a possible molecular target for epilepsy management, trying to identify research gaps and future directions. Overall, several converging lines of evidence implicate TRPV1 channel as a potentially attractive target in epilepsy research but more studies are needed to exploit the possible role of TRPV1 in seizures/epilepsy and to evaluate the value of TRPV1 ligands as candidates for new antiseizure drugs.

Novel Alaninamide Derivatives with Drug-like Potential for Development as Antiseizure and Antinociceptive Therapies─In Vitro and In Vivo Characterization.

In the present study, a series of original alaninamide derivatives have been designed applying a combinatorial chemistry approach, synthesized, and characterized in the in vivo and in vitro assays. The obtained molecules showed potent and broad-spectrum activity in basic seizure models, namely, the maximal electroshock (MES) test, the 6 Hz (32 mA) seizure model, and notably, the 6 Hz (44 mA) model of pharmacoresistant seizures. Most potent compounds 26 and 28 displayed the following pharmacological values: ED50 = 64.3 mg/kg (MES), ED50 = 15.6 mg/kg (6 Hz, 32 mA), ED50 = 29.9 mg/kg (6 Hz, 44 mA), and ED50 = 34.9 mg/kg (MES), ED50 = 12.1 mg/kg (6 Hz, 32 mA), ED50 = 29.5 mg/kg (6 Hz, 44 mA), respectively. Additionally, 26 and 28 were effective in the ivPTZ seizure threshold test and had no influence on the grip strength. Moreover, lead compound 28 was tested in the PTZ-induced kindling model, and then, its influence on glutamate and GABA levels in the hippocampus and cortex was evaluated by the high-performance liquid chromatography (HPLC) method. In addition, 28 revealed potent efficacy in formalin-induced tonic pain, capsaicin-induced pain, and oxaliplatin- and streptozotocin-induced peripheral neuropathy. Pharmacokinetic studies and in vitro ADME-Tox data proved favorable drug-like properties of 28. The patch-clamp recordings in rat cortical neurons showed that 28 at a concentration of 10 µM significantly inhibited fast sodium currents. Therefore, 28 seems to be an interesting candidate for future preclinical development in epilepsy and pain indications.

Preclinical common data elements for general pharmacological studies (pharmacokinetic sample collection, tolerability, and drug administration). A report of the TASK3-WG1A General Pharmacology Working Group of the ILAE/AES Joint Translational Task Force.

Growing concerns over rigor and reproducibility of preclinical studies, including consistency across laboratories and translation to clinical populations, have triggered efforts to harmonize methodologies. This includes the first set of preclinical common data elements (CDEs) for epilepsy research studies, as well as Case Report Forms (CRFs) for widespread use in epilepsy research. The General Pharmacology Working Group of the ILAE/AES Task Force (TASK3-WG1A) has continued in this effort by adapting and refining CDEs/CRFs to address specific study design areas as they relate to preclinical drug screening: general pharmacology, pharmacokinetics (PK) and pharmacodynamics (PD), and tolerability. This work has expanded general pharmacology studies to include dose records, PK/PD, tolerability, and elements of rigor and reproducibility. Tolerability testing CRFs included rotarod and Irwin/Functional Observation Battery (FOB) assays. The material provided in the form of CRFs can be delivered for widespread use within the epilepsy research community.

Assessment of glyceride-structured oleogels as an injectable extended-release delivery system of bupivacaine.

This manuscript systematically assesses three different glycerides (tripalmitin, glyceryl monostearate, and a blend of mono-, di- and triesters of palmitic and stearic acids (Geleol™)) as potential gelator structuring agents of medium-chain triglyceride oil to form an oleogel-based injectable long-acting local anesthetic formulation for postoperative pain management. Drug release testing, oil-binding capacity, injection forces, x-ray diffraction, differential scanning calorimetry, and rheological testing were serially performed to characterize the functional properties of each oleogel. After benchtop assessment, the superior bupivacaine-loaded oleogel formulation was compared to bupivacaine HCl, liposomal bupivacaine, and bupivacaine-loaded medium-chain triglyceride oil in a rat sciatic nerve block model to assess in vivo long-acting local anesthetic performance. In vitro drug release kinetics were similar for all formulations, indicating that drug release rate is primarily dependent on the drug's affinity to the base oil. Glyceryl monostearate-based formulations had superior shelf-life and thermal stability. The glyceryl monostearate oleogel formulation was selected for in vivo evaluation. It was found to have a significantly longer duration of anesthetic effect than liposomal bupivacaine and was able to provide anesthesia twice as long as the equipotent bupivacaine-loaded medium-chain triglyceride oil, indicating that the increased viscosity of the oleogel provided enhanced controlled release over the drug-loaded oil alone.

Synergistic effects of the galanin analog 810-2 with the antiseizure medication levetiracetam in rodent seizure models.

OBJECTIVE: The use of many antiseizure medications (ASMs) is limited due to pharmacoresistance and dose-limiting side effects, suggesting an unmet need for novel therapeutic approaches. The neuropeptide galanin reduces seizures in several preclinical seizure and epilepsy models, but its clinical utility is limited due to rapid metabolism and poor blood-brain barrier penetration. The lead galanin analog 810-2 is systemically bioavailable and reduces seizures when administered alone. Further development of this analog, with the potential for use as an add-on therapy in patients with epilepsy, requires a better understanding of the use of this analog in combination with approved ASMs. We sought to evaluate 810-2 in combination with commonly used ASMs in rodent models of seizures. METHODS: The mouse 6-Hz seizure assay was used to test efficacy of 810-2 in combination with levetiracetam (LEV), valproic acid (VPA), or lacosamide (LCM) using a 1:1 dose ratio in isobolographic studies. Further characterization was performed for the combination of 810-2 and LEV in the mouse corneal kindling and rat 6-Hz assays. RESULTS: Whereas the combination of 810-2 with VPA and LCM yielded additive interactions, the combination of 810-2 with LEV demonstrated a synergistic interaction in the mouse 6-Hz assay. Supra-additive effects were also observed in the mouse corneal kindling and rat 6-Hz assays for this combination. SIGNIFICANCE: The combination of 810-2 with LEV suggests the potential for this galanin analog to be further developed as an add-on therapy for patients with epilepsy, particularly when coadministered with LEV.

Galanin analogs prevent mortality from seizure-induced respiratory arrest in mice.

Objective: Sudden Unexpected Death in Epilepsy (SUDEP) accounts for 20% of mortality in those with recurrent seizures. While risk factors, monitoring systems, and standard practices are in place, the pathophysiology of SUDEP is still not well understood. Better knowledge of SUDEP and its potential mechanisms of action is crucial to reducing risk in this patient population and developing potential treatment options. Clinical studies and animal models of SUDEP suggest that diminished post-ictal respiratory control may be the dominant mechanism contributing to mortality. Recently, it was demonstrated that the depletion of the neuropeptide galanin in the amygdala occurs in human SUDEP. The amygdala plays a key role in the central integration of respiratory signaling; the depletion of galanin may represent a critical change that predisposes individuals to SUDEP. Materials and methods: To evaluate the impact of enhancing galaninergic signaling to potentially protect against SUDEP, we studied seizure-induced respiratory arrest (S-IRA) following central (intracerebroventricular, intra-amygdala) and systemic (intraperitoneal, subcutaneous) administration of galanin analogs. Seizure naïve and seizure experienced (fully kindled) mice were tested. Results: Central and systemically administered galanin analogs protect against S-IRA in naïve C57Bl/6J mice. Differential efficacy between receptor subtype-selective analogs varied based on the route of administration. Sub-chronic systemic administration at doses that reduced 6 Hz seizures also protected against S-IRA. Acute treatment benefits also extended to fully kindled mice experiencing tonic extension. Significance: These data demonstrate that galanin analogs may be protective against post-ictal respiratory collapse.

Evaluating the efficacy of prototype antiseizure drugs using a preclinical pharmacokinetic approach.

OBJECTIVE: Pharmacokinetics (PK) of a drug drive its exposure, efficacy, and tolerability. A thorough preclinical PK assessment of antiseizure medications (ASMs) is therefore essential to evaluate the clinical potential. We tested protection against evoked seizures of prototype ASMs in conjunction with analysis of plasma and brain PK as a proof-of-principle study to enhance our understanding of drug efficacy and duration of action using rodent seizure models. METHODS: In vivo seizure protection assays were performed in adult male CF-1 mice and Sprague Dawley rats. Clobazam (CLB), N-desmethyl CLB (NCLB), carbamazepine (CBZ), CBZ-10,11-epoxide (CBZE), sodium valproate (VPA), and levetiracetam (LEV) concentrations were quantified in plasma and brain using liquid chromatography-tandem mass spectrometry. Mean concentrations of each analyte were calculated and used to determine PK parameters via noncompartmental analysis in Phoenix WinNonLin. RESULTS: NCLB concentrations were approximately 10-fold greater than CLB in mice. The antiseizure profile of CLB was partially sustained by NCLB in mice. CLB concentrations were lower in rats than in mice. CBZE plasma exposures were approximately 70% of CBZ in both mice and rats, likely contributing to the antiseizure effect of CBZ. VPA showed a relatively short half-life in both mice and rats, which correlated with a sharp decline in efficacy. LEV had a prolonged brain and plasma half-life, associated with a prolonged duration of action in mice. SIGNIFICANCE: The study demonstrates the utility of PK analyses for understanding the seizure protection time course in mice and rats. The data indicate that distinct PK profiles of ASMs between mice and rats likely drive differences in drug efficacy between rodent models.

A Model for Epilepsy of Infectious Etiology using Theiler's Murine Encephalomyelitis Virus.

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop epilepsy as a consequence, with rates being significantly higher in less economically developed countries. This work provides an overview of modeling epilepsy of infectious etiology and using it as a platform for novel antiseizure compound testing. A protocol of epilepsy induction by non-stereotactic intracerebral injection of Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice is presented, which replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. The clinical evaluation of mice during encephalitis to monitor seizure activity and detect the potential antiseizure effects of novel compounds is described. Furthermore, histopathological consequences of viral encephalitis and seizures such as hippocampal damage and neuroinflammation are shown, as well as long-term consequences such as spontaneous epileptic seizures. The TMEV model is one of the first translational, infection-driven, experimental platforms to allow for the investigation of the mechanisms of epilepsy development as a consequence of CNS infection. Thus, it also serves to identify potential therapeutic targets and compounds for patients at risk of developing epilepsy following a CNS infection.

New Phenylglycinamide Derivatives with Hybrid Structure as Candidates for New Broad-Spectrum Anticonvulsants.

In the present study, a focused combinatorial chemistry approach was applied to merge structural fragments of well-known TRPV1 antagonists with a potent anticonvulsant lead compound, KA-104, that was previously discovered by our group. Consequently, a series of 22 original compounds has been designed, synthesized, and characterized in the in vivo and in vitro assays. The obtained compounds showed robust in vivo antiseizure activity in the maximal electroshock (MES) test and in the 6 Hz seizure model (using both 32 and 44 mA current intensities). The most potent compounds 53 and 60 displayed the following pharmacological profile: ED50 = 89.7 mg/kg (MES), ED50 = 29.9 mg/kg (6 Hz, 32 mA), ED50 = 68.0 mg/kg (6 Hz, 44 mA), and ED50 = 73.6 mg/kg (MES), ED50 = 24.6 mg/kg (6 Hz, 32 mA), and ED50 = 56.3 mg/kg (6 Hz, 44 mA), respectively. Additionally, 53 and 60 were effective in the ivPTZ seizure threshold and had no influence on the grip strength and body temperature in mice. The in vitro binding and functional assays indicated a multimodal mechanism of action for 53 and 60. These molecules, beyond TRPV1 antagonism, inhibited calcium currents and fast sodium currents in patch-clamp assays. Further studies proved beneficial in vitro ADME-Tox properties for 53 and 60 (i.e., high metabolic stability, weak influence on CYPs, no neurotoxicity, etc.). Overall, 53 and 60 seem to be interesting candidates for future preclinical development in epilepsy and pain indications due to their interaction with the TRPV1 channel.

Precision medicine for genetic epilepsy on the horizon: Recent advances, present challenges, and suggestions for continued progress.

The genetic basis of many epilepsies is increasingly understood, giving rise to the possibility of precision treatments tailored to specific genetic etiologies. Despite this, current medical therapy for most epilepsies remains imprecise, aimed primarily at empirical seizure reduction rather than targeting specific disease processes. Intellectual and technological leaps in diagnosis over the past 10 years have not yet translated to routine changes in clinical practice. However, the epilepsy community is poised to make impressive gains in precision therapy, with continued innovation in gene discovery, diagnostic ability, and bioinformatics; increased access to genetic testing and counseling; fuller understanding of natural histories; agility and rigor in preclinical research, including strategic use of emerging model systems; and engagement of an evolving group of stakeholders (including patient advocates, governmental resources, and clinicians and scientists in academia and industry). In each of these areas, we highlight notable examples of recent progress, new or persistent challenges, and future directions. The future of precision medicine for genetic epilepsy looks bright if key opportunities on the horizon can be pursued with strategic and coordinated effort.

Anticonvulsive properties of soticlestat, a novel cholesterol 24-hydroxylase inhibitor.

OBJECTIVE: The formation of 24S-hydroxycholesterol is a brain-specific mechanism of cholesterol catabolism catalyzed by cholesterol 24-hydroxylase (CYP46A1, also known as CH24H). CH24H has been implicated in various biological mechanisms, whereas pharmacological lowering of 24S-hydroxycholesterol has not been fully studied. Soticlestat is a novel small-molecule inhibitor of CH24H. Its therapeutic potential was previously identified in a mouse model with an epileptic phenotype. In the present study, the anticonvulsive property of soticlestat was characterized in rodent models of epilepsy that have long been used to identify antiseizure medications. METHODS: The anticonvulsive property of soticlestat was investigated in maximal electroshock seizures (MES), pentylenetetrazol (PTZ) acute seizures, 6-Hz psychomotor seizures, audiogenic seizures, amygdala kindling, PTZ kindling, and corneal kindling models. Soticlestat was characterized in a PTZ kindling model under steady-state pharmacokinetics to relate its anticonvulsive effects to pharmacodynamics. RESULTS: Among models of acutely evoked seizures, whereas anticonvulsive effects of soticlestat were identified in Frings mice, a genetic model of audiogenic seizures, it was found ineffective in MES, acute PTZ seizures, and 6-Hz seizures. The protective effects of soticlestat against audiogenic seizures increased with repetitive dosing. Soticlestat was also tested in models of progressive seizure severity. Soticlestat treatment delayed kindling acquisition, whereas fully kindled animals were not protected. Importantly, soticlestat suppressed the progression of seizure severity in correlation with 24S-hydroxycholesterol lowering in the brain, suggesting that 24S-hydroxycholesterol can be aggressively reduced to produce more potent effects on seizure development in kindling acquisition. SIGNIFICANCE: The data collectively suggest that soticlestat can ameliorate seizure symptoms through a mechanism distinct from conventional antiseizure medications. With its novel mechanism of action, soticlestat could constitute a novel class of antiseizure medications for treatment of intractable epilepsy disorders such as developmental and epileptic encephalopathy.

Screening of prototype antiseizure and anti-inflammatory compounds in the Theiler's murine encephalomyelitis virus model of epilepsy.

OBJECTIVE: Infection with Theiler's murine encephalomyelitis virus (TMEV) in C57Bl/6J mice results in handling-induced seizures and is useful for evaluating compounds effective against infection-induced seizures. However, to date only a few compounds have been evaluated in this model, and a comprehensive study of antiseizure medications (ASMs) has not yet been performed. Furthermore, as the TMEV infection produces marked neuroinflammation, an evaluation of prototype anti-inflammatory compounds is needed as well. METHODS: Male C57Bl/6J mice were inoculated with TMEV (day 0) followed by daily administrations of test compounds (day 3-7) and subsequent handling sessions (day 3-7). Doses of ASMs, comprising several mechanistic classes, were selected based on previously published data demonstrating the effect of these compounds in reducing seizures in the 6 Hz model of pharmacoresistant seizures. Doses of anti-inflammatory compounds, comprising several mechanistic classes, were selected based on published evidence of reduction of inflammation or inflammation-related endpoints. RESULTS: Several prototype ASMs reduced acute seizures following TMEV infection: lacosamide, phenytoin, ezogabine, phenobarbital, tiagabine, gabapentin, levetiracetam, topiramate, and sodium valproate. Of these, phenobarbital and sodium valproate had the greatest effect (>95% seizure burden reduction). Prototype anti-inflammatory drugs celecoxib, dexamethasone, and prednisone also moderately reduced seizure burden. SIGNIFICANCE: The TMEV model is utilized by the Epilepsy Therapy Screening Program (ETSP) as a tool for evaluation of novel compounds. Compounds reducing seizures in the TMEV comprise distinct mechanistic classes, some with mechanisms of action that extend beyond traditional ASMs.

Spontaneous recurrent seizures in an intra-amygdala kainate microinjection model of temporal lobe epilepsy are differentially sensitive to antiseizure drugs.

The discovery and development of novel antiseizure drugs (ASDs) that are effective in controlling pharmacoresistant spontaneous recurrent seizures (SRSs) continues to represent a significant unmet clinical need. The Epilepsy Therapy Screening Program (ETSP) has undertaken efforts to address this need by adopting animal models that represent the salient features of human pharmacoresistant epilepsy and employing these models for preclinical testing of investigational ASDs. One such model that has garnered increased interest in recent years is the mouse variant of the Intra-Amygdala Kainate (IAK) microinjection model of mesial temporal lobe epilepsy (MTLE). In establishing a version of this model, several methodological variables were evaluated for their effect(s) on pertinent quantitative endpoints. Although administration of a benzodiazepine 40 min after kainate (KA) induced status epilepticus (SE) is commonly used to improve survival, data presented here demonstrates similar outcomes (mortality, hippocampal damage, latency periods, and 90-day SRS natural history) between mice given midazolam and those that were not. Using a version of this model that did not interrupt SE with a benzodiazepine, a 90-day natural history study was performed and survival, latency periods, SRS frequencies and durations, and SRS clustering data were quantified. Finally, an important step towards model adoption is to assess the sensitivities or resistances of SRSs to a panel of approved and clinically used ASDs. Accordingly, the following ASDs were evaluated for their effects on SRSs in these mice: phenytoin (20 mg/kg, b.i.d.), carbamazepine (30 mg/kg, t.i.d.), valproate (240 mg/kg, t.i.d.), diazepam (4 mg/kg, b.i.d.), and phenobarbital (25 and 50 mg/kg, b.i.d.). Valproate, diazepam, and phenobarbital significantly attenuated SRS frequency relative to vehicle controls at doses devoid of observable adverse behavioral effects. Only diazepam significantly increased seizure freedom. Neither phenytoin nor carbamazepine significantly altered SRS frequency or freedom under these experimental conditions. These data demonstrate that SRSs in this IAK model of MTLE are pharmacoresistant to two representative sodium channel-inhibiting ASDs (phenytoin and carbamazepine) and partially sensitive to GABA receptor modulating ASDs (diazepam and phenobarbital) or a mixed-mechanism ASD (valproate). Accordingly, this model is being incorporated into the NINDS-funded ETSP testing platform for treatment resistant epilepsy.

Development of an antiepileptogenesis drug screening platform: Effects of everolimus and phenobarbital.

OBJECTIVE: The kainic acid (KA)-induced status epilepticus (SE) model in rats is a well-defined model of epileptogenesis. This model closely recapitulates many of the clinical and pathological characteristics of human temporal lobe epilepsy (TLE) that arise following SE or another neurological insult. Spontaneous recurrent seizures (SRS) in TLE can present after a latent period following a neurological insult (traumatic brain injury, SE event, viral infection, etc.). Moreover, this model is suitable for preclinical studies to evaluate the long-term process of epileptogenesis and screen putative disease-modifying/antiepileptogenic agents. The burden of human TLE is highly variable, similar to the post-KA SE rat model. In this regard, this model may have broad translational relevance. This report thus details the pharmacological characterization and methodological refinement of a moderate-throughput drug screening program using the post-KA-induced SE model of epileptogenesis in male Sprague Dawley rats to identify potential agents that may prevent or modify the burden of SRS. Specifically, we sought to demonstrate whether our protocol could prevent the development of SRS or lead to a reduced frequency/severity of SRS. METHODS: Rats were administered either everolimus (2-3 mg/kg po) beginning 1, 2, or 24 h after SE onset, or phenobarbital (60 mg/kg ip) beginning 1 h after SE onset. All treatments were administered once/day for 5-7 days. Rats in all studies (n = 12/treatment dose/study) were then monitored intermittently by video-electroencephalography (2 weeks on, 2 weeks off, 2 weeks on epochs) to determine latency to onset of SRS and disease burden. RESULTS: Although no adverse side effects were observed in our studies, no treatment significantly modified disease or prevented the presentation of SRS by 6 weeks after SE onset. SIGNIFICANCE: Neither phenobarbital nor everolimus administered at several time points after SE onset prevented the development of SRS. Nonetheless, we demonstrate a practical and moderate-throughput screen for potential antiepileptogenic agents in a rat model of TLE.

Development of an antiseizure drug screening platform for Dravet syndrome at the NINDS contract site for the Epilepsy Therapy Screening Program.

OBJECTIVE: Dravet syndrome (DS) is a rare but catastrophic genetic epilepsy, with 80% of patients carrying a mutation in the SCN1A gene. Currently, no antiseizure drug (ASD) exists that adequately controls seizures. In the clinic, individuals with DS often present first with a febrile seizure and, subsequently, generalized tonic-clonic seizures that can continue throughout life. To facilitate the development of ASDs for DS, the contract site of the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) has evaluated a mouse model of DS using the conditional knock-in Scn1aA1783V/WT mouse. METHODS: Survival rates and temperature thresholds for Scn1aA1783V/WT were determined. Prototype ASDs were administered via intraperitoneal injections at the time-to-peak effect, which was previously determined, prior to the induction of hyperthermia-induced seizures. ASDs were considered effective if they significantly increased the temperature at which Scn1aA1783V/WT mice had seizures. RESULTS: Approximately 50% of Scn1aA1783V/WT survive to adulthood and all have hyperthermia-induced seizures. The results suggest that hyperthermia-induced seizures in this model of DS are highly refractory to a battery of ASDs. Exceptions were clobazam, tiagabine, levetiracetam, and the combination of clobazam and valproic acid with add-on stiripentol, which elevated seizure thresholds. SIGNIFICANCE: Overall, the data demonstrate that the proposed model for DS is suitable for screening novel compounds for the ability to block hyperthermia-induced seizures and that heterozygous mice can be evaluated repeatedly over the course of several weeks, allowing for higher throughput screening.