Structural basis for antiepileptic drugs and botulinum neurotoxin recognition of SV2A.

More than one percent of people have epilepsy worldwide. Levetiracetam (LEV) is a successful new-generation antiepileptic drug (AED), and its derivative, brivaracetam (BRV), shows improved efficacy. Synaptic vesicle glycoprotein 2a (SV2A), a putative membrane transporter in the synaptic vesicles (SVs), has been identified as a target of LEV and BRV. SV2A also serves as a receptor for botulinum neurotoxin (BoNT), which is the most toxic protein and has paradoxically emerged as a potent reagent for therapeutic and cosmetic applications. Nevertheless, no structural analysis on AEDs and BoNT recognition by full-length SV2A has been available. Here we describe the cryo-electron microscopy structures of the full-length SV2A in complex with the BoNT receptor-binding domain, BoNT/A2 HC, and either LEV or BRV. The large fourth luminal domain of SV2A binds to BoNT/A2 HC through protein-protein and protein-glycan interactions. LEV and BRV occupy the putative substrate-binding site in an outward-open conformation. A propyl group in BRV creates additional contacts with SV2A, explaining its higher binding affinity than that of LEV, which was further supported by label-free spectral shift assay. Numerous LEV derivatives have been developed as AEDs and positron emission tomography (PET) tracers for neuroimaging. Our work provides a structural framework for AEDs and BoNT recognition of SV2A and a blueprint for the rational design of additional AEDs and PET tracers.

Nanotechnological advances in the treatment of epilepsy: a comprehensive review.

Epilepsy is one of the most prevalent chronic neurological disorders characterized by frequent unprovoked epileptic seizures. Epileptic seizures can develop from a broad range of underlying abnormalities such as tumours, strokes, infections, traumatic brain injury, developmental abnormalities, autoimmune diseases, and genetic predispositions. Sometimes epilepsy is not easily diagnosed and treated due to the large diversity of symptoms. Undiagnosed and untreated seizures deteriorate over time, impair cognition, lead to injuries, and can sometimes result in death. This review gives details about epilepsy, its classification on the basis of International League Against Epilepsy, current therapeutics which are presently offered for the treatment of epilepsy. Despite of the fact that more than 30 different anti-epileptic medication and antiseizure drugs are available, large number of epileptic patients fail to attain prolonged seizure independence. Poor onsite bioavailability of drugs due to blood brain barrier poses a major challenge in drug delivery to brain. The present review covers the limitations with the state-of-the-art strategies for managing seizures and emphasizes the role of nanotechnology in overcoming these issues. Various nano-carriers like polymeric nanoparticles, dendrimers, lipidic nanoparticles such as solid lipid nanoparticles, nano-lipid carriers, have been explored for the delivery of anti-epileptic drugs to brain using oral and intranasal routes. Nano-carries protect the encapsulated drugs from degradation and provide a platform to deliver controlled release over prolonged periods, improved permeability and bioavailability at the site of action. The review also emphasises in details about the role of neuropeptides for the treatment of epilepsy.

Biallelic variants in the synaptic vesicle glycoprotein 2 A are associated with epileptic encephalopathy.

Synaptic Vesicle Glycoprotein 2 A (SV2A) is a membrane protein of synaptic vesicles and the binding site of antiepileptic drug levetiracetam. Biallelic Arg383Gln is reported in a family with intractable epilepsy earlier. Here, we report on the second family with early onset drug resistant epilepsy. We identified homozygous Arg289Ter variant by exome sequencing that segregated with the phenotype in the family. The affected children in these two families are normal at birth and developed recurrent seizures beginning in the second month of life and developed secondary failure of growth and development. Knock out mice models earlier had replicated the human phenotype observed in these two families. These findings support that biallelic loss of function variants in SV2A result in early onset intractable epilepsy in humans.

Pharmacokinetics of the Recalcitrant Drug Lamotrigine: Identification and Distribution of Metabolites in Cucumber Plants.

Treated wastewater is an important source of water for irrigation. As a result, irrigated crops are chronically exposed to wastewater-derived pharmaceuticals, such as the anticonvulsant drug lamotrigine. Lamotrigine is known to be taken up by plants, but its plant-derived metabolites and their distribution in different plant organs are unknown. This study aimed to detect and identify metabolites of lamotrigine in cucumber plants grown for 35 days in a hydroponic solution by using LC-MS/MS (Orbitrap) analysis. Our data showed that 96% of the lamotrigine taken up was metabolized. Sixteen metabolites possessing a lamotrigine core structure were detected. Reference standards confirmed two; five were tentatively identified, and nine molecular formulas were assigned. The data suggest that lamotrigine is metabolized via N-carbamylation, N-glucosidation, N-alkylation, N-formylation, N-oxidation, and amidine hydrolysis. The metabolites LTG-N2-oxide, M284, M312, and M370 were most likely produced in the roots and were translocated to the leaves. Metabolites M272, M312, M314, M354, M368, M370, and M418 were dominant in leaves. Only a few metabolites were detected in the fruits. With an increasing exposure time, lamotrigine leaf concentrations decreased because of continuous metabolism. Our data showed that the metabolism of lamotrigine in a plant is fast and that a majority of metabolites are concentrated in the roots and leaves.

Neuroplastin exerts antiepileptic effects through binding to the α1 subunit of GABA type A receptors to inhibit the internalization of the receptors.

BACKGROUND: Seizures are associated with a decrease in γ-aminobutyric type A acid receptors (GABAaRs) on the neuronal surface, which may be regulated by enhanced internalization of GABAaRs. When interactions between GABAaR subunit α-1 (GABRA1) and postsynaptic scaffold proteins are weakened, the α1-containing GABAaRs leave the postsynaptic membrane and are internalized. Previous evidence suggested that neuroplastin (NPTN) promotes the localization of GABRA1 on the postsynaptic membrane. However, the association between NPTN and GABRA1 in seizures and its effect on the internalization of α1-containing GABAaRs on the neuronal surface has not been studied before. METHODS: An in vitro seizure model was constructed using magnesium-free extracellular fluid, and an in vivo model of status epilepticus (SE) was constructed using pentylenetetrazole (PTZ). Additionally, in vitro and in vivo NPTN-overexpression models were constructed. Electrophysiological recordings and internalization assays were performed to evaluate the action potentials and miniature inhibitory postsynaptic currents of neurons, as well as the intracellular accumulation ratio of α1-containing GABAaRs in neurons. Western blot analysis was performed to detect the expression of GABRA1 and NPTN both in vitro and in vivo. Immunofluorescence co-localization analysis and co-immunoprecipitation were performed to evaluate the interaction between GABRA1 and NPTN. RESULTS: The expression of GABRA1 was found to be decreased on the neuronal surface both in vivo and in vitro seizure models. In the in vitro seizure model, α1-containing GABAaRs showed increased internalization. NPTN expression was found to be positively correlated with GABRA1 expression on the neuronal surface both in vivo and in vitro seizure models. In addition, NPTN overexpression alleviated seizures and NPTN was shown to bind to GABRA1 to form protein complexes that can be disrupted during seizures in both in vivo and in vitro models. Furthermore, NPTN was found to inhibit the internalization of α1-containing GABAaRs in the in vitro seizure model. CONCLUSION: Our findings provide evidence that NPTN may exert antiepileptic effects by binding to GABRA1 to inhibit the internalization of α1-containing GABAaRs.

Potential impact of mesenchymal stem cells on nephrotoxicity induced by gamma irradiation and antiepileptic drugs cotherapy in rats.

The goal of this study was to assess the influence of bone marrow-derived mesenchymal stem cells (BM-MSCs) on the nephrotoxicity induced by fractionated doses of gamma irradiation (Rad) and the cotherapy of levetiracetam and oxcarbazepine in male rats. Adult rats were randomly divided into four groups. Group I: Control, Group II: antiepileptic drugs (AEDs), Group III: AEDs +Rad and Group IV: AEDs + Rad + MSCs. Rats treated with AEDs and exposed to fractionated doses of γ-irradiation displayed a discernible increase in serum urea, creatinine, kidney injury marker, kidney malondialdehyde, transforming growth factor beta (TGF-ß) and the relative expression of Smad3 along with a decrease in the relative expression of Smad7 and glutathione level. Alternatively, groups treated with BM-MSCs with AEDs and Rad showed a substantial modification in the majority of the evaluated parameters and looked to be successful in reducing the hazards of the combination therapy of AEDs and radiation. The reno-histopathological study supports the biochemical analysis. In conclusion, BM-MSCs exhibited therapeutic potential against nephrotoxicity induced by fractionated doses of γ-irradiation and AEDs. The outcome was brought about by the downregulation of the TGF-ß/Smad pathway. BM-MSCs might be suggested as a valuable therapeutic strategy to overcome kidney injury induced by gamma irradiation during AEDs cotherapy.

EGR1-Driven METTL3 Activation Curtails VIM-Mediated Neuron Injury in Epilepsy.

Uncovering mechanisms underlying epileptogenesis aids in preventing further epilepsy progression and to lessen seizure severity and frequency. The purpose of this study is to explore the antiepileptogenic and neuroprotective mechanisms of EGR1 in neuron injuries encountered in epilepsy. Bioinformatics analysis was conducted to identify the key genes related to epilepsy. The mice were rendered epileptic using the kainic acid protocol, followed by measurement of seizure severity, high amplitude and frequency, pathological changes of hippocampal tissues and neuron apoptosis. Furthermore, an in vitro epilepsy model was constructed in the neurons isolated from newborn mice, which was then subjected to loss- and gain-of-function investigations, followed by neuron injury and apoptosis assessment. Interactions among EGR1, METTL3, and VIM were analyzed by a series of mechanistic experiments. In the mouse and cell models of epilepsy, VIM was robustly induced. However, its knockdown reduced hippocampal neuron injury and apoptosis. Meanwhile, VIM knockdown decreased inflammatory response and neuron apoptosis in vivo. Mechanistic investigations indicated that EGR1 transcriptionally activated METTL3, which in turn downregulated VIM expression through m6A modification. EGR1 activated METTL3 and reduced VIM expression, thereby impairing hippocampal neuron injury and apoptosis, preventing epilepsy progression. Taken together, this study demonstrates that EGR1 alleviates neuron injuries in epilepsy by inducing METTL3-mediated inhibition of VIM, which provides clues for the development of novel antiepileptic treatments.

Intracerebroventricular administration for delivery of antiseizure therapeutics: Challenges and opportunities.

Intracerebroventricular (ICV) administration is increasingly being explored as a means for delivering antiseizure and antiepileptic therapies to epileptic brain tissue. This route bypasses the blood-brain barrier, thus enabling the delivery of therapeutics that are restricted from the brain, while reducing the risk of systemic adverse reactions. Nevertheless, projections from studies in patients with other diseases suggest that efficacy of some ICV-delivered therapeutics may be limited when the epileptogenic tissue or network circuits are localized more than a few millimeters away from the ventricles. In this article, we present the characteristics of the cerebrospinal fluid as a drug administration site, the brain barriers, and their relevance to treating focal and generalized epilepsies. We refer to ICV delivery of advanced therapies for treating neurodevelopmental disorders with epilepsy. We describe properties of therapeutic compounds, from small molecules to RNA-based therapeutics, proteins, and viral vectors, which can make them either fitting or poor candidates for ICV administration in epilepsy. We additionally provide an overview of preclinical studies and clinical trials involving the ICV route of delivery. Finally, we compare ICV delivery with other routes of administration that bypass the cerebral circulation. This review aims to provide information that will hopefully help investigators select candidate patients and therapeutics for ICV therapies, and to highlight advantages and challenges inherent to this approach.

Deregulation of Astroglial TASK-1 K+ Channel Decreases the Responsiveness to Perampanel-Induced AMPA Receptor Inhibition in Chronic Epilepsy Rats.

Tandem of P domains in a weak inwardly rectifying K+ channel (TWIK)-related acid sensitive K+-1 channel (TASK-1) is activated under extracellular alkaline conditions (pH 7.2-8.2), which are upregulated in astrocytes (particularly in the CA1 region) of the hippocampi of patients with temporal lobe epilepsy and chronic epilepsy rats. Perampanel (PER) is a non-competitive α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) antagonist used for the treatment of focal seizures and primary generalized tonic-clonic seizures. Since AMPAR activation leads to extracellular alkaline shifts, it is likely that the responsiveness to PER in the epileptic hippocampus may be relevant to astroglial TASK-1 regulation, which has been unreported. In the present study, we found that PER ameliorated astroglial TASK-1 upregulation in responders (whose seizure activities were responsive to PER), but not non-responders (whose seizure activities were not responsive to PER), in chronic epilepsy rats. ML365 (a selective TASK-1 inhibitor) diminished astroglial TASK-1 expression and seizure duration in non-responders to PER. ML365 co-treatment with PER decreased spontaneous seizure activities in non-responders to PER. These findings suggest that deregulation of astroglial TASK-1 upregulation may participate in the responsiveness to PER, and that this may be a potential target to improve the efficacies of PER.

Wastewater-derived organic contaminants in fresh produce: Dietary exposure and human health concerns.

Irrigation with reclaimed wastewater is a growing practice aimed at conserving freshwater sources, especially in arid and semiarid regions. Despite the apparent advantages to water management, the practice of irrigation with reclaimed wastewater exposes the agroenvironment to contaminants of emerging concern (CECs). In this report, we estimated the unintentional dietary exposure of the Israeli population (2808 participants) to CECs from consumption of produce irrigated with reclaimed wastewater using detailed dietary data obtained from a National Health and Nutrition Survey (Rav Mabat adults; 2014-2016). Human health risk analyses were conducted based on acceptable daily intake (ADI) and threshold of toxicological concern (TTC) approaches. The highest unintentional exposure to wastewater-borne CECs was found to occur through the consumption of leafy vegetables. All analyzed CECs exhibited hazard quotients <1 for the mean- and high-exposure scenarios, indicating no human health concerns. However, for the extreme exposure scenario, the anticonvulsant agents lamotrigine and carbamazepine, and the carbamazepine metabolite epoxide-carbamazepine exhibited the highest exposure levels of 29,100, 27,200, and 19,500 ng/person (70 kg) per day, respectively. These exposure levels exceeded the TTC of lamotrigine and the metabolite epoxide-carbamazepine, and the ADI of carbamazepine, resulting in hazard quotients of 2.8, 1.1, and 1.9, respectively. According to the extreme estimated scenario, consumption of produce irrigated with reclaimed wastewater (leafy vegetables in particular) may pose a threat to human health. Minimizing irrigation of leafy vegetables using reclaimed wastewater and/or improving the quality of the reclaimed wastewater using an advanced treatment would significantly reduce human dietary exposure to CECs.

Uptake of antiepileptic drugs in forskolin-induced differentiated BeWo cells: alteration of gabapentin transport.

Previous studies have indicated that the expression levels of several transporters are altered during placental trophoblast differentiation. However, changes in the transport activities of therapeutic agents during differentiation must be comprehensively characterised. Antiepileptic drugs, including gabapentin (GBP), lamotrigine (LTG), topiramate, and levetiracetam, are increasingly prescribed during pregnancy. The objective of this study was to elucidate differences in the uptake of antiepileptic drugs during the differentiation process.Human placental choriocarcinoma BeWo cells were used as trophoblast models. For differentiation into syncytiotrophoblast-like cells, cells were treated with forskolin.The uptake of GBP and LTG was lower in differentiated BeWo cells than in undifferentiated cells. In particular, the maximum uptake rate of GBP transport was decreased in differentiated BeWo cells. Furthermore, GBP transport was trans-stimulated by the amino acids His and Met. We investigated the profiles of amino acids in undifferentiated and differentiated BeWo cells. Supplementation with His and Met, which demonstrated trans-stimulatory effects on GBP uptake, restored GBP uptake in differentiated cells. The findings of this study suggest that drug transport in BeWo cells can be altered before and after differentiation, and that the altered GBP uptake could be mediated by the intracellular amino acid status.

Brivaracetam and Levetiracetam Suppress Astroglial L-Glutamate Release through Hemichannel via Inhibition of Synaptic Vesicle Protein.

To explore the pathophysiological mechanisms of antiseizure and adverse behavioural/psychiatric effects of brivaracetam and levetiracetam, in the present study, we determined the effects of brivaracetam and levetiracetam on astroglial L-glutamate release induced by artificial high-frequency oscillation (HFO) bursts using ultra-high-performance liquid chromatography. Additionally, the effects of brivaracetam and levetiracetam on protein expressions of connexin43 (Cx43) and synaptic vesicle protein 2A (SV2A) in the plasma membrane of primary cultured rat astrocytes were determined using a capillary immunoblotting system. Acutely artificial fast-ripple HFO (500 Hz) burst stimulation use-dependently increased L-glutamate release through Cx43-containing hemichannels without affecting the expression of Cx43 or SV2A in the plasma membrane, whereas acute physiological ripple HFO (200 Hz) stimulation did not affect astroglial L-glutamate release or expression of Cx43 or SV2A. Contrarily, subchronic ripple HFO and acute pathological fast-ripple HFO (500 Hz) stimulations use-dependently increased L-glutamate release through Cx43-containing hemichannels and Cx43 expression in the plasma membrane. Subchronic fast-ripple HFO-evoked stimulation produced ectopic expression of SV2A in the plasma membrane, but subchronic ripple HFO stimulation did not generate ectopic SV2A. Subchronic administration of brivaracetam and levetiracetam concentration-dependently suppressed fast-ripple HFO-induced astroglial L-glutamate release and expression of Cx43 and SV2A in the plasma membrane. In contrast, subchronic ripple HFO-evoked stimulation induced astroglial L-glutamate release, and Cx43 expression in the plasma membrane was inhibited by subchronic levetiracetam administration, but was not affected by brivaracetam. These results suggest that brivaracetam and levetiracetam inhibit epileptogenic fast-ripple HFO-induced activated astroglial transmission associated with hemichannels. In contrast, the inhibitory effect of therapeutic-relevant concentrations of levetiracetam on physiological ripple HFO-induced astroglial responses probably contributes to the adverse behavioural/psychiatric effects of levetiracetam.

Magnetic resonance imaging features of hippocampus and mechanism of neurocognitive dysfunction for antiepileptic drugs in treatment of depression rats.

To explore the effects of antiepileptic drug sodium valproate on magnetic resonance imaging (MRI) images, neurological cognition, and JAK1/STAT3 pathway in hippocampus of rats with depression, 30 Sprague Dawley (SD) rats were included. The depression model (DM) was prepared through the chronic stress restraint test. Some model rats were injected with 10 mg/kg sodium valproate into abdominal cavity before modeling (RT group)), and healthy rats were selected as controls (healthy control (HC) group). Depth of split brain was greatly increased in DM group, and nitrogen-acetyl aspartic acid (NAA)/creatine (Cr), glutamic acid (Glu)/Cr, and choline (Cho)/Cr ratios were greatly reduced (P < 0.05). Behavioral test results showed that sugar water preference rate, escape latency, and divergence index in DM group were greatly reduced (P < 0.05), and cumulative immobility time, target quadrant stay time, and number of crossings in forced swimming and tail suspension were prolonged dramatically (P < 0.05), with no difference between the two groups (P > 0.05). Expression levels of interleukin 1ß (IL-1ß) and interleukin 6 (IL-6) in hippocampus of DM group were obviously increased (P < 0.05), and expression levels of JAK1 and STAT3 were decreased visibly (P < 0.05), with no difference between the two (P > 0.05). In summary, anti-epileptic drug sodium valproate effectively improves hippocampal volume characteristics and memory and neurocognitive dysfunction of depression models.

Elevation of brain gamma-aminobutyric acid levels is associated with vigabatrin-associated brain abnormalities on magnetic resonance imaging.

OBJECTIVE: Vigabatrin (VGB) is an effective antiseizure medication for West syndrome. It works by irreversibly inhibiting gamma-aminobutyric acid (GABA) transaminase and increasing central GABA levels. Vigabatrin-associated brain abnormalities on magnetic resonance imaging (VABAM) are an adverse effect of VGB that has only been reported in children, but the pathophysiology of this effect is unknown. In this study, we evaluated the relationship of VGB and brain GABA levels as well as the association between VABAM and GABA. METHODS: For the 15 consecutive pediatric patients treated with VGB, free GABA, glutamine, and glutamate in cerebrospinal fluid (CSF) and plasma, GABA to creatine and phosphocreatine (Cr) peak ratio (GABA/Cr), glutamine (Gln)/Cr, and glutamate (Glu)/Cr as quantified by proton MR spectroscopy (1H-MRS) were retrospectively examined. GABA/Cr was compared with in-house normal pediatric controls. Differences in the levels of each metabolite in VABAM and non-VABAM cases were also examined. RESULTS: Thirteen of the included subjects underwent magnetic resonance imaging (MRI) and 1H-MRS, and two of them presented VABAM; both cases were very-low-birth-weight preterm infants with post-hemorrhagic hydrocephalus. CSF levels of free GABA were significantly higher in VABAM (5.26 ± 1.95 µmol/L) than in non-VABAM (0.59 ± 0.63 µmol/L) cases (P = 0.03). The GABA/Cr was significantly higher in patients with VGB (0.34 ± 0.16) than in pediatric controls (0.20 ± 0.05) (P = 0.02). The GABA/Cr tended to be higher in VABAM (0.48 ± 0.10) than in non-VABAM cases (0.31 ± 0.15) (P = 0.31). SIGNIFICANCE: Elevated brain GABA levels were observed in VABAM patients, suggesting its involvement in the pathogenesis of this condition. In particular, a marked increase in CSF-free GABA was characteristic. Although the elevation of the GABA/Cr is mild, it may be useful for early identification of patients with risk of VABAM.

Group I metabotropic glutamate receptors contribute to the antiepileptic effect of electrical stimulation in hippocampal CA1 pyramidal neurons.

Low-frequency deep brain stimulation (LFS) inhibits neuronal hyperexcitability during epilepsy. Accordingly, the use of LFS as a treatment method for patients with drug-resistant epilepsy has been proposed. However, the LFS antiepileptic mechanisms are not fully understood. Here, the role of metabotropic glutamate receptors group I (mGluR I) in LFS inhibitory action on epileptiform activity (EA) was investigated. EA was induced by increasing the K+ concentration in artificial cerebrospinal fluid (ACSF) up to 12 mM in hippocampal slices of male Wistar rats. LFS (1 Hz, 900 pulses) was delivered to the bundles of Schaffer collaterals at the beginning of EA. The excitability of CA1 pyramidal neurons was assayed by intracellular whole-cell recording. Applying LFS reduced the firing frequency during EA and substantially moved the membrane potential toward repolarization after a high-K+ ACSF washout. In addition, LFS attenuated the EA-generated neuronal hyperexcitability. A blockade of both mGluR 1 and mGluR 5 prevented the inhibitory action of LFS on EA-generated neuronal hyperexcitability. Activation of mGluR I mimicked the LFS effects and had similar inhibitory action on excitability of CA1 pyramidal neurons following EA. However, mGluR I agonist's antiepileptic action was not as strong as LFS. The observed LFS effects were significantly attenuated in the presence of a PKC inhibitor. Altogether, the LFS' inhibitory action on neuronal hyperexcitability following EA relies, in part, on the activity of mGluR I and a PKC-related signaling pathway.

Reactive metabolites of the anticonvulsant drugs and approaches to minimize the adverse drug reaction.

Several generations of antiepileptic drugs (AEDs) are available in the market for the treatment of seizures, but these are amalgamated with acute to chronic side effects. The most common side effects of AEDs are dose-related, but some are idiosyncratic adverse drug reactions (ADRs) that transpire due to the formation of reactive metabolite (RM) after the bioactivation process. Because of the adverse reactions patients usually discontinue the medication in between the treatment. The AEDs such as valproic acid, lamotrigine, phenytoin etc., can be categorized under such types because they form the RM which may prevail with life-threatening adverse effects or immune-mediated reactions. Hepatotoxicity, teratogenicity, cutaneous hypersensitivity, dizziness, addiction, serum sickness reaction, renal calculi, metabolic acidosis are associated with the metabolites of drugs such as arene oxide, N-desmethyldiazepam, 2-(1-hydroxyethyl)-2-methylsuccinimide, 2-(sulphamoy1acetyl)-phenol, E-2-en-VPA and 4-en-VPA and carbamazepine-10,11-epoxide, etc. The major toxicities are associated with the moieties that are either capable of forming RM or the functional groups may itself be too reactive prior to the metabolism. These functional groups or fragment structures are typically known as structural alerts or toxicophores. Therefore, minimizing the bioactivation potential of lead structures in the early phases of drug discovery by a modification to low-risk drug molecules is a priority for the pharmaceutical companies. Additionally, excellent potency and pharmacokinetic (PK) behaviour help in ensuring that appropriate (low dose) candidate drugs progress into the development phase. The current review discusses about RMs in the anticonvulsant drugs along with their mechanism vis-a-vis research efforts that have been taken to minimize the toxic effects of AEDs therapy.

UDP-glucuronosyltransferase 1A4-mediated N2-glucuronidation is the major metabolic pathway of lamotrigine in chimeric NOG-TKm30 mice with humanised-livers.

Lamotrigine is a phenyltriazine anticonvulsant used to treat epilepsy and bipolar disorder, with species-dependent metabolic profiles. In this study, we investigated the metabolism of lamotrigine in chimeric NOG-TKm30 mice transplanted with human hepatocytes (humanised-liver mice).Substantial lamotrigine N2-glucuronidation activities were observed in the liver microsomes from humanised-liver mice, humans, marmosets, and rabbits, compared to those from monkeys, minipigs, guinea pigs, rats, and mice. Lamotrigine N2-glucuronidation activities in the liver microsomes from humanised-liver mice were dose-dependently inhibited by hecogenin, a specific inhibitor of the human UGT1A4.The major metabolite in the hepatocytes from humanised-liver mice and humans was lamotrigine N2-glucuronide, whereas that in mouse hepatocytes was lamotrigine N2-oxide. After a single oral dose of lamotrigine (10 mg/kg), the plasma levels of N2-glucuronide, N5-glucuronide, and N2-methyl were higher in humanised-liver mice compared to that in NOG-TKm30 mice. Lamotrigine N2-glucuronide was the most abundant metabolite in the urine in humanised-liver mice, similar to that reported in humans; whereas, lamotrigine N2-oxide was predominantly excreted in the urine in NOG-TKm30 mouse.These results suggest that humanised-liver mice may be a suitable animal model for studying the UGT1A4 mediated-lamotrigine metabolism.

Identification and Characterization of Cannabidiol as an OX1R Antagonist by Computational and In Vitro Functional Validation.

The potential, multifaceted therapeutic profile of cannabidiol (CBD), a major constituent derived from the Cannabis sativa plant, covers a wide range of neurological and psychiatric disorders, ranging from anxiety to pediatric epilepsy and drug addiction. However, the molecular targets responsible for these effects have been only partially identified. In this view, the involvement of the orexin system, the key regulator in arousal and the sleep/wake cycle, and in motivation and reward processes, including drug addiction, prompted us to explore, using computational and experimental approaches, the possibility that CBD could act as a ligand of orexin receptors, orexin 1 receptor of type 1 (OX1R) and type 2 (OX2R). Ligand-binding assays showed that CBD is a selective ligand of OX1R in the low micromolar range (Ki 1.58 ± 0.2 µM) while in vitro functional assays, carried out by intracellular calcium imaging and mobilization assays, showed that CBD acts as an antagonist at this receptor. Finally, the putative binding mode of CBD has been inferred by molecular docking and molecular dynamics simulations and its selectivity toward the OX1R subtype rationalized at the molecular level. This study provides the first evidence that CBD acts as an OX1R antagonist, supporting its potential use in addictive disorders and/or body weight regulation.

Effects of antiseizure medications on placental cells: Focus on heterodimeric placental carriers.

OBJECTIVE: Appropriate placental nutrient transfer is essential for optimal fetal development. We have previously shown that antiseizure medications (ASMs) can alter the expression of placental carriers for folate and thyroid hormones. Here we extended our analysis to heterodimeric carriers that mediate the placental uptake of amino acids and antioxidant precursors. We focused on the L-type amino acid transporter (LAT)2/SLC7A8, the cystine/glutamate antiporter xCT/SLC7A11, and their chaperone 4F2hc/SLC3A2. METHODS: BeWo cells were exposed for two or five days to therapeutic concentrations of valproate, levetiracetam, carbamazepine, lamotrigine, or lacosamide. Transcript levels were measured by quantitative PCR. Levetiracetam effects on placental carriers were further explored using a tailored gene array. RESULTS: At five days, 30 µg/mL levetiracetam (high therapeutic concentrations) significantly reduced the expression of all studied genes (p < 0.05). Carbamazepine treatment was associated with lower SLC7A8 (LAT2) expression (p < 0.05), whereas valproate increased the transcript levels of this transporter by up to 2.0-fold (p < 0.01). Some of these effects were already observed after two incubation days. Lamotrigine did not alter gene expression, and lacosamide slightly elevated SLC3A2 levels (p < 0.05). The array analysis confirmed the trends observed for levetiracetam and identified additional affected genes. SIGNIFICANCE: Altered expression of placental heterodimeric transporters may represent a mechanism by which ASM affect fetal development. The placental effects are differential, with valproate, carbamazepine and levetiracetam as the more active compounds. The concentration-dependence of those ASM effects are in line with established dose-dependent teratogenicity implying that ASM doses should be adjusted during pregnancy with caution.

Efficacy of Fenfluramine and Norfenfluramine Enantiomers and Various Antiepileptic Drugs in a Zebrafish Model of Dravet Syndrome.

Dravet syndrome (DS) is a rare genetic encephalopathy that is characterized by severe seizures and highly resistant to commonly used antiepileptic drugs (AEDs). In 2020, FDA has approved fenfluramine (FFA) for treatment of seizures associated with DS. However, the clinically used FFA is a racemic mixture (i.e. (±)-FFA), that is substantially metabolized to norfenfluramine (norFFA), and it is presently not known whether the efficacy of FFA is due to a single enantiomer of FFA, or to both, and whether the norFFA enantiomers also contribute significantly. In this study, the antiepileptic activity of enantiomers of FFA (i.e. (+)-FFA and (-)-FFA) and norFFA (i.e. (+)-norFFA and (-)-norFFA) was explored using the zebrafish scn1Lab-/- mutant model of DS. To validate the experimental conditions used, we assessed the activity of various AEDs typically used in the fight against DS, including combination therapy. Overall, our results are highly consistent with the treatment algorithm proposed by the updated current practice in the clinical management of DS. Our results show that (+)-FFA, (-)-FFA and (+)-norFFA displayed significant antiepileptic effects in the preclinical model, and thus can be considered as compounds actively contributing to the clinical efficacy of FFA. In case of (-)-norFFA, the results were less conclusive. We also investigated the uptake kinetics of the enantiomers of FFA and norFFA in larval zebrafish heads. The data show that the total uptake of each compound increased in a time-dependent fashion. A somewhat similar uptake was observed for the (+)-norFFA and (-)-norFFA, implying that the levo/dextrotation of the structure did not dramatically affect the uptake. Significantly, when comparing (+)-FFA with the less lipophilic (+)-norFFA, the data clearly show that the nor-metabolite of FFA is taken up less than the parent compound.