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1.  Epoxy Fatty Acids and Inhibition of the Soluble Epoxide Hydrolase Selectively Modulate GABA Mediated Neurotransmission to Delay Onset of Seizures 
PLoS ONE  2013;8(12):e80922.
In the brain, seizures lead to release of large amounts of polyunsaturated fatty acids including arachidonic acid (ARA). ARA is a substrate for three major enzymatic routes of metabolism by cyclooxygenase, lipoxygenase and cytochrome P450 enzymes. These enzymes convert ARA to potent lipid mediators including prostanoids, leukotrienes and epoxyeicosatrienoic acids (EETs). The prostanoids and leukotrienes are largely pro-inflammatory molecules that sensitize neurons whereas EETs are anti-inflammatory and reduce the excitability of neurons. Recent evidence suggests a GABA-related mode of action potentially mediated by neurosteroids. Here we tested this hypothesis using models of chemically induced seizures. The level of EETs in the brain was modulated by inhibiting the soluble epoxide hydrolase (sEH), the major enzyme that metabolizes EETs to inactive molecules, by genetic deletion of sEH and by direct administration of EETs into the brain. All three approaches delayed onset of seizures instigated by GABA antagonists but not seizures through other mechanisms. Inhibition of neurosteroid synthesis by finasteride partially blocked the anticonvulsant effects of sEH inhibitors while the efficacy of an inactive dose of neurosteroid allopregnanolone was enhanced by sEH inhibition. Consistent with earlier findings, levels of prostanoids in the brain were elevated. In contrast, levels of bioactive EpFAs were decreased following seizures. Overall these results demonstrate that EETs are natural molecules which suppress the tonic component of seizure related excitability through modulating the GABA activity and that exploration of the EET mediated signaling in the brain could yield alternative approaches to treat convulsive disorders.
doi:10.1371/journal.pone.0080922
PMCID: PMC3862847  PMID: 24349022
2.  Tetramethylenedisulfotetramine Alters Ca2+ Dynamics in Cultured Hippocampal Neurons: Mitigation by NMDA Receptor Blockade and GABAA Receptor-Positive Modulation 
Toxicological Sciences  2012;130(2):362-372.
Tetramethylenedisulfotetramine (TETS) is a potent convulsant that is considered a chemical threat agent. We characterized TETS as an activator of spontaneous Ca2+ oscillations and electrical burst discharges in mouse hippocampal neuronal cultures at 13–17 days in vitro using FLIPR Fluo-4 fluorescence measurements and extracellular microelectrode array recording. Acute exposure to TETS (≥ 2µM) reversibly altered the pattern of spontaneous neuronal discharges, producing clustered burst firing and an overall increase in discharge frequency. TETS also dramatically affected Ca2+ dynamics causing an immediate but transient elevation of neuronal intracellular Ca2+ followed by decreased frequency of Ca2+ oscillations but greater peak amplitude. The effect on Ca2+ dynamics was similar to that elicited by picrotoxin and bicuculline, supporting the view that TETS acts by inhibiting type A gamma-aminobutyric acid (GABAA) receptor function. The effect of TETS on Ca2+ dynamics requires activation of N-methyl-d-aspartic acid (NMDA) receptors, because the changes induced by TETS were prevented by MK-801 block of NMDA receptors, but not nifedipine block of L-type Ca2+ channels. Pretreatment with the GABAA receptor-positive modulators diazepam and allopregnanolone partially mitigated TETS-induced changes in Ca2+ dynamics. Moreover, low, minimally effective concentrations of diazepam (0.1µM) and allopregnanolone (0.1µM), when administered together, were highly effective in suppressing TETS-induced alterations in Ca2+ dynamics, suggesting that the combination of positive modulators of synaptic and extrasynaptic GABAA receptors may have therapeutic potential. These rapid throughput in vitro assays may assist in the identification of single agents or combinations that have utility in the treatment of TETS intoxication.
doi:10.1093/toxsci/kfs244
PMCID: PMC3529644  PMID: 22889812
Ca2+; oscillations; GABAA receptors; microelectrode array; NMDA receptors; rapid throughput assay; tetramethylenedisulfotetramine.
3.  Adjunctive perampanel for refractory partial-onset seizures 
French, Jacqueline A. | Krauss, Gregory L. | Biton, Victor | Squillacote, David | Yang, Haichen | Laurenza, Antonio | Kumar, Dinesh | Rogawski, Michael A. | Campanille, Verónica | Floridia, Jorge | Ilari, Rita | Consalvo, Damián E. | Thomson, Alfredo | Sfaello, Ignacio | Pociecha, Juan | Nieto, Flavia | Firstenfeld, Alfredo | Zuin, Daniel | Mesri, Jacobo | Silva, Walter | Nofal, Pedro | Cristalli, Diana | Clement, Jean-Francois | Hwang, Paul | McLachlan, Richard | Pillay, Neelan | Lasso, Jorge | Peralta, Balduin Lawson | Hernandez, Marcelo Leiva | Tenhamm, Eugenio | Barroso, Noe | Milian, Albino Contreras | Morales, Sarug Reyes | Rodríguez, Ildefonso | Jain, Sanjay | Wilfong, Angus | Kalra, Arun | Renfroe, Ben | Moore, David | Flamini, J. | Klapper, Jack | Szaflarski, Jerzy | Teasley, Jean | Halford, Jonathan | Ferreira, Jose | Goodpasture, Hewitt | Sperling, Michael | Klein, Pavel | Ayala, Ricardo | Brower, Richard | Leroy, Robert | Davis, Ronald | Elterman, Roy | Enlow, Thomas | Sosa, Veronica | Puri, Vinay | Miller, John | Shneker, Bassel | Bautista, Ramon | Chung, Steve | Lesch, David | Steiner, David | Webb, Randall | Armstrong, Robert | Biton, Victor | Krauss, Gregory | Mitchell, Wendy | Hogan, Robert | Knowlton, Robert | Wheless, James | Aung-Din, Ronald | Ali, Imran | Brown, Lawrence | Gruenthal, Michael | Kankirawatana, Pongkiat | Charlet, Michael | Conry, Joan | Owen, Meriem Bensalem | Yerby, Mark | Herrman, Craig | Fessler, Albert | Modur, Pradeep | Miranda, Fernando | King-Stephens, David
Neurology  2012;79(6):589-596.
Objective:
To assess efficacy and safety of once-daily 8 or 12 mg perampanel, a noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor antagonist, when added to concomitant antiepileptic drugs (AEDs) in the treatment of drug-resistant partial-onset seizures.
Methods:
This was a multicenter, double-blind, placebo-controlled trial (ClinicalTrials.gov identifier: NCT00699972). Patients (≥12 years, with ongoing seizures despite 1–3 AEDs) were randomized (1:1:1) to once-daily perampanel 8 mg, 12 mg, or placebo. Following baseline (6 weeks), patients entered a 19-week double-blind phase: 6-week titration (2 mg/week increments to target dose) followed by a 13-week maintenance period. Percent change in seizure frequency was the primary endpoint; 50% responder rate was the primary endpoint for EU registration.
Results:
Of 388 patients randomized and treated, 387 provided seizure frequency data. Using this intent-to-treat population over the double-blind phase, the median percent change in seizure frequency was −21.0%, −26.3%, and −34.5% for placebo and perampanel 8 and 12 mg, respectively (p = 0.0261 and p = 0.0158 for 8 and 12 mg vs placebo, respectively). Fifty percent responder rates during the maintenance period were 26.4%, 37.6%, and 36.1%, respectively, for placebo, perampanel 8 mg, and perampanel 12 mg; these differences were not statistically significant for 8 mg (p = 0.0760) or 12 mg (p = 0.0914). Sixty-eight (17.5%) patients discontinued, including 40 (10.3%) for adverse events. Most frequent treatment-emergent adverse events were dizziness, somnolence, irritability, headache, fall, and ataxia.
Conclusions:
This trial demonstrated that once-daily, adjunctive perampanel at doses of 8 or 12 mg improved seizure control in patients with uncontrolled partial-onset seizures. Doses of perampanel 8 and 12 mg were safe, and tolerability was acceptable.
Classification of evidence:
This study provides Class I evidence that once-daily 8 and 12 mg doses of adjunctive perampanel are effective in patients with uncontrolled partial-onset seizures.
doi:10.1212/WNL.0b013e3182635735
PMCID: PMC3413761  PMID: 22843280
4.  Clustered burst firing in FMR1 premutation hippocampal neurons: amelioration with allopregnanolone 
Human Molecular Genetics  2012;21(13):2923-2935.
Premutation CGG repeat expansions (55–200 CGG repeats; preCGG) within the fragile X mental retardation 1 (FMR1) gene cause fragile X-associated tremor/ataxia syndrome (FXTAS). Defects in neuronal morphology and migration have been described in a preCGG mouse model. Mouse preCGG hippocampal neurons (170 CGG repeats) grown in vitro develop abnormal networks of clustered burst (CB) firing, as assessed by multielectrode array recordings and clustered patterns of spontaneous Ca2+ oscillations, neither typical of wild-type (WT) neurons. PreCGG neurons have reduced expression of vesicular GABA and glutamate (Glu) transporters (VGAT and VGLUT1, respectively), and preCGG hippocampal astrocytes display a rightward shift on Glu uptake kinetics, compared with WT. These alterations in preCGG astrocytes and neurons are associated with 4- to 8-fold elevated Fmr1 mRNA and occur despite consistent expression of fragile X mental retardation protein levels at ∼50% of WT levels. Abnormal patterns of activity observed in preCGG neurons are pharmacologically mimicked in WT neurons by addition of Glu or the mGluR1/5 agonist, dihydroxyphenylglycine, to the medium, or by inhibition of astrocytic Glu uptake with dl-threo-β-benzyloxyaspartic acid, but not by the ionotropic Glu receptor agonists, α-2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid or N-methyl-d-aspartic acid. The mGluR1 (7-(hydroxyimino)cyclopropa [b]chromen-1a-carboxylate ethyl ester) or mGluR5 (2-methyl-6-(phenylethynyl)pyridine hydrochloride) antagonists reversed CB firing. Importantly, the acute addition of the neurosteroid allopregnanolone mitigated functional impairments observed in preCGG neurons in a reversible manner. These results demonstrate abnormal mGluR1/5 signaling in preCGG neurons, which is ameliorated by mGluR1/5 antagonists or augmentation of GABAA receptor signaling, and identify allopregnanolone as a candidate therapeutic lead.
doi:10.1093/hmg/dds118
PMCID: PMC3373240  PMID: 22466801
5.  11β-Hydoxylase Inhibitors Protect Against Seizures in Mice by Increasing Endogenous Neurosteroid Synthesis 
Neuropharmacology  2011;61(1-2):133-137.
Steroid 11β-hydroxylase (CYP11B1; EC 1.14.15.4) is a mitochondrial enzyme located in the zona fasciculata of the adrenal cortex and also in the brain that mediates the conversion of 11-deoxycortisol to cortisol and 11-deoxycorticosterone (DOC) to corticosterone. Inhibitors of CYP11B1, such as metyrapone and etomidate, reduce glucocorticoid synthesis and raise levels of DOC providing greater availability for metabolic conversion to the GABAA receptor modulating neurosteroid allotetrahydrodeoxycorticosterone (THDOC). Because THDOC is a potent anticonvulsant, it is plausible that CYP11B1 inhibitors could protect against seizures. Here we demonstrate that metyrapone affords dose-dependent protection against 6-Hz seizures 30 min after injection (ED50, 191 mg/kg), but is markedly more potent at 6 h (ED50, 30 mg/kg). Similarly, etomidate is also protective at 30 min and 6 h (ED50 values, 4.5 and 1.7 mg/kg). Finasteride, an inhibitor of neurosteroid synthesis, attenuated the anticonvulsant effects of both CYP11B1 inhibitors at 6 h, but not 30 min following their injection. Plasma THDOC levels measured by liquid chromatography-mass spectrometry were markedly increased 6 h after injection of both CYP11B1 inhibitors and this increase was attenuated by finasteride pretreatment. We conclude that inhibition of CYP11B1 causes delayed seizure protection due to slow build-up of neurosteroids. Early seizure protection is independent of neurosteroids.
doi:10.1016/j.neuropharm.2011.03.019
PMCID: PMC3105122  PMID: 21458468
Neurosteroid; Allotetrahydrodeoxycorticosterone; Steroid 11β-hydroxylase (CYP11B1) inhibitor; Metyrapone; Etomidate; Finasteride
6.  Ganaxolone Suppression of Behavioral and Electrographic Seizures in the Mouse Amygdala Kindling Model 
Epilepsy research  2010;89(2-3):254-260.
Summary
Ganaxolone (3α-hydroxy-3β-methyl-5α-pregnan-20-one), a synthetic analog of the endogenous neurosteroid allopregnanolone and a positive allosteric modulator of GABAA receptors, may represent a new treatment approach for epilepsy. Here we demonstrate that pretreatment with ganaxolone (1.25–20 mg/kg, s.c.) causes a dose-dependent suppression of behavioral and electrographic seizures in fully amygdala kindled female mice, with nearly complete seizure protection at the highest dose tested. The ED50 for suppression of behavioral seizures was 6.6 mg/kg. The seizure suppression produced by ganaxolone was comparable to that of clonazepam (ED50, 0.1 mg/kg, s.c.). To the extent that amygdala kindling represents a model of mesial temporal lobe epilepsy, this study supports the utility of ganaxolone in the treatment of patients with temporal lobe seizures.
doi:10.1016/j.eplepsyres.2010.01.009
PMCID: PMC2854307  PMID: 20172694
ganaxolone; neurosteroid; clonazepam; GABAA receptor; amygdala kindling; epilepsy; seizure; female mice
7.  Revisiting AMPA Receptors as an Antiepileptic Drug Target 
Epilepsy Currents  2011;11(2):56-63.
In the 1990s there was intense interest in ionotropic glutamate receptors as therapeutic targets for diverse neurological disorders, including epilepsy. NMDA receptors were thought to play a key role in the generation of seizures, leading to clinical studies of NMDA receptor blocking drugs in epilepsy. Disappointing results dampened enthusiasm for ionotropic glutamate receptors as a therapeutic target. Eventually it became appreciated that another type of ionotropic glutamate receptor, the AMPA receptor, is actually the predominant mediator of excitatory neurotransmission in the central nervous system and moreover that AMPA receptors are critical to the generation and spread of epileptic activity. As drugs became available that selectively target AMPA receptors, it was possible to demonstrate that AMPA receptor antagonists have powerful antiseizure activity in in vitro and in vivo models. A decade later, promising clinical studies with AMPA receptor antagonists, including the potent noncompetitive antagonist perampanel, are once again focusing attention on AMPA receptors as a drug target for epilepsy therapy.
doi:10.5698/1535-7511-11.2.56
PMCID: PMC3117497  PMID: 21686307
10.  Neurosteroids on the Epilepsy Chessboard—Keeping Seizures in Check 
Epilepsy Currents  2010;10(6):161-163.
doi:10.1111/j.1535-7511.2010.01388.x
PMCID: PMC3001027  PMID: 21157546
11.  Pathological alterations in GABAergic interneurons and reduced tonic inhibition in the basolateral amygdala during epileptogenesis 
Neuroscience  2009;163(1):415-429.
An acute brain insult such as traumatic head/brain injury, stroke, or an episode of status epilepticus can trigger epileptogenesis, which, after a latent, seizure-free period, leads to epilepsy. The discovery of effective pharmacological interventions that can prevent the development of epilepsy requires knowledge of the alterations that occur during epileptogenesis in brain regions that play a central role in the induction and expression of epilepsy. In the present study, we investigated pathological alterations in GABAergic interneurons in the rat basolateral amygdala (BLA), and the functional impact of these alterations on inhibitory synaptic transmission, on days 7 to 10 after SE induced by kainic acid. Using design-based stereology combined with GAD67 immunohistochemistry, we found a more extensive loss of GABAergic interneurons compared to the loss of principal cells. Fluoro-Jade C staining showed that neuronal degeneration was still ongoing. These alterations were accompanied by an increase in the levels of glutamate decarboxylase and the α1 subunit of the GABAA receptor, and a reduction in the GluK1 (previously known as GluR5) subunit, as determined by Western blots. Whole-cell recordings from BLA pyramidal neurons showed a significant reduction in the frequency and amplitude of action potential-dependent spontaneous IPSCs, a reduced frequency but not amplitude of miniature IPSCs, and impairment in the modulation of IPSCs via GluK1-containing kainate receptors (GluK1Rs). Thus, in the BLA, GABAergic interneurons are more vulnerable to seizure-induced damage than principal cells. Surviving interneurons increase their expression of glutamate decarboxylase and the α1 GABAA receptor subunit, but this does not compensate for the interneuronal loss; the result is a dramatic reduction of tonic inhibition in the BLA circuitry. As activation of GluK1Rs by ambient levels of glutamate facilitates GABA release, the reduced level and function of these receptors may contribute to the reduction of tonic inhibitory activity. These alterations at a relatively early stage of epileptogenesis may facilitate the progress towards the development of epilepsy.
doi:10.1016/j.neuroscience.2009.06.034
PMCID: PMC2733834  PMID: 19540312
basolateral amygdala; interneurons; epileptogenesis; status epilepticus; kainate receptors; inhibitory synaptic transmission; glutamate decarboxylase; GluR5 subunit; GluK1 subunit; GABAA receptor subunits
12.  Neurosteroid Replacement Therapy for Catamenial Epilepsy 
Summary
Perimenstural catamenial epilepsy, the cyclical occurrence of seizure exacerbations near the time of menstruation, affects a high proportion of women of reproductive age with drug refractory epilepsy. Enhanced seizure susceptibility in perimenstrual catamenial epilepsy is believed to be due to the withdrawal of the progesterone-derived GABAA receptor modulating neurosteroid allopregnanolone as a result of the fall in progesterone at the time of menstruation. Studies in a rat pseudopregnancy model of catamenial epilepsy indicate that following neurosteroid withdrawal there is enhanced susceptibility to chemoconvulsant seizures. There is also a transitory increase in the frequency of spontaneous seizures in epleptic rats that had experienced pilocarpine-induced status epilepticus. In the catamenial epilepsy model, there is a marked reduction in the antiseizure potency of anticonvulsant drugs, including benzodiazepines and valproate, but an increase in the anticonvulsant potency and protective index of neurosteroids such as allopregnanolone and the neurosteroid analog ganaxolone. The enhanced seizure susceptibility and benzodiazepine-resistance following neurosteroid withdrawal may be related to reduced expression and altered kinetics of synaptic GABAA receptors and increased expression of GABAA receptor subunits (such as α4) that confer benzodiazepine insensitivity. The enhanced potency of neurosteroids may be due to a relative increase following neurosteroid withdrawal in the expression of neurosteroid-sensitive δ-subunit-containing perisynaptic/extrasynaptic GABAA receptors. Positive allosteric modulatory neurosteroids and synthetic analogs such as ganaxolone may be administered to prevent catamenial seizure exacerbations, which we refer to as “neurosteroid replacement therapy.”
doi:10.1016/j.nurt.2009.01.006
PMCID: PMC2682439  PMID: 19332335
catamenial epilepsy; progesterone; neurosteroid; allopregnanolone; ganaxolone; GABAA receptor
13.  Convection-Enhanced Delivery in the Treatment of Epilepsy 
Summary
Convection-enhanced delivery (CED) is a novel drug-delivery technique that uses positive hydrostatic pressure to deliver a fluid containing a therapeutic substance by bulk flow directly into the interstitial space within a localized region of the brain parenchyma. CED circumvents the blood-brain barrier and provides a wider, more homogenous distribution than bolus deposition (focal injection) or other diffusion-based delivery approaches. A potential use of CED is for the local delivery of antiseizure agents, which would provide an epilepsy treatment approach that avoids the systemic toxicities of orally administered antiepileptic drugs and bystander effects on non-epileptic brain regions. Recent studies have demonstrated that brief CED infusions of nondiffusible peptides that inhibit the release of excitatory neurotransmitters, including ω-conotoxins and botulinum neurotoxins, can produce long-lasting (weeks to months) seizure protection in the rat amygdala-kindling model. Seizure protection is obtainable without detectable neurological or behavioral side effects. Although conventional diffusible antiepileptic drugs do confer seizure protection when administered locally by CED, the effect is transitory. CED is a potential approach for seizure protection that could represent an alternative to resective surgery in the treatment of focal epilepsies that are resistant to orally-administered antiepileptic drugs. The prolonged duration of action of nondiffusible toxins would allow seizure protection to be maintained chronically with infrequent reinfusions.
doi:10.1016/j.nurt.2009.01.017
PMCID: PMC2753495  PMID: 19332329
Convection-enhanced delivery; drug delivery; ω-conotoxin; botulinum neurotoxin; kindling; antiepileptic drug; epilepsy; seizure
14.  Obituary 
Epilepsy Currents  2009;9(5):141.
doi:10.1111/j.1535-7511.2009.01314.x
PMCID: PMC2759046
15.  New Molecular Targets for Antiepileptic Drugs: α2δ, SV2A, and Kv7/KCNQ/M Potassium Channels 
Many currently prescribed antiepileptic drugs (AEDs) act via voltage-gated sodium channels, through effects on γ-aminobutyric acid–mediated inhibition, or via voltage-gated calcium channels. Some newer AEDs do not act via these traditional mechanisms. The molecular targets for several of these nontraditional AEDs have been defined using cellular electrophysiology and molecular approaches. Here, we describe three of these targets: α2δ, auxiliary subunits of voltage-gated calcium channels through which the gabapentinoids gabapentin and pregabalin exert their anticonvulsant and analgesic actions; SV2A, a ubiquitous synaptic vesicle glycoprotein that may prepare vesicles for fusion and serves as the target for levetiracetam and its analog brivaracetam (which is currently in late-stage clinical development); and Kv7/KCNQ/M potassium channels that mediate the M-current, which acts a brake on repetitive firing and burst generation and serves as the target for the investigational AEDs retigabine and ICA-105665. Functionally, all of the new targets modulate neurotransmitter output at synapses, focusing attention on presynaptic terminals as critical sites of action for AEDs.
PMCID: PMC2587091  PMID: 18590620
16.  Intrinsic Severity as a Determinant of Antiepileptic Drug Refractoriness 
Epilepsy Currents  2008;8(5):127-130.
For the most part, resistance to medications in epilepsy is independent of the choice of antiepileptic drug. This simple clinical observation constrains the possible biological mechanisms for drug refractory epilepsy by imposing a requirement to explain resistance for a diverse set of chemical structures that act on an even more varied group of molecular targets. To date, research on antiepileptic drug refractoriness has been guided by the “drug transporter overexpression” and the “reduced drug-target sensitivity” hypotheses. These concepts posit that drug refractoriness is a condition separate from the underlying epilepsy. Inadequacies in both hypotheses mandate a fresh approach to the problem. In this article, we propose a novel approach that considers epilepsy pharmacoresistance in terms of intrinsic disease severity. We suggest that neurobiological factors that confer increased disease severity lead to drug intractability. The occurrence of frequent seizures at disease onset is an important factor that signals increased severity.
doi:10.1111/j.1535-7511.2008.00272.x
PMCID: PMC2566613  PMID: 18852835
17.  Efficacy of the ketogenic diet in the 6-Hz seizure test 
Epilepsia  2007;49(2):334-339.
SUMMARY
Purpose
Since the ketogenic diet is effective in drug-resistant epilepsies, we sought to determine whether it is active in the 6-Hz seizure test, which identifies agents with a broader spectrum of activity than conventional antiepileptic screening tests.
Methods
Male (3–4 week old) NIH Swiss mice were fed a normal or ketogenic diet ad libitum for 2–21 days. The intensity of the corneal stimulation current required to elicit seizures in the 6-Hz test was measured. Blood glucose and β-hydroxybutyrate were measured on the day of seizure testing.
Results
CC50 (current intensity producing seizures in 50% of mice tested) was 50.6 mA and 15 mA in mice fed for 12 days with a ketogenic or normal diet, respectively (p < 0.001). CC50 was elevated in separate experiments after 16, but not 2, 5, and 21 days of ketogenic diet exposure. CC50 values of growing mice fed the normal diet does not differ, indicating CC50 does not vary with mouse weight during a rapid growth phase. β-Hydroxybutyrate was significantly higher, and glucose was significantly lower in mice fed the ketogenic diet than those fed the normal diet. Blood glucose and β-hydroxybutyrate levels did not correlate with CC50.
Discussion
The ketogenic diet significantly elevates the seizure threshold in the 6-Hz test in a time-specific manner. Protection from seizures in this model was not related to level of ketosis. CC50 was insensitive to body weight in mice fed the normal diet, demonstrating that the 6-Hz model can assess anticonvulsant regimens where weight is a confounding factor.
doi:10.1111/j.1528-1167.2007.01430.x
PMCID: PMC2390715  PMID: 18070095
Ketogenic diet; 6-Hz seizure model; Seizure; Intractable epilepsy
18.  Neuroprotective and disease-modifying effects of the ketogenic diet 
Behavioural pharmacology  2006;17(5-6):431-439.
The ketogenic diet has been in clinical use for over 80 years, primarily for the symptomatic treatment of epilepsy. A recent clinical study has raised the possibility that exposure to the ketogenic diet may confer long-lasting therapeutic benefits for patients with epilepsy. Moreover, there is evidence from uncontrolled clinical trials and studies in animal models that the ketogenic diet can provide symptomatic and disease-modifying activity in a broad range of neurodegenerative disorders including Alzheimer’s disease and Parkinson’s disease, and may also be protective in traumatic brain injury and stroke. These observations are supported by studies in animal models and isolated cells that show that ketone bodies, especially β-hydroxybutyrate, confer neuroprotection against diverse types of cellular injury. This review summarizes the experimental, epidemiological and clinical evidence indicating that the ketogenic diet could have beneficial effects in a broad range of brain disorders characterized by the death of neurons. Although the mechanisms are not yet well defined, it is plausible that neuroprotection results from enhanced neuronal energy reserves, which improve the ability of neurons to resist metabolic challenges, and possibly through other actions including antioxidant and anti-inflammatory effects. As the underlying mechanisms become better understood, it will be possible to develop alternative strategies that produce similar or even improved therapeutic effects without the need for exposure to an unpalatable and unhealthy, high-fat diet.
PMCID: PMC2367001  PMID: 16940764
Alzheimer’s disease; cellular energetics; epilepsy; ketone bodies; ketogenic diet; mitochondria; neuroprotection; Parkinson’s disease; stroke; traumatic brain injury
19.  Prolonged Attenuation of Amygdala-Kindled Seizure Measures in Rats by Convection-Enhanced Delivery of the N-Type Calcium Channel Antagonists ω-Conotoxin GVIA and ω-Conotoxin MVIIA 
Convection-enhanced delivery (CED) permits the homogeneous distribution of therapeutic agents throughout localized regions of the brain parenchyma without causing tissue damage as occurs with bolus injection. Here, we examined whether CED infusion of the N-type calcium channel antagonists ω-conotoxin GVIA (ω-CTX-G) and ω-conotoxin MVIIA (ω-CTX-M) can attenuate kindling measures in fully amygdala-kindled rats. Rats were implanted with a combination infusion cannula-stimulating electrode assembly into the right basolateral amygdala. Fully kindled animals received infusions of vehicle, ω-CTX-G (0.005, 0.05, and 0.5 nmol), ω-CTX-M (0.05, 0.15, and 0.5 nmol), proteolytically inactivated ω-CTX-M (0.5 nmol), or carbamazepine (500 nmol) into the stimulation site. CED of ω-CTX-G and ω-CTX-M over a 20-min period resulted in a dose-dependent increase in the afterdischarge threshold and a decrease in the afterdischarge duration and behavioral seizure score and duration during a period of 20 min to 1 week after the infusion, indicating an inhibitory effect on the triggering and expression of kindled seizures. The protective effects of ω-conotoxins reached a maximum at 48 h postinfusion, and then they gradually resolved over the next 5 days. In contrast, carbamazepine was active at 20 min but not at 24 h after the infusion, whereas CED of vehicle or inactivated ω-CTX-M had no effect. Except for transient tremor in some rats receiving the highest toxin doses, no adverse effects were observed. These results indicate that local CED of high-molecular-weight presynaptic N-type calcium channel blockers can produce long-lasting inhibition of brain excitability and that they may provide prolonged seizure protection in focal seizure disorders.
doi:10.1124/jpet.107.125047
PMCID: PMC2257985  PMID: 17717191
20.  Molecular Targets for Antiepileptic Drug Development 
Summary
This review considers how recent advances in the physiology of ion channels and other potential molecular targets, in conjunction with new information on the genetics of idiopathic epilepsies, can be applied to the search for improved antiepileptic drugs (AEDs). Marketed AEDs predominantly target voltage-gated cation channels (the α subunits of voltage-gated Na+ channels and also T-type voltage-gated Ca2+ channels) or influence GABA-mediated inhibition. Recently, α2–δ voltage-gated Ca2+ channel subunits and the SV2A synaptic vesicle protein have been recognized as likely targets. Genetic studies of familial idiopathic epilepsies have identified numerous genes associated with diverse epilepsy syndromes, including genes encoding Na+ channels and GABAA receptors, which are known AED targets. A strategy based on genes associated with epilepsy in animal models and humans suggests other potential AED targets, including various voltage-gated Ca2+ channel subunits and auxiliary proteins, A- or M-type voltage-gated K+ channels, and ionotropic glutamate receptors. Recent progress in ion channel research brought about by molecular cloning of the channel subunit proteins and studies in epilepsy models suggest additional targets, including G-protein-coupled receptors, such as GABAB and metabotropic glutamate receptors; hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits, responsible for hyperpolarization-activated current Ih; connexins, which make up gap junctions; and neurotransmitter transporters, particularly plasma membrane and vesicular transporters for GABA and glutamate. New information from the structural characterization of ion channels, along with better understanding of ion channel function, may allow for more selective targeting. For example, Na+ channels underlying persistent Na+ currents or GABAA receptor isoforms responsible for tonic (extrasynaptic) currents represent attractive targets. The growing understanding of the pathophysiology of epilepsy and the structural and functional characterization of the molecular targets provide many opportunities to create improved epilepsy therapies.
doi:10.1016/j.nurt.2006.11.010
PMCID: PMC1852436  PMID: 17199015
Epilepsy; channelopathy; antiepileptic drug; sodium channel; calcium channel; potassium channel; GABA receptor; glutamate receptor; GABA transporter; glutamate transporter; gap junction
21.  Diverse Mechanisms of Antiepileptic Drugs in the Development Pipeline 
Epilepsy research  2006;69(3):273-294.
There is a remarkable array of new chemical entities in the current antiepileptic drug (AED) development pipeline. In some cases, the compounds were synthesized in an attempt improve upon the activity of marketed AEDs. In other cases, the discovery of antiepileptic potential was largely serendipitous. Entry into the pipeline begins with the demonstration of activity in one or more animal screening models. Results from testing in a panel of such models provide a basis to differentiate agents and may offer clues as to the mechanism. Target activity may then be defined through cell-based studies, often years after the initial identification of activity. Some pipeline compounds are believed to act through conventional targets, whereas others are structurally novel and may act by novel mechanisms. Follow-on agents include the levetiracetam analogs brivaracetam and seletracetam that act as SV2A-ligands; the valproate-like agents valrocemide, valnoctamide, propylisopropyl acetamide, and isovaleramide; the felbamate analog flurofelbamate, a dicarbamate, and the unrelated carbamate RWJ-333369; the oxcarbazepine analog licarbazepine, which probably acts as a use-dependent sodium channel blockers, and its prodrug acetate BIA 2-093; and various selective partial benzodiazepine receptor agonists, including ELB139, which is a positive allosteric modulator of α3-containing GABAA receptors. A variety of AEDs that may act through novel targets are also in clinical development: lacosamide, a functionalized amino acid; talampanel, a 2,3-benzodiazepine selective noncompetitive AMPA receptor antagonist; NS1209, a competitive AMPA receptor antagonist; ganaxolone, a neuroactive steroid that acts as a positive modulator of GABAA receptors; retigabine, a KCNQ potassium channel opener with activity as a GABAA receptor positive modulator; the benzanilide KCNQ potassium channel opener ICA-27243 that is more selective than retigabine; and rufinamide, a triazole of unknown mechanism.
doi:10.1016/j.eplepsyres.2006.02.004
PMCID: PMC1562526  PMID: 16621450
antiepileptic drug; drug discovery; epilepsy models; maximal electroshock test; pentylenetetrazol test; kindling model
22.  Evidence for Low GluR2 AMPA Receptor Subunit Expression at Synapses in the Rat Basolateral Amygdala 
Journal of neurochemistry  2005;94(6):1728-1738.
Fast excitatory synaptic responses in basolateral amygdala (BLA) neurons are mainly mediated by ionotropic glutamate receptors of the AMPA subtype. AMPA receptors containing an edited GluR2 subunit are calcium impermeable, whereas those that lack this subunit are calcium permeable and also inwardly rectifying. Here we sought to determine the extent to which synapses in the rat BLA have AMPA receptors with GluR2 subunits. We assessed GluR2 protein expression in the BLA by immunocytochemistry with a GluR2 subunit-specific antiserum at the light and electron microscopic level; for comparison a parallel examination was carried out in the hippocampus. We also recorded from amygdala brain slices to examine the voltage-dependent properties of AMPA receptor-mediated evoked synaptic currents in BLA principal neurons. At the light microscopic level, GluR2 immunoreactivity was localized to the perikarya and proximal dendrites of BLA neurons; dense labeling was also present over the pyramidal cell layer of hippocampal subfields CA1 and CA3. In electron micrographs from the BLA, most of the synapses were asymmetrical with pronounced postsynaptic densities (PSD). They contained clear, spherical vesicles apposed to the PSD and were predominantly onto spines (86%), indicating that they are mainly with BLA principal neurons. Only 11% of morphological synapses in the BLA were onto postsynaptic elements that showed GluR2 immunoreactivity in contrast to hippocampal subfields CA1 and CA3 in which 76% and 71% of postsynaptic elements were labeled (p < 0.001). Synaptic staining in the BLA and hippocampus, when it occurred, was exclusively postsynaptic, and particularly heavy over the PSD. In whole-cell voltage clamp recordings, 72% of BLA principal neurons exhibited AMPA receptor-mediated synaptic currents evoked by external capsule stimulation that were inwardly rectifying. Although BLA principal neurons express perikaryal and proximal dendritic GluR2 immunoreactivity, few synapses onto these neurons express GluR2 and a preponderance of principal neurons have inwardly rectifying AMPA-mediated synaptic currents, suggesting that targeting of GluR2 to synapses is restricted. Many BLA synaptic AMPA receptors are likely to be calcium permeable and could play roles in synaptic plasticity, epileptogenesis and excitoxicity.
doi:10.1111/j.1471-4159.2005.03334.x
PMCID: PMC1352164  PMID: 16045445
AMPA receptor; GluR2 subunit; basolateral amygdala; hippocampus; electron microscopy; patch clamp recording; BLA, basolateral amygdala
23.  Molecular Targets Versus Models for New Antiepileptic Drug Discovery 
Epilepsy research  2006;68(1):22-28.
Animal models have played a key role in the discovery and characterization of all marketed antiepileptic drugs (AED). The conventional wisdom is that the standard animal screening models are becoming obsolete because they fail to identify compounds that act in mechanistically new ways and as a result do not offer therapeutic advantages over presently available agents. In fact, far from only detecting me-too drugs, the models often uncover compounds with distinctive profiles of activity in various types of epilepsy and in addition have unexpected efficacy in non-epilepsy conditions, such as neuropathic pain, bipolar disorder, and migraine. Moreover, the animal models—because they are unbiased with respect to mechanism—provide an opportunity to uncover drugs that act in new ways and through new targets, such as α2δ and SV2A. In vitro testing is not likely to replace screening in animal models because in vitro systems cannot model the specific pharmacodynamic actions required for seizure protection, and do not assess bioavailability and brain accessibility.
doi:10.1016/j.eplepsyres.2005.09.012
PMCID: PMC1373807  PMID: 16377151
24.  Anticonvulsant Activity of Androsterone and Etiocholanolone 
Epilepsia  2005;46(6):819-827.
Summary
Purpose
Men with epilepsy often have sexual or reproductive abnormalities that are attributed to alterations in androgen levels, including subnormal free testosterone. Levels of the major metabolites of testosterone – androsterone (5α-androstan-3α-ol-17-one; 5α, 3α-A), a neurosteroid that acts as a positive allosteric modulator of GABAA receptors, and its 5β-epimer etiocholanolone (5β-androstan-3α-ol-17-one; 5β, 3α-A) – may also be reduced in epilepsy. 5α 3α-A has been found in adult brain and both metabolites, which can also be derived from androstenedione, are present in substantial quantities in serum along with their glucuronide and sulfate conjugates. This study sought to determine whether these endogenous steroid metabolites can protect against seizures.
Methods
The anticonvulsant activity of 5α 3α-A and 5β, 3α-A was investigated in electrical and chemoconvulsant seizure models in mice. The steroids were also examined for activity against extracellularly-recorded epileptiform discharges in the CA3 region of the rat hippocampal slice induced by perfusion with 55 μM 4-aminopyridine (4-AP).
Results
Intraperitoneal injection of 5α, 3α-A protected mice in a dose-dependent fashion from seizures in the following models (ED50, dose in mg/kg protecting 50% of animals): 6 Hz electrical stimulation (29.1), pentylenetetrazol (43.5), pilocarpine (105), 4-AP (215), and maximal electroshock (224). 5β, 3α-A was also active in the 6 Hz and pentylenetetrazol models, but was less potent (ED50 values, 76.9 and 139 mg/kg, respectively), whereas epiandrosterone (5α,3β-A) was inactive (ED50, ≤300 mg/kg). 5α, 3α-A (10–100 μM) also inhibited epileptiform discharges in a concentration-dependent fashion in the in vitro slice model, whereas 5β, 3α-A was active but of lower potency and 5α, 3β-A was inactive.
Conclusions
5α, 3α-A and 5β, 3α-A have anticonvulsant properties. Although of low potency, the steroids are present in high abundance and could represent endogenous modulators of seizure susceptibility.
doi:10.1111/j.1528-1167.2005.00705.x
PMCID: PMC1181535  PMID: 15946323
Androsterone; Etiocholanolone; Epiandrosterone; Pentylenetetrazol; Pilocarpine; 4-Aminopyridine; 6-Hz model; Seizure; Mouse
25.  Calcium Channel α2–δ Subunit, A New Antiepileptic Drug Target 
Epilepsy research  2006;69(3):183-272.
doi:10.1016/j.eplepsyres.2006.03.014
PMCID: PMC1574365  PMID: 16835945

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