Rafal M. Kaminski

Allopregnanolone Analogs That Positively Modulate GABAA Receptors Protect against Partial Seizures Induced by 6‐Hz Electrical Stimulation in Mice

Summary:  Purpose: Low‐frequency (6 Hz), long‐duration (3 s) electrical stimulation in mice produces seizures characterized by immobility, focal clonus, and automatic behaviors reminiscent of human limbic epilepsy. Renewed interest has been expressed in this seizure model with the recognition that it is sensitive to a broad spectrum of anticonvulsants (AEDs) and may have distinct pharmacologic responsiveness from other in vivo tests of AED efficacy. Here we sought to determine whether the progesterone‐derived neuroactive steroid allopregnanolone (5α,3α‐P) and several structural analogues with varying degrees of activity as positive allosteric modulators of γ‐aminobutyric acid (GABA)A receptors are protective in the 6‐Hz seizure model.

SV2A protein is a broad-spectrum anticonvulsant target: functional correlation between protein binding and seizure protection in models of both partial and generalized epilepsy.

SV2A, a synaptic vesicle protein, has been recently identified as a binding target for levetiracetam (Keppra). The specific mechanism by which SV2A binding leads to seizure protection has not yet been fully elucidated. However, a functional correlation between SV2A binding affinity and anticonvulsant potency has been observed in the mouse audiogenic seizure model. The present study was undertaken to test whether similar correlations exist in rodent models of partial and generalized epilepsies. As expected, there was a high degree of correlation between anticonvulsant potency and SV2A binding affinity in the mouse audiogenic seizure model (r(2)=0.77; p<0.001). A similar correlation was also observed in the mouse corneal kindling (r(2)=0.80; p<0.01) and in the rat model of generalized absence epilepsy (GAERS) (r(2)=0.72; p<0.01). Moreover, there were no significant differences between the slopes and intercepts of regression lines in these models. Interestingly, the protective potencies in these three epilepsy models were also well correlated with each other. As such, protective doses of a given SV2A ligand in one model could be easily predicted based on the data obtained in another model. Taken together, these results support the concept that SV2A protein is an important target for both partial and generalized epilepsies and thereby relevant for the generation of new antiepileptic drugs with potential broad-spectrum efficacy.

Topiramate selectively protects against seizures induced by ATPA, a GluR5 kainate receptor agonist

Although the mechanism of action of topiramate is not fully understood, its anticonvulsant properties may result, at least in part, from an interaction with AMPA/kainate receptors. We have recently shown that topiramate selectively inhibits postsynaptic responses mediated by GluR5 kainate receptors. To determine if this action of topiramate is relevant to the anticonvulsant effects of the drug in vivo, we determined the protective activity of topiramate against seizures induced by intravenous infusion of various ionotropic glutamate receptor agonists in mice. Topiramate (25-100 mg/kg, i.p.) produced a dose-dependent elevation in the threshold for clonic seizures induced by infusion of ATPA, a selective agonist of GluR5 kainate receptors. Topiramate was less effective in protecting against clonic seizures induced by kainate, a mixed agonist of AMPA and kainate receptors. Topiramate did not affect clonic seizures induced by AMPA or NMDA. In contrast, the thresholds for tonic seizures induced by higher doses of these various glutamate receptor agonists were all elevated by topiramate. Unlike topiramate, carbamazepine elevated the threshold for AMPA- but not ATPA-induced clonic seizures. Our results are consistent with the possibility that the effects of topiramate on clonic seizure activity are due to functional blockade of GluR5 kainate receptors. Protection from tonic seizures may be mediated by other actions of the drug. Together with our in vitro cellular electrophysiological results, the present observations strongly support a unique mechanism of action of topiramate, which involves GluR5 kainate receptors.Although the mechanism of action of topiramate is not fully understood, its anticonvulsant properties may result, at least in part, from an interaction with AMPA/kainate receptors. We have recently shown that topiramate selectively inhibits postsynaptic responses mediated by GluR5 kainate receptors. To determine if this action of topiramate is relevant to the anticonvulsant effects of the drug in vivo, we determined the protective activity of topiramate against seizures induced by intravenous infusion of various ionotropic glutamate receptor agonists in mice. Topiramate (25-100 mg/kg, i.p.) produced a dose-dependent elevation in the threshold for clonic seizures induced by infusion of ATPA, a selective agonist of GluR5 kainate receptors. Topiramate was less effective in protecting against clonic seizures induced by kainate, a mixed agonist of AMPA and kainate receptors. Topiramate did not affect clonic seizures induced by AMPA or NMDA. In contrast, the thresholds for tonic seizures induced by higher doses of these various glutamate receptor agonists were all elevated by topiramate. Unlike topiramate, carbamazepine elevated the threshold for AMPA- but not ATPA-induced clonic seizures. Our results are consistent with the possibility that the effects of topiramate on clonic seizure activity are due to functional blockade of GluR5 kainate receptors. Protection from tonic seizures may be mediated by other actions of the drug. Together with our in vitro cellular electrophysiological results, the present observations strongly support a unique mechanism of action of topiramate, which involves GluR5 kainate receptors.

Anticonvulsant Activity of Androsterone and Etiocholanolone

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)—also may be reduced in epilepsy. 5α,3α‐A has been found in adult brain, and both metabolites, which also can 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.

Proepileptic phenotype of SV2A-deficient mice is associated with reduced anticonvulsant efficacy of levetiracetam

Purpose:  Synaptic vesicle protein 2A (SV2A) constitutes a distinct binding site for an antiepileptic drug levetiracetam (Keppra). In the present study we characterized SV2A (+/−) heterozygous mice in several seizure models and tested if the anticonvulsant efficacy of levetiracetam is reduced in these mice.

Benefit of combination therapy in epilepsy: A review of the preclinical evidence with levetiracetam

Levetiracetam (Keppra) is an antiepileptic drug (AED) characterized by a novel mechanism of action, unique profile of activity in seizure models, and broad‐spectrum clinical efficacy. The present report critically reviews several preclinical studies focused on combination therapy with levetiracetam and other anticonvulsants in various seizure and epilepsy models. Administration of levetiracetam together with many different clinically used AEDs or other anticonvulsants generally enhances their protective activity and, among existing AEDs, this was particularly prevalent with valproate. The protective activity of other AEDs was also enhanced by levetiracetam, which seems to be a universal finding that is independent of seizure model or drug combination studied. However, particularly strong enhancement was observed when levetiracetam was combined with agents either enhancing GABAergic or reducing glutamatergic neurotransmission. Importantly, these combinations were not associated with exacerbation of side effects or pharmacokinetic interactions. Based on the available preclinical data, it appears that combination treatment with levetiracetam and other anticonvulsants provides additional therapeutic benefit that may be attributed to its novel and distinct mechanism of action. Moreover, combinations of levetiracetam with clinically used AEDs that enhance GABAergic inhibition may be considered for rational polytherapy, which is often necessary in drug‐resistant patients.

Issues related to development of antiepileptogenic therapies.

Several preclinical proof‐of‐concept studies have provided evidence for positive treatment effects on epileptogenesis. However, none of these hypothetical treatments has advanced to the clinic. The experience in other fields of neurology such as stroke, Alzheimers disease, or amyotrophic lateral sclerosis has indicated several problems in the design of preclinical studies, which likely contribute to failures in translating the positive preclinical data to the clinic. The Working Group on “Issues related to development of antiepileptogenic therapies” of the International League Against Epilepsy (ILAE) and the American Epilepsy Society (AES) has considered the possible problems that arise when moving from proof‐of‐concept antiepileptogenesis (AEG) studies to preclinical AEG trials, and eventually to clinical AEG trials. This article summarizes the discussions and provides recommendations on how to design a preclinical AEG monotherapy trial in adult animals. We specifically address study design, animal and model selection, number of studies needed, issues related to administration of the treatment, outcome measures, statistics, and reporting. In addition, we give recommendations for future actions to advance the preclinical AEG testing.

Nrf2 defense pathway: Experimental evidence for its protective role in epilepsy

Epigenetic mechanisms involved in transcriptional regulation of multiple molecular pathways are potentially attractive therapeutic interventions for epilepsy, because single target therapies are unlikely to provide both anticonvulsant and disease‐modifying effects.

Influence of melatonin upon the protective action of conventional anti-epileptic drugs against maximal electroshock in mice

Melatonin (50 mg/kg; 60 min before the test) significantly raised the electroconvulsive threshold in mice. The protective action of melatonin (50 mg/kg) in the electroconvulsive threshold test was reversed by aminophylline, picrotoxin and bicuculline. Melatonin at the subconvulsive dose of 25 mg/kg potentiated the anticonvulsive activity of carbamazepine and phenobarbital (ED50 values were significantly decreased from 12.1 to 8.3 and from 18.9 to 11.8 mg/kg, respectively). No potentiation was observed in the case of valproate and diphenylhydantoin (their ED50s were changed from 253 to 249 and from 10.3 to 9.7 mg/kg, respectively). Melatonin did not influence the plasma or brain levels of anti-epileptics studied, so a pharmacokinetic interaction is not probable. Melatonin (25 mg/kg) alone and its combinations with carbamazepine or phenobarbital, providing a 50% protection against maximal electroshock, were devoid of significant motor adverse effects, but caused strong long-term memory deficit. Consequently, it does not seem to be a good candidate for the treatment of epilepsy.

Systems genetics identifies Sestrin 3 as a regulator of a proconvulsant gene network in human epileptic hippocampus

Gene-regulatory network analysis is a powerful approach to elucidate the molecular processes and pathways underlying complex disease. Here we employ systems genetics approaches to characterize the genetic regulation of pathophysiological pathways in human temporal lobe epilepsy (TLE). Using surgically acquired hippocampi from 129 TLE patients, we identify a gene-regulatory network genetically associated with epilepsy that contains a specialized, highly expressed transcriptional module encoding proconvulsive cytokines and Toll-like receptor signalling genes. RNA sequencing analysis in a mouse model of TLE using 100 epileptic and 100 control hippocampi shows the proconvulsive module is preserved across-species, specific to the epileptic hippocampus and upregulated in chronic epilepsy. In the TLE patients, we map the trans-acting genetic control of this proconvulsive module to Sestrin 3 (SESN3), and demonstrate that SESN3 positively regulates the module in macrophages, microglia and neurons. Morpholino-mediated Sesn3 knockdown in zebrafish confirms the regulation of the transcriptional module, and attenuates chemically induced behavioural seizures in vivo.