Zebrafish – a valuable animal model

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The zebrafish (Danio rerio), approximately 2-4 cm in length, has become a valuable animal model for investigations of neurological diseases and for use in high intensity screening to discover disease-ameliorating drugs.  In particular, this tiny non-mammalian animal has been of use in research into the genetics and pathophysiology of epilepsy and in the evaluation of anti-seizure drugs.  This blog will discuss this model and some of its contributions to understanding epilepsy.

Animal Models

There are many experimental models of epilepsy.  The greatest research effort has focused on rodent models of epilepsy (chemically or electrically-induced seizure, acute or chronic).  Interest in both experimental and natural models of epilepsy in the non-human primates (monkeys) has risen in recent years as has interest in smaller, non-mammalian models (amoeba, roundworm, fruit fly, zebrafish).  To be of scientific value, scientists must rigorously validate all models.  They must demonstrate neuronal electrical activity, neurotransmitter release, gene expression and response to antiseizure drugs comparable to that observed in patients with epilepsy.  As a result, animal models have contributed significantly to the identification of the multiple causes of epilepsy, genetic and environmental and are absolutely essential to the development of safe and effective antiseizure drugs.

Zebrafish Attributes that Make for a Successful Animal Model

Although rodent models of epilepsy have contributed considerably to our understanding of this disease,  rodents are expensive to breed, house and treat.  In contrast, the zebrafish is inexpensive to breed, maintain and treat.  Thus, large numbers are readily cared for with minimal expense.  Zebrafish mature quickly (~3 months), are highly fertile, giving rise to large numbers of offspring and can live in captivity up to 5 years.  The immature form, the larva, is transparent facilitating visual experimentation especially in observing gene expression and nerve activity.  Larvae and embryos are ideal for screening of large numbers of drugs for antiseizure activity.

Additionally and importantly, the zebrafish model relates well to rodent models and patients with epilepsy.  Firstly, 77% of zebrafish genes are similar to those in humans and approximately 80% of disease-associated genes are also present in zebrafish.  EEG recordings in drug-induced seizures in larvae and adult fish are similar to those observed in patients with epilepsy. 

Zebrafish mutants exist with alterations in genes involved in a number of human epileptic syndromes e.g. Dravet’s syndrome (see blog 17). Zebrafish mutants also show similar electrophysical activity as well as responses to anti-seizure drugs as evident in humans.  Additionally, zebrafish express brain neurotransmitters such as gamma-aminobutyric acid, glycine and acetylcholine. These are the same ones involved in human epilepsy. Neurotransmitters in zebrafish increase or decrease in response to seizure-producing and anti-seizure drugs as reported for rodents.

Contribution from Zebrafish Mutants

Many rare and not so rare genetically driven epilepses in humans have been successfully developed in zebrafish mutants.  One epilepsy of interest is Dravet syndrome, a grave pediatric epilepsy with severe seizures and associated cognitive deficits.  The disease origin is attributed in large part to a mutated neuronal sodium channel.  Zebrafish mutants lacking key elements of the sodium channel as in Dravet syndrome were developed and characterized (Baraban et al., 2013).  Mutant zebrafish exhibit abnormal brain electrical activity, related hyperactive locomotion and convulsions.  Furthermore, anti-seizure drugs that reduce seizures in Dravet syndrome also reduce seizures in zebrafish. Moreover, those that are ineffective in Dravet syndrome fail in mutated zebrafish. 

Zebrafish Mutants and Drug Evaluation

Using this model, over 300 chemicals and known drugs were tested (Baraban et al., 2017).  One drug, chemizole, an FDA approved antihistamine with a good safety profile, was highly effective.  This is a surprise but significant finding.   Since other antihistamines were ineffective, the mechanism of action of chemizole in seizure reduction suggests a unique unknown target. This is an opportunity to develop a new potentially novel antiseizure drug for Dravet syndrome.

Contribution from the proconvulsant-treated Zebrafish

The proconvulsant-treated zebrafish (also shortened to the PTZ-treated zebrafish) is a validated and reliable model of epilepsy.  It is used to identify the initial neuronal changes in epilepsy, to identify new anti-seizure drugs and recently to study the negative role of seizures on cognition.  PTZ (pentylenetetrazole) is a proconvulsant. Researchers treat zebrafish with PTZ to generate seizure activity comparable to generalized absence and myoclonic seizures in humans (Gawel et al., 2020).  Specifically, scientists anesthetize the zebrafish and inject the zebrafish intraperitonally (space between skin and intestines) with PTZ and/or an anti-seizure drug. Scientists then return the zebrafish to the testing tank for observations depending on the experimental protocol.   PTZ-induced seizures in zebrafish are dose and time-dependent and ameliorated by anti-seizure drugs such as valporic acid and diazepam (see blog 5).  Changes in locomotor activity following PTZ administration correlates with EEG activity.  

PTZ (proconvulsant)-treated Zebrafish results

As with Zebrafish mutants, researchers use the PTZ-treated zebrafish to screen for novel anti-seizure drugs.  Another interesting use is the study of the negative role of seizures in memory and learning.  Kundap et al., (2017) approached this by developing a T-maze for fish to evaluate memory and behavior.  In this study, researchers collected data on seizure activity, memory, neurotransmitter release and gene expression.  Interestingly, not only did seizure activity affect performance in the T-maze but the anti-seizure drugs that reduced seizure activity also reduced memory.  Tested drugs were phenytoin (Dilantin) oxcarbazepin (Trileptal), gabapentin (Gralise), diazepam (Diastat), rivastigmine (used in dementia ).  The authors conclude that this study provides “proof-of-concept” that the PTZ-treated zebrafish is a valuable model to study the negative effects of repetitive seizures on cognition.  These results emphasize the need to use cautiously those anti-seizure drugs that are additive to the harmful effects of seizure on memory.

Zebrafish Rendition

Conclusions

Investigations with validated animal models are an essential strategy to reveal the genetics and pathophysiology of epilepsy. Also, validated animal models are necessary to develop candidate drugs to eliminate seizures and cure epilepsy.  Although the most frequently used models for epilepsy are rodent models, the smaller, less expensive and equally useful model is the zebrafish.  Its many attributes and its validated relevance to genetics and etiology of human epilepsies may make it the model of choice for anti-seizure drug screening.

References

Baraban SC, Dinday MT, Hortopan GA. Drug screening in Scn1a mutant zebra-fish identifies clemizole as a potential Dravet syndrome treatment. Nat Com-mun 2013;4:2410.

Cunliffe VT et al., Epilepsy research methods update. Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms Seizure 24 (2015) 44–51.

Gawel K et al., Seizing the moment: Zebrafish epilepsy models. Neuroscience and Biobehavioral Reviews 116 (2020) 1–20.

Kandratavicius L et al.,  Animal models of epilepsy: use and limitations.   Neuropsychiatric Disease and Treatment (2014):10 1693–1705.

Kundap UP et al., Zebrafish as a Model for Epilepsy-Induced Cognitive Dysfunction: A Pharmacological, Biochemical and Behavioral Approach Front. Pharmacol. 8:515, 2017.

Niemyer JE et al., Seizures initiate in zones of relative hyperexcitation in a zebrafish epilepsy model Brain (2022): 145; 2347–2360.

Precision Medicine – Promising Future

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Introduction

There is a rising number of epilepsies that have been associated with genetic mutations called genetic variants (see Blog 14) .  For example, some of these epilepsies result from gene variants in ion channels that produce a loss of function or a gain of function.  Such changes promote seizures and other neurological deficits.  Specifically, the common gene variants account for 30% of adult general epilepsies. As more genetic expertise is developed, this number is expected to significantly increase.  Ongoing research from Ingo Helbig and collaborators at the Children’s Hospital of Philadelphia, Perelman School of Medicine and other University neurological departments) suggests that precision medicine is the promising future therapy of genetic-based epilepsies.

Precision Medicine

As reviewed by Knowles et al., (2022), precision medicine is an encompassing approach to optimal treatment.  In particular, it is based on classifying patients according to measurable biology, susceptibility to disease, and response to treatment to assure an efficacious intervention for those who would benefit and avoid those who would not.  For genetic epilepsies, “ideal precision treatment would correct a well-defined genetic mechanism in the context of individualized factors, to impart freedom from seizures and comorbidities” (Knowles et al., 2022).  However, this calls for integration of genetic analysis, natural history and clinical data into searchable databases.  Already, genetic testing alone yields better treatment and less hospitalizations.  Adding natural history and clinical data to genetic analysis would achieve considerably more treatment success.

Achievements Toward Precision Medicine

Identification of genetic variants in certain epilepsies allows for the application of molecular biology techniques to interfere with unwanted mRNA produced by genetic variants.  Therefore, appropriately designed oligonucleotides as well as vector-promoter-gene complexes successfully treat animal models of epilepsy. Currently, evaluation of the latter is ongoing in a clinical trial (NCT05419492) (https://clinicaltrials.gov/ct2/show/) for a genetic epilepsy. 

The discovery of the gene termed SCN8A exemplifies the feasibility of  rapid trajectory from gene identification to precision medicine.  Specifically, SCN8A (voltage-gated sodium channel gene) was discovered in 1995 in mice. Later, its pathological variants were associated with epileptic encephalopathies (seizures with neurological pathology).  By 2019, following intense research, the testing of a specific sodium channel inhibitor (NBI-921352) for the genetic variant was initiated.  Presently, “NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A-developmental epileptic encephalopathy (SCN8A-DEE) and adult focal-onset seizures” (Johnson et al., 2022).  Although this is impressive, “coordinated and systematic streamlining of the epilepsy precision medicine pipeline” from gene discovery to effective therapy is the essential goal (Knowles et al., 2022).

Challenges for Precision Medicine

Technological improvements in genome-wide association studies (GWAS) have facilitated the ability to screen large groups of patients to detect genetic variants associated with disease.  The result is the creation of large available, searchable databases of genetic variants.  However, not only must the gene variant associated with the disease be considered but more importantly, the unique expression of the gene variant in a particular patient must be defined. 

The expression of a gene variant is termed the phenotype of the disease. The phenotype includes all of the clinical observations and measurements in the patient relevant to the epilepsy. Therefore, gene expression may take many forms creating multiplicities and complexities of expression (heterogeneity) from patient to patient. Hence, the correct association of a gene variant with a defined phenotype poses a serious challenge.  According to Helbig and Tayoun, (2016) accurately defining the phenotype of a rare genetic variant, e.g. STXBP1 producing an epileptic encephalopathy represents a “hurdle” due to the phenotypic spectrum that it produces. 

To address this challenge, Helbig et al., (2019) characterized the phenotype of patients with “missense variant in AP2M1” (coding a protein needed for uptake mechanisms on the cell membrane).  The use of the searchable database, Human Phenotype Ontology, set up in 2008 to formalize the phenotypes of all diseases, enhanced this work.  More recently, the International League Against Epilepsy added neurological data and guidelines to it (Kohler et al., 2021).  This database presents “a standardized format to provide both terminology and semantics to a broad range of phenotypic features, including neurological features” (Helbig and Tayoun, 2016).  Consequently, with this type of analysis, researchers (Helbig and Tayou) were able to show “significant phenotypic overlap in individuals with the recurrent AP2M1”. 

Conclusions

To successfully treat epilepsy and its comorbidities both the genotype and phenotype of the epilepsy requires accurate assessment.  Researchers at Children’s Hospital of Pennsylvania and the Perelman School of Medicine and their collaborators are focused on this assessment.  The challenge lies in the interpretation of the epilepsy phenotype since both severe and less severe epilepsies are driven by the same genetic variants.  Sorting out this phenotypic heterogeneity will be worthwhile and lead to benefits of precision medicine for all patients.

References (pubmed)

Clatot J et al., SCN1A gain-of-function mutation causing an early onset epileptic encephalopathy. Epilepsia.64(5):  1318-1330, 2023.

Ganesan S, et al., A longitudinal footprint of genetic epilepsies using automated electronic medical record interpretation. Genet Med. 22(12):  2060-2070, 2020.

Helbig I et al., A Recurrent Missense Variant in AP2M1 Impairs Clathrin-Mediated Endocytosis and Causes Developmental and Epileptic Encephalopathy.  Am J Hum Genet .104(6):  1060-1072, 2019.

Helbig I, Tayoun ANA. Understanding Genotypes and Phenotypes in Epileptic Encephalopathies. Mol Syndromol  7:  172–181, 2016.

Kohler S et al.,  Human Phenotype Ontology in 2021, Nucleic Acids Research  49, Database issue D1207–D1217, 2021.

Knowles JK et al., Precision medicine for genetic epilepsy on the horizon: Recent advances, present challenges, and suggestions for continued progress. Epilepsia. 63(10):   2461–2475, 2022.

Lewis-Smith D et al., Phenotypic homogeneity in childhood epilepsies evolves in gene-specific patterns across 3251 patient-years of clinical data. Eur J Hum Genet. 29(11):  1690-1700, 2021.

Seiffert S et al., KCNC2 variants of uncertain significance are also associated to various forms of epilepsy. Front Neurol. 14: 1212079, 2023.

Xian J et al., Delineating clinical and developmental outcomes in STXBP1-related disorders. medRxiv. 2023 May 11;2023.05.10.23289776. doi: 10.1101/2023.05.10.23289776. Preprint

Early Detection of Seizures

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For decades,  the early detection of a seizure was a serious research effort.  It was obvious that if a seizure could be detected before its initiation, the patient would be better prepared to seek help to suppress the seizure.  However, early detection of a seizure with a lead time of hours proved to be unattainable.  Over the past decade, early detection of a seizure within minutes not hours of its initiation seemed a more reasonable objective.  This blog will focus on the state of research on early detection of seizures with advances developed by the epilepsy laboratory at Johns Hopkins University.

Introduction

As reviewed by Jouny et al., (2011), “The window of opportunity is the time of the earliest detectable changes in the EEG and the onset of disabling clinical symptoms”.  Thus, this provides a definition of early detection.  It may sound straightforward but there are numerous challenges to early detection of seizures. Jouny et al., reviews some of them.

1. Early detection of seizures –  Sensitivity and specificity

Early detection is just that.  Firstly, it must detect the earliest change in the electrical (neuronal) activity that leads to a seizure (called a true positive) and not an electrical event that would not lead to a seizure (called false positive).  Stated another way, the seizure is termed the ictal event and non seizure activity is the interictal activity.  Secondly, early detection must distinguish between lots of interictal activity and a smaller amount of ictal activity.  Many times initially there is little neuronal difference in the two.  Therefore, both sensitivity and specificity are required elements of any measurement targeting early detection of a seizure.

2.  Early detection of seizures – Capacity of microelectrodes.  

Microelectrodes in high density arrays are useful in obtaining detailed data of abnormal neuronal activity (termed pathological high frequency oscillations) indicative of  localized seizures. Specifically, researchers attach microelectrodes to the EEG or intracellular electrodes to capture as much information as possible.  Seizures that spread over extensive areas, unfortunately, surpass the present day recording capabilities of the microelectrode arrays.

3.  Early detection of seizures – Sampling rate.

Higher sampling rates are desirable but this produces more data for analysis.  This requires more powerful computers and better electronics.  Improvements here are needed to detect unique potential changes that distinguish the initiation of the seizure and the seizure itself from all the electrical activity in between.

Current Advances in Early Detection of Seizures.

Early detection devices depend on what is called support vector machines (SVM) which are a type of machine learning algorithms.  “Several algorithms have been developed for early-seizure-onset detection with real-time capabilities for scalp-EEG” (Ehrens et al., 2022).  In particular, they include early detection algorithms worldwide from labs in the Netherlands (Bogaarts et al., 2016); in India (Sridevi et al., 2019); in Italy (Chisci et al., 2010); in Austria/Netherlands (Fürbass et al., 2015); in Germany (Meier et al., 2008; Manzouri et al., 2021); in Canada (Saab and Gotman, 2005) and in the US, (Minasyan et al., 2010, Yidiz et al., 2022, Ehrens et al., 2022). 

Application of early detection algorithms

Early detection devices are appropriately evaluated in an epilepsy monitoring unit (EMU).  The EMU needs to gather reams of data for precise surgical intervention in patients with epilepsy that are resistant to drugs.  The Johns Hopkins Epilepsy Laboratory developed an “dynamical and self-adapting algorithm” (Ehrenss et al., 2022) which detects in real time the seizure phase of early onset as well as the seizure itself.  It is patient specific and does not require prior EEG training.  Basically, the ” algorithm uses a dynamic training set, based on a 20-min dataset that is updating every second” and ” requires no prior training because the algorithm compares the past 20 minutes of activity with the current activity and looks for novelty” (Ehrens et al., 2022)  A processing time of 0.5 seconds allows  algorithm processing, removal of artifacts, and extraction of essential features in the 20 minute learning session.

One-class SVM in early detection of seizures.

This algorithm, referred to as a one-class SVM, was tested in 35 patients with epilepsy (male: female, 62%:38%, 34 years average age) in the Johns Hopkins Hospital EMU.  “Patients were implanted intracranially with a combination of subdural grids, strips and/or depth electrodes to record cortical EEG and stereo EEG signals respectively ” (Ehrens et la., 2022).   For other information on intracranial recordings, check out Blog 10.

As presented above, the algorithm collected EEG data, eliminated non-neural artifacts and identified features essential to seizure identification  This preprocessing trains the one-class SVM and determines novelty.  The output is “post processed” such that it will then identify and signal the detection of an epileptiform event in real time. 

Although multiple SVM configurations were evaluated, the single SVM configuration yielded the best results.  The John Hopkins Laboratory SVM achieved a rate of 87% for true positives and a rate of 1.25% for false positive/hour.  That is, the SVM algorithm detected a seizure correctly 87% of the time and was wrong 1.25% of the time.  The true positive rate increased another 6% points and the false positives declined by half with removal of artifacts post analysis.  The mean detection latency (early detection) was 10.4 seconds. 

Conclusions

There is definitely a need for devices capable of early detection of seizures.  In the EMU, early detection alerts the staff and enables immediate treatment of the patient with epilepsy.  Outside the EMU, an early detection device would dramatically enhance the quality of life of patients with epilepsy.  The algorithm (one class SVM) is moving closer to achieving these goals.  Presently, 100% sensitivity of early detection is achievable in 74% of patients in the EMU setting with the unique algorithm developed at Johns Hopkins Laboratory (Ehrens et al., 2022). 

References (pubmed)

Bogaarts JG et al.,. Improved epileptic seizure detection combining dynamic feature normalization with EEG novelty detection. Med Biol Eng Comput  54:1883–92, 2016.

Chisci L. et al., Real-time epileptic seizure prediction using AR models and support vector machines. IEEE Trans Biomed Eng. 57(5):1124-32, 2010.

Ehrens et al., Dynamic training of a novelty classifier algorithm for real-time detection of early seizure onset.  Clin Neurophysiol.  135: 85–95, 2022.

Furbass F. et al., Prospective multi-center study of an automatic online seizure detection system for epilepsy monitoring units Clin Neurophysiol. 126(6):1124-1131, 2015.

Jouny CC. et al., Improving Early Seizure Detection Epilepsy Behav.  22(Suppl 1): S44–S48, 2011.

Manzouri F et al., A Comparison of Energy-Efficient Seizure Detectors for Implantable Neurostimulation Devices Front Neurol 12:703797, 2022.

Meier R et al., Detecting epileptic seizures in long-term human EEG: a new approach to automatic online and real-time detection and classification of polymorphic seizure patterns J Clin Neurophysiol. 25(3):119-31, 2008.

Minasyan GR et al., -specific early seizure detection from scalp electroencephalogram J Clin Neurophysiol. 27(3):163-78, 2010.

Saab ME, Gotman J. A system to detect the onset of epileptic seizures in scalp EEG Clin Neurophysiol. 116(2):427-42, 2005.

Sridevi V, et al.,. Improved Patient-Independent System for Detection of Electrical Onset of Seizures. J Clin Neurophysiol 36:14–24, 2019.

Yidiz I et al., Unsupervised seizure identification on EEG Comput Methods Programs Biomed. 215:106604, 2022.

Anti-Seizure Drugs of the Future

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Introduction

There are now more than 30 anti-seizure drugs available to treat seizures, the manifestation of epilepsy.  Despite the ongoing effort to develop novel drugs over the past century, none of the drugs in use today cure epilepsy.  Additionally, approximately 30% of patients with epilepsy are not helped with these drugs and many patients taking anti-seizure medications experience adverse reactions.  Thus, there is a need for future anti-seizure drugs with specificity, with no adverse side effects, with greater tolerability and with a potential to cure epilepsy.  This blog will briefly review the development of present day anti-seizure drugs and describe the pipeline of future anti-seizure drugs.

History of Anti-seizure Drugs

A detailed history of the development of anti-seizure drugs has been reviewed by Loscher and Klein (2021).   Potassium bromide  (1850s) and phenobarbital (1910) were the first two drugs to treat epilepsy.  Modification of the barbital structure gave rise to the first generation of anti-seizure drugs (e.g. phenytoin, acetazolamide, ethosuximide of the 13 available by 1958).

Second generation anti-seizure drugs appeared from 1960 to 1975 with structures different from first generation anti-seizure drugs and included (valproate, carbamazepine and the benzodiazepines).  Subsequently, third generation anti-seizure drugs were developed with the objective of “targeting” a specific molecular component of excessive neuronal activity e.g. sodium channel or a neurotransmitter.  Third generation drugs of which there are 20 or more include for example, lamotrigine (lamictal), vigabatrin (sabril), levetiracetam (Kappra), and cenobamate (xcopri).

The specific CNS targets on which the third generation of drugs exert their anti-seizure effects have been reviewed in Assessment 6 – Epilepsy Medication – Need to Know Information.  Some of the third generation anti-seizure drugs exhibit fewer side effects such as reduced potential for teratogenicity (harm to fetal formation), reduced skin sensitivities (allergies), less interference with other drugs and hence greater tolerability.  However, “most novel (third-generation) anti-seizure medications are not more effective than older drugs” Losher and Klein, (2021).  Consequently, the number of patients failing to experience seizure suppression has not changed despite considerable drug discovery effort. 

Experimental Identification of Anti-seizure drugs

The majority, if not all, of the anti-seizure drugs on the market were identified in select rodent models of seizures.  The main ones are 1) MES or maximal electroshock useful in identification of drugs effective against generalized tonic-clonic seizure e.g. grand mal seizure, 2) 6-Hz seizure test and chronic kindling models important in the identification of  drugs suppressing focal-onset seizures and 3) genetic rat models for identification of drugs affecting generalized absence seizures (Loscher and Klein, 2021).  Significant drug discovery assistance has been provided by the Epilepsy Therapy Screening Program supported by National Institute of Neurological Disorders and Stroke (Kehne et al., 2017). 

Why third generation drugs are not more efficacious than older drug?

The main answer relates to preclinical drug discovery.  The standard rodent models of epilepsy have been undeniably important in anti-seizure drug development.  But as emphasized by Loscher and Schmidt (2011) there is now a clear need for additional animal models and new innovative models that can identify more efficacious anti-seizure drugs in general and anti-seizure drugs effective in patients resistant to current drugs.  More importantly, newer more relevant models, although more complex and time-consuming are needed to identify drugs capable of the prevention and cure of epilepsy.   

Another reason for the failure of third generation anti-seizure drugs to achieve greater efficacy relates to the clinical trial protocols.  For drug approval, new drugs are compared to either placebo or a low dose of an approved drug.  This lowers the bar to development of  greatly improved drugs.  The result is that comparison of efficacy head-to-head of a new drug and an approved drug remains unknown, stalling progress.   Ideally, the best clinical trial compares an approved drug with a new drug at several doses.  Expense and large number of required recruits limit this trial type.  Sadly, several  large pharmaceutical firms, which could handle large clinical trials, have discontinued their epilepsy programs; remaining smaller companies are focusing on rare genetic epilepsies.

Future Drugs in the Pipeline

There are over 20 drugs currently in Phase I, II and a few in Phase III trials.  There are 8 promising compounds still in preclinical studies but nearing a phase I clinical trial.  Many of these novel compounds have mechanisms of action different from approved drugs on the market.  The following mechanisms are of interest.

Positive Allosteric Modulators

There are 8 compounds in this group.  Five of the eight new compounds act on the GABA-A receptor.  This receptor exerts inhibitory effects on neuronal activity.  Many available drugs already target the GABA-A receptor, either through augmentation of this receptor, prevention of the GABA-A neurotransmitter degradation or reuptake.  In contrast, future compounds in development seek to stimulate a regulatory site on the receptor so as to modulate its effects on nerve transmission.  Other compounds in this group target the excitatory glutamate receptors (mGlu2 or AMPA) via positive allosteric modulation.  In this case, modulation serves to suppress nerve activity.

Potassium Channel Openers

Despite the withdrawal of the first generation potassium channel opener, retigabine, due to limited use and toxicities, there are two second generation potassium channel openers in phase II trials and one in preclinical studies.  The expectation is that enhancing the opening of potassium channels will hyperpolarize the nerve membrane and dampen excessive nerve activity.

Calcium Channel Inhibitors/Sodium Channel Modulators

Two novel compounds are in phase I and II that block T-type calcium channels and one in preclinical studies that is considered broad spectrum in blocking both calcium channels and sodium channels.  There are already several approved calcium channel blockers such as ethosuximide (zarontin), methsuximide (celontin), eslicarbazepine (aptiom) and possibly valproate (Loscher and Klein, 2021).

One novel inhibitor of the sodium channel (NaV 1.6) is in phase I trial and another compound which acts as an modulator of sodium channel activity is in preclinical studies.  There are also several sodium channel inhibitors already on the market.

Anti-inflammatory Mechanisms

Compounds in development are focused on different inflammatory mechanisms.  These include general anti-inflammatory activity, inhibition of specific inflammatory enzymes, inhibition of leukocyte migration into the brain, and antagonism of an inflammatory receptor e.g. Il-1 or its production.  Unfortunately,  inhibition of leukocyte migration has already failed in a Phase IIa  trial and data from others are considered mediocre.

Multiple Mechanisms

Compounds in this group modulate multiple targets.  For example, one compound will modulate specific proteins that carry the neurotransmitter to the nerve terminal.  The same compound will also exert benzodiazepine-like effects.  Another compound stimulates serotonin receptors and inhibits sigma-1 receptors involved in protein synthesis in nerves.  Others block sodium and calcium channels plus modulating other receptors.  Thus far this approach has not been successful with generally poor efficacy or poor reproducibility in different trials.

Other Mechanisms

Several compounds will treat rare but devastating infancy and early childhood-onset epilepsies such as Dravet and Lennox-Gastaut syndromes.  Two of these compounds seek to reduce brain metabolism, with either inhibition of the breakdown of glucose or the formation of cholesterol.  Others act by stimulating protein formation or inhibiting formation of acetylcholine, a neurotransmitter involved in memory.

Fate of Future Drugs

Future anti-seizure drugs include compounds with novel mechanisms of action (positive allosteric modulation) and known mechanisms of action (T-type calcium channel inhibitors).  Dampening inflammation or combining multiple mechanisms in one compound does not seem of value.  Whether these unique compounds will succeed in becoming an antiseizure drug to help those currently without drug therapy or become fourth generation drugs no better than previous drugs, remains to be determined.

As emphasized in several reviews, the major problem of understanding epilepsy remains unresolved.  That is, there are a wide variety of epilepsies, most with unknown causes.  It is, therefore, impossible to match a specific drug with a known mechanism of action to a person with epilepsy for which this  is only a general description of the seizure.  Drug selection by the physician is and remains empirical.  Rational drug selection even from a large assortment does not exist for the patient with epilepsy. 

Better Efficacy and Tolerability?

Will these future drugs exhibit a better efficacy and safety profile than the third generation drugs?  There is an awareness for improved safety and tolerability.  This was true for third generation drugs.  Some of the third generation drugs exhibit different pharmacokinetics and interact less if at all with other drugs. 

The fact remains that all these drugs, current and future are developed to suppress seizures. This dampens CNS activity.  And they all were screened in standard preclinical seizure models.  As a result, all will influence numerous other pathways in the brain to cause minor to major unwanted changes.  Specificity in anti-seizure drug discovery is needed but this is impossible since epilepsy itself remains a black box.  Perhaps better drugs will come with more relevant animal models.  At present correcting genetic defects responsible for some epilepsies is the most promising approach and will benefit a few that desperately need it.

References

Loscher W. Kleins P. The Pharmacology and Clinical Efficacy of Antiseizure Medications:

From Bromide Salts to Cenobamate and Beyond. CNS Drugs (2021) 35:935–963

Kehne JH, Klein BD, Raeissi S, Sharma S. The National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) Neurochem Res (2017) 42:1894–1903

Loscher W Schmidt D. Modern antiepileptic drug development has failed to deliver: Ways out of the current dilemma Epilepsia, 52(4):657–678, 2011

Gene Therapy for Epilepsy

Future Therapy, , , ,

Introduction

Epilepsy is treated today with anti-seizure medications and in select cases, with surgery.  Although there are over 25 possible anti-seizure drugs, approximately 30% of patients with epilepsy do not respond to drug treatment and those who do, frequently endure adverse side effects.  Thus, there is an urgency to find better therapies.  One potential future strategy is gene therapy for epilepsy (see other strategies, Noncoding RNAs: diagnosis/cure for epilepsy?).

Gene therapy for diseases other than epilepsy have shown success (https://learn.genetics.utah.edu/ content/genetherapy/success/).  These diseases include immune deficiency diseases, hereditary blindness, hemophila and Parkinson’s disease.  In the future, epilepsy may also benefit from gene therapy.   This blog is based on several excellent reviews (see References below) that describe the reality of this future therapy.

What is gene therapy?

Gene therapy is a special technique whereby a gene carried by a vector is injected into a patient.  The selected gene is one that would replace, optimize or inhibit the disease-producing gene.  The vector can be viral or synthetic.  Viral vectors are either adeno-associated viruses, lentiviruses or a herpes simplex viruses.  These viruses are modified so that they can no longer divide and infect.  Therefore, they serve only as carriers.  Synthetic vectors are non viral vectors such as lipids and polymers.  Although not as efficient as viral vectors, synthetic vectors get into the brain readily and are easily manufactured.

What genes are important in epilepsy?

Researchers have identified several genes that influence different aspects of epilepsy.  Genes identified thus far are

a)  membrane channels that conduct ions e.g. sodium and potassium;

b)  receptors at the junction of nerves e.g. the NMDA receptor;

c)  neuromodulators influencing nerve excitability e.g. neuropeptide Y;

d)  genes involved in influencing DNA function e.g. adenosine and associated enzyme, adenosine kinase.

Ideally, gene therapy for epilepsy has three areas of focus.  This includes gene therapy for prevention of  disease initiation, eradication of seizures and amelioration of those brain areas subsequently changed by epilepsy.  It is thought that seizure inhibition is likely to be the first successful target for gene therapy.

Assessment of gene therapy

Animal Models

Gene therapy has been primarily investigated in animal (mouse, rat) models of epilepsy.  Most models evaluate one aspect of epilepsy.  Accordingly, there exists the PTZ model in which an injection of convulsant-inducing pentylenetetrazol causes generalized seizures.  This acute model has been helpful in screening for anti-convulsant drugs.  Another model (termed latent) requires a waiting period for seizure development following a particular insult (pilocarpine, kainic acid).  The initial phase is thought to mimic known injuries e.g. stroke, fever, trauma in man.  This type of model has considerable value in that the initiating changes, the seizures and associated cognitive changes may be investigated.  There also exist models of spontaneous seizures. 

Promising results in animal models

Ion Channels – target of gene therapy

Most of the current anti-seizure drugs block the defective sodium channel located on nerves. Drug-induced inhibition reduces the nerve impulse and dampens the unwanted excitability of the seizure.  Conversely, super activation of the potassium channel has a similar effect.  Therefore, gene therapy to correct defective sodium and potassium channels is reasonable. 

In mouse models analogous to infant epilepsies, several studies showed that gene therapy for the sodium channel inhibited seizures and improved other brain deficiencies.   In two rat models of epilepsy (focal neocortical and temporal lobe), gene therapy of a modified potassium channel effectively suppressed seizures.

Neurotransmitter Receptors – target of gene therapy

Nerves communicate with one another via release of small molecules called neurotransmitters that act on specific receptors.  Two neurotransmitters of interest in epilepsy are gamma-aminobutyric acid (GABA)  and N-methyl-D-aspartic acid (NMDA).  The former acts on its receptor to inhibit nerve activity and the latter acts on its receptor to enhance it.  Thus, these neurotransmitter receptors are potential gene therapy targets. 

Enhancing the number and/or activity of  the receptors stimulated by GABA  decreases seizure activity in animal models.  Conversely, modification of the receptors responding to the excitatory stimulation of NMDA attenuates seizures in animal models. 

Neuromodulators – target of gene therapy

There exist an abundance of neuromodulators that not only influence the extent of neuronal traffic but also affect nerve cell integrity.  Neuromodulators are thought to play a role in collateral changes with epilepsy.  

Several modulators of nerve excitability such as neuropeptide Y (NPY), dynorphin, fibroblast growth factor 2, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor have been evaluated in animal models.  Vector carriers varied from adenovirus (NPY) to synthetic carriers (glial cell neurotrophin) injected directly into the brain.  Depending on the models, diminished seizures or reduced abnormal nerve cell growth  was reported. The effects were stable for months to one year.

DNA modulators – target of gene therapy

Adenosine and its metabolic enzyme, adenosine kinase play a role in epilepsy.  An increase in the enzyme activity results in a decline in the concentration of adenosine.  Adenosine is an important player in gene expression.  Hence, it influences what genes are “on” and which are “off”.  Patients with epilepsy are deficient in adenosine.  In rat models, implantation of a synthetic vector-releasing adenosine into the epileptic brain beneficially influences gene expression, retards abnormal neuronal growth and decreases seizure frequency.

Clinical trial using gene therapy

There is one clinical trial in progress to evaluate the safety and efficacy of gene therapy in epilepsy. This is the ENDEAVOR trial.  Its protocol is to test adenoviral-delivered sodium channel gene to infants 6-36 months with Dravet Syndrome.  Dravet syndrome is a severe form of epilepsy with seizures, mental and growth retardation and sudden death potential.  Many with Dravet Syndrome have a single mutation in the sodium channel making gene therapy possible.  This trial will evaluate the safety and efficacy of ETX101 (vector-promoter-gene complex) injected into a cerebral ventricle (https://clinicaltrials.gov/ct2/show/NCT05419492).

Challenges

Gene therapy has the potential to cure some forms of epilepsy. The ENDEAVER trial is one to follow. 

However, there remain considerable challenges to overcome.  These challenges include the complexity of genetic mutations.   Although there are a few epilepsies tied to one gene,  most epilepsies involve multiple genes..  Even when only one gene is identified, there is no guarantee that the defective gene acts the same way in each patient with that particular type of epilepsy.  Vector selection is also a challenge since the vector must deliver the gene to the correct cell type, gain access to the brain when given intravenously, persist for an adequate amount of time and not induce an unwanted immunological response.  Much of this has been and continues to be resolved in animal models, but gene therapy still awaits well-designed safety and efficacy studies in man.

Gene Therapy

References

Balestrini S, SisodiyaSM. Pharmacogenomics in epilepsy.  Neuroscience Letters 667: 27–39, 2018.

Bouza AA, Isom LL. Chapter 14: Voltage-gated sodium channel β subunits and their related diseases. Handb Exp Pharmacol. 246:   423–450, 2018.

Thakran S et al., Genetic Landscape of Common Epilepsies:  Advancing towards Precision in Treatment Int. J. Mol. Sci. 21: 7784, 2020.

Zhang L, Wang Y. Gene therapy in epilepsy Biomedicine & Pharmacotherapy 143: 112075, 2021.

Noncoding RNAs: diagnosis/cure for epilepsy?

Epilepsy Topics, , ,

Assessment 13

Introduction

Several reviews present data to support the hypothesis that noncoding RNAs have the potential to serve as a diagnostic marker for epilepsy. Additionally, because noncoding RNAs play an integral function in the disease process, they may also serve as a therapeutic target to cure epilepsy.  In these two respects, noncoding RNAs are worth discussing.

In the US, there are approximately 3 million Americans living with epilepsy and each year approximately 150,000 more are diagnosed with this disease (https://www.epilepsyallianceamerica.org).  Of these, roughly 30-50%, depending on the type of epilepsy, are not helped with medications and many on medications are dissatisfied due to drug-related side effects.  Therefore, it is postulated that this embarrassing gap in effective therapy is largely due to a lack of noninvasive biomarkers coupled with a modest understanding of the molecular instigators of epilepsy.  Here is where research may help.

Noncoding RNAs

High school biology taught that one gene produces one protein and that genes via this principle dictate our heredity e.g. who we are.  Furthermore, genes (actually DNA molecules) transcribe their information to another smaller molecule, RNA, which then translates DNA’s instructions precisely into a specific protein.  Scientists now know that this pathway is tightly regulated by many factors, one of which is noncoding RNA, the subject of this blog. 

Thus noncoding RNA is a RNA molecule but as its name implies, it does not code for a protein.  Instead, noncoding RNAs control which proteins are made and how much is made. Interestingly, certain classes of noncoding RNAs regulate other noncoding RNAs.  The bottom line is that noncoding RNAs are essential for the normal health of all cells.  However, when they exist in excess or in lesser amounts compared to normal, gene regulation falls apart, essential proteins are not made and pathology ensues.

Role in epilepsy

The role of noncoding RNAs in epilepsy has been extensively studied in animal models of epilepsy and largely confirmed with tissues surgically removed in patients with intractable, drug-resistant epilepsy. In animal models and human tissues, the majority of disease-associated regulating RNAs are present in abnormally high amounts. Therefore, they are instrumental in supporting a pro-inflammatory milieu, damaging neuronal structure and encouraging neuronal cell death.  These are changes essential for initiation and continuance of epilepsy (see Blog 12).

Noncoding RNAs as a noninvasive biomarker for epilepsy

There is a desperate need for noninvasive biomarkers of epilepsy.  Briefly, a biomarker is a measurement of a pathological process and/or a biomarker indicates the response to a therapeutic/surgical intervention.   To date, the seizure, observed clinically or by EEG, is the only validated biomarker (see Blog 7 for issues) for epilepsy.   Because noncoding RNAs are small molecules, they exit the cell and appear in body fluids e.g. blood.  Present day instrumentation is capable of detecting very low levels of circulating noncoding RNAs.  Studies confirm that variations in blood levels of disease-associated noncoding RNAs mimic changes in the brain, making them a potentially valuable noninvasive biomarker for detection/diagnosis of epilepsy.

There have been a number of studies in man with the objective of assessing the role of noncoding RNAs as a diagnostic noninvasive biomarker.  Many show that certain regulating RNAs measured in serum, plasma or cerebrospinal fluid are indicative of specific epilepsies compared to normal controls.  Some studies measured noncoding RNAs as a biomarker for drug-resistant epilepsy.  In all studies, the select or disease-associated noncoding RNAs were statistically significantly changed from controls and hence have potential to accurately characterize the epilepsy.

Noncoding RNAs as a target to cure epilepsy

Data show that certain disease-associated noncoding RNAs create instability severe enough to ferment inflammation and cause neuronal death. This ignites the beginning stages of epilepsy. Thus, noncoding RNAs represent potential targets for future therapy.  Several anti-noncoding RNA therapies e.g. antisense oligonucleotides, anti-microRNA, microRNA mimics, microRNA sponges (Manna et al., 2022), are currently in development in animal models and could prove valuable to patients with epilepsy.

Issues to overcome

There is little doubt that noncoding RNAs will become useful diagnostic biomarkers and therapeutic targets in the future.  However, several hurdles remain.  One is standardization.  There is the need to develop valid universal methods to allow critical comparison of results between separate studies and different laboratories.  Studies also need to improve on sensitivity and specificity.  “Sensitivity measures the proportion of correctly identified subjects having the specific condition or disease. Specificity measures the proportion of correctly identified subjects not having the disease (Pitkanen et al., 2019).  From well-designed studies, scientists can use these measurement to determine acceptable values. Also, larger studies in man are clearly essential.

Conclusions

Understanding the  role of noncoding RNAs in health and disease is an important endeavor.  Noncoding RNAs are key regulators of gene expression and aberrant regulation leads to dysfunction and disease.  Results from animal models of epilepsy and various studies in patients with epilepsy strongly indicate epilepsy-associated noncoding RNAs could become reliable diagnostic biomarkers for the disease as well as future therapeutic targets.

Biological principle of noncoding RNAs
noncoding RNAs regulate gene function

Select References (Pubmed)

Manna I et al., Non-Coding RNAs: New Biomarkers and Therapeutic Targets for Temporal Lobe Epilepsy. Int. J. Mol. Sci. 23, 3063, 2022.

Papadelis C, Perry MS. Localizing the Epileptogenic Zone with Novel  Biomarkers. Semin Pediatr Neurol 39:100919,  2021.

Pitkanen A. et al., Epilepsy biomarkers – Toward etiology and pathology specificity. Neurobiol Dis. 123: 42–58, 2019.

Some studies in man

Wang X et al., Serum microRNA-4521 is a potential biomarker for focal cortical dysplasia with refractory epilepsy. Neurochem Res 41  :905-12, 2016.

Yan S et al., Altered microRNA profiles in plasma exosomes from mesial temporal lobe epilepsy with hippocampal sclerosis. Oncotarget 8:  4136–4146, 2017.

An N et al,. Elevated serum miR-106b and miR- 146a in patients with focal and generalized epilepsy. Epilepsy Res 127:  311–316, 2016.

Sun J. et al.,  Identification of serum miRNAs differentially expressed in human epilepsy at seizure onset and post-seizure. Mol. Med. Rep 14:  5318–5324, 2016.

Oxidative Stress and Epilepsy

Pathophysiology of epilepsy, , , ,

Blog 12 – Oxidative Stress and Epilepsy

Oxidative stress is thought to be the underlying cause of major brain diseases including epilepsy.  In association with neuroinflammation (Blog 11 Sleep Disturbances and Epilepsy – Adversaries), oxidative stress is considered an important facilitator in epilepsy that eventually leads to brain damage.  On this point, there exist numerous reviews and animal studies supporting a causative role of oxidative stress in epilepsy.  Sadly, there are only a handful of clinical trials that have tested this role.

Scientists know very little about the development and progression of epilepsy.  It is clear that abnormal repetitive neuronal activity called seizures, are the major symptom of the disease.  A small percentage of  epilepsies are due to known genetic mutations.  The remainder, termed acquired epilepsies, have possible origins in known neurological insults e.g. trauma, illness. However, the cause of acquired epilepsy for most, remains unknown.  Therefore, studies that might identify a common biological pathway such as oxidative stress, for the majority of epilepsies, may lead to better therapies and a possible cure.

Recall that approximately 30-40 percent of patients with epilepsy do not respond to anti-seizure medication.   Those that do, however, must endure side effects, some of which cause patients to stop their medication.  Furthermore, anti-seizure medication treats the symptoms and not the underlying pathological processes.  Thus, it is vital that research into the basic mechanism(s) of epilepsy is front and center.  Evaluation of the role of oxidative stress in epilepsy is a reasonable place to start.

What is Oxidative Stress?

Metabolic processes in our cells, whether dependent on enzymes or not, constantly produce substances (small molecules, particles) that are highly reactive with nearby essential compounds.  These reactive species (RS) contain oxygen, nitrogen or carbon that promote oxidative changes.  Importantly, there is an abundance of RS in our cells and at high concentrations, all RS may directly harm neighboring indispensable compounds, such as proteins, DNA and fats.  Specifically, if the damaged compound is not repaired, these random oxidative changes hinder and subsequently prohibit normal cellular functions.  The outcome is that cells die prematurely and create a state of inflammation.

Fortunately, humans possess a wealth of anti-oxidative defense mechanisms to prevent random oxidative destruction.  These mechanisms include the well-known anti-oxidative compounds e.g. vitamin C and E, selenium, polyphenols, lycopene and flavonoids, all found in fruits and vegetables.  Lesser known mechanisms but of higher overall benefits are the anti-oxidative enzymes e.g. superoxide dismutase (SOD), catalase and glutathione peroxidase.  Additionally, sophisticated repair mechanisms exist that exclusively identify and repair oxidized DNA and proteins.

Oxidative stress occurs when the amount of RS overwhelms the antioxidative systems.  Therefore, key components are not repaired and become dysfunctional.  Fortunately, our anti-oxidative mechanisms are so effective that oxidative stress is minimal.  However, with age, illness, injury, extreme stress, and initial stages of chronic disease, oxidative stress is evident and harmful.

Role of Oxidative Stress in Epilepsy

Several recent reviews summarize the experimental data supportive of a role of oxidative stress in seizures and progression of epilepsy (Lin et al., 2020; Parsons et al., 2022).  Mainly, oxidative stress as defined above is considered “one of the early cellular events and a critical factor to determine the fate of neurons in epilepsy.” (Lin et al., 2020). 

Supportive Data that Oxidative Stress is a Key Mechanism in Epilepsy

1.  Epilepsies due to genetic mutations and genetic animal models of epilepsy show production of reactive species, deficiency of anti-oxidative mechanisms and subsequent neuronal damage (Pearson-Smith et al., 2017). 

2.  The amount of oxidative metabolites and antioxidative potential in children and adults with severe epilepsy correlate with seizure frequency (Morimoto et al., 2016).  Additionally, altered anti-oxidative systems have been identified in patients with temporal lobe epilepsy (Keskin et al., 2016).   Analysis of resected human brain tissue (those undergoing surgery) show evidence of oxidative damage and neuronal cell damage and death (Pecorelli et al., 2015).

3.  Results of numerous experiments in experimental models of epilepsy report that anti-oxidants e.g. N-acetyl-cysteine, melatonin, vitamin C and vitamin E inhibit seizure-induced oxidative damage.  Also, artificial activation of the genetic network that promotes anti-oxidative mechanisms prevents oxidative stress, preserves neurons and reduces seizures in animal models of epilepsy.

Clinical Trials

Armed with aforementioned findings, clinicians began to assess the efficacy of anti-oxidant therapy in patients with epilepsy.  Nineteen antioxidant compounds have been successfully evaluated in animal models of epilepsy (Lin et al.,2020).   Of these, 7 compounds (N-acetyl cysteine; ubiquinone; vitamin C, selenium/sildenafil; vitamin E and melatonin) have undergone clinical trials in man.  Despite the fact that many trials ended more than 7 years ago, results for only two of these compounds, vitamin E and melatonin, are publically available. 

Vitamin E

Vitamin E is a fat soluble anti-oxidant found in nuts, plant-based oils (e.g. olive, sunflower) and avocados, for example.  Two clinical trials using vitamin E as an add-on to anti-seizure medication produced contrasting results.  The first study of 12 patients with epilepsy reported that vitamin E, daily for six months significantly reduced seizure frequency in 10 of the 12 patients (Ogunmekan and Hwang, 1989).   In contrast, daily use of vitamin E for 3 months in 43 patients was without effect on seizure frequency or severity (Raju et al., 1994).  Both studies used what clinicians consider to be very high doses of vitamin E (more than 15 times the recommended daily allowance).

Melatonin

Melatonin is a sleep regulating hormone produced by the pineal gland in the brain.  It is commercially available in extended release form to enhance sleep duration and quality.  Additionally, due to its structure, it acts as an anti-oxidant.  Since epilepsy is associated with sleep disturbances (Sleep Disturbances and Epilepsy – Adversaries), the use of melatonin as an add-on therapy for drug resistant (intractable epilepsy) is reasonable.

There have been six published clinical trials evaluating melatonin in epilepsy.  Five of the 6 studies pertain to children and evaluated melatonin efficacy in 6-37 youngsters with epilepsy for generally a 3 month period.  Melatonin significantly improved sleep characteristics and improved seizure severity in 2 of the 5 studies.  Due to the small number of participants, it is impossible to know why positive results occurred in some but not all studies.  One study in adults with generalized epilepsy found melatonin added on to therapy of valproate for 8 weeks improved both sleep characteristics and reduced the seizure frequency and severity (Verma et al., 2021).  Although larger than trials with children, this trial evaluated only 52 patients.

Yin-Yang of Oxidative Stress

Conclusions

There is considerable experimental data supporting the hypothesis that oxidative stress is instrumental in creating an abnormal environment that continues to perpetuate seizures and maintain the disease state.  Clinical evaluations of more effective antioxidative compounds would be worthwhile.  The positive results of melatonin in adults is a good start that needs confirmation with a larger study.

Select References

Keskin Guler, S et al., Antioxidative-oxidative balance in epilepsy patients on antiepileptic therapy: A prospective case-control study. Neurol. Sci. 5:  763–767, 2016.

Lin T-K et al., Seizure-Induced Oxidative Stress in Status Epilepticus: Is Antioxidant Beneficial? Antioxidants 9: 1029, 2020

Morimoto M et al., Oxidative Stress Measurement and Prediction of Epileptic Seizure in Children and Adults With Severe Motor and Intellectual Disabilities. J Clin Med Res. 8(6):  437-444,2016.

Ogunmekaan AO, Hwang PA.  A randomized, double-blind, placebo-controlled, clinical trial of D-alpha-tocopheryl acetate (vitamin E), as add-on therapy, for epilepsy in children. Epilepsia. 30(1):  84-9, 1989.

Parsons ALM et al., The Interconnected Mechanisms of Oxidative Stress and Neuroinflammation in Epilepsy. Antioxidants  11:  157, 2022.

Pecorelli A et al., NADPH oxidase activation and 4-hydroxy-2-nonenal/aquaporin-4 adducts as possible new players in oxidative neuronal damage presents in drug-resistant epilepsy. Biochim Biophys Acta.  1852(3):  507-19, 2015..

Raju GB, Behari M, Prasad K, Ahuja GK.  Randomized, double-blind, placebo-controlled, clinical trial of D-alpha-tocopherol (vitamin E) as add-on therapy in uncontrolled epilepsy. Epilepsia. 35(2):  368-72,1994.

Verma N et al.,  Effect of add-on melatonin on seizure outcome, neuronal damage, oxidative stress, and quality of life in generalized epilepsy with generalized onset motor seizures in adults: A randomized controlled trial.  J Neurosci Res. 99(6):  1618-1631, 2021.

Sleep Disturbances and Epilepsy – Adversaries

Epilepsy Topics, Sleep disturbances, , , , ,

Assessment 11 – A complex relation exists between sleep disturbances and epilepsy.  Despite recognizing this reciprocity for well over a century, researchers are just now defining the adversarial effects of sleep disturbances on seizure frequency and severity of epilepsy on the one hand, and, on the other hand, the equally harmful effects of epilepsy on quality of sleep.  This blog will present the current understanding on the bidirectional relation of sleep disturbances and epilepsy

Introduction

Many important biological activities exhibit a cyclic function, known as circadian rhythms.  The most widely known circadian rhythm is the sleep-wake cycle.  This cycle exhibits a regularity of  7-9 hours of sleep, alternatively considered a transient unconscious state, and 14+ hours of conscious wakefulness.  The cyclic nature is tightly regulated by a plethora of genes, some aptly called clock genes.

Sleep is essential.  It provides a restorative benefit for our organs. Furthermore, It is necessary for the consolidation of long term memories. It is critical for removal of toxic metabolic waste products that accumulate in the brain during the day.  

Measurements of brain wave activity show that sleep occurs in several stages numbered 1-4. Light sleep spans stages 1 and 2; deep sleep develops in stages 3 and 4.  Stage 4 is followed by a period of rapid eye movement (REM) that is associated with dreaming.   Humans experience these stages (1-4, REM) over 90 minutes or so, resulting in about 7 repeats per night.  The more time spent in stages 3, 4 and REM, the fewer awakenings, the better rested one feels, and the greater the health benefit.

Sleep Disturbances

Sleep disturbances may take several forms.  They are 1) obstructive sleep apnea (OSA), a condition of reduced airway support and subsequent serious perturbations of oxygen and carbon dioxide levels, 2) insomnia, an inability to maintain sleep and 3) poor quality of sleep with frequent awakenings and reduced deep sleep (stages 3,4) and REM sleep. 

Sleep disturbances lead to complaints of excessive daytime sleepiness, reduced cognition and accidents.   Untreated OSA results in additional negative changes such as an increased risk for hypertension, diabetes, and heart disease.  Sleep disturbances also may exacerbate epilepsy and vice versa, epilepsy may worsen sleep disturbances.

Research Findings

Effects of Sleep Disturbances on Epilepsy

Sleep disturbances and epilepsy are intertwined.  To what extent and why is not entirely clear.  The most well-known finding supporting a relation of sleep disruptions and epilepsy is one used clinically to provoke seizures.  That is the state of sleep deprivation.  A night without sleep increases the likelihood of recording a daytime seizure with the 20 minute clinical EEG test.  In other words, sleep deprivation provokes seizures. It is used clinically to verify their presence. However, unraveling the mechanism of this empirical observation represents a challenge that is just beginning to be met.

Prospective Trial – Seizure Susceptibility Influenced By Sleep Deprivation

Sleep disturbances may initiate the onset of epilepsy.  A 10 year prospective population study (Harnod et al., 2017) that tracked medical records of  those (~ 277,000 individuals) half with and half without sleep disturbed breathing support this observation.  Harnod et al (2017) showed “the cumulative incidence of epilepsy was higher in the sleep disturbed breathing cohort than in the comparison cohort”.  The authors also concluded  that “Sleep deprivation increases seizure susceptibility and often provokes epileptic seizures”.  The data are impressive but regrettably they lack key information relating to patient confirmation, epilepsy type, seizure frequency and other confounding factors such as smoking.

A polysomnographic study is the medical term for a sleep study.  Polysomnography measures brain waves (EEG), blood gases (oxygen and carbon dioxide) and quantifies the number of reduced/absent breathing episodes per night (see detail at https://www.mayoclinic.org).  Therefore, polysomnographic studies provide quantitative data.

Result from these studies, reported individually or summarized in a meta-analysis(Lin et al., 2017) indicate that the prevalence of one of the most serious sleep disturbances, obstructive sleep apnea (OSA), is about 30% in patients with epilepsy treated with antiseizure drugs and higher in those with drug-resistant epilepsy.  Importantly, treatment of OSA in patients with epilepsy with continuous positive airway pressure (CPAP) not only corrects OSA but also reduces seizure frequency.   

Effects of Epilepsy on Sleep

According to several studies using a questionnaire, patients with epilepsy report a higher number of sleep complaints compared to those without epilepsy.  Complaints included insomnia, excessive daytime sleepiness and disturbed breathing such as obstructive sleep  apnea.  The complaints were 25-50% higher in patients with epilepsy compared to those without epilepsy

Night Time Seizures

Epilepsy impacts sleep in several ways.  Firstly, night time seizures, most common in frontal lobe epilepsy but occasionally occurring in other types of epilepsy, directly disrupt sleep.  It may cause insomnia or poor quality sleep. 

Discharges Between Seizures

Secondly, data from patients with an implantable brain recorder/stimulator device to reduce seizure frequency and severity, shed light on the indirect effects of seizures on sleep.  Recall from Assessment 8, continuous brain recordings in patients with epilepsy revealed the cyclic nature of epilepsy with seizure reoccurrence in intervals of days, weeks, months. Data show that abnormal brain waves termed interictal epileptiform discharges (IEDs) occur in between seizures in patient with epilepsy.  Interestingly, IEDs peak during sleep hours, specifically appearing during stages 1-4.  Their presence modifies normal sleep waves in those stages. In contrast, REM sleep suppresses IEDs.  It is unknown why this is.  One suggestion is that the character of normal brain waves in sleep stages 1-4 (synchronous and slow) is permissive for reception of irregular discharges such as IEDs. 

Anti-Seizure Medications

Thirdly, there is early recognition that some antiseizure drugs in certain types of epilepsy may enhance sleep disturbances and hence indirectly worsen the epilepsy by disturbing sleep character.  This issue is correctable with an alternative medication, shifting time of drug use and defining the sleep disturbance with a polysomnographic study.

Link between sleep and epilepsy

The results of numerous studies in animal models and in humans with epilepsy point to a common link between sleep disturbances and epilepsy.  Specifically, Bonilla-Jaime H. et al.,  (2021) reviewed an extensive body of evidence that supports the hypothesis that the common factor between sleep disturbances and epilepsy may be neuroinflammation.

Neuroinflammation

Neuroinflammation refers to a profusion of harmful changes in the brain initiated and maintained by a wealth of pro-inflammatory mediators.  Dying neurons and associated neurons called astrocytes and glial cells are the source of these mediators.  In measuring pro-inflammatory mediators, scientists conclude that both sleep disturbances and epilepsy promote neuroinflammation and the two together create a vicious cycle.  In other words, epilepsy worsens sleep disturbances and sleep disturbances worsen epilepsy through this pathway of neuroinflammation.

This is a milestone discovery with opportunities for new therapies for epilepsy.  Several therapies already have the backing of clinical trial results.  For example, use of continuous positive airway pressure (CPAP) restores normal breathing in patients with epilepsy and  OSA, and importantly, decreases seizure frequency and severity.  Another therapy, the ketone diet (see Assessment 9) decreases neuroinflammation and reduces seizure frequency in certain types of epilepsies.  Possible future therapies are anti-inflammatory medications to prevent and/or minimize neuroinflammation and additionally, therapies for insomnia and poor sleep such as cognitive behavioral therapy.

Conclusions

Unraveling the relation between sleep disturbances and epilepsy is important.  The modest amount of work to date defines an adversarial reciprocity between sleep disturbances and epilepsy with each worsening the other.  However, defining a common pathway such as neuroinflammation offers hope of someday diminishing both sleep disturbances and epilepsy. Clearly, future large clinical trials could provide definitive progress.

Proposal: Sleep Disturbances and Epilepsy related through neuroinflammation

Select References

Bonilla-Jaime H. et al., Sleep Disruption Worsens Seizures: Neuroinflammation as aPotential Mechanistic Link. Int. J. Mol. Sci. 22:  12531, 2021.

Harnod T, Wang Y-C, Lin C-L, Tseng C-H.  High risk of developing subsequent epilepsyin patients with sleep-disordered breathing.  PLoS ONE 12(3):  e0173491, 2017.

Lin Z, Si Q, Xiaoyi Z. Obstructive sleep apnoea in patients with epilepsy: a meta-analysis. Sleep Breath 21(2): 263-270, 2017.

Marlow BA et al., Treating obstructive sleep apnea in adults with epilepsy A randomized pilot trial. Neurology 71:  572–577, 2008.

Pavlova MK et al., Proceedings of the Sleep and Epilepsy Workshop: Section 2 Comorbidities: Sleep Related Comorbidities of Epilepsy.  Epilepsy Currents 21(3):  210-214, 2021.

Scharf MT et al., Obstructive sleep apnea risk in patients with focal versus generalized epilepsy. Epilepsy & Behavior 111:  107190, 2020.

The Subscalp EEG Recording – promising technology 

Diagnostics, Epilepsy Topics, ,

Introduction

Subscalp EEG recording is a technological advancement positioned to revolutionize diagnostics, treatment and understanding of epilepsy.

Over the past 7 years or so, several investigatory groups have developed subscalp EEG recording systems. These systems consist of electrodes that can be implanted under the skin of the scalp.  Electrodes are attached to external data collection and analytical systems.  These systems allow continuous EEG monitoring 24/7 for months and potentially for years. 

Unmet Need

There is a unmet need for accurate long term measurements of seizures.  To provide a correct diagnosis of epilepsy and develop optimal therapy, the physician needs information on exact seizure frequency, location, and brain wave activity prior to and after a seizure. As discussed in Blog 8 (Intracranial EEG recording), implantation of electrodes directly in the brain continually assesses brain wave activity and has provided significant insights in the cyclic nature of the disease.  However, intracranial recordings necessitate major surgery and, therefore, are available for only a small number of patients with epilepsy.  On the other hand, scalp EEG (electrodes placed on top of the scalp) coupled with video monitoring, although not invasive, supply continuous seizure assessment for no more than 14 days, at best.

Ultra-long term EEG recording lies between intracranial recording and scalp recording.  It has potential to not only determine seizure frequency and location but to confirm the cyclic nature of epilepsy, thereby significantly adding to insights from intracranial EEG recordings.  Furthermore, subscalp EEG systems appear capable of predicting future seizures, thus allowing for preemptive suppression.

However, keep in mind, this technology is in its initial stages but has great promise.

Validation of Subscalp EEG Recording

While there are many foreseeable uses of continuous subscalp EEG recordings, the first use is for long term acquisition of seizures and pre/post seizure activity in patients with epilepsy. 

There are 6 systems in clinical validation phases (clinical trial completion, ongoing or about to begin).  Two systems, the 24/7EEG SubQ (https:// www.uneeg. com/ en/ epilepsy/products/subq) and the Minder have completed small clinical safety/feasibility studies (see Weisdorf et al., 2018; 2019; Sterling et al., 2021). 

The 24/7EEG SubQ system

The 24/7EEG SubQ system consists of  a) subcutaneously implanted electrodes and b) a data collection system composed of a transceiver (transmitter/receiver combo), data storage device and power supply.  The first study with this system (4 patients with temporal lobe epilepsy) was a feasibility (proof-of-concept) study.  Results showed that SubQ EEG recordings were similar to standard scalp EEG recordings.  Specifically, the SubQ system measured seizures, pre-seizure activity, sleep brain wave activity and some unrelated activity.  These are all findings comparable to standard EEG recordings. 

The most outstanding characteristic of the subscalp recording is, unlike standard scalp EEG measurements, its ability to gather data over long periods of time.  The second clinical trial with the SubQ device implanted in 9 patients with temporal epilepsy and monitored EEG activity for the target time of 3 months (achieving a minimum of 9 weeks of recording for each patient).  This is clearly a milestone since it is the first study to demonstrate “ultra-long” EEG monitoring with a minimally invasive device and without disruption of daily activities of living.

The Minder System

Another subscalp EEG system, the Minder completed an 8 months study with 5 patients with epilepsy. The components of the Minder system are electrodes capable of monitoring brain activity in both hemispheres and collecting data by telemetry to a processor placed behind the ear.  A smart phone receives the data and stores it in the cloud. 

Subscalp EEG data from the Minder compared favorably with standard video/scalp EEG data obtained at specified times during the study.  These results confirm the feasibility of a minimally invasive implanted subscalp EEG system such as the Minder for long term monitoring of focal seizures.

Adverse Effects of Subscalp EEG Recording

Adverse effects from subscalp devices described above are mild.  Expected pain and soreness at the site of surgical implantation lasting about a week occurred in most patients.  However, few experienced pain and soreness beyond one week.  Some experienced mild headaches unrelated to the surgery.

Limitations of Subscalp EEG Recordings

Firstly, the collection of thousands of hours of brain wave activity creates an analytical nightmare.  In summarizing the data to reasonably handle it, some data may be lost.  Efforts to develop intelligent algorithms are in development so that all data can be treated equally.  Secondly, the position of the implanted electrodes may miss seizures originating deep in the brain. Thus some patients with epilepsy may not benefit from subscalp EEG devices.  Thirdly, completed safety and feasibility studies necessarily involved a small number of select patients (those with temporal lobe epilepsy).  Therefore, for confirmation of the generalized benefit of subscalp EEG for most patients with epilepsy, larger scale studies with patients with diverse types of epilepsy are required.

Conclusions

Subscalp EEG recording systems represent a relevant and dearly needed advancement over the short term EEG recordings of the standard scalp EEG.  Subscalp EEG systems provide ultra-long term recordings. These recordings provide accurate determinations of seizure frequency and location, information essential for optimal drug therapy or future surgical decisions.  Especially attractive is the fact that subscalp EEG systems require minimal surgery for implantation and once implanted do not disrupt everyday activities.

There are at present 6 subscalp EEG systems, 2 of which successfully demonstrated safety and feasibility  in patients with temporal lobe epilepsy.  This is a significant start.  The limited results thus far are indeed very promising.  Continued development and clinical assessment can promote and expand a transforming new technology to better serve patients with epilepsy.  This blog will provide updates on subscalp EEG systems from future clinical trials.

Comparison of EEG Technologies

References (http://pubmed)

Duun-Henriksen J et al., A new era in electroencephalographic monitoring? Subscalp devices for ultra–long-term recordings. Epilepsia.61:  1805–1817, 2020.

Stirling RE et al., Seizure forecasting using a novel sub-scalp ultra-long term EEG monitoring system. Front. Neurol. 12:713794, 2021.

Weisdort S et al., High similarity between EEG from subcutaneous and proximate scalp electrodes in patients with temporal lobe epilepsy. J Neurophysiol 120: 1451–1460, 2018.

Weisdort S. et al., Ultra‐long‐term subcutaneous home monitoring of epilepsy—490 days of EEG from nine patient. Epilepsia. 60:  2204–2214, 2019.

Ketogenic Diet: Can It Cure Epilepsy?

Epilepsy Topics, Ketogenic Diet, , , ,

Introduction to the Ketogenic Diet

Assessment 9 – Anti-seizure medication is the standard therapy for patients with epilepsy as discussed in an earlier blog ( Assessment 6 – Epilepsy Medication – Need to Know Information). Despite the availability of over 20 some anti-seizure drugs, approximately 30-35% of patients with epilepsy gain no benefit from these medications.  These patients are considered to have resistant epilepsy.  Options for resistant epilepsy are few.  However, one such option is the ketogenic diet.  This blog will describe the ketogenic diet, its effect on seizures and its limitations.

Background

The composition and efficacy of the ketogenic diet was published close to 100 years ago in a paper by Peterman (1928).  It is named the classic ketogenic diet (CKD) to distinguish it from the more recently developed variations of this diet.  In many respects, the CKD is similar to starvation/fasting diets in vogue to treat epilepsy some 2000 years ago. 

Classic Ketogenic Diet (CKD)

Components of the Classic Ketogenic Diet

CKD is a diet that is very high in fats and very low in carbohydrates and proteins.  Adherence to the CKD requires consumption (as a percentage of daily calories) of 80-90% fats, 6-8% protein and 3-4% carbohydrates.  The dietary goal of the CKD is to convert normal metabolism driven by the use of sugars for energy to one driven by the use of fats for energy.  The result is a decline in blood levels of sugar and insulin and an elevation in blood levels of fats and their metabolic products, termed ketone bodies.  Ketone bodies (e.g. hydroxybutyrate, acetoacetate and acetone) are fats readily utilized by the brain.  Although poorly understood, the outcome is seizure suppression.

Modified Ketogenic Diets

As you can imagine, the CKD with its incredibly high fat content requirement is unappealing and to some quite unpalatable.  Patients that do not immediately respond to the diet, readily give up on it.  Generally after 2 years, only 30% of patients remain on the diet.  Hence, over the years, scientists developed modifications of the CKD. 

The established variations are a) modified Atkins Diet (MAD), b) the low glycemic index treatment (LGIT), and c) the medium chain triglyceride diet (MCTD).  Compared to CKD, these modified diets permit a significant reduction in consumption of fats and allow an increase in protein and carbohydrate consumption.  Specifically, the percentage of calories from fat is reduced to 60% or less. Protein is increased to 10-25 %. Similarly, carbohydrates is increased to 10-19%.  Although not studied as extensively as the CKD, these diets are more palatable. So far, the results of studies to date indicate that efficacy is close to that shown with the CKD.

Actual Dietary Benefits – Seizure Reduction

Children/Adolescents with Resistant Epilepsy  

Since 1928, as reviewed by Wells et al., (2020)  results of many studies (observational trials, randomized clinical trials and meta-analyses) show that adherence to the ketogenic diet significantly decreases seizure frequency in children and adolescents with resistant epilepsy.  In particular, from 2008 to 2017, 7 randomized clinical trials tested the efficacy of CKD or one of its variants.  The trials differed in number of patients (48-145), age of participants (1-18 years) and duration (3- 16 months).  Nevertheless, patients consuming ketogenic diets compared to those on a normal diet experienced significant reductions in seizure frequency.  Depending on the trial, 38% to 100% of patients on ketogenic diets experienced greater than a 50% reduction in seizure frequency.  Additionally, a smaller number  of patients became seizure free.

Adult Patients with Resistant Epilepsy  

There are few studies on the use of ketogenic diets in adult patients with resistant epilepsy.  A summary analysis of several small clinical trials with adults (18-86 yrs) with drug resistant epilepsy, concluded that CKD was efficacious in adults.  In particular, 53%  of patients experienced greater than 50% reduction in seizure frequency. Thirteen percent of patients were seizure free on the diet.  There is a need for confirmation with larger trials.

Mechanism of Seizure Reduction

There is no convincing evidence as to how an increase in ketone bodies serves to suppress seizures and in some cases to cure epilepsy.  Study results in animals suggest that ketone metabolism unlike sugar metabolism promotes

a) anti-inflammatory activity and neuronal protection and repair,

b) suppresses production of mediators that excite nerves and enhances production of mediators that inhibit abnormal nerve activity,

c) influences gut bacteria to produce effective brain relevant compounds, and changes brain metabolism to optimize function of favorable nerve genes. 

 At present, there is no evidence in man that these pathways predominate during adherence to ketogenic diets.

Adverse Effects Of the Ketogenic Diet

Appearance of adverse effects is one reason patients discontinue the ketogenic diet.  To be fair, this is not the only reason. Lack of efficacy and poor palatability of the diet are generally more important. Not surprisingly, a series of gastrointestinal-related changes are the main complaints with consumption of ketogenic diets.  Specific adverse effects include constipation, diarrhea, abdominal pain, gastroesophageal reflux, and hunger.  Other adverse effects, not as common occur with long term use (greater than one year). They are kidney stones, reduction of bone mineral content, and abnormal blood lipid profile e.g. elevation of triglycerides/low density lipoprotein (LDL) and decrease in high density lipoprotein (HDL).  However, abnormal blood lipids did not result in cardiovascular disease.  As expected, modified CKDs e.g. diets noted above, result in fewer and less severe adverse effects. Hence patients achieve greater tolerance and adherence

Summary

Ketogenic diets remain an option for patients of all ages with resistant epilepsy.  For those responding to these diets, seizure frequency declined by half or more and some patients become seizure free.  Compared to the classic ketogenic diet, the modified diets encourage greater adherence. Patients experience fewer adverse effects and efficacy is nearly equal to the classic diet.  Considering these observations, it seems incredibly important for scientists to determine exactly how ketogenic diets work to suppress seizures.

Reference used for this blog are available on request and found on pubmed.com.