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Discovery of Antiseizure Compounds in Zebrafish Models for the Treatment of Drug-Resistant Epilepsy

Boek - Dissertatie

With 70 million people affected worldwide is epilepsy one of the most common neurological diseases characterized by unpredictable, unprovoked, recurrent seizures. Moreover, the disease is often accompanied by psychiatric and cognitive comorbidities, affecting dramatically the quality of life of patients. So far, pharmacological intervention is the first-line treatment for epilepsy. Unfortunately, about 30% of patients experience seizures that cannot be well controlled with the current marketed antiseizure drugs (ASDs).To find novel compounds a wide variety of preclinical animal epilepsy and epileptic seizure models has been generated and employed in phenotypic drug discovery projects. For instance, in 2015, the updated Epilepsy Therapy Screening Program (ETSP) (NIH, USA) has incorporated several rodent seizure models with drug-resistant signature in its working flow. Although those models have the potential to identify innovative ASDs with efficacy in as yet ASDs-resistant patients, the newly screening pipeline is labor-intensive, and has limited throughput. More recently, zebrafish models, especially those with a drug-resistant profile and high-throughput capacity, have gained an increasing popularity in drug discovery. Zebrafish display an excellent compromise between system complexity of the vertebrate organism and the practical simplicity of the in vitro models. Moreover, they allow to find candidate ASDs at a lower cost and time, and thus can be used in the early-stage drug screening to speed up innovative ASDs discovery.In this doctoral research two distinct zebrafish models, a chemically-induced and genetic zebrafish seizure and epilepsy model that proved to exhibit high pharmacoresistant profiles were used for the discovery of new hits and the identification of compounds of interest.
Compared to random screening of compounds, a medicinal plant-based approach has been suggested to result in a faster and cheaper identification of active ingredients, as these plants have been pre-selected through centuries of use by ethnomedical practitioners. Traditional Chinese Medicine (TCM) is one of the most widely practiced forms of botanical therapy in the world, including also multiple recipes of medicinal plants against epilepsy and seizures.Therefore, in the first project 42 extracts of medicinal extracts from fourteen neuroprotective and antiseizure TCM plants were prepared, and a phenotype-based screening was performed using a combination of acute zebrafish seizure models (PTZ and EKP) and a rodent seizure model (mouse 6-Hz psychomotor seizure model).Both the zebrafish EKP seizure and the mouse 6 Hz (44 mA) psychomotor seizure models represent a high potential to identify ASDs with a novel mechanism-of-action. Our strategy led to the identification of magnolol and honokiol, main constituents Magnolia officinalis, as well as the structurally related allyl biphenolic methylhonokiol as potent antiseizure agents. In addition, magnolol was able to protect mice from seizures induced by 6-Hz electrical stimulation in a dose-dependent manner, thereby confirming its antiseizure activity in a mammalian model.
Dravet syndrome (DS) is a catastrophic genetic epilepsy of childhood characterized by a variety of drug-resistant seizures initially often induced by fever. Of interest, distinct de novo mutations in the SCN1A gene are found in about 85% patients with DS. A genetic zebrafish Dravet model based on scn1Lab mutants, has been developed to accurately reflect the genetic basis and characteristics of DS. This model was used in our second study with the aim to explore the antiseizure activity of enantiomers of fenfluramine (FFA) and its presumedly active metabolite norfenfluramine (norFFA), expanding on the clinical treatment of DS patients with racemic FFA. Firstly, we validated the model pharmacologically by using both single and combined ASDs. The results were in accordance with the updated clinical treatment algorithm for DS. Thereafter, the antiseizure activities of the compounds were confirmed showing a concentration-response relationship, thereby demonstrating that both the enantiomers of FFA but also their norFFA metabolites contribute to the antiseizure activity of racemic FFA in DS patients.Taken together, our work discovered three potential hits against therapy-resistant epilepsies and revealed the value of medicinal plants as an interesting resource for ASDs discovery. Furthermore, evidence is provided that the use of zebrafish as high-throughput model of treatment-resistant seizures can be deployed in innovative ASDs discovery.
Jaar van publicatie:2020