In 2013, the Baraban Lab published their work on creating and characterizing an scn1lab mutant model of zebrafish that results in many symptoms of the human disease Dravet syndrome. In that initial report, they described how they used the model to screen thousands of compounds for both anti-seizure effects and non-toxicity, identifying several targets for potential treatment of DS including fenfluramine, lorcaserin, trazodone, and clemizole. Fenfluramine, lorcaserin, and trazodone are known to act via serotonin receptors (“5-HT” receptors) and are all in clinical development for Dravet syndrome. Clemizole, also in clinical development for Dravet syndrome, was initially surprising because it is an old antihistamine known to affect histamine (H1) receptors, and antihistamines are often contraindicated in treatment of epilepsy. Subsequent studies identified a serotonergic (5-HT) mechanism of action of clemizole, and suggested it was this effect on serotonin receptors, and not histamine receptors, that produced the apparent anti-seizure activity of clemizole. In this study, the authors investigated exactly which 5-HT receptors were key to reducing seizures in the zebrafish model of Dravet syndrome by synthesizing several analogs of the compound clemizole, adding and removing portions of the molecule as needed to increase affinity to specific serotonin receptors, and assessed their efficacy in the zebrafish model of DS.
Although there are several 5-HT receptors (labeled “5-HT1-7” with further divisions labeled “5-HT2A, B, C, etc“), they focused on 5-HT2 receptors because of studies linking these receptors to seizures and premature death in animals, as well as previous work linking fenfluramine, lorcaserin, and trazodone to the 5-HT2 group of receptors. Clemizole itself was previously found to show affinity for the 5-HT2A and 5-HT2B receptors. Here, the authors created and tested 28 of their synthesized clemizole analogs, throwing out any that proved toxic failed to reduce seizure-like movement in the larvae. Three of the compounds successfully suppressed seizure-like activity in the zebrafish larvae and showed a definite preference for 5-HT2B over 5-HT2A or 5-HT2C receptors. The authors then screened several commercially available compounds known to be attracted to the 5-HT2B receptors and found three that also decreased seizure-like movements in the larvae, supporting the hypothesis that the 5-HT2B receptor is important in reducing seizures in the zebrafish.
The compounds that successfully decreased seizure-like movements then went on to the second phase of testing, which is like an EEG recording. The authors immobilize the larvae in a jelly-like substance and insert a microelectrode into the brain to measure abnormal electrical activity. The three analogs of clemizole as well as two other available compounds passed the EEG phase of testing, providing further evidence that the 5-HT2B receptors are indeed important in reducing seizures in this model.
The authors summarize that 5 of the most promising candidates showed preference for 5-HT2B receptors, suggesting this could be a focus for future treatment, and examine the evidence for expression of these receptors in various types of neurons in the brain. However, they note that increasing specificity decreased affinity over the parent compound clemizole, which could present a challenge to development, and discuss other challenges affinity for 5-HT2B may present.