Efficacy of Fenfluramine and Norfenfluramine Enantiomers and Various Antiepileptic Drugs in a Zebrafish Model of Dravet Syndrome

Abstract

Dravet syndrome (DS) is a rare genetic encephalopathy that is characterized by severe seizures and highly resistant to commonly used antiepileptic drugs (AEDs). In 2020, FDA has approved fenfluramine (FFA) for treatment of seizures associated with DS. However, the clinically used FFA is a racemic mixture (i.e. (±)-FFA), that is substantially metabolized to norfenfluramine (norFFA), and it is presently not known whether the efficacy of FFA is due to a single enantiomer of FFA, or to both, and whether the norFFA enantiomers also contribute significantly. In this study, the antiepileptic activity of enantiomers of FFA (i.e. (+)-FFA and (−)-FFA) and norFFA (i.e. (+)-norFFA and (−)-norFFA) was explored using the zebrafish scn1Lab−/− mutant model of DS. To validate the experimental conditions used, we assessed the activity of various AEDs typically used in the fight against DS, including combination therapy. Overall, our results are highly consistent with the treatment algorithm proposed by the updated current practice in the clinical management of DS. Our results show that (+)-FFA, (−)-FFA and (+)-norFFA displayed significant antiepileptic effects in the preclinical model, and thus can be considered as compounds actively contributing to the clinical efficacy of FFA. In case of (−)-norFFA, the results were less conclusive. We also investigated the uptake kinetics of the enantiomers of FFA and norFFA in larval zebrafish heads. The data show that the total uptake of each compound increased in a time-dependent fashion. A somewhat similar uptake was observed for the (+)-norFFA and (−)-norFFA, implying that the levo/dextrotation of the structure did not dramatically affect the uptake. Significantly, when comparing (+)-FFA with the less lipophilic (+)-norFFA, the data clearly show that the nor-metabolite of FFA is taken up less than the parent compound.

Clemizole and trazodone are effective antiseizure treatments in a zebrafish model of STXBP1 disorder

“…As described, 13,14,29 a zebrafish model of STXBP1‐related disorders was generated using CRISPR‐Cas9 gene editing. LFP recordings in homozygous stxbp1b mutant larvae (4 dpf) confirmed the presence of spontaneous ictal‐like (Type II) epileptiform events (Figures 1A,B).…”

“…Drug solutions (~0.5 mL) were bath applied using a polyethylene disposable transfer pipette (Fisherbrand #13–711‐7 M) to agarose‐embedded larvae at room temperature. All drugs were tested at a concentration of 1 mM as previous studies empirically indicate millimolar (mM) drug concentrations are appropriate for acute pharmacology studies in agarose‐embedded larvae 16,24–30 . Experiments were performed on at least three independent clutches of larvae for each drug.…”

“…LFP analysis focused on long‐duration (>1 s), large‐amplitude (>0.5 mV) Type II ictal‐like multi‐ or polyspike events as these were previously shown to correlate with whole‐body convulsive seizure behaviors consisting of high‐velocity head and tail movements 29 and brain‐wide network hypersynchronization 30 . Type I electrical events are defined as low‐amplitude interictal‐like sharp waveforms, with voltage deflections at least three times above baseline (Figure 1C).…”

“…Baseline noise level was measured as 0.017 ± 0.005 mV (n = 75). Quantification of Type II event frequency was performed using threshold detection and custom MATLAB software (see Figure 1C), as described previously 29 . Briefly, a binning method combined with a sliding window algorithm was used to calculate the active level of the signal within the current time window.…”

A Review of Targeted Therapies for Monogenic Epilepsy Syndromes

“…Fenfluramine ( 36 ), cannabidiol ( 37 ), and stiripentol ( 35 ) have all been found to provide clinical benefit for this severe epilepsy syndrome in randomized clinical trials ( 52 ). These clinical findings are buttressed by animal studies, including a study of fenfluramine and norfenfluramine in a zebrafish model of DS ( 53 ) and a study of cannabidiol and stiripentol in hyperthermia-induced seizures in a mouse model of DS ( 54 ). We include these medications as “precision therapies” given their efficacy, even though the precise mechanism of action in DS patients is not fully understood.…”