An Examination of the Mechanism of Action of Fenfluramine in Dravet Syndrome: A Look Beyond Serotonin Parthena Martin, Brooks Boyd, Gail Farfel, Arnold Gammaitoni, Brad Galer Zogenix, Inc., Emeryville, CA, USA

ff Sigma (non-selective) activity was assessed using a twitch Table 3: Enantiomers of FFA and Norfenfluramine: Ki Values (mol/L) INTRODUCTION contraction in guinea pig vas deferens tissue using Demonstrating >30% Binding Based on Percent Inhibition DISCUSSION ff Racemic fenfluramine (FFA) has provided unique, (+)-SKF-10,047 as the standard Receptor (+) FFA (-) FFA (+) Norfen (-) Norfen Positive Control ff FFA is known as a “serotonergic” drug unexpected seizure control with a prolonged duration of b Adrenergic 3.53 x 10-9 1.61 x 10-5 1.36 x 10-5 9.76 x 10-6 8.48 x 10-6 –– FFA releases serotonin by disrupting vesicles and reversing effect in children and young adults with Dravet syndrome,1 (non-selective) (propranolol) the serotonin transporter function so that there is an -10 which is unusual in the treatment of epilepsy. RESULTS -6 -5 -6 -6 3.97 x 10 increase in serotonin in the synapse b2 Adrenergic 8.84 x 10 1.40 x 10 8.60 x 10 5.56 x 10 (propranolol) –– Zebrafish data have shown that FFA has some activity on ff FFA’s serotonergic effects are produced through two Literature Search 7.71 x 10-10 4 M Muscarinic 8.30 x 10-6 1.15 x 10-5 3.27 x 10-6 4.00 x 10-6 5-HT 1D, 2A, and 2C to exert an anticonvulsant effect mechanisms: release of serotonin (5-HT) by reversal of ff Forty-seven receptors were identified from the literature 1 (atropine) 2 ff The sigma 1 receptor may play a role in the serotonin transporter flux, and direct activation of serotonin Sigma 1.84 x 10-9 (Table 1) 1.63 x 10-7 3.51 x 10-7 1.80 x 10-6 2.30 x 10-6 receptors.3,4 There are limited data in the literature in animal (non-selective) (haloperidol) anticonvulsant activity of FFA 5,6 7-10 Table 1: Receptors Identified in the Literature as Associated 1.20 x 10-8 – Activation of the sigma 1 receptor attenuates seizures13,14 models and in humans using serotonin reuptake inhibitors, Sigma 1 1.09 x 10-7 5.02 x 10-7 2.61 x 10-6 4.60 x 10-6 – (pentazocine) suggesting serotonin agonism might provide some benefit With Seizures –– Concentrations of FFA that elicited activity in the assays 5.18 x 10-9 Adenosine A Cannabinoid CB Glutamate Non-selective Nicotinic Sigma 2 4.31 x 10-7 8.00 x 10-7 2.98 x 10-6 3.21 x 10-6 were close to predicted concentrations in brain tissue against seizures. Conversely, antidepressants have also been 1 1 (haloperidol) 11 –– The sigma 1 receptor dimerizes with other receptors, reported to lower seizure threshold in some instances. As b-Adrenergic Glycine - strychnine . CGRP Orexin 1 including the muscarinic M receptor, and it modulates (non-selective) sensitive 1 the incidence of depression and anxiety is reported as high Agonist and Antagonist Assays serotonergic neurotransmission15,16 12 as 24% in people with epilepsy and treatment with SSRIs is Chemoattractant a B-Adrenergic Histamine H PAF ff Functional activity was observed for the b adrenergic –– FFA did not act as a simple agonist or antagonist in 1 C 3 2 common, if indirect 5-HT agonism were a potent antiepileptic 3A the functional assay but had more complex behavior, receptor, the muscarinic M1 receptor, and the sigma mechanism, it can be assumed the effects would frequently b -Adrenergic Chemokine CCR IP Prokineticin 1 suggesting the involvement of other receptors 1 1 3 (non-selective) receptor be noted in clinical practice, and this is not the case. ff b -adrenergic and muscarinic M receptors are not b -Adrenergic CCK K Channel KAPT Prostanoid DP –– b Adrenergic receptor 2 1 ff Recently, Zhang6, Sourbron,4 and colleagues investigated 2 A 2 thought to be likely to play a role in the antiepileptic Angiotension AT Cl Channel Leukotriene D Serotonin 1A • (±) FFA and both enantiomers, as well as (±) norfenfluramine and the effects of direct agonism by FFA on specific serotonin 1 4 the (-) enantiomer acted as antagonists with IC ’s ranging from effect of FFA 6 50 receptors using the zebrafish model of Dravet syndrome, and Sigma non- Apelin APJ CRF Muscarinic M 49 to 70 µM –– Literature reports of the effect of FFA on these receptors they found that the antiepileptic activity in the zebrafish was 1 1 selective are sparse and suggest that any effect may be weak17 –– Muscarinic M1 receptor related to activation of 3 specific receptor subtypes: 5-HT 1D, Benzodiazepine BZ Dopamine D Na Channel Somatostatin 1 –– The binding and functional activity found in our assays 1 • (-) norfenfluramine acted as an antagonist with an IC50 of 95 µM 2A, and 2C. – Sigma receptor occurred at concentrations exceeding the plasma levels Bradykinin B EGF Neurokinin 1 Substance P – 18 1 in patients or predicted concentrations in brain tissue ff The efficacy contribution of each receptor was partial, • Agonist assay (conducted in absence of (+)-SKF-10,047) Bombesin BB GABA Neuropeptide Y Vasopressin 1B –– Therefore it is unlikely that these receptors contribute to the and the homology of the human serotonin receptors to 1 A 1 »» None of the compounds increased the twitch contraction anticonvulsant activity of FFA in Dravet Syndrome the zebrafish is not exact. Given this, and the unusual Vasoactive amplitude Calcitonin CALR Galanin GA1R1 Neuropeptide FF NPY Intestinal effectiveness of FFA, we decided to explore whether other 1 »» Racemic norfenfluramine as well as enantiomers produced Polypeptide 1 a concentration-dependent decrease in the baseline twitch neuronal systems might be involved in the unique efficacy HYPOTHESIS: In addition to its serotonergic effects, CA Channel Type L Glucocorticoid Neurotensin 1 contraction amplitude seen to date for FFA administered to control seizures in FFA may also act through the sigma 1 receptor to CALR, calretinin; CCK, cholecystokinin; CGRP, calcitonin gene-related peptide; CRF, corticotropin-releasing factor; EGF, • Antagonist assay (conducted in presence of (+)-SKF-10,047) patients with Dravet syndrome. epidermal growth factor; GA1R1, galanin receptor-1; GABA, γ-aminobutyric acid; IP3, inositol 1,4,5-trisphosphate; PAF, reduce seizure activity in Dravet Syndrome platelet-activating factor. »» Racemic FFA slightly increased the (+)-SKF-10,047-induced increase in twitch contraction amplitude METHODS Receptor Binding »» Racemic norfenfluramine and its enantiomers induced a concentration-dependent inhibition of the (+)-SKF-induced ff Searches on PubMed and MEDLINE were performed ff All receptors in Table 1 were tested for binding with FFA, increase in the twitch contraction amplitude December, 2014 by using the following terms: epilepsy, seizure, norfenfluramine, and their enantiomers REFERENCES receptor, receptor binding, CNS receptor, and synonyms ff Receptors that showed binding >30% as measured by percent 1. Ceulemans B, et al. Epilepsia. 2012;53:1131-9. ff All studies showing preclinical evidence of certain receptors inhibition are shown in Table 2 (FFA and norfenfluramine) and 2. Baumann MH, et al. Neuropsychopharmacoly. 2014;39(6):1355-65. potentially being involved in epileptogenesis, seizures, or 3. Vickers SP, et al. Neuropharmacoly. 2001;41:200-9. Table 3 (enantiomers of FFA and norfenfluramine) SUMMARY OF RESULTS 4. Sourbron J, et al. ACS Chem Neurosci. 2016;7:588-98. epilepsy were included ff Pharmacologically meaningful binding (defined as 10 µM ff Our preliminary data suggest that FFA, 5. Bagdy G, et al. J Neurochem. 2007;100(4):857-73. or less to allow identification of secondary effects of known 6. Zhang Y, et al. PLoS ONE. 2015;10(5):e0125898.doi:10.1371/journal.pone.0125898. Receptor Binding norfenfluramine, and their enantiomers bind to and 7. Favale E, et al. Seizure. 2003;12(5):316-18. ff Binding of FFA and norfenfluramine and their enantiomers antiepileptic mechanisms that might be involved in seizure have effects at noradrenergic, muscarinic, and 8. Specchio LM, et al. Clin Neuropharmacol. 2004;27(3):133-6. activity) was obtained at b and b -adrenergic, muscarinic M , 9. Favale E, et al. Neurology. 1995;45:1926-7. was conducted using radioligand binding assays with 2 1 sigma receptors, which are identified in the literature recombinant human receptors or with rodent, bovine, or and non-selective sigma receptors 10. Meador K. Epilepsy Behav Case Rep. 2014;2:54-6. as having a potential role in epilepsy and seizure 11. Pisani F, et al. Epilepsia. 1999; 40(suppl 10):S48-S56. rabbit receptors when human receptors were not available Table 2: Racemic FFA and Norfenfluramine: Ki Values (mol/L) for activity 12. Muzerengi S and Moor CC. Prog Neurol Psychia. March/April, 2013:20-4. (Sekisui XenoTech,LLC, Kansas City, KS) Receptors Demonstrating >30% Binding Based on Percent Inhibition 13. Shin EJ, et al. Br J Pharmacol. 2005;144(7):908-18. f 14. Guo L, et al. Br J Pharmacol. 2015;172:4052-65. –– Data were expressed as Ki values in molar concentration Receptor (±) Fenfluramine (±) Norfenfluramine Positive Control f The binding activity of the racemic compounds and enantiomers are similar 15. Bermack JE and Debonnel G. Br J Pharmacol. 2001;134:691-9. Agonist and Antagonist Activity b Adrenergic 3.90 x 10-9 16. Maurice T. Personal communication. 1.75 x 10-5 1.20 x 10-5 ff FFA, norfenfluramine, and their enantiomers were tested (non-selective) (propranolol) 17. Rowland NE and Carlton J. Prog Neurobiol. 1986;27(1):13-62. ff Although FFA, norfenfluramine, and their enantiomers 18. Zogenix. Data on File. in functional assays for those receptors where binding was 5.12 x 10-10 b Adrenergic 1.26 x 10-5 8.77 x 10-6 elicited activity in the sigma receptor assays, the 2 (propranolol) 10 μM or lower (Eurofins Cerep SA, Celle L’Everscault, France) pattern of activity was not a simple agonist or 9.80 x 10-10 ff Agonist and antagonist activity of G-protein coupled M Muscarinic 1.13 x 10-5 3.74 x 10-6 1 (atropine) antagonist effect, but was more complex and may ACKNOWLEDGMENTS receptors were assessed using human recombinant receptors Sigma 1.31 x 10-9 suggest the interaction of sigma with other receptors Medical writing assistance was provided by PharmaWrite (Princeton, in Chinese hamster ovary (CHO) cells 2.66 x 10-7 2.92 x 10-6 (non-selective) (haloperidol) NJ) and funded by Zogenix, Inc.

Presented as part of the Zogenix Scientific Exhibit during the 70th Annual Meeting of the American Epilepsy Society, Houston, Texas, December 2-6, 2016.