EA040818B1 - METHOD FOR TREATMENT OF EPILEPTIC DISORDER USING 5HT RECEPTOR AGONIST - Google Patents

Description

This application claims priority to U.S. Application No. 62/120726, filed Feb. 25

2015, the contents of which are incorporated herein by reference in their entirety.

Statement of rights to an invention carried out as part of government-sponsored research and development

The present invention was made with government support under grant number R01 NS 07921403 from the National Institutes of Health. The government has certain rights to the invention.

Field of invention

The present invention relates to methods for treating an epileptic disorder using a 5HT receptor agonist or a pharmaceutically acceptable salt thereof.

The state of the art to which the invention relates

Dravet syndrome (DS) is a catastrophic pediatric epilepsy with severe mental retardation, impaired social development, and prolonged drug-resistant seizures. One of its main causes is mutations in Nav1.1 (SCN1A), a voltage-gated sodium channel. Seizures experienced by individuals with DS and other epileptic disorders are not adequately controlled using available antiepileptic drugs (AEDs), and children with DS are candidates for neurosurgical resection. Thus, there is a need in the art for treatment options for epilepsy, especially DS and related catastrophic pediatric epilepsies. The present description provides solutions to these problems and other problems of the prior art.

Brief description of the essence of the invention

Provided herein are methods for treating epileptic disorders using a 5HT agonist or a pharmaceutically acceptable salt thereof. In one aspect, the method includes administering to an individual in need thereof a therapeutically effective amount of a 5HT agonist, a 5HT agonist analog, or a pharmaceutically acceptable salt thereof.

According to an aspect of the invention, a method of treating an epileptic disorder may include administering to a patient having an epileptic disorder a therapeutically effective amount of a 5HT receptor agonist or a pharmaceutically acceptable salt thereof.

In an exemplary embodiment, the 5HT agonist may be a 5HT _2A receptor agonist or a 5HT2B receptor agonist. In some exemplary embodiments, it is contemplated that the 5HT agonist may be one or more of ACP-104, ACP-106, AR-116081, AR-116082, ATTH-105, belladonna in combination with ergotamine tartrate, BW 723C86, cisapride, cisa -MPS, cizapa, cisap-Mps, CSC-500 Series, DOI or its salts (eg HCl), ergotamine tartrate and caffeine, MPS esoride, flibanserin, icaran LP, manotaka plus, migrila, mirtazapine, rimafar, mirtazapine, naratriptan, nelotanserin , norfenfluramine, normagut tab., nefadosone hydrochloride, OSU-6162, pridofine, sensiflu, PRX-00933, RP-5063, small molecular weight 5HT _2A agonist against inflammatory diseases, small molecular weight 5-HT _2C agonist against schizophrenia and obesity, small molecular weight 5-HT receptor agonist HT2C against obesity, small molecule compounds for 5-HT2C and 5-HT6 receptor targeting against schizophrenia, small molecule compounds for modulating 5HT2 against CNS and metabolic disorders, TGBA-01AD, t radodone hydrochloride, temanogrel hydrochloride, vabicaserin hydrochloride, virdex, VR-1065, ziprasidone hydrochloride and/or ziprasidone sigillata.

In some exemplary embodiments, it is contemplated that the 5HT receptor agonist is not clemisole or fenfluramine. In some exemplary embodiments, it is further provided that the 5HT receptor agonist is not acetazolamide, benzodiazepine (diazepam; clobazam), cannabadiol, carbamazepine, clemizol, ethosuximide, felbamate, fenfluramine, fluoxetine, gabapentin, ganaxolone, lacosamide, lamotirine, levetrigin, nitrazepam, oxcarbazepine, perampenel, phenytoin, phenobarbital, piracetam, potassium bromide, pregabalin, primidone, retigabine, rufinamide, styripentol, tiagabine, topiramate, valproic acid, verapamil, vigabatrin and/or zonisamide.

In an exemplary embodiment, the 5HT receptor agonist binds directly to the 5HT receptor. In an exemplary embodiment, the 5HT receptor agonist specifically activates the 5HT receptor.

In an exemplary embodiment, the 5HT receptor agonist provides increased activity mediated by the 5HT _2A receptor or 5HT _2B receptor with equivalent or reduced activity mediated by the 5HT _2C receptor.

In an exemplary embodiment, the 5HT receptor agonist is a dual 5HT _2A receptor and 5HT2B receptor agonist.

In an exemplary embodiment, the 5HT receptor agonist is not a serotonin reuptake inhibitor.

In an exemplary embodiment, the 5HT receptor agonist does not bind or significantly modulate the activity of at least one of the receptor 5HT _1A , 5HT _1B , 5HT1D, 5HT _2C ,

- 1 040818

P \ 5HT ₄ (for example, 5HT _4e ), 5HT ₆ , 5HT7, NPY Y1, L-type Ca-channel, N-type Ca-channel, SK-Ca-channel,

GABA-dependent Cl channel, GABA transporter, GABA-A1 receptor, GABA-Blb receptor, Na channel, 5HT transporter, CB1 receptor, CB2 receptor, BZD, or ER estrogen alpha.

In an exemplary embodiment, the 5HT receptor agonist is any one or more of flibanserin, 2,5-dimethoxy-4-iodomamphetamine monohydrochloride (DOI HCl), norfenfluramine, or BW 723C86.

In an exemplary embodiment, the epileptic disorder is Dravet's syndrome, Lennox-Gastaut syndrome, infantile convulsions, or Otahara's syndrome. In an exemplary embodiment, the epileptic disorder is Dravet's syndrome. In an exemplary embodiment, the epileptic disorder is a pediatric epileptic disorder.

In an exemplary embodiment, the individual has a cardiovascular disease.

In an exemplary embodiment, the subject is refractory to treatment with a serotonin reuptake inhibitor.

In an exemplary embodiment, the subject is subject to side effects when administered with a serotonin reuptake inhibitor. In an exemplary embodiment, the serotonin reuptake inhibitor is fenfluramine.

In an exemplary embodiment, the subject is on a ketogenic diet.

In an exemplary embodiment, the 5HT receptor agonist inhibits compulsive behavior or electrographic seizures in an individual with epilepsy, an individual with Alzheimer's disease, an individual with autism, or an individual with Parkinson's disease.

In an exemplary embodiment, a 5HT receptor agonist reduces the incidence of unprovoked seizures in said individual compared to no said 5HT receptor agonist.

In an exemplary embodiment, administration of said 5HT receptor agonist reduces or prevents myoclonic seizures or status epilepticus in said individual, as compared to no 5HT receptor agonist.

In an exemplary embodiment, the 5HT receptor agonist is administered to said individual in an amount of from about 0.1 to about 1000 mg per kg of body weight.

In an exemplary embodiment, the 5HT receptor agonist is administered to said individual at a daily dose of from about 0.1 mg to about 1000 mg per kg of body weight of said individual.

In an exemplary embodiment, the 5HT receptor agonist is co-administered with an antiepileptic drug (AED). In an exemplary embodiment, the 5HT receptor agonist is an adjuvant therapy for an antiepileptic drug (AED). In an illustrative embodiment, the AED is acetazolamide, benzodiazepine, cannabadiols, carbamazepine, clobazam, clonezepam, eslicarbazepine acetate, ethosuximide, ettoin, felbamate, fenfluramine, fosphenytoin, gabapentin, ganaxolone, huperzine A, lacosamide, lamotrigine, perepampanempine, levnitrazelacetam , piracetam, phenobarbital, phenytoin, potassium bromide, pregabalin, primidone, retigabine, rufinamide, valproic acid, sodium valproate, styripentol, tiagabine, topiramate, vigabatrin, or zonisamide. In an exemplary embodiment, the AED is valproic acid, sodium valproate, clonezepam, ethosuximide, felbamate, gabapentin, carbamazepine, oxcarbazepine, lamotrigine, levetiracetam, benzodiazepine, phenobarbital, pregabalin, primidone, tiagabine, topiramate, potassium bromide, phenytoin, styripentol, or vigarinthol. zonisamide. In an exemplary embodiment, the AED is valproic acid, sodium valproate, gabapentin, topiramate, carbamazepine, oxcarbazepine, or vigabatrin.

In an exemplary embodiment, the AED is not topiramate.

In an exemplary embodiment, the AED is not fenfluramine.

In an exemplary embodiment, the AED is administered simultaneously or sequentially with said 5HT receptor agonist.

In another aspect, the invention relates to a method for treating an epileptic disorder, said method comprising administering to an individual in need thereof a therapeutically effective amount of a 5HT receptor agonist or a pharmaceutically acceptable salt thereof, wherein said individual has a cardiovascular disease, is a treatment-resistant reuptake inhibitor serotonin levels or is susceptible to side effects when administered with a serotonin reuptake inhibitor.

In an exemplary embodiment, the 5HT receptor agonist is a 5HT ₂ a receptor agonist or a 5HT2 receptor agonist. In an exemplary embodiment, the 5HT receptor agonist is clemizol, a clemizol analog, or a pharmaceutically acceptable salt thereof. In an exemplary embodiment, the pharmaceutically acceptable salt is Clemizol HCl.

In embodiments, the 5HT receptor agonist is sumatriptan, naratriptap,

- 2 040818 rizatriptan, zolmitriptan, urapidil, BRL-54443 (3-(1-methylpiperidin-4-yl)-1H-indol-5-ol), lorcaserin, buspirone, ziprasidone, TSV-2 ((4-bromo-3 ,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide), BRL-15572 (3-(4-(4-chlorophenyl)piperazin-1-yl)-1,1-diphenyl-2-propanol), trazodone, BMY 7378 ( 8(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decan-7,9-dione), atomoxetine and venlafaxine. In embodiments, the 5HT receptor agonist is sumatriptan. In embodiments, the 5HT receptor agonist is naratriptap. In embodiments, the 5HT receptor agonist is rizatriptan. In embodiments, the 5HT receptor agonist is zolmitriptan. In embodiments, the 5HT receptor agonist is urapidil. In embodiments, the 5HT receptor agonist is BRL-54443 (3(1-methylpiperidin-4-yl)-1H-indol-5-ol). In embodiments, the 5HT receptor agonist is lorcaserin. In embodiments, the 5HT receptor agonist is buspirone. In embodiments, the 5HT receptor agonist is ziprasidone. In embodiments, the 5HT receptor agonist is TCB-2 ((4-bromo-3,6dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide). In embodiments, the 5HT receptor agonist is BRL-15572 (3-(4-(4-chlorophenyl)piperazin-1-yl)-1,1-diphenyl-2-propanol). In embodiments, the 5HT receptor agonist is trazodone. In embodiments, the 5HT receptor agonist is BMY 7378 (8-(2-[4-(2-methoxyphenyl)-1piperazinyl]ethyl)-8-azaspiro[4.5]decan-7,9-dione). In embodiments, the 5HT receptor agonist is atomoxetine. In embodiments, the 5HT receptor agonist is venlafaxine.

In embodiments, the 5HT receptor agonist is trazodone or a pharmaceutically acceptable salt thereof.

In an exemplary embodiment, Clemizol, said Clemizol analog, or said pharmaceutically acceptable salts thereof, form part of a pharmaceutical composition. In an exemplary embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In an exemplary embodiment, the pharmaceutical composition comprises a therapeutically effective amount of Clemizol, said Clemizol analog, or said pharmaceutically acceptable salt thereof.

In an exemplary embodiment, the pharmaceutical composition is co-administered with an antiepileptic drug (AED). In an exemplary embodiment, the pharmaceutical composition comprises clemizol, said clemizol analogue or said pharmaceutically acceptable salt thereof, and an AED. In an exemplary embodiment, the 5HT receptor agonist is not fenfluramine. In an exemplary embodiment, the 5HT receptor agonist binds directly to the 5HT receptor.

In an exemplary embodiment, the 5HT receptor agonist specifically activates the 5HT receptor. In an exemplary embodiment, the 5HT receptor agonist provides increased activity mediated by the 5HT _2A receptor or 5HT _2B receptor with equivalent or reduced activity mediated through the 5HT2C receptor. In an exemplary embodiment, the 5HT receptor agonist is a dual 5HT2A receptor and 5HT _2B receptor agonist.

In an exemplary embodiment, the 5HT receptor agonist is not a serotonin reuptake inhibitor.

In an exemplary embodiment, the 5HT receptor agonist does not significantly bind to at least one of the receptor 5HT _1A , 5HT _1B , 5HT1D, 5HT _2C , 5HT ₃ , 5HT ₄ (e.g., 5HT _4e ), 5HT6, 5HT ₇ , NPY Y1, L-type Ca-channel, N-type Ca-channel, SK-Ca-channel, GABA-dependent Cl-channel, GABA transporter, GABA-A1 receptor, GABA-B1b receptor, Na-channel, 5HT transporter, CB1 receptor, CB2 receptor , BZD or ER estrogen alpha.

In another aspect, the invention relates to a method for modulating 5HT receptor activity, comprising contacting the 5HT receptor with clemizol, a clemizol analog, or a pharmaceutically acceptable salt thereof.

In an exemplary embodiment, the modulation is an activation.

In an exemplary embodiment, the 5HT receptor is a 5HT _2A receptor or a 5HT _2B receptor.

In another aspect, the invention relates to a method of treating a disease or disorder due to a deficiency of serotonin in the brain or the activity of one or more 5HT receptors, comprising administering to an individual in need thereof, a therapeutically effective amount of clemizol, a clemizol analog, or a pharmaceutically acceptable salt thereof.

In an exemplary embodiment, the disease or disorder is not epilepsy.

In an exemplary embodiment, the disease or disorder is not Dravet's syndrome.

In an exemplary embodiment, the disease or disorder is selected from the group consisting of migraine, fragile X syndrome, Prader-Willi syndrome, schizophrenia, depression, Alzheimer's disease, autism, neuropathic pain, Parkinson's disease, irritable

- 3 040818 intestines and dementia.

In one aspect, a pharmaceutical composition is provided that includes clemizol, a clemizol analog, or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease or disorder caused by a deficiency of serotonin in the brain or the activity of one or more 5HT receptors.

In embodiments of the aspect described above, the 5HT receptor agonist does not bind or modulate (e.g., inhibit) to a significant extent the activity of at least one of 5HT1A, 5HT1B, 5HT1D, 5HT3, 5HT4e, GABAA1, GABAB _(1b) , BZD. CB1, CB2, GABA-dependent α channel, SK-Ca channel, or GABA transporter. In embodiments, the 5HT receptor agonist does not bind or modulate (e.g., inhibit) to a significant extent the activity of 5HT1A, 5HT1B, 5HT1D, 5HT3, 5HT4e, GABAA1, GABAB( _W BZD. CB1, CB2, GABA-dependent Cl channel, SKCa- channel and GABA transporter.

In embodiments of the aspect described above, the 5HT receptor agonist modulates the target by less than about 50% relative to a reference modulator (eg, a natural or known modulator commonly used in the art). In embodiments, the 5HT receptor agonist modulates the target by less than about 40% relative to a reference modulator (eg, a natural or known modulator commonly used in the art). In embodiments, the 5HT receptor agonist modulates the target by less than about 30% relative to a reference modulator (eg, a natural or known modulator commonly used in the art). In embodiments, the 5HT receptor agonist modulates the target by less than about 20% relative to a reference modulator (eg, a natural or known modulator commonly used in the art). In embodiments, the 5HT receptor agonist modulates the target by less than about 10% relative to a reference modulator (eg, a natural or known modulator commonly used in the art).

In embodiments of the above aspect, the 5HT receptor agonist does not significantly modulate activity when the 5HT receptor agonist does not modulate (e.g., inhibit) the target by less than about 50% relative to a reference inhibitor (e.g., a natural or known modulator commonly used in the art). ). In embodiments, the 5HT receptor agonist does not significantly modulate activity when the 5HT receptor agonist does not modulate (e.g., inhibit) the target by less than about 40% relative to a reference inhibitor (e.g., a natural or known modulator commonly used in the art). In embodiments, the 5HT receptor agonist does not significantly modulate activity when the 5HT receptor agonist does not modulate (e.g., inhibit) the target by less than about 30% relative to a reference inhibitor (e.g., a natural or known modulator commonly used in the art). In embodiments, the 5HT receptor agonist does not significantly modulate activity when the 5HT receptor agonist does not modulate (e.g., inhibit) the target by less than about 20% relative to a reference inhibitor (e.g., a natural or known modulator commonly used in the art). In embodiments, the 5HT receptor agonist does not significantly modulate activity when the 5HT receptor agonist does not modulate (e.g., inhibit) the target by less than about 10% relative to a reference inhibitor (e.g., a natural or known modulator commonly used in the art). In embodiments, the 5HT receptor agonist does not significantly modulate activity when the 5HT receptor agonist does not modulate (e.g., inhibit) the target by less than about 5% relative to a reference inhibitor (e.g., a natural or known modulator commonly used in the art).

Brief description of the drawings

Fig. 1. Molecular characterization of scn1Lab zebrafish mutants. (A) Sequencing confirmed a T to G mutation in the cDNA of the scn1Lab mutant. (B) Confirmation of reduced expression in scn1Lab mutants compared to sibling controls at 3, 5 and 7 dpf using qPCR. Data are presented as mean ±8.U.M; *significance is determined using Student's t-test as p<0.05. Data were normalized to the internal reference gene for β-actin. Values are the median of five independent biological samples (1 sample=10 pooled larvae) for each of the 3 developmental stages. Data are presented as mean±S.E.M; * Significance is determined using Student's t-test as p<0.05. (C) Relative expression of scn8aa and scn8ab in Navl mutants. 1 (n=5) and sibling controls (n=5) at 5 dpf. Data are presented as in B. (D) Total preparation in situ hybridization against scn1Lab in zebrafish larvae at 3, 5 and 7 dpf. Wild-type larvae are shown in side images; expression is shown in dark purple. For comparison, Scn1Laa expression is shown after 3 dpf. The heart is indicated by an arrow in the 5 and 7 dpf panels. (E) Rear view of scb1Laa expression at 3 dpf; it should be noted a pronounced expression in the areas corresponding to the CNS of zebrafish larvae. Abbreviated designations: 4 040818 Tel, brain bladder; TeO, optic tectum; Cb, cerebellum. Scale marks=0.35 mm on

D, 0.2 mm on E.

Fig. 2. Microarray analysis of zebrafish scn1Lab mutants. (A) Heat maps depicting the expression of genes differentially expressed between scn1Lab mutant larvae and sibling control larvae at 5 dpf. Rows correspond to individual genes. The columns correspond to different larvae. Genes that are highly expressed in scn1Lab mutants relative to controls are as shown. (B) MA plot of normalized microarray data for all 44,000 genes. The logarithmic ratio M and the mean fluorescence intensity A were calculated as averages for all replicas. (C) List of 30 genes showing the most differences in expression between scn1Lab mutants and sibling controls.

Fig. 3. Quantitative analysis of zebrafish scn1Lab mutants by RT-PCR. (A) Comparison of fold changes in gene expression obtained by microarray (array) analysis and real-time qPCR analysis. The y-axis corresponds to the average fold change in the expression of each zebrafish gene after 5 dpf. The x-axis corresponds to different genes. (B) qPCR analysis of three genes involved in epileptogenesis. Relative gene expression is presented as ratios of log2 to the least abundant transcript (log2AAct). Data were normalized to the internal reference gene for β-actin. The values correspond to the average values for five independent biological samples (1 sample - 10 pooled larvae). Planks point to S.E.M; *p<0.05, t-criterion. (C) Gene ontology classification for differentially expressed genes found in scn1Lab mutants at 5 dpf (p<0.05, one-way ANOVA and >1.5 fold change). Biological processes are shown corresponding to the annotations of at least 5 genes in at least one category.

Figure 4. Spontaneous seizures in zebrafish scn1Lab mutants. (A) Immobilized and agar-stained larvae are shown. Images were acquired using a 4x objective and 2x magnifier on an Olympus upright microscope during electrophysiological forebrain recording of sibling control larvae (A, left) and scn1Lab mutant larvae (A, middle) at 5 dpf. The dark pigmentation of the mutants should be noted. The recording electrodes can be seen in panels A1-2, and the approximate region of the end of the recording electrode in the forebrain (red circle) is shown using representative HuC:GFP labeled larvae in A, right. Scale mark: 100 µm. (B) Plot tracing movement in the sample for sibling control larvae (B, left) and scn1Lab mutant larvae (B, right) at 5 dpf. (C) Representative 10 min recording intervals obtained in the forebrain of paralyzed, immobilized and agar-embedded scn1Lab mutant larvae between 3 and 7 dpf. It should be noted the presence of a spontaneous pulsed discharge of small and large amplitude; additional temporal extension of seizure activity. Representative registration under identical registration conditions in sibling control larvae at 5 dpf is also shown. Scale mark: 2 mV; 3 0 s.

Fig. 5. Pharmacological confirmation of zebrafish scn1Lab mutants. (A) Heat map showing response to nine different AEDs. Each bar corresponds to the percentage change in pulse rate (baseline drug/baseline x 100) for one individual zebrafish mutant. Drugs that inhibit seizures are shown in dark blue. All drugs were tested at a concentration of 1 mM. It should be noted that in some trials, carbamazepine and vigabatrin increased the pulse rate from initial baseline levels. (B) Plot of mean change in pulse rate and standard error of data is plotted on a heat map. Paired t-test or signed Wilcoxon rank-sum test for data that failed the normality test demonstrated significance as follows: diazepam (p=0.002; n=7), potassium bromide (p=0.016; n=7), stripentol (p =0.024; n=7) and valproate (p=0.004; n=7). (C) Pulse duration plot for all tests shown in A. Data are presented as mean±S.E.M. for electrographic seizures at baseline (black bars) and after drug exposure (white bars). The inset shows a representative recording of 2-min intervals during the test with styripentol; scale marks: large line, 1 mV, 1s; small line, 1 mV, 100 ms. (D) Graph of the proportion of time spent on seizures for all trials shown in A. Data are presented as mean±S.E.M. for electrographic seizures at baseline (black bars) and after drug exposure (white bars). Student's t-test or Mann-Whitney rank sum test for data that failed the normality test demonstrated significance as follows: diazepam (p=0.001; n=1); potassium bromide (p=0.043; n=1); styripentol (p=0.007; n=1) and valproate (p=0.007; n=1 (E) Movement tracking plots for 10 individual mutant larvae grown in embryonic medium (top row) or on a ketogenic diet for 48 hours. Plots show swimming speed and travel lines, with darker colors indicating higher speeds, 10 min trials shown (F) Representative recording of 10 min intervals for the same fish shown in E; representative examples indicated * on the movement plots Scale mark: 1 mV, 30 s Inset shows pulse at higher temporal resolution (indicated as #), scale mark: 1 mV, 100 ms.

Fig. 6. Screening to identify drugs that rescue the scn1Lab mutant epileptic phenotype. (A) Plot of mean velocity (in mm/s) for two consecutive recordings of mutant larvae in embryonic medium. Experiments were performed by first placing the mutant larvae in embryonic medium and obtaining the initial movement response, then the embryonic medium was replaced with new embryonic medium (to mimic the procedure used for the test compounds) and a second movement response was obtained. The percentage change in speed from baseline (registration #1) versus experimental level (registration #2) is shown. In a box chart, the bottom and top of the box correspond to the 25th and 75th percentiles, respectively. The line through the rectangle corresponds to the median value and the vertical lines cover the entire range of values. This graph corresponds to normal changes in activity tracking in the absence of drug loading. (B) Plot of the effect of eleven known antiepileptic drugs on the behavior of motor seizures in scn1Lab mutants at 5 dpf. Phenotype-based analysis was performed in a 96-well format (eg, see panel 5C1). Bars correspond to percent change in mean rate comparing baseline seizure activity data of the mutant with the same mutant after drug application. For all drug studies, 6-12 fish per experiment were used. Drugs were tested at a concentration of 1 mM; diazepam (Dzp; p<0.001), carbamazepine (Carb, p=0.024), ganaxolone (Gan; p=0.003), styripentol (Stp; p=0.001), valproate (Vpa, p=0.026) and exposure for 48 h ketogenic diet (KD; p=0.003) reduced seizure activity, measured as change in rate, by more than 34% (dashed line at B; corresponds to a fold change greater than the standard deviation in control data). Acetazolamide (Acet, p<0.001) and ethosuximide (Etx; p=0.250) aggravated seizures; levetiracetam (Lev; p=0.243) and lamotrigine (Ltg; p=0.058) had no effect. (C) Plot of seizure behavior for Scn1Laab mutants at 5 dpf for 320 compounds tested. Colored circles correspond to positive results; compounds that reduced activity by 100% were mostly toxic; 6-12 fish per trial. The arrow pointer indicates the first test with Clemizol. It should be noted that some compounds increased seizure activity as expected. (D) Schedule of drug retests on selected groups of scn1Lab mutants at 5 dpf; 100 μM per drug; 10 fish to test. Abbreviations: Clem, Klemizol; Clem+PTZ, Clemizol+15 mM PTZ; Clorg, clorgyline; Tolp, tolperisone; Zox, zoxazolamine. The effect of emergency clemizol on PTZ-induced seizure is shown in wild-type larvae. Bars correspond to mean ± S.E.M. For panels B and D: paired Student's t-test or Mann-Whitney rank sum test with significance set to p=0.01 (*) or p<0.001 (**). (E) Electrophysiological recording of Clemisol-treated scn1Lab mutant samples, first in a locomotion assay (panel D) and then monitored using an extracellular forebrain recording electrode (top curve; inset shows an ictal-like pulse). Similar curves are shown for the untreated Navl mutant. 1 (middle curve) and the zoxazolamine-treated mutant (lower curve). Pulse analysis for untreated mutants (n=3): pulse rate=1.5±0.3 pulses/min; pulse duration=926±414 ms; fractional time spent on seizure=0.73±0.17% vs. clemizol-treated mutants (n=7): pulse rate=0.2±0.01 pulses/min; pulse duration=154±127 ms; time in fractions spent on seizure=0.03±0.02%; p=0.001 for all comparisons, Kruskal-Wallis ANOVA with Dunn's pairwise multiple comparison test). Scale marks: large curve: 0.5 mV, 10 s; insert: 0.5 mV, 100 ms.

Fig. 7: Confirmation of Clemizol activity in Scn1Laa mutants. (A) Representative 10 min recording intervals taken in the forebrain of paralyzed, immobilized, and agar-embedded Scn1Laa mutant larvae at 6 dpf. The presence of a spontaneous pulsed discharge of small and large amplitude should be noted. (B) Movement tracking plots for 10 individual mutant larvae and wild-type sibling larvae. The graph shows swim speed and travel lines, with darker colors indicating higher speed; 10 min tests are shown. Seizures were assessed by the staging system described in Baraban et al. (Neuroscience 2005). SO, little or no swimming activity; S1, increased mobility; S2 whirlpool-like swimming activity and S3; convulsions of the whole body with phenomena of rapid swimming and loss of body position. (C) Bar plot of mean velocity (in mm/s) for 96 zebrafish, with fish sorted into putative Scn1Laa and sibling controls based on the seizure stages described above. Experiments were performed by first placing mutant larvae in embryonic medium and obtaining an initial locomotion response, then the embryonic medium was replaced with new embryonic medium and a second locomotion response was obtained. The percentage change in speed from baseline (registration #1) versus experimental level (registration #2) is shown. On the rectangular chart, the lower and upper parts of the rectangle correspond to the 25th and 75th percentiles, respectively. The line through the rectangle corresponds to the median value and the vertical lines cover the entire range of values. Plots are shown for all 96 fish (left), putative zebrafish Scn1Laa (middle), and sibling controls (left). Subsequent PCR analysis was performed to confirm mutant and control pooled groups. (D) Plot of the effect of stripentol (Stp), diazepam (Dzp), clemizol (Clem), and lamotrigine (Ltg) on seizure locomotion behavior in Scn1Laa mutants at 5 dpf. The average speed is shown before and after drug administration. N=7 fish per drug. Bars correspond to mean±S.E.M. Paired Student's t-test or Mann-Whitney rank sum test with significance at p=0.01 (*) or p < 0.001 (**).

Fig. 8: Antihistamines do not have antiepileptic properties in scn1Lab mutants. Plot of the effect of various antihistamines in a movement assay for seizures using scn1Lab mutants at 5 dpf. The average speed is shown before and after drug administration. N=7 fish per drug. Additional connections are shown on the right. Bars correspond to mean±S.E.M. Paired Student's t-test or Mann-Whitney rank sum test with significance at p=0.01 (*) or p<0.001 (**). Note: Some antihistamines increased seizure activity in this assay.

Fig. 9: Clemizol concentration-response studies in scn1Lab. Graphs from two different concentration-response studies showing percent inhibition of mean velocity from baseline. N=7 fish per concentration and tests were performed on separate groups of mutant larvae.

Fig. 10: Shelf screening was performed using 34 different compounds with antihistamine properties. All compounds were tested against spontaneous seizures in scn1Lab larvae at 5 dpf; 6-10 fish per remedy. The results are shown as a change in average speed from a graph of motion tracking data. Compounds were tested at concentrations between 0.1 and 1 mM. The threshold value for a positive match is indicated by the dotted line. Three compounds reached this threshold, however all 3 were identified as toxic (arrows).

Fig. 11: 5HT library screening. Plot of locomotion behavior in convulsions for scn1Lab mutants at 5 dpf for 62 compounds tested. The cut-off value for inhibition of seizure activity (positive results) was chosen as a decrease in mean swimming speed >38%; The threshold for proconvulsive or hyperexcitability effects was chosen as an increase in mean swimming speed >44% (green dotted lines). Compounds were tested at a concentration of 250 μM and 6 fish were tested per drug. The list of compounds is presented with positive results indicated in gray (on the map) or black circles (on the graph).

Fig. 12A-12B: FIG. 12A. Plot of mean rate change for scn1Lab mutants at 5 dpf exposed to different concentrations of trazodone during drug exposure periods of 30 min (black bars) or 90 min (grey bars). The threshold for inhibition of seizure activity (positive results) was chosen as a >40% reduction in mean swimming speed. Trazodone was toxic at 750 µM (dashed lines). Compounds were tested in groups of 6 fish per drug. Fig. 12V. EEG recordings of scn1Lab mutant larvae samples treated with trazodone (seizure suppression) or control drug MK-801 (no seizure suppression).

Detailed description of the invention

By 5HT receptor or 5-hydroxytryptamine receptor is meant a group of G-protein coupled receptors (GPCRs) and ligand-gated ion channels (LGICs) that occur in the central (CNS) and peripheral (PNS) nervous system and belong mainly to group of serotonin receptors. 5HT receptors can be classified into seven families of receptors including: 5HT1 (eg, G _i /G ₀ protein coupled receptors), 5HT ₂ (eg, G _q /Gn protein coupled receptors), 5HT ₃ (eg, ligand-dependent Na ⁺ and K ⁺ cation channel), 5HT ₄ (eg, Gs-coupled receptors), 5HT ₅ (eg, G _i /G ₀ protein-coupled receptors), 5HT6 (eg, Gs-coupled receptors), and 5HT ₇ (eg, Gs-coupled receptors). In addition, the seven families of 5HT receptors can be further subdivided into a number of subfamilies. For example, the 5HT1 family also includes the following subfamilies: 5HT _1A (for example, which is known to function in the blood vessels and CNS and may be involved in addiction, aggression, anxiety, appetite, autoreceptor function, blood pressure, cardiovascular function, vomiting, heart rate, impulsivity, memory, mood, nausea, nociception, penile erection, pupil dilation, respiration, sexual behavior, sleep, sociability, thermoregulation, and vasoconstriction), 5HT _1B (eg, known to function in blood vessels and CNS and may be involved in addiction, aggression, anxiety, autoreceptor

- 7 040818 function, learning, motor activity, memory, mood, penile erection, sexual behavior and vasoconstriction), 5HT1D (for example, which is known to function in the blood vessels and CNS and may be involved in anxiety, autoreceptor function , locomotor activity and vasoconstriction), 5HT1E (eg, which is known to function in the CNS and may be involved in migraine). As another example, the 5HT2 family can be subdivided into the following subfamilies: 5HT2A (for example, known to function in blood vessels, CNS, gastrointestinal tract, platelets, PNS, and smooth muscle, and may be involved in addiction, anxiety, appetite, cognition, imagination, learning, memory, mood, sensory perception, sexual behavior, sleep, thermoregulation, and vasoconstriction), 5HT2 _B (eg, known to function in blood vessels, CNS, gastrointestinal tract, platelets, PNS, and smooth muscle and may be involved in anxiety, appetite, cardiovascular function, gastrointestinal motility, sleep, and vasoconstriction) and _5HT2C (eg, known to function in blood vessels, CNS, gastrointestinal tract, platelets, PNS, and smooth muscle, and may be involved in cravings, anxiety, appetite, gastrointestinal motility, locomotor activity spine, mood, penile erection, sexual behavior, sleep, thermoregulation and vasoconstriction). In addition, the 5HT ₅ family can further be subdivided into the following subfamilies: 5HT _5A (eg, which may function in the CNS and play a role in locomotion and sleep, as well as function as an autoreceptor) and 5HT _5B (eg, which can function in rodents and appears to be a pseudogene in humans).

By 5HT receptor agonist is meant any agent that activates the 5HT receptor relative to the absence of a 5HT receptor agonist or analogously to serotonin. Exemplary 5HT receptor agonists include, but are not limited to, any one or more of: ACP-104, ACP106, AR-116081, AR-116082, ATTH-105, belladonna in combination with ergotamine tartrate, BW 723C86, cisapride, cisa-MPS , cizapa, cisap-Mps, CSC-500 Series, DOI or its salts, ergotamine tartrate and caffeine, MPS esoride, flibanserin, icaran LP, manotaka plus, migrila, mirtazapine rimafar, mirtazapine, naratriptan, nelotanserin, norfenfluramine, normaguta tab., nefadosone hydrochloride, OSU-6162, pridophine, sensiflu, PRX-00933, RP-5063, small molecular weight 5-HT2A agonist against inflammatory diseases, small molecular weight 5-HT2C agonist against schizophrenia and obesity, small molecular weight 5-HT2C receptor agonist against obesity, small molecular weight compounds for 5-HT _2C and 5-HT6 receptor targeting against schizophrenia, small molecule compounds to modulate 5HT2 against CNS and metabolic disorders, TGBA-01AD, trazodone hydrochloride, temanogrel hydrochloride , vabicaserin hydrochloride, virdex, VR-1065, ziprasidone hydrochloride and/or ziprasidone sigillata.

In embodiments, the 5HT receptor agonist is sumatriptan, naratriptap, rizatriptan, zolmitriptan, urapidil, BRL-54443 (3-(1-methylpiperidin-4-yl)-1H-indol-5-ol), lorcaserin, buspirone, ziprasidone, TCB -2 ((4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide), BRL-15572 (3-(4-(4-chlorophenyl)piperazin-1-yl)-1,1-diphenyl-2 -propanol), trazodone, BMY 7378 (8(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decan-7,9-dione), atomoxetine or venlafaxine.

In embodiments, the 5HT receptor agonist is trazodone.

It is further contemplated that, in some embodiments, the 5HT receptor agonist does not include one or more of the following: acetazolamide, benzodiazepine (diazepam; clobazam), cannabadiol, carbamazepine, clemizol, ethosuximide, felbamate, fenfluramine, fluoxetine, gabapentin, ganaxolone , lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine, perampenel, phenytoin, phenobarbital, piracetam, potassium bromide, pregabalin, primidone, retigabine, rufinamide, styripentol, tiagabine, topiramate, valproic acid, verapamil, vigabatrin and zonisamide.

By agent is meant any small chemical compound, antibody, nucleic acid molecule or polypeptide, or fragments thereof.

Cardiac condition refers to related diseases, including but not limited to coronary heart disease (CHD), cardiomyopathy, cardiovascular disease (CVD), coronary artery disease, heart failure, hypertension, inflammatory heart disease, valvular heart disease, atherosclerosis and cardiac hypertrophy. A cardiac condition can be a systemic disease that can affect the heart, brain, vital organs, and limbs. By coronary heart disease (CHD) is meant a disease that causes insufficient coronary blood flow to provide sufficient blood flow to the heart muscle and surrounding tissues. By cardiovascular disease (CVD) is meant any of a number of specific diseases that affect the heart itself or the circulatory system, especially myocardial tissue, and the veins and arteries leading to and from the heart. For example, CVD may include, but is not limited to, acute coronary syndrome, arrhythmias, atherosclerosis, heart failure, myocardial infarction, neointimal hyperplasia, pulmonary hypertension, stroke, valvular disease, and cardiac hypertrophy. Heart disease can

- 8 040818 be diagnosed by various methods known in the art. For example, such methods may include assessing the subject for dyspnea, orthopnea, paroxysmal nocturnal dyspnea, intermittent claudication, angina pectoris, chest pain, in which any of a number of symptoms known in the art may be present, such as exercise intolerance, edema, palpitations, weakness, loss of consciousness or cough; heart disease can be diagnosed through a biochemical blood test. As described above and as used herein, a heart disease refers to a disorder that directly affects the heart or circulatory system.

Analogue or analogue is used according to its usual meaning in chemistry and biology, and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called reference compound) but differs in composition, such as the substitution of one atom for another element, or the presence of a particular functional group, or the substitution of one functional group for another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Thus, an analog is a compound that is similar or comparable in function and appearance, but not in structure or origin, to a reference compound.

Klemizol refers to a compound having the formula

Clemizol includes pharmaceutically acceptable salts and formulations of Clemizol as described herein (eg, Clemizol salt). Exemplary salts of clemizol include, but are not limited to, clemizol-HCl, clemizolpenicillin, clemizol sulfate, or clemizol undecylate.

The clemizol analog as described herein refers to compounds of similar structure. Such compounds include, for example, those described in PCT/US 2008/076804 and US Pat. No. 4,011,322, which are incorporated herein by reference in their entirety. The following illustrative analogues of clemizol are indicated, for example, in US 2012/0232062; PCT Publications No. 2009/038248; US 2010/107739; US 2010/107742, WO 2002/089731. WO 2005/032329, WO 2009/039248, WO 2010/039195, WO 2010/107739 and WO 2010/107742, each of which is incorporated herein by reference in its entirety. Clemizol analogs described herein (including the compounds described in the above references) may be substituted (ie, modified) at 1 or 2 positions as indicated in formula (I) below (boxes Y and Z). Clemizol analogs may be substituted (ie, modified) at the 4, 5, 6 or 7 positions as indicated by box X in formula (I).

Flibanserin refers to a compound having the following formula (II):

Flibanserin includes pharmaceutically acceptable salts and formulations of flibanserin (eg flibanserin salt).

Norfenfluramine refers to a compound having the following formula (III):

Norfenfluramine includes pharmaceutically acceptable salts and formulations of norfenfluramine (eg, norfenfluramine salt).

DOI refers to 2,5-dimethoxy-4-iodamphetamine. DOI includes pharmaceutically acceptable salts and formulations of DOI. Exemplary DOI salts include, but are not limited to, 2,5-dimethoxy-4iodomamphetamine monohydrochloride (DOI HCl) having the following formula (IV):

- 9 040818

Ύ .... ο.

(IV).

BW 723C86 refers to a tryptamine derivative drug that acts as a 5HT _2B receptor agonist and has the following formula (V):

νη ₂

G\

TA n (V) .

BW 723C86 includes pharmaceutically acceptable salts and formulations of BW 723C86 (eg BW 723C86 salt).

The term pharmaceutically acceptable salts includes salts of active compounds that are prepared with relatively non-toxic acids or bases, depending on the particular substituents on the compounds described herein. When the 5-HT agonist contains relatively acidic functionalities, base addition salts can be prepared by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or a similar salt. Where the 5-HT agonist contains relatively basic functional groups, acid addition salts can be prepared by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrocarboxylic, phosphoric, monohydrophosphoric, dihydrophosphoric, sulfuric, monohydrosulfuric, hydroiodic or phosphorous acids, and the like, and salts derived from relatively non-toxic organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, etc. . Amino acid salts are also included. such as arginate and the like, and organic acid salts such as glucuronic or galacturonic acid and the like. (See, for example, Berge et al., Pharmaceutical Salts, Journal of Pharmaceutical Science, 1977, 66, 1-19). The 5-HT agonist may contain both basic and acidic functional groups which allow conversion to either base addition or acid addition salts.

Thus, a 5-HT agonist may exist as a salt with, for example, pharmaceutically acceptable acids. The present invention includes such salts. Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, propionates, tartrates (e.g. (+)-tartrates, (-)-tartrates or mixtures thereof, including racemic mixtures) , succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (eg, methyl iodide, ethyl iodide, and the like). These salts can be obtained by methods known to those skilled in the art.

The neutral forms of the 5-HT agonist are preferably reduced by contacting the salt with a base or acid and isolating the parent compound in the usual manner. The parent form of a compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.

In addition to salt forms, the 5-HT agonist may be provided in prodrug form. Prodrugs are compounds that readily undergo chemical changes under physiological conditions to form the compounds of the present invention. 5-HT agonist prodrugs can be converted in vivo after administration. In addition, 5-HT agonist prodrugs can be converted to active compounds by chemical or biochemical means under ex vivo conditions, for example, upon contact with a suitable enzyme or chemical.

The 5-HT agonist may exist in both unsolvated and solvated forms, including hydrated forms. Generally, solvated forms are equivalent to unsolvated forms and are within the scope of the present invention. The 5-HT agonist may exist in polycrystalline or amorphous form. In general, all physical forms are equivalent for the uses covered by the present invention and are within the scope of the present invention.

An effective amount is an amount sufficient to cause the agonist to achieve

- 10 040818

5-HT (including its pharmaceutically acceptable salts) for the intended purpose of not having a 5-HT agonist (including its pharmaceutically acceptable salts) (e.g., achieving the effect for which it is administered, treating a disease, reducing protein/enzyme activity, increasing protein/enzyme activity , inhibition of the signal transduction cascade, or reduction of one or more symptoms of the disease or condition). An example of an effective amount is an amount of a 5-HT agonist (including pharmaceutically acceptable salts thereof) that is sufficient to provide treatment, prevention or reduction of a symptom or symptoms of a disease, which may also be referred to as a therapeutically effective amount. Reduction of a symptom or symptoms (and the grammatical equivalents of this expression) means a reduction in the severity or frequency of the symptom(s) or elimination of the symptom(s) (eg, seizures). A prophylactically effective amount of a drug is the amount of a drug that, when administered to an individual, has an intended prophylactic effect, such as preventing or delaying the occurrence (or recurrence) of an injury, disease, disease, or condition, or reducing the likelihood of the occurrence (or recurrence) of an injury, disease, pathology or condition, or their symptoms (for example, seizures). The full prophylactic effect does not necessarily occur with the administration of a single dose, and it may occur only after a series of doses is administered. Thus, a prophylactically effective amount may be administered in one or more administrations. The exact amounts depend on the goal of treatment and can be determined by one of skill in the art using known methods (see, for example, Lieberman, Pharmaceutical Dosage Forms (vol. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

A therapeutically effective amount of a 5-HT agonist (including pharmaceutically acceptable salts thereof) can initially be determined in cell culture assays. Target concentrations are those concentrations of active compound(s) that are capable of delivering the methods described herein, as determined using the methods described herein or known in the art.

As is well known in the art, therapeutically effective amounts for use in humans can also be determined in animal models. For example, the human dose may be formulated to achieve a concentration found to be effective in animals. Dosage in humans can be adjusted by monitoring the effectiveness of the compounds and adjusting the dosage up or down as described above. Dose adjustment to achieve maximum efficacy in humans based on the methods described above and other methods is within the ability of a person skilled in the art.

Dosages may vary depending on the needs of the patient and the compound used. The dose administered to the patient, in the context of the present invention, should be sufficient to provide a favorable therapeutic response in the patient over time. The size of the dose will also be determined by the existence, nature and extent of any adverse side effects. Determining the proper dosage for a particular situation is within the skill of the practitioner. Typically, treatment is initiated using smaller dosages that are less than the optimum dose of the compound. Thereafter, the dosage is increased in small increments until the optimum effect under the circumstances is reached.

Dosages and intervals may be adjusted individually to provide levels of compound administered that are effective for the particular clinical indication being treated. This may provide a therapeutic regimen appropriate to the severity of the disease state of the individual.

Using the ideas described herein, an effective prophylactic or therapeutic treatment regimen can be planned that does not cause significant toxicity and yet is effective in treating the clinical symptoms that a particular patient exhibits. This planning should involve careful selection of the active compound, taking into account factors such as compound potency, relative bioavailability, patient body weight, the presence and severity of adverse side effects, the preferred route of administration, and the toxicity profile of the chosen agent.

A control or control experiment is used according to its usual meaning, and refers to an experiment in which the subjects or reagents of the experiment are exposed to the same treatment as in a parallel experiment, except for some procedure, reagent, or experimental variable. In some cases, the control is used as a standard for comparison when evaluating the effects of the experiment. In some embodiments, the control is indicative of the activity of the 5-HT agonist in the absence of the 5-HT agonist (including pharmaceutically acceptable salts thereof).

Test compound, as used herein, refers to an experimental compound used in a screening process to identify activity, inactivity, or

- 11 040818 other modulation of a specific biological target or cascade, for example, the 5-HT receptor. The test compound may be the 5-HT agonist described herein, including pharmaceutically acceptable salts thereof.

The term modulation, modulate or modulator is used according to its ordinary meaning and refers to the act of changing or varying one or more properties. A modulator refers to a composition or compound that increases or decreases the level of a target molecule, or the function of a target molecule, or the physical state of a target molecule. Modulation refers to the process of changing or varying one or more properties. For example, as used in relation to the effects of a biological target modulator, to modulate means to alter by increasing or decreasing the property or function of a biological target, or the amount of a biological target (eg, 5-HT receptor).

As defined herein, the term inhibition, inhibit, inhibitory, and the like. in relation to protein-inhibitor interaction means a negative effect (eg, reduction) on the activity or function of the protein relative to the activity or function of the protein in the absence of an inhibitor. In some embodiments, inhibition refers to a reduction in a disease or symptoms of a disease. In some embodiments, inhibition refers to a decrease in the activity of a particular target protein or target nucleic acid. Thus, inhibition includes, at least in part, partial or complete blocking of stimulation, reduction, prevention or delay of activation, or inactivation, desensitization or suppression of signal transmission or protein/enzyme activity or protein amount.

The term activation or activating, etc. refers to protein-compound interactions that positively affect (eg, increase) the activity or function of a protein relative to the activity or function of a protein in the absence of an activator compound. Activation may refer to increased activity of a particular target protein. Activation may refer to the restoration of the lost function of the mutant target protein. Activation, as used herein, may refer to the activation of one or more 5-HT receptors.

Bringing into contact is used according to its usual meaning, and refers to the process of bringing two different structures (eg, chemical compounds, including biomolecules, or cells) close enough that they react, interact, or physically touch. However, it should be understood that the end product of the reaction can be obtained directly from the reaction between the added reactants or from an intermediate compound of one or more added reactants that may be formed in the reaction mixture.

The term bringing into contact may include providing a reaction, interaction or physical contact between two structures, where the two structures may be a compound, as described in the present description, and a protein or enzyme. In some embodiments, contacting includes causing a compound described herein to interact with a receptor, such as a 5-HT receptor.

The term associated or associated with in the context of a substance, or activity or function of a substance associated with a disease, means that the disease is caused (in whole or in part), or a symptom of the disease is caused (in whole or in part) by the substance, or the activity or function of the substance.

The term patient or individual in need thereof refers to a living organism suffering from or predisposed to a disease or condition that can be treated by administering a pharmaceutical composition described herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammalian animals such as zebrafish. The patient may be a human.

The term disease or condition refers to the condition or health status of a patient or individual that may be treated with the compounds or methods described herein.

The terms epileptic disorder, epileptic disorder, convulsive disorder or epilepsy as used herein refer to a spectrum of chronic neurological disorders most commonly characterized by the presence of unprovoked seizures. See, for example, Noebels et. al., Jasper's Basic Mechanisms of the Epilepsies, 4th edition, Bethesda (MD) : National Center for Biotechnology Information (US); 2012. Epilepsy, as used herein, can refer to brain damage (eg, due to trauma, stroke, or cancer) or a genetic mutation. The symptoms of epileptic disorders may be the result of abnormal electrochemical signaling between neurons in the brain. Patients who have experienced two or more unprovoked seizures can be considered to have epilepsy.

Types of epileptic disorders include, for example, benign rolandic epilepsy, anterior lobe epilepsy, infantile seizures, juvenile myoclonic epilepsy (JME), juvenile absence epilepsy, childhood absence epilepsy (eg, pycnolepsy),

- 12 040818 febrile convulsions, Lafort progressive myoclonic epilepsy, Lennox-Gastaut syndrome, Landau-Klefner syndrome, Dravet syndrome (DS), generalized epilepsy with febrile convulsions (GEFS+), severe myoclonic epilepsy of infants (SMEI), benign neonatal familial convulsions (BFNC), West syndrome, Otahara syndrome, early myoclonic encephalopathies, migratory partial epilepsy, infantile epileptic encephalopathies, tuberous sclerosis complex (TSC), focal cortical dysplasia, type I lissencephaly, Miller-Dyker syndrome, Angelman syndrome, fragile X syndrome, disordered epilepsy autism spectrum disorder, subcortical band heterotopia, Walker-Warburg syndrome, Alzheimer's disease, post-traumatic epilepsy, progressive myoclonic epilepsy, reflex epilepsy, Rasmussen's syndrome, temporal lobe epilepsy, limbic epilepsy, status epilepticus, abdominal epilepsy, massive bilateral myoclonus, menstrual epilepsy, Jacksonian epileptic disorder, Unferricht-Lundborg disease or photosensitive epilepsy.

A pharmaceutically acceptable excipient and a pharmaceutically acceptable carrier or carrier portion refer to a substance that facilitates administration of a 5-HT agonist (including pharmaceutically acceptable salts thereof) and absorption in an individual and can be incorporated into compositions without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, Ringer's lactate solution, normal sucrose solution, normal glucose solution, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution). ), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine and dyes, and the like. Such preparations can be sterilized and, if desired, mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants and/or fragrances, etc., which do not react adversely with the compounds of the invention. The person skilled in the art will understand that other pharmaceutically acceptable excipients are suitable within the scope of the present invention.

The term preparation includes the formulation of a 5-HT agonist (including pharmaceutically acceptable salts thereof) with an encapsulating material as a carrier, providing a capsule in which the active ingredient, with or without other carriers, is surrounded by a carrier, which is thus associated with it. Similarly, cachets and lozenges are included. As dosage forms suitable for oral administration, tablets, powders, capsules, pills, cachets and lozenges can be used.

As used herein, the term administration means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intrathecal, intranasal or subcutaneous administration, or implantation of a sustained release device such as an osmotic mini- pump, individual. Administration is by any route, including parenteral or transmucosal (eg, buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intramuscular, oral arterioles, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

The 5-HT agonist (including its pharmaceutically acceptable salts) and its pharmaceutical compositions can be delivered transdermally, topically, as a formulation in the form of applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders and aerosols. Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by a patient. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Liquid form preparations include solutions, suspensions and emulsions, for example aqueous or water/propylene glycol solutions. In addition, 5-HT agonists (including their pharmaceutically acceptable salts) may include components that provide sustained release and/or comfort. Such components include high molecular weight anionic mucus-mimic polymers, gelling polysaccharides, and finely divided drug carrier substrates. These components are discussed in more detail in US patent No. 4911920; 5403841; 5,212,162 and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The 5-HT agonist (including pharmaceutically acceptable salts thereof) can also be delivered as sustained release microspheres in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres that release slowly subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995) as biodegradable and injectable gel formulations.

- 13 040818 (see, for example, Gao Pharm. Res. 12:857-863, 1995); or as microspheres for oral administration (see, for example, Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). 5-HT agonist formulations (including pharmaceutically acceptable salts thereof) can be delivered using liposomes that fuse with the cell membrane or undergo endocytosis, i. using liposome-coupled receptor ligands that bind to protein receptors on the cell membrane surface, resulting in endocytosis. Using liposomes, in particular when the surface of the liposomes contains ligands specific to target cells or otherwise preferentially targeted to a particular organ, it is possible to focus the delivery of 5-HT agonist compositions (including pharmaceutically acceptable salts thereof) to target cells in vivo (see e.g., Al-Muhammed, J. Microencapsul 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576- 1587, 1989). The compositions can also be delivered as nanoparticles.

By co-administration is meant that the composition described in the present description. administered at the same time, immediately before or immediately after one or more additional therapies. The 5-HT agonist (including pharmaceutically acceptable salts thereof) may be administered alone or may be co-administered to a patient. Co-administration includes the simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (for example, to reduce metabolic degradation). The 5-HT agonist (including its pharmaceutically acceptable salts) can be delivered transdermally, topically, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

The terms adjunctive therapy, adjuvant therapy, adjuvant therapy, and adjuvant therapy are used interchangeably herein and refer to the combination of a 5-HT agonist or a pharmaceutically acceptable salt thereof with another anticonvulsant drug for the treatment of epilepsy.

The anticonvulsant drug, antiepileptic drug, AED, or anticonvulsant drug is used interchangeably herein and according to their common and customary meaning, and includes compositions for reducing or eliminating seizures. Anticonvulsant drugs include, but are not limited to, acetazolamide, benzodiazepine, cannabadiols, carbamazepine, clobazam, clonezepam, eslicarbazepine acetate, ethosuximide, ettoin, felbamate, fenfluramine, fosphenytoin, gabapentin, ganaxolone, huperzine A, lacosamide, lamotrigine, levenitrazepinacetam, , perampanel, piracetam, phenobarbital, phenytoin, potassium bromide, pregabalin, primidone, retigabine, rufinamide, valproic acid, sodium valproate, styripentol, tiagabine, topiramate, vigabatrin, or zonisamide.

As used in the context of the invention, the term resistant refers to a decrease in the effectiveness of an agent or drug. For example, an individual who is resistant to treatment with a serotonin reuptake inhibitor (eg, such as fenfluramine) exhibits a reduced response relative to the response observed when the individual was first treated with the serotonin reuptake inhibitor.

As used herein, the term does not significantly bind means that the 5HT receptor agonist of the invention exhibits 10-fold, or 20-fold, or 50-fold, or 60-fold, or 70-fold, or 80-fold or 90-fold or 100-fold less binding to another transmembrane protein such as 5HT receptor, NPY YP receptor, Ca ⁺ channel, C1 channel, GABA transporter or GABA-A1 receptor, Na channel, 5HT transporter and/or a CB1 or CB2 receptor. This reduced level of binding can be measured by ELISA or biosensor assay (eg Biacore). In one example, the 5HT receptor agonist of the invention does not bind at a detectable level to the 5HT _1A receptor, 5HT1B, 5HT _{1D , 5HT 2C ,} 5HT3, 5HT4, 5HT6, 5HT ₇ , NPY Y1, L-type Ca-channel, Ca-channel ( N-type), SK-Ca channel, GABA-dependent C1 channel, GABA transporter, GABA-A1 receptor, GABA-B1b receptor, Na-channel, 5HT transporter, CB1 receptor, CB2 receptor, BZD, or ER alpha estrogen, when measured by ELISA or Biacore or Western blotting or FACS. In this regard, not binding at a detectable level means that the level of binding does not substantially exceed the background level. In exemplary embodiments, the 5-HT agonist does not significantly modulate the activity of at least one of the receptor 5HT1A, 5HT _1B , 5HT1D, 5HT _2C , 5HT3, 5HT4, 5HT ₆ , 5HT ₇ , NPY Y1, L-type Ca channel, N-type Ca-channel, SK-Ca-channel, GABA-dependent Cl-channel, GABA transporter, GABA-A1 receptor, GABA-B1b receptor, Na-channel, 5HT transporter, CB1 receptor, CB2 receptor, BZD or estrogen ER alpha. As used herein, the term does not significantly modulate activity means that the 5HT receptor agonist of the invention induces a receptor-mediated biological response that is substantially similar to its activity in the absence of an activating or inhibitory ligand.

Methods of treatment

The present invention provides methods for treating epileptic disorder

- 14 040818 stva. In one aspect, the method is a method of treating an epileptic disorder by administering to an individual in need thereof a therapeutically effective amount of a 5-HT receptor agonist, or a pharmaceutically acceptable salt thereof. In another aspect, the method is a method of treating an epileptic disorder by administering to an individual in need thereof a pharmaceutical composition as described herein, wherein the pharmaceutical composition comprises a 5-HT receptor agonist or a pharmaceutically acceptable salt thereof. The individual may be on (eg, may be eating according to) a ketogenic diet. The individual may have a cardiovascular disease. The subject may be resistant to treatment with a serotonin reuptake inhibitor. The individual may be subject to side effects when the serotonin reuptake inhibitor is administered. The individual may be a child (eg, an individual having a pediatric epileptic condition).

The epileptic disorder may be benign rolandic epilepsy, anterior lobe epilepsy, infantile seizures, juvenile myoclonic epilepsy (JME), juvenile absence epilepsy, childhood absence epilepsy (eg, pycnolepsy), febrile seizures, Lafort progressive myoclonic epilepsy, Landau-Gasteau syndrome, Klefner syndrome, Dravet syndrome, generalized epilepsy with febrile seizures (GEFS+), severe myoclonic epilepsy of infants (SMEI), benign neonatal familial seizures (BFNC), West syndrome, Otahara syndrome, early myoclonic encephalopathies, migratory partial epilepsy, infantile epileptic encephalopathies, tuberose complex sclerosis (TSC), focal cortical dysplasia, type I lissencephaly, Miller-Dyker syndrome, Angelman syndrome, fragile X syndrome, autism spectrum disorder epilepsy, subcortical ribbon-like heterotopia, Walker-Warburg syndrome , Alzheimer's disease, post-traumatic epilepsy, progressive myoclonic epilepsy, reflex epilepsy, Rasmussen's syndrome, temporal lobe epilepsy, limbic epilepsy, status epilepticus, abdominal epilepsy, massive bilateral myoclonus, catamenial epilepsy, Jacksonian epileptic disorder, Unferricht-Lundborg's disease, or photosensitive epilepsy. Epilepsy Epilepsy can include generalized seizures or partial (ie focal) seizures.

The epileptic disorder may be Dravet's syndrome, Lennox-Gastaut syndrome, infantile convulsions, or Otahara's syndrome. The epileptic disorder may be Dravet's syndrome, Lennox-Gastaut syndrome, infantile convulsions or Otahara's syndrome, or pediatric epileptic disorder. Pediatric epileptic disorder can be benign childhood epilepsy, benign neonatal familial seizures (BFNC), febrile seizures, Dravet syndrome, Lennox-Gastaut syndrome, infantile seizures, Otahara syndrome, juvenile myoclonic epilepsy, juvenile absence epilepsy, childhood absence epilepsy (eg, pycnolepsy ), infantile convulsions. The epileptic disorder may be Dravet's syndrome.

Pediatric epileptic disorder may be benign childhood epilepsy. Pediatric epileptic disorder may be benign neonatal familial seizures (BFNC). Pediatric epileptic disorder may be febrile seizures. Pediatric epileptic disorder may be Dravet's syndrome. Pediatric epileptic disorder may be Lennox-Gastaut syndrome. Pediatric epileptic disorder may be infantile seizures. Pediatric epileptic disorder may be Otahara's syndrome. The pediatric epileptic disorder may be juvenile myoclonic epilepsy. Pediatric epileptic disorder may be juvenile absence epilepsy.

The pediatric epileptic disorder may be childhood absence epilepsy (eg, pycnolepsy).

Pediatric epileptic disorder may be infantile seizures.

An epileptic disorder may result from a neurological disease or injury, such as, for example, encephalitis, cerebritis, abscess, stroke, tumor, trauma, genetic damage, tuberous sclerosis, cerebral dysgenesis, or hypoxic-ischemic encephalopathy.

An epileptic disorder may be associated with a neurodegenerative disease such as, for example, Alzheimer's disease or Parkinson's disease. Epileptic disorder may be associated with autism. An epileptic disorder may be associated with a mutation in a gene. Epileptic illness may be associated with compulsive behavior or electrographic seizures. The administration of a 5-HT receptor agonist or a pharmaceutically acceptable salt thereof can inhibit compulsive behavior or electrographic seizures in epileptic disorder, in an individual with Alzheimer's disease (for example, an individual suffering from Alzheimer's disease), in an individual with autism (for example, in an individual with AU

- 15 040818 tism) or in an individual with Parkinson's disease (for example, in an individual suffering from Parkinson's disease). Thus, a 5-HT receptor agonist or a pharmaceutically acceptable salt thereof can inhibit seizure behavior or electrographic seizures in epileptic disorder. A 5-HT agonist or a pharmaceutically acceptable salt thereof can inhibit seizure behavior or electrographic seizures in an individual with Alzheimer's disease. A 5-HT receptor agonist, or a pharmaceutically acceptable salt thereof, can inhibit seizure behavior or electrographic seizures in an individual with autism. A 5-HT receptor agonist, or a pharmaceutically acceptable salt thereof, can inhibit seizure behavior or electrographic seizures in an individual with Parkinson's disease.

The administration of a 5-HT receptor agonist or a pharmaceutically acceptable salt thereof can reduce the incidence (eg, number of occurrences) of unprovoked seizures in an individual compared to the absence of a 5-HT receptor agonist or a pharmaceutically acceptable salt thereof. Thus, it is possible to monitor the patient's response to administration of a 5HT receptor agonist or a pharmaceutically acceptable salt thereof gradually from a time point prior to administration of the compounds described herein (eg, control or time point).

Administration of a 5-HT receptor agonist or a pharmaceutically acceptable salt thereof can reduce or prevent myoclonic seizures or status epilepticus in an individual when compared to no 5-HT receptor agonist or a pharmaceutically acceptable salt thereof. Administration of a 5-HT receptor agonist or a pharmaceutically acceptable salt thereof can reduce or prevent myoclonic seizures in an individual when compared to no 5-HT receptor agonist or a pharmaceutically acceptable salt thereof. Administration of a 5-HT receptor agonist or a pharmaceutically acceptable salt thereof can reduce or prevent status epilepticus in an individual when compared to no 5-HT receptor agonist or a pharmaceutically acceptable salt thereof. Thus, it is possible to monitor a patient's response to administration of a 5-HT receptor agonist or a pharmaceutically acceptable salt thereof gradually from a point in time prior to administration of the compounds described herein (eg, control or time point).

An epileptic disorder may be an epileptic disorder that does not respond to treatment with an antiepileptic drug (AED). The individual may be on a ketogenic diet. The epileptic disorder may be an epileptic disorder in an adult (eg, over 16 years of age).

An epileptic disorder may be an epileptic disorder in children. Thus, the epileptic disorder may be a pediatric epileptic disorder. The baby may be less than about 1 week old. The baby may be less than about 1 month old.

The child may be less than about 6 months of age. The child may be less than about 12 months old. The child may be less than about 2 years old. The child may be less than about 3 years old. The child may be less than about 4 years old. The child may be less than about 5 years old. The child may be less than about 6 years of age. The child may be less than about 7 years old. The child may be less than about 8 years old. The child may be less than about 9 years of age. The child may be less than about 10 years old. The child may be less than about 12 years of age.

The baby may be more than about 1 week old. The baby may be more than about 1 month old. The baby may be more than about 6 months old. The child may be more than about 12 months old. The child may be more than about 2 years old. The child may be more than about 3 years old. The child may be more than about 4 years old. The child may be more than about 5 years old. The child may be more than about 5 years old. The child may be more than about 6 years of age. The child may be more than about 7 years old. The child may be more than about 8 years old. The child may be more than about 9 years old. The child may be more than about 10 years old. The child may be more than about 11 years of age. The child may be more than about 12 years of age.

The child may have an epileptic disorder that is treated with the administration of an AED as described herein. Thus, a 5-HT receptor agonist, or a pharmaceutically acceptable salt thereof, may be administered to such a child (eg, as adjuvant therapy).

In another aspect, a method for treating Dravet's syndrome is provided. A method for treating Dravet's syndrome includes administering to an individual in need thereof a therapeutically effective amount of a 5-HT receptor agonist or a pharmaceutically acceptable salt thereof. A method for treating Dravet's syndrome may include administering to an individual in need thereof a pharmaceutical composition of an agonist

- 16 040818 5-HT receptor, or a pharmaceutically acceptable salt thereof, as described herein. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) can be co-administered to an individual in need thereof with an AED as described herein.

In the methods described herein, a 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be co-administered with an antiepileptic drug (AED). The AED may be acetazolamide, benzodiazepine, cannabadiols, carbamazepine, clobazam, clonezepam, eslicarbazepine acetate, ethosuximide, ettoin, felbamate, fenfluramine, fosphenytoin, gabapentin, ganaxolone, huperzine A, lacosamide, lamotrigine, levetiracetam, nitrazepam, perampanzepine, oxcarazepam phenobarbital, phenytoin, potassium bromide, pregabalin, primidone, retigabine, rufinamide, valproic acid, sodium valproate, styripentol, tiagabine, topiramate, vigabatrin, or zonisamide. The AED may be valproic acid, sodium valproate, clonezepam, ethosuximide, felbamate, gabapentin, carbamazepine, oxcarbazepine, lamotrigine, levetiracetam, benzodiazepine, phenobarbital, pregabalin, primidone, tiagabine, topiramate, potassium bromide, phenytoin, styripentol, vigabatrin, or zonisamide. The AED may be valproic acid, sodium valproate, gabapentin, topiramate, carbamazepine, oxcarbazepine, or vigabatrin.

The AED may be acetazolamide. The AED may be a benzodiazepine. The AED may be cannabadiols. The AED may be carbamazepine. The AED may be clobazam. The AED may be clonezepam. The AED may be eslicarbazepine acetate.

The AED may be ethosuximide. The AED may be etotoin. The AED may be felbamate. The AED may be fenfluramine. The AED may be fosphenytoin. The AED may be gabapentin. The AED may be ganaxolone. The AED may be Huperzine A. The AED may be lacosamide. The AED may be lamotrigine. The AED may be levetiracetam. The AED may be nitrazepam. The AED may be oxcarbazepine. The AED may be perampanel. The AED may be piracetam. The AED may be phenobarbital. The AED may be phenytoin. The AED may be potassium bromide. The AED may be pregabalin. The AED may be a primidone. The AED may be retigabine. The AED may be rufinamide. The AED may be valproic acid. The AED may be sodium valproate. The AED may be styripentol. The AED may be tiagabine. The AED may be topiramate. The AED may be vigabatrin. The AED may be zonisamide. Clemizol or a Clemizol analog (including pharmaceutically acceptable salts thereof) or a pharmaceutical composition of Clemizol or a Clemizol analog can be administered as an adjuvant therapy for one or more of the AEDs described herein.

The 5-HT receptor agonist, or a pharmaceutically acceptable salt thereof, can thus be administered as an adjunct to (eg, in combination with) therapy for AED for the treatment of seizures, including seizures associated with the epileptic disorders described herein. The 5-HT receptor agonist, or a pharmaceutically acceptable salt thereof, may be administered as an adjuvant therapy (eg, in combination with) for an AED for the treatment of seizures, including seizures associated with the epileptic disorders described herein.

An epileptic disorder may be characterized by partial seizures or generalized seizures.

An epileptic disorder may be characterized by partial seizures. An epileptic disorder may be characterized by generalized seizures. A partial seizure may be a simple focal seizure, a complex focal seizure, or a partial focal seizure with secondary generalization. A generalized seizure may be a generalized tonic-clonic seizure, an absence seizure (ie, a minor seizure), myoclonic seizures, clonic seizures, tonic seizures, or atonic seizures.

When co-administered with the AEDs described herein, the 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) and the AED may be administered simultaneously. When administered concomitantly, the 5-HT receptor agonist (including its pharmaceutically acceptable salts) can be formulated with an AED (ie, in a single dosage unit). The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) can be formulated to be administered separately from the AED but administered at the same time. When co-formulated with the AEDs described herein, the 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered sequentially (eg, before or after) administration of the AED. As noted in the art, the person skilled in the art can easily determine the sequential order of administration.

The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of about 1 to about 1000 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of about 10 to about 1000 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of about 10 to about 600 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of about 25 to about 500 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of about 25 to about 400 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of about 25 to about 350 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of about 25 to about 300 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of about 25 to about 250 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of about 25 to about 200 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of about 25 to about 150 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of about 25 to about 100 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of about 25 to about 75 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of about 25 to about 50 mg/kg. As used herein, mg/kg refers to mg per kg of an individual's body weight. The dosages described herein include the administration of a 5-HT receptor agonist (including its pharmaceutically acceptable salts) as the sole therapeutic active, or the administration of a 5-HT receptor agonist (including its pharmaceutically acceptable salts) as the therapeutic active in combination (as described in the present description) with the AED described in the present description.

The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 1 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 5 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 10 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 20 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 25 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 30 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 40 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 50 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 75 mg/kg. The 5HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 100 mg/kg.

The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 125 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 150 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 175 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 200 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 225 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 250 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 275 mg/kg.

The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 300 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 325 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 350 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 375 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 400 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 425 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 450 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 475 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 500 mg/kg.

The 5-HT receptor agonist analogue (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 600 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 700 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 800 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 900 mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at a dose of approximately 1000 mg/kg.

The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at the dosages described herein at least once daily (e.g., once every

- 18 040818 for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours). The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) can be administered every day at the dosages described herein. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at least twice a week at the dosages described herein. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered at least three times per week as described herein. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be administered once a month as described herein.

The present invention provides methods for treating a disease or disorder caused by a deficiency of serotonin in the brain or the activity of one or more 5HT receptors by administering a therapeutically effective amount of clemizol, a clemizol analog, or a pharmaceutically acceptable salt thereof. A disease or disorder caused by a deficiency of serotonin in the brain or the activity of one or more 5HT receptors may be Fragile X Syndrome. A disease or disorder caused by a deficiency of serotonin in the brain or the activity of one or more 5HT receptors may be Prader-Willi syndrome. A disease or disorder caused by a deficiency of serotonin in the brain or the activity of one or more 5HT receptors may be schizophrenia. A disease or disorder caused by a deficiency of serotonin in the brain or the activity of one or more 5HT receptors may be depression. A disease or disorder caused by a deficiency of serotonin in the brain or the activity of one or more 5HT receptors may be Alzheimer's disease. A disease or disorder caused by a deficiency of serotonin in the brain or the activity of one or more 5HT receptors may be autism. A disease or disorder caused by a deficiency of serotonin in the brain or the activity of one or more 5HT receptors may be neuropathic pain. A disease or disorder caused by a deficiency of serotonin in the brain or the activity of one or more 5HT receptors may be Parkinson's disease. A disease or disorder caused by a deficiency of serotonin in the brain or the activity of one or more 5HT receptors may be irritable bowel syndrome. A disease or disorder caused by a deficiency of serotonin in the brain or the activity of one or more 5HT receptors may be dementia.

In addition, within the scope of the present invention, there is provided a method for modulating 5HT receptor activity, comprising contacting the 5HT receptor with clemizol, a clemizol analog, or a pharmaceutically acceptable salt thereof.

The clemizol analog may include compounds of formula (I) described herein and may include compounds of similar structure as indicated, for example, in PCT/US 2008/076804, WO 10107739, WO 2009039248 or US patent No. 4011322, which are included in this description as links.

In embodiments, the invention relates to methods for treating epilepsy or Dravet's syndrome by administering a therapeutically effective amount of trazadone, or a pharmaceutically acceptable salt thereof. In embodiments, the therapeutically effective amount is from about 10 to about 600 mg/day. In embodiments, the methods are for the treatment of epilepsy. In embodiments, the epilepsy is pediatric epilepsy. In embodiments, the methods are for the treatment of Dravet's syndrome.

Pharmaceutical compositions

Within the scope of the present invention are provided pharmaceutical compositions containing a 5-HT receptor agonist or a pharmaceutically acceptable salt thereof, useful in the treatment of the aforementioned diseases or disorders. The pharmaceutical composition may be formulated as a tablet, powder, capsule, pill, cachet or lozenge as described herein. The pharmaceutical composition may be formulated as a tablet, capsule, pill, cachet or lozenge for oral administration. The pharmaceutical composition may be formulated to dissolve in solution for administration by means such as, for example, intravenous administration. The pharmaceutical composition can be formulated for oral administration, suppository administration, topical administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intranasal administration, subcutaneous administration, implantation, transdermal administration, or transmucosal administration, as described in the present description.

When administered as a pharmaceutical composition, the pharmaceutical compositions may include optical isomers, diastereomers, enantiomers, isoforms, polymorphs, hydrates, solvates or products, or pharmaceutically acceptable salts of the 5-HT receptor agonist. The 5HT receptor agonist (including pharmaceutically acceptable salts thereof) included in a pharmaceutical composition may be covalently linked to a carrier portion as described above. Alternatively, the 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) included in the pharmaceutical composition is not covalently linked to the carrier moiety.

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The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) can be administered or co-administered to an individual in need thereof with an AED as described herein. Co-administration is intended to include the simultaneous or sequential administration, as described herein, of the 5-HT receptor agonist alone or in combination (eg, more than one compound - eg, the AED described herein). The preparations can also be combined, when desired, with other active substances (for example, to prevent seizures).

Lineups

The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or the pharmaceutical composition described herein can be prepared and administered in a wide variety of oral, parenteral and topical dosage forms. Thus, a 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or a pharmaceutical composition described herein may be administered by injection (eg, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal). Also, the 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or the pharmaceutical composition described herein can be administered via inhalation, eg intranasally. In addition, the 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or pharmaceutical composition may be administered transdermally. It is also contemplated that a variety of routes of administration (eg, intramuscular, oral, transdermal) may be used to administer a 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or a pharmaceutical composition containing it. The pharmaceutical compositions described herein may include a pharmaceutically acceptable carrier or excipient and one or more 5-HT receptor agonists (including their pharmaceutically acceptable salts). The pharmaceutical compositions described herein may include a pharmaceutically acceptable carrier or excipient, one or more 5-HT receptor agonists (including their pharmaceutically acceptable salts), and one or more AEDs as described herein.

The formulation may include pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. The solid carrier may be one or more substances which may act as diluents, flavoring agents, binders, preservatives, tablet disintegrants, or an encapsulating material.

In powders, the carrier may be a finely divided solid in admixture with the finely divided active ingredient. In tablets, the active ingredient may be mixed with a carrier having the desired binding properties in suitable proportions and compressed into the desired shape and size.

Powders and tablets preferably contain 5 to 70% active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like. The term preparation includes the formulation of the active compound with an encapsulating material, such as a carrier, providing a capsule in which the active ingredient, with or without other carriers, is surrounded by a carrier, which is thus associated with it. Similarly, cachets and lozenges are included. As solid dosage forms suitable for oral administration, tablets, powders, capsules, pills, cachets and lozenges can be used.

Suitable solid excipients include, but are not limited to, magnesium carbonate; magnesium stearate; talc; pectin; dextrin; starch; tragacanth; fusible wax; cacao butter; carbohydrates; sugars, including but not limited to lactose, sucrose, mannitol or sorbitol, starch from corn, wheat, rice, potatoes or other plants; cellulose such as methylcellulose, hydroxypropylmethylcellulose or sodium carboxymethylcellulose; and gums, including gum arabic and tragacanth; as well as proteins, including but not limited to gelatin and collagen. If desired, disintegrating or solubilizing agents such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate can be added.

Dragee cores are provided with suitable coatings such as concentrated sugar solutions which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to tablets and dragee coatings to identify the product or to characterize the amount of the 5-HT receptor agonist (including its pharmaceutically acceptable salts) or pharmaceutical composition (ie dosage). The pharmaceutical preparations described herein can be used orally using, for example, hard capsules made from gelatin, as well as soft sealed capsules made from gelatin and a coating such as glycerol or sorbitol.

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To prepare suppositories, a low-melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active ingredient is homogeneously dispersed therein, for example by stirring. The molten homogeneous mixture is then poured into molds of suitable size, allowed to cool and thereby solidify.

Liquid form preparations include solutions, suspensions and emulsions, such as aqueous or water/propylene glycol solutions. For parenteral injection, liquid preparations may be formulated in solution as an aqueous polyethylene glycol solution.

When parenteral administration is required or desired, particularly suitable additives for a 5-HT receptor agonist (including its pharmaceutically acceptable salts) or a pharmaceutical composition containing it are injectable sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions or implants, including suppositories. In particular, carriers for parenteral administration include aqueous dextrose solutions, saline, pure water, ethanol, glycerin, propylene glycol, peanut oil, sesame oil, polyoxyethylene block polymers, and the like. Convenient unit dosages are ampoules. The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or a pharmaceutical composition containing it can also be liposomal or administered via transdermal pumps or patches. Pharmaceutical additives suitable for use in the present invention include those described in, for example, Pharmaceutical Sciences (17th Ed., Mack Pub. Co., Easton, PA) and WO 96/05309, both of which are incorporated herein. as links.

Aqueous solutions suitable for oral administration can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizers and thickeners, if desired. Aqueous suspensions suitable for oral administration can be prepared by dispersing the finely divided active ingredient in water with a viscous material such as natural or synthetic gums, gums, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g. lecithin), a condensate of alkylene oxide with a fatty acid (e.g. polyoxyethylene stearate), a condensate of ethylene oxide with a long chain aliphatic alcohol (e.g. heptadecaethylene oxycetanol), a condensate of ethylene oxide with a partial an ester formed from a fatty acid and hexite (for example, polyoxyethylene sorbitan monooleate) or a condensation product of ethylene oxide with a partial ester formed from a fatty acid and hexite anhydride (for example, polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more colorants, one or more flavors, and one or more sweeteners such as sucrose, aspartame or saccharin. The formulations can be adjusted for osmolarity.

The present invention also encompasses solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions and emulsions. These preparations may contain, in addition to the active ingredient, coloring agents, flavoring agents, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solibilizers, and the like.

Oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweeteners such as glycerol, sorbitol or sucrose may be added to provide a palatable oral formulation. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. For an example of an injectable oily carrier, see Minto, J. Pharmacol. Exp. Ther. 281:93-102, 1997. The pharmaceutical formulations described herein may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil or a mineral oil as described above, or a mixture thereof. Suitable emulsifiers include naturally occurring gums such as gum arabic and gum tragacanth, naturally occurring phosphatides such as soy lecithin, esters or partial esters formed from fatty acids and hexitan anhydrides such as sorbitan monooleate, and the condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents, for example in syrup and elixir formulations. Such formulations may also contain an emollient, preservative or coloring agent.

Preferably the pharmaceutical is in unit dosage form. In this form, the preparation is subdivided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form may be a packaged preparation, packages containing discrete amounts of the drug, such as packaged tablets, capsules and powders in vials or ampoules. Also, the unit dosage form may be a capsule, tablet, cachet or lozenge itself, or it may be a suitable amount of any of these in packaged form.

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The amount of the active ingredient in a unit dose formulation may vary, or may be adjusted to between 0.1 and 10,000 mg, depending on the particular application and efficacy of the active ingredient. If desired, the composition may also contain other compatible drugs.

The compositions may include a surfactant or other suitable co-solvent in the composition. Such co-solvents include Polysorbate 20, 60 and 80; Pluronic F-68, F-84 and R-103; cyclodextrin; and polyoxyl 35 castor oil. Such co-solvents are typically used at a level of from about 0.01 to about 2% by weight. Viscosity greater than that of simple aqueous solutions may be desirable to reduce variability in formulation distribution, to reduce physical separation of the components of a suspension or emulsion formulation, and/or otherwise to improve formulation. Such thickeners include, for example, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylcellulose, chondroitin sulfate and its salts, hyaluronic acid and its salts, and combinations of the foregoing. Such agents are typically used at a level of from about 0.01 to about 2% by weight.

Viscosity greater than that of simple aqueous solutions may be desirable to reduce variability in formulation distribution, to reduce physical separation of the components of a suspension or emulsion formulation, and/or otherwise to improve formulation. Such thickeners include, for example, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylcellulose, chondroitin sulfate and salts thereof, hyaluronic acid and salts thereof, and combinations of the foregoing, and others known to those skilled in the art. Such agents are typically used at a level of from about 0.01 to about 2% by weight. Determination of acceptable amounts of any of the above adjuvants can be easily done by a person skilled in the art.

Pharmaceutical compositions may also include components to provide sustained release and/or comfort. Such components include high molecular weight, anionic, mucus-mimicking polymers, gelling polysaccharides, and finely divided drug carrier substrates. These components are discussed in more detail in US patent No. 4911920; 5403841; 5,212,162 and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes.

The pharmaceutical composition may be intended for intravenous use. The pharmaceutically acceptable excipient may include buffers to adjust the pH to the desired range for intravenous use. Many buffers are known, including inorganic acid salts such as phosphate, borate and sulfate.

A 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or a pharmaceutical composition thereof can be delivered transdermally for the treatment of the epileptic disorders described herein, topically formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders and aerosols.

The 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) may be provided as a salt in the pharmaceutical compositions described herein and may be formed with a variety of acids including, but not limited to, hydrochloric, sulfuric, acetic, lactic, tartar, apple, amber, etc. Salts tend to be more soluble in aqueous or other protic solvents than the corresponding free base forms.

A 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or a pharmaceutical composition thereof, administered for the treatment of the epileptic disorders described herein, may be administered via parenteral administration such as intravenous (IV) administration or administration into a body cavity or lumen organ. Formulations for administration typically contain a solution of the compositions of the present invention dissolved in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that may be used are water and Ringer's solution, isotonic sodium chloride solution. Also, sterile fatty oils can usually be used as the solvent or suspending medium. Any bland fixed oil can be used for this purpose, including synthetic mono- or diglycerides. In addition, fatty acids such as as oleic acid can be used similarly for the preparation of injectables. These solutions are sterile and usually free of unwanted materials. These formulations can be sterilized by conventional well known sterilization techniques. The formulations may contain pharmaceutically acceptable excipients necessary to approximate physiological conditions, such as pH adjusters and buffering agents, toxicity correctors, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the compositions of the present invention in these formulations can vary widely and will be selected based primarily on fluid volumes, viscosity, body weight, and the like, in accordance with the particular route of administration chosen and the needs of the patient. For intravenous administration composition

- 22 040818 may be a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension. This suspension can be formulated according to the state of the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent such as 1,3-butanediol solution.

Pharmaceutical formulations of a 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) for the treatment of epileptic disorder can be delivered using liposomes that fuse with the cell membrane or undergo endocytosis, i. using ligands bound to a liposome or bound directly to an oligonucleotide that binds to protein receptors on the cell membrane surface, which can lead to endocytosis. Using liposomes, in particular when the surface of the liposomes contains ligands specific to target cells, or otherwise preferentially directed to a specific organ, it is possible to focus the delivery of compositions of the present invention to target cells in vivo (see, for example, AlMuhammed, J. Microencapsul 13: 293-306, 1996; Chonn, Curr. Opin. Biotechnoi 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989).

Co-administration involves the administration of one active substance (for example, clemizol or an analog of clemizol (including its pharmaceutically acceptable salts)) within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours from the second active substance (for example, an anticonvulsant drug). Co-administration may include administering one active agent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of the second active agent. Co-administration may include administering the two active substances at the same time, approximately simultaneously (eg, within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in any order. Co-administration can be carried out by co-formulation, i. e. obtaining one pharmaceutical composition comprising both active substances. In other embodiments, the active substances may be formulated separately. The active and/or excipients may be linked or conjugated to each other.

Co-administration also includes combination with treatments for epileptic disorders such as dietary requirements or dietary changes. Thus, a 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or a pharmaceutical composition thereof may be administered to individuals on specialized diets, including, but not limited to, a ketogenic diet (e.g., a diet high in fat, sufficient in protein, low in carbohydrates).

Effective dosages

The pharmaceutical composition may include a 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) in a therapeutically effective amount, i. in an amount effective to achieve its intended purpose. The true amount effective for a particular application depends, inter alia, on the condition being treated. For example, when administered in methods of treating an epileptic disorder (eg, Dravet's syndrome), such compositions contain amounts of a 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or a pharmaceutical composition thereof effective to achieve the desired result (eg, seizure inhibition).

The dosage and frequency of administration (single or multiple doses) of the 5-HT receptor agonist (including its pharmaceutically acceptable salts) or pharmaceutical composition thereof may vary depending on various factors, including the route of administration; size, age, sex, health status, body weight, body mass index and diet of the recipient; the nature and severity of the symptoms of the disease being treated; the presence of other diseases or health-related problems; type of concomitant treatment; and complications of any disease or treatment regimen. Other therapeutic regimens and agents may be used in conjunction with the methods described herein.

A therapeutically effective amount of a 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or a pharmaceutical composition thereof for the treatment of epileptic disorders described herein can initially be determined in cell culture assays. Target concentrations are concentrations of a 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or a pharmaceutical composition thereof capable of inhibiting or otherwise reducing seizures experienced by a patient.

Therapeutically effective amounts of a 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or a pharmaceutical composition thereof for use in humans can be determined in animal models. For example, the human dose may be formulated to achieve a concentration found to be effective in animals. Dosage in humans can be adjusted by monitoring the patient's response to treatment and adjusting the dosage up or down as described above.

- 23 040818

Dosages may vary depending on the needs of the individual and the compound used. The dose administered to an individual, in the context of the pharmaceutical compositions described herein, should be sufficient to provide a favorable therapeutic response in the individual over time. The size of the dose will also be determined by the existence, nature and extent of any adverse side effects. Typically, treatment is started with smaller dosages less than the optimum dose of the compound. Thereafter, the dosage is increased in small increments until the optimum effect under the circumstances is reached.

Dosages and intervals may be adjusted individually to provide levels of the 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) or pharmaceutical composition thereof effective for the particular epileptic disorder being treated. This may provide a therapeutic regimen appropriate to the severity of the disease state of the individual.

Using the ideas described herein, an effective prophylactic or therapeutic treatment regimen can be planned that does not cause significant toxicity and yet is effective in treating the clinical symptoms that a particular patient exhibits. This planning should involve careful selection of the 5-HT receptor agonist (including its pharmaceutically acceptable salts) or its pharmaceutical composition, taking into account factors such as efficacy, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred route of administration, and profile. toxicity of the selected agent.

Toxicity

The ratio between toxicity and therapeutic effect for a particular compound is its therapeutic index, and it can be expressed as the ratio between LD ₅₀ (amount of compound lethal in 50% of the population) and ED ₅₀ (amount of compound effective in 50% of the population). Compounds that exhibit high therapeutic indices are preferred. Therapeutic index data obtained from cell culture assays and/or animal studies can be used in formulating a dosage range for human use. The dosage of such compounds is preferably in the range of plasma concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. See, for example, Fingl et al., THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch.1, pl, 1975. The exact composition, route of administration, and dosage may be chosen by the individual's physician, taking into account the condition of the patient and the particular method in which the compound is used.

When a parenteral formulation is necessary or desirable, particularly suitable additives for a 5-HT receptor agonist (including pharmaceutically acceptable salts thereof) included in the pharmaceutical composition may be injectable sterile solutions, oily or aqueous solutions, as well as suspensions, emulsions or implants, including suppositories. In particular, carriers for parenteral administration include aqueous dextrose solutions, saline, pure water, ethanol, glycerin, propylene glycol, peanut oil, sesame oil, polyoxyethylene block polymers, and the like. Convenient unit dosages are ampoules.

Pharmaceutical additives for use in the pharmaceutical compositions described herein may include those described in, for example, Pharmaceutical Sciences (17th Ed., Mack Pub. Co., Easton, PA) and WO 96/05309, the ideas of both of which are included to the present description as references.

Examples

Example 1

Epilepsy can be acquired as a result of brain damage or a genetic mutation. Among genetic epilepsy, 650 variants in the SCN1A gene have been identified (Harkin, L.A. et al. The spectrum of SCN1A-related infantile epileptic encephalopathies. Brain 130, 843-852 (2007); Mulley J. C, et al., SCN1A mutations and epilepsy Hum Mutat 25, 535-542 (2005)). Missensmutations or frameshift mutations in this gene are associated with the presence of generalized epilepsy with febrile seizures (GEFS+) (Ceulemans, B. P., et al., Clinical correlations of mutations in the SCN1A gene: from febrile seizures to severe myoclonic epilepsy in infancy Pediatric Neurol 30, 236-243 (2004)), as well as the more severe disorder known as Dravet's syndrome. Children with DS initially display normal development but often experience episodes of febrile seizures during the first year of life, eventually progressing to severe spontaneous recurrent seizures, mental retardation, ataxia, and psychomotor dysfunction. Seizures are not well controlled with available antiepileptic drugs (AEDs), and these children are candidates for neurosurgical resection (Bender, A. C, et al., SCN1A mutations in Dravet syndrome: Impact of interneiron dysfunction on neural networks and cognitive outcome. Epilepsy Beh. 23, 177-186 (2012)).

There are four main subtypes of poten alpha subunits in the mammalian brain.

- 24 040818 cyal-dependent sodium channels: NaVl.1, NaV1.2, NaVl.3 and NaV1.6, encoded by the SCN1A, SCN2A, SCN3A and SCN8A genes, respectively. The opening of these channels allows for sodium conduction and rapid depolarization of the cell membrane, for example, which are features necessary for the initiation of an action potential (Catterall, WA, et al., Na _v l. 1 channels and epilepsy. J. Physiol. 588, 1849-1859 (2010)). In mice, Nav1.1 is widely expressed in the central nervous system, including the initial axon segment of parvalbumin-positive hippocampal interneurons and excitatory chief cells (Kim, DY, et al., Reduced sodium channel Na(v)ll levels in BACEl-null mice. J. Biol Chem 286, 8106-8116 (2011) Chen, C, et al., Mice lacking sodium channel betal subunits display defects in neuronal excitability, sodium channel expression, and nodal architecture J. Neurosci 24, 4030 -4042 (2004)). Heterozygous deletion of Navl.1 in mice results in reduced firing capacity of highly dissociated, rapidly discharging interneurons (Yu, FH, et al., Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat. Neurosci. 9 , 1142-1149 (2006)). Mice with a global or interneuron-specific heterozygous Nav1.1 deletion exhibit temperature-induced and spontaneous seizures, mild ataxia, autism-like behavior, and premature death (Yu FH, et al., Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy Nat Neurosci 9 1142-1149 (2006) Oakley J C et al Temperature- and age-dependent seizures in a mouse model of severe myoclonic epilepsy in infancy Proc Natl Acad. Sci. USA 106, 3994-3999 (2009); Cheah, CS, et al., Specific deletion of Nav1. 1 sodium channels in inhibitory interneurons causes seizures and premature death in a mouse model of Dravet syndrome. Proc. Natl. Acad. Sci. USA 109, 14646-14651 (2012)). Knockin mice containing a premature stop codon in domain III of the Nav1.1 channel also show a decrease in peak amplitude during prolonged firing of interneurons and an increased sensitivity to temperature-induced seizures (Ogiwara, I., et al., Na _v 1.1 localizes to axons of parvalbuminpositive inhibitory interneurons: a circuit basis for epileptic seizures in mice carrying an Scn1a gene mutation J. Neurosci 27, 5903-5914 (2007)).

The establishment and characterization of working animal models are important for understanding the pathophysiology of DS and for facilitating the identification of novel therapies. While considerable attention has been focused on modeling SCN1A mutations in mice, these animals have been shown to be difficult to breed and epileptic phenotypes are strongly influenced by lineage background genetics. Induced pluripotent stem cells can be obtained from DS patients, however, individual neurons do not replicate the network conditions required to induce seizures in vivo. The Danio rerio (zebrafish), a simple vertebrate species, provides an alternative model system with significant advantages of genetic manipulation, economical breeding, and in vivo drug development (Lessman, C. A., The developing zebrafish (Danio rerio): a vertebrate model for high-throughput screening of chemical libraries. Birth Defects Res. C Embryo Today 93, 268-280 (2011); Delvecchio, C, et al., The zebrafish: a powerful platform for in vivo, HTS drug discovery. Assay Drug Dev. Technol. 9, 354-361 (2011) Rinkwitz, S., et al., Zebrafish: an integrative system for neurogenomics and neurosciences. Prog. Neurobiol. 93, 231-243 (2011)). Ideally, an animal model should be based on a known genetic cause of the disease (SCN1A mutation), accurately reproduce key features of the disease (epilepsy), and respond or not respond to therapies commonly used in patients with the disease (pharmacological validation). If successful, such a model could provide insight into the disease process and catalyze research into new therapies.

In zebrafish, the voltage-gated sodium channel family consists of four groups of duplicated genes: Scn1Laa and scn1Lab, scn4aa and scn4ab, scn5Laa and scn5Lab and scn8aa and scn8ab (Novak, A. E., et al., Embryonic and larval expression of zebrafish voltage-gated sodium channel alpha-subunit genes Dev Dyn 235 1962-1973 (2006)). The zebrafish scn1Lab gene has 77% identity with human SCN1A and is expressed in the central nervous system. A homozygous zebrafish mutant for this gene (originally named didys552) was detected by chemical mutagenesis screening using an optokinetic assay reaction (Schoonheim, P. J., Arrenberg, A. B., Del Bene, F., & Baier H., Optogenetic localization and genetic perturbation of saccade-generating neurons in zebrafish J. Neurosci 30, 7111-7120 (2010)). These types of screens are based on the induction of random point mutations using an N-ethyl-N-nitrosourea (ENU) alkylating agent, the resulting mutations are generally loss of function and are recessive. Although this mutation is homozygous, the scn1Lab zebrafish mutants are consistent with the autosomal dominant Dravet syndrome in humans, given the duplication of the zebrafish genome and the presence of an additional Nav1.1 (Scn1Laa) homologue.

The scn1Lab mutants were characterized at the molecular and behavioral level, the mutants were shown to exhibit spontaneous drug-resistant seizures, and were then used in a new high-throughput screening program to identify compounds that attenuate the epilepsy phenotype. Phenotype-based screening identified clemizol, an FDA-approved compound, as an effective inhibitor of spontaneous seizure behavior and electrographic seizures in these mutants.

- 25 040818

scnlLab expression and characterization of mutant zebrafish. Zebrafish with a mutation in domain III of the voltage-gated sodium channel were identified by Dr. Herwig Baier in screening using chemical mutagenesis (Schoonheim, P. J., Arrenberg, A. B., Del Bene, F., & Baier H., Optogenetic localization and genetic perturbation of saccade-generating neurons in zebrafish. J. Neurosci. 30 , 7111-7120 (2010)). The original scn1Lab mutants were backcrossed to a Tupfel long (TL) background for 7-10 generations and the methionine (M) to arginine (R) mutation was confirmed in the colony (FIG. 1A). Quantitative (q) PCR and reverse transcriptase (RT) PCR revealed a decrease in scn1Lab mRNA expression in mutant larvae at 3, 5, and 7 days post-fertilization (dpf) (Fig. 1B); antibodies that recognize this protein in zebrafish are not available. As expected (Novak, A. E., et al., Embryonic and larval expression of zebrafish voltage-gated sodium channel alpha-subunit genes. Dev. Dyn. 235, 1962-1973 (2006)), scn1Lab is prominently expressed early larval development (Fig. 1B) and, in particular, in the central nervous system after 3 dpf (Fig. 1D, E). Hybridization of the total preparation in situ revealed diffuse but pronounced expression in brain regions corresponding to the forebrain (telencephalon), optic tectum, and cerebellum. A similar expression profile was observed for Scn1Laa at 3 dpf. After 5 and 7 dpf, expression in the CNS remained strong, and a weak signal of scn1Lab was also noted in the heart (Fig. 1D). Relative expression of scn8aa or scn8ab (Nav1.6), for example, a subunit putatively acting as a genetic modifier of DS (Martin M. S., et al., The voltage-gated sodium channel Scn8a is a genetic modifier of severe myoclonic epilepsy of infancy. Hum. Mol. Gen. 16, 2892-2899 (2007)), showed no significant difference in expression between mutants and sibling controls at 5 dpf (Fig. 1C). Similarly, microarray analysis at 5 dpf also found no compensatory change in mRNA expression of thirteen different zebrafish scn subunits (Table I) including another homologue (Scn1Laa). These results demonstrate a selective defect in the zebrafish Nav1.1 gene, which is expressed in the CNS at an early stage of development.

Large-scale transcriptomic analysis of scn1Lab mutants. Although hereditary disorders of voltage-gated ion channels are recognized as an etiology of epilepsy, the study of transcriptional changes has not been described for any epilepsy-related channelopathies. An Agilent Danio rerio chip covering ~44,000 probes was used to detect differences in gene expression without distortion (Fig. 2A, B). Hierarchical cluster analysis demonstrated that ~2.5% (1099) of these probes were differentially expressed between mutants and sibling controls at 5 dpf (p<0.01, t-test; 674 activated and 425 suppressed); 405 were categorized as unknown feature. A list of 30 repressed and activated known genes showing the most significant differences in expression is shown in FIG. 2C. These differences were modest as 90% (990/1099) of the identified genes showed a fold change between 0.8 and 2.0. Similar to microarray analysis of mice with a mutation in one Mecp2 gene (Jordan, C, et al., Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets. BMC Med. Genet. 8, 36 (2007)), many of identified had no obvious CNS-related function and/or expression.

The two genes with the highest fold change, the somatolactin β gene and Na,K-ATPase, have expression mostly restricted to the hypophysis (smtlb) (Lopez, M., et al., Expression of the somatolactin β gene during zebrafish embryonic development. Gene Expr. Patterns 6, 156-161 (2006)) or ear, intestinal bulb and pronephric duct (atp1a1a.5) (Blasiole, B., et al., Cloning, mapping, and developmental expression of a sixth zebrafish Na, K -ATPase alpha1 subunit gene (atp1a1a.5) Mech Dev 119 Suppl 1:S211-S214 (2002)). Probes for several genes associated with apoptosis (casp8, casp8b and casp3b) did not reveal any statistically significant changes in microarray studies. Among the genes with altered expression in scn1Lab mutants, six have previously been shown to be involved in neurological disorders, e.g. Parkinson's disease) and wasp2 (Angelman's syndrome). Microarray-based gene expression measurements were validated for 14 randomly selected genes using qPCR (Fig. 3A).

Biological functions were assigned to all genes using gene ontology (GO) annotations and 482 genes showing at least a 1.5-fold change in expression and a p<0.01 value were further categorized (FIG. 3C). Calcium ion binding genes include the genes for annexin A1c, A1b and 2a, spectrin α2, neurexin 2a, calsintein 1, and parvalbumin 3. Significant changes were also noted in the gap junction channel gene (cx43), which is involved in the clustering of voltage-gated sodium channels in the initial segment of the axon (spna2) and the ubiquitin domain of the GABA receptor (tap1lc3b). Three additional genes not found on the microarray were selected for analysis by qPCR (Fig. 3B): the hcnl gene, shown to be correlated with SCN1A using data acquisition, and downregulated in several seizure models (Noam, Y. , et al., Towards an integrated view of HCN channel role in epilepsy Curr Opin Neurobiol 21 873-879 (2011)) was significantly suppressed in scn1Lab mutants compared to sibling control (p<0.05 2- third-party Student's t-test).

- 26 040818

However, for example, the homer and bdnf genes involved in synaptogenesis associated with the formation of recurrent excitatory synapses and epilepsy (Avedissian M., et al., Hippocampal gene expression analysis using the ORESTES methodology shows that homer 1a mRNA is upregulated in the acute period of the pilocarpine epilepsy model Hippocampus 17, 130-136 (2007) Tongiorgi E., et al., Brain-derived neurotrophic factor mRNA and protein are targeted to discrete dendritic laminas by events that trigger epileptogenesis J. Neurosci 24, 6842 -6852 (2004)) were unchanged.

Spontaneous seizures in scn1Lab mutant zebrafish.

Scn1Lab mutants were monitored for signs of spontaneous electrographic seizures beginning at 3 dpf, such as in the first larval stage when an epileptiform discharge can be detected (Baraban S.C., et al., A large-scale mutagenesis screen to identify seizureresistant zebrafish. Epilepsia 48 , 1151-157 (2007); Hortopan G. A., et al., Spontaneous seizures and altered gene expression in GABA signaling pathways in a mind bomb mutant zebrafish. J. Neurosci. 30, 13718-13728 (2010); Hunt R. F., Hortopan G. A. , Gillespie A., & Baraban S. C., A novel zebrafish model of hyperthermiainduced seizures reveals a role for TRPV4 channels and NMDA-type glutamate receptors, Exp Neurol 237, 199-206 (2012); Baraban S. C, Taylor M. R., Castro P. A., & Baier H., Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression Neuroscience 131, 759-768 (2005) Chege S. W., Hortopan G. A., Dinday M. T., & Baraban S. C. , Expression and function of KCNQ channels in larval zebrafish. dev. neurobiol. 72, 18 6-198 (2012)). Mutant larvae were identified by their black appearance (Fig. 4A), which indicates a defect in pigment aggregation, and they die prematurely between 10 and 12 dpf, as previously described (Novak A. E., et al., Embryonic and larval expression of zebrafish voltagegated sodium channel alpha-subunit genes Dev Dyn 235 1962-1973 (2006)). Registration of extracellular forebrain fields from paralyzed and agar-immobilized scn1Lab mutants was noted by frequent short-term similar epileptic discharges and long-term similar ictal phenomena with large amplitude, starting after 3 dpf (n=4) and gradually becoming more pronounced between 4 and 7 dpf (n= 132) (FIG. 2C). These phenomena were confirmed in 100% of the mutants at 3 dpf, 100% at 4 dpf, 97% at 5 dpf, 98% at 6 dpf and 100% at 7 dpf.

Abnormal electrical phenomena were not observed in age-matched sibling controls at any developmental stage (n=36). Hunt R. F., Hortopan G. A., Gillespie A., & Baraban S. C. A novel zebrafish model of hyperthermia-induced seizures reveals a role for TRPV4 channels and NMDA-type glutamate receptors. Exp. Neurol. 237, 199- 206 (2012)) could be induced after 5 dpf in scn1Lab mutants and controls at approximately similar temperature thresholds (mutant: 26.9±0.5°C; n=14; control: 25.9±0.5°C; n=14; p=0.164 t-test). However, these measurements were complicated in mutants by the simultaneous occurrence of high-frequency spontaneous epileptiform discharges. The mutants had increased levels of swimming activity and exhibited unprovoked convulsive-like behavior consisting of whole-body convulsions and rapid non-directional movement starting at 4 dpf (n=36). A representative motion tracking plot of a scn1Lab mutant exhibiting hyperactivity and seizure behavior is shown in FIG. 4B. This behavior is similar to that classified as stage III seizures in pentylepetetrazole-exposed larvae (Baraban S. C, Taylor M. R., Castro P. A., & Baier H., Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c- fos expression Neuroscience 131, 759-768 (2005)). Convulsive behavior was never observed in the controls at any stage of development (n=36). In pooled mutant and sibling control larvae, scn1Lab mutants remain near the edges of petri dishes, which is considered a form of thigmotaxis in fish (Ellis, L. D., Seibert, J., & Soanes, K. H., Distinct modes of induced hyperactivity in zebrafish larvae.

Brain Res. 1449, 46-59 (2012)). These results demonstrate a striking epileptic phenotype in scn1Lab zebrafish mutants.

Pharmacological evaluation of scn1Lab mutant zebrafish.

Seizures associated with SCN1A mutations respond poorly to most AEDs. To evaluate pharmacological sensitivity, spontaneous electrographic seizures were recorded in agar-embedded scn1Lab mutants (5-6 dpf) at baseline and again after application of commercially available AED. All drugs were added to the container at a concentration of 1 mm; seven fish were examined for each drug. The frequency of epileptiform events (including similar interictal and ictal discharges) and fractional time spent in seizures were reduced in scn1Lab mutants by valproate, diazepam, potassium bromide, and styripentol (Fig. 5A, B, D). The pulse duration did not change to a significant level for any of these drug exposures (FIG. 5C).

As expected, most AEDs had no effect and epileptiform activity became more frequent after exposure to carbamazepine (in 2 of 7 fish), ethosuximide (in 4 of 7 fish) or vigabatrin (in 6 of 7 fish). Since children with DS often respond to the ketogenic diet (KD) (Dravet C, et al., Severe myoclonic epilepsy in infancy: Dravet syndrome. Adv. Neurol. 95, 71-102 (2005)) a separate group of scn1Lab mutants, sibs and controls WT was exposed to this form of diet for 48 h starting at 4 dpf. Movement tracking data in KD-exposed larvae at 6 dpf

- 27 040818 confirm reduction of seizure-like behavior to control levels in 7 of 10 mutants (Fig. E; mean rate, treated mutants=0.43 ± 0.09 mm/h, n=16; untreated mutants=0.81 ± 0 .05 mm/s, n=28, p<0.05, Kruskal-Wallis rank ANOVA with Dunn's pairwise multiple comparison). There were no significant differences in swimming behavior in KD-treated sibling controls (mean speed=0.63 ± 0.05 mm/s, n=20) compared to untreated WT larvae at 6 dpf (mean speed=0.62 ± 0 .07 mm/s; n=20). Short-term exposure (20 min) to the diet had no effect on seizure behavior in mutants in the locomotion assay (n=14; mean speed change <34%). Subsequent recording of the forebrain fields performed on the same zebrafish used in the locomotion analysis (Fig. 5E, top line) confirmed the onset of spontaneous epileptiform discharge in the scn1Lab embryonic media-exposed mutants and suppression of impulse activity in the KD-exposed mutants. within 48 h (Fig. 5E, bottom line). These results demonstrate that the pharmacological profile for scn1Lab mutants resembles that seen in children with DS.

High-throughput drug screening in scn1Lab mutants. Behavioral seizure activity can be easily and quickly monitored using a movement tracking format (Baraban S.C., et al., A large-scale mutagenesis screen to identify seizure-resistant zebrafish. Epilepsia 48, 1151-157 (2007); Hortopan G. A., et al. , Spontaneous seizures and altered gene expression in GABA signaling pathways in a mind bomb mutant zebrafish J. Neurosci 30, 13718-13728 (2010) Baraban S. C, Taylor M. R., Castro P. A., & Baier H., Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression Neuroscience 131, 759-768 (2005); Chege S. W., Hortopan G. A., Dinday M. T., & Baraban S. C., Expression and function of KCNQ channels in larval zebrafish. Dev. Neurobiol 72, 186-198 (2012) Berghmans S, Hunt J, Roach, A, & Goldsmith P, Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants Epilepsy Res 75 , 18-28 (2007) Baxendale S., et al., Identification of compound s with anti-convulsant properties in a zebrafish model of epileptic seizures. Dis. Model. Mech. 5, 773-774 (2012); Cario C L., Farrell T. C, Milanese C., & Burton E. A., Automated measurement of zebrafish larval movement. J Physiol. 589, 3703-3708 (2011); Winter M. J., et al., Validation of a larval zebrafish locomotor assay for assessing the seizure liability of early-stage development drugs. J Pharm. Tox. Methods 5, 176-187 (2008); Orellana-Paucar A. M., et al., Anticonvulsant activity of bisabolene sesquiterpenoids of Curcuma longa in zebrafish and mouse seizure models. Epilepsy Beh. 24, 14-22 (2012) (FIGS. 4B and 5B1).

A high-throughput phenotype-based strategy has been developed to screen chemical libraries for compounds that reduce mutant behavior to stage 0 (very little swimming activity) or stage I (increased but not jerky swimming activity), e.g. to a behavior equivalent to that observed in normal WT mutants. Automated measurement of larval activity was performed using EthoVision tracking software (Noldus Information Technology) and a high speed camera. Previous studies have confirmed that high speed movement >20 mm/s is consistent with paroxysmal seizure-like seizures (stage III) (Winter M. J., et al., Validation of a larval zebrafish locomotor assay for assessing the seizure liability of early-stage development drugs. J Pharm Tox Methods 5, 176-187 (2008) Orellana-Paucar A M et al Anticonvulsant activity of bisabolene sesquiterpenoids of Curcuma longa in zebrafish and mouse seizure models Epilepsy Beh 24 14-22 (2012 )).

Using the 96-well format, the swimming activity of the mutant was monitored automatically at baseline and then again after the addition of test compound (100 μl); each compound was tested on 6-12 individual larvae at 5 dpf. The change in mutant swim activity between two successive recording intervals in embryonic medium was taken as baseline and is shown in FIG. 6A (n=28). Based on a standard deviation of 17.3 for recording at baseline associated simply with fluid change, compounds were screened for that inhibited locomotion (measured as a change in mean speed) by >34%. To validate this approach, eleven AEDs and KDs were first screened using this assay. As expected from the electrophysiological analysis (Fig. 5), diazepam, potassium bromide, styripentol, valproate, and 48 h KD exposure effectively inhibited seizure behavior in the movement-based assay (Fig. 6B); ganaxolone, a neuroactive steroid related to allopregnalone, was also effective. Test compounds were then screened at an initial concentration of 667 μM from a library that included US Food and Drug Administration (FDA) approved and toxicologically tested drugs.

Of the 320 compounds screened in vivo, 18 were found to significantly inhibit spontaneous convulsions in scn1Lab mutants to levels comparable to stage 0 or stage I behavior and/or reduce mean swimming speed (red circles in Figure 6C). These 18 compounds were then re-examined on a separate group of scn1Lab mutants at concentrations of 667, 67, and 6.7 μM. In the initial screening, 81 compounds were identified as lethal, ie. no noticeable heartbeat or movement in response to touch after exposure for 30 min, and they were re-evaluated at a dilution of 1:100; none of them reached the continuation of the study. Beeb

- 28 040818 The drug library included a number of additional compounds with putative anticonvulsant properties (beclamid, aminohydroxybutyric acid, and tiletamine) that were also ineffective in the 96-well movement assay at 667 μM. Of the retested compounds, 14 either did not successfully inhibit convulsive behavior on a second set of scn1Lab mutants or only inhibited the behavior at higher drug concentrations. The following 4 (out of 18) compounds were selected that were effective in reducing seizure-induced swimming activity and mean speed at all three drug concentrations for further study: zoxazolamine, clemizol HCl, clogilin HCl, and tolperisone HCl (Fig. 6D). Each of these compounds was evaluated a third time in a movement assay at 100 μM and then monitored for forebrain electrographic activity. Clologin (a monoamine oxidase A inhibitor) and the muscle relaxants zoxazolamine (Hadra, R. & Millichap J. G., Quantitative assessment of motor function in cerebral palsy: evaluation of zoxazolamine (flexin), a new muscular relaxant agent. Neurology 6, 843-852 (1956) ) and tolperisone (Sakitama, K., The effects of centrally acting muscle relaxants on the intrathecal noradrenaline-induced facilitation of the flexor reflex mediated by group II afferent fibers in rats. Jpn. J. Pharmacol. 63, 369-736 (1993) ) were identified as false positives as they reduced swim activity at this concentration, however, when the same mutant was flooded with agar, electrographic seizure events were still observed (see FIG. 6E).

Only one compound, clemizole (an antihistamine and an inhibitor of NS4B RNA binding) (Finkelstein, M., Kromer C. M., Sweeney S. A., & Delahunt C. S., Some aspects of the pharmacology of clemizole hydrochloride. J. Am Einav S., Sobol H. D., Gehrig E., & Glenn J. S., Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis. Nat Biotechnol 26, 1019-1027 (2008)), was effective in suppressing spontaneous seizure activity in both assays (Fig. 6D-E). Clemizol had no significant effect on seizure behavior in the locomotion assay at concentrations between 6.25 and 50 µM (n=33). As an additional evaluation of the therapeutic potential of short-term treatment with clemizol, 100 μM clemizol was demonstrated to be effective in reducing seizure behavior in WT zebrafish exposed to 15 mM pentilepetetrazole (Fig. 6D; n=10) i.e. in a GABA receptor antagonist model of acute seizures. These results indicate that scn1Lab mutants can be used in high-throughput screening to identify lead compounds for Dravet syndrome.

The scn1Lab zebrafish mutant described herein is the first simple vertebrate model for sodium channel mutation that reproduces features of Dravet syndrome, a devastating form of drug-resistant epilepsy in children. These mutants exhibit hyperactivity, including seizure behavior, spontaneous electrographic seizures, shortened lifespan, and a pharmacological profile similar to that in humans. Additional molecular analysis of the scn1Lab mutants indicates no significant changes in global gene expression and no compensation, at the RNA level, by other subunits of the voltage-gated Na+ channel. A two-stage phenotype-based drug screening strategy to identify lead compounds with the potential to mitigate the epileptic phenotypes associated with the SCN1A mutation identified one FDA-approved drug (Clemisol).

Electroencephalographic (EEG) activity is usually normal in the first year of life in DS patients, and change to abnormal paroxysmal or multi-peak activity occurs between 1 and 9 years of age. This age-dependent pattern was mimicked in developing zebrafish larvae at ages when Scn1a expression was significant. Extracellular forebrain recordings from very young larvae (3 dpf) appeared mostly normal with occasional small pulses of multi-peak activity. Frequent short-term similar interictal activity with multi-peak pulsed discharges of large amplitude becomes more pronounced as the larvae mature. The architecture of these electrical phenomena resembled those previously described for wild-type larvae exposed to pentilepetetrazole (Baraban, S. C, Taylor, M. R., Castro, P. A., & Baier H., Pentylenetetrasol induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience 131, 759-768 (2005)), 4-aminopyridine (Baraban, S.C., et al., A large-scale mutagenesis screen to identify seizure-resistant zebrafish. Epilepsia 48, 1151-157 (2007)), linopyridine (Chege S. W., Hortopan G. A., Dinday M. T., & Baraban S. C., Expression and function of KCNQ channels in larval zebrafish. Dev. Neurobiol. 72, 186-198 (2012)) or hyperthermia (Hunt R. F., Hortopan G. A., Gillespie A., & Baraban S. C, A novel zebrafish model of hyperthermia-induced seizures reveals a role for TRPV4 channels and NMDA-type glutamate receptors Exp Neurol 237, 199-206 (2012)).

The form of electrographic convulsive activity corresponds to hyperactivity, convulsions of the whole body with associated activity of swimming at high speed and short-term loss of body position in mutants with free behavior. These types of spontaneous behaviors are never seen in wild-type larvae and again resemble behaviors seen only when exposed to

- 29 040818 convulsive drugs. This behavior is an indirect indication of seizure activity and can be used to rapidly evaluate in vivo drug treatment and lethality in a multiwell format using automated motion tracking software (Berghmans, S., Hunt, J., Roach, A., & Goldsmith Baxendale, S., et al., Identification of compounds with anti-convulsant properties Epilepsy Res. 75, 18-28 (2007) Winter, M. J., et al., Validation of a larval zebrafish locomotor assay for assessing the seizure liability of early-stage development drugs. J. Pharm. Tox. Methods 5, 176-187 (2008)). Seizures in scn1Lab zebrafish mutants responded to a ketogenic diet and four AEDs (eg, valproate, benzodiazepine, potassium bromide, and styripentol) administered in clinics to patients with DS.

Interestingly, the electrographic activity of seizures in scn1Lab mutants remained unchanged (or possibly worsened) in response to several commercially available AEDs. While it is possible that drug concentrations in excess of 1 mM may be required to eliminate electrical phenomena, they can be considered high and potentially non-selective concentrations. In drug trials using a model of acute PTZ-induced seizures in zebrafish larvae (Baraban, S.C., et al., A large-scale mutagenesis screen to identify seizure-resistant zebrafish. Epilepsia 48, 1151-157 (2007); Berghmans, S., Hunt, J., Roach, A., & Goldsmith, P., Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants Epilepsy Res. 75, 18-28 (2007); S., et al., Afrikanova, T., et al., Validation of the zebrafish pentylenetetrazol seizure model: locomotor versus electrographic responses to antiepileptic drugs PLoS One 8, e54166 (2013)), AED concentrations of 1 mM and below are often sufficient to assess antiepileptic activity. While failing to respond to seven different AEDs, this model fits the clinical definition of drug-resistant epilepsy (de Toffol, B., et al., ESPERA study: Applicability of the new ILAE criteria for antiepileptic drug resistance of focal epilepsies in current clinical practice Epilepsy Beh 25, 166-169 (2012)).

For nearly 40 years, the discovery and identification of new AEDs have been based almost entirely on preclinical animal models of acquired or acute seizures in rodents (Loscher, W. & Schmidt, D., Modern antiepileptic drug development has failed to deliver: Ways out of the current dilemma Epilepsia 52, 657-658 (2011)). This approach has successfully identified drugs that block generalized tonic-clonic seizures in humans (Bialer, M. & White H. S., Key factors in the discovery and development of new antiepileptic drugs. Nat. Rev. Drug Discov. 9, 10-19 ( 2012)), but remains time-consuming, resource-intensive, costly and labor-intensive. While testing against PTZ or other types of acquired seizures in zebrafish larvae may be more effective than similar tests in rodents (Berghmans, S., Hunt, J., Roach, A., & Goldsmith, P., Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants Epilepsy Res. 75, 18-28 (2007) Baxendale, S., et al., Identification of compounds with anticonvulsant properties in a zebrafish model of epileptic seizures Dis. Model Mech 5, 773-774 (2012) Afrikanova, T., et al., Validation of the zebrafish pentylenetetrazol seizure model: locomotor versus electrographic responses to antiepileptic drugs, PLoS One 8, e54166 (2013)), they must eventually identify the same classes of compounds.

In contrast, this disclosure describes an alternative screening strategy using a 96-well format for rapid automated behavioral monitoring followed by sensitive electrophysiological analysis of spontaneous electrographic seizure activity in mutant fish mimicking a known human genetic disorder. This in vivo strategy simultaneously monitors lethality and is not limited to SCN1A, but can be used for any epileptic disorder.

Indeed, this phenotype-based approach may form the basis for a genetically informed or personalized approach for drug development. While genetically engineered mice have been developed that mimic known SCN1A mutations and exhibit epilepsy, breeding can be difficult, background strains can modify seizure phenotypes, and AEDs are rarely investigated in these animals. For example, in Scn1aRX/+ mutant mice, only stiripentol and clobazam were evaluated for effects on hyperthermia-induced seizure thresholds (Cao D., et al., Efficacy of stiripentol in hyperthermia-induced seizures in a mouse model of Dravet syndrome. Epilepsia 53, 1140 -1145 (2012)). Treatment of mutant mice with Scn1a+/clonezepam, an allosteric GABA-A receptor modulator, partially restored autism-like behavior, but it was not evaluated as antiepileptic (de Toffol, B., et al., ESPERA study: Applicability of the new ILAE criteria for antiepileptic drug resistance of focal epilepsies in current clinical practice Epilepsy Beh 25, 166-169 (2012)).

When rodent models of drug-resistant epilepsy have been described, such as a subgroup of wild-type rats selected from kindling or post-epileptic status models

- 30 040818 (Han S., et al., Autistic-like behavior in Scnla+/- mice and rescue by enhanced GABA-mediated neurotransmission. Nature 489, 385-390 (2012)), they remain very poorly characterized and are not suitable for initial high-throughput drug screening steps. In contrast, using the scn1Lab zebrafish mutant with over 75% sequence identity to the human sodium channel mutation, large-scale profiling with over 44,000 honds was performed, developmental progression of scn1Lab channel expression and epileptic phenotypes were demonstrated, and effects were analyzed. available antiepileptic therapies and a library of 320 compounds was screened against spontaneous unprovoked seizures. Although this first screening to test the principle was done with only one fish per well, 6-12 fish per trial, and one trial per week, the ease with which zebrafish can be scaled up (especially under commercial conditions) to study hundreds thousand larvae per week, makes this an attractive system for the first stage of a rapid large scale in vivo drug development program. Simultaneously, in vivo toxicity assessment, one of the biggest sources of failure in moving leading compounds from laboratory to clinic, is a key advantage of this approach over available screening strategies based on organotypic hippocampal culture or in silica screening strategies.

Although any animal model drug development data should be handled with caution, clemizol, a compound with H1 antagonist and NS4B RNA inhibitor properties, is an FDA-approved drug with a safe toxicological profile found in this screening and provides an impressive starting point for further research. For example, although it has recently become clear that antihistamines inhibit induced seizures in neonatal rats (Yamada, K., Takizawa, F., Tamura, T., & Kanda T., The effect of antihistamines on seizures induced by increasing-current electroshocks: ketotifen , but not olopatadine, promotes the seizures in infant rats Biol Pharm Bull 35, 693-697 (2012)), without linking to any particular theory, this is probably not the mechanism of action in this case. The inventors demonstrated that four other H1 antihistamines (pimethixene maleate, chlorpyramine HCl, mebhydroline naphthalenesulfonate and iproheptin) did not suppress seizure behavior in scn1Lab mutants. Moreover, data indicate the potential of H1 antihistamines to adversely modify seizures in children (Miyata, I., Saegusa, H., & Sakurai, M., Seizure-modifying potential of histamine H1 antagonists: a clinical observation. Pediatr. Int. 53 , 706-708 (2011)), indicating that more detailed analysis is required to identify the mechanism of action. Given that clemizol was also effective in the zebrafish version of the metrazol test, it may be worthwhile to conduct an additional preclinical trial in the NIH-sponsored anticonvulsant drug development program at the University of Utah. Most importantly, these studies indicate that in vivo drug screening and experimental analysis of scn1Lab zebrafish mutants may prove extremely valuable in understanding (and treating) Dravet syndrome.

Animals.

scn1Lab (didys552) zebrafish embryos were a courtesy gift from Herwig Baier. HuC:GFP adult zebrafish were a kind gift from Stephen Ekker. Zebrafish have been bred and maintained in accordance with the guidelines of the University of California, San Francisco Committee on the Use and Care of Animals. Zebrafish larvae were maintained in embryonic medium consisting of 0.03% Instant Ocean (Aquarium Systems, Inc., Mentor, OH, USA) in deionized water containing 0.002% methylene blue as a fungicide. Broods of zebrafish larvae were bred from scn1Lab heterozygous animals that were backcrossed to wild-type TL or HuC:GFP zebrafish for at least 7 generations. Homozygous mutants (sorted on the basis of pigmentation) and age-matched sibling larvae were used. Although the exact genetic defect responsible for skin pigmentation problems is unknown, interestingly, activation of the gene encoding the melanocortin 5a receptor by 1.5-fold was noted in microarray data.

Seizure monitoring.

Movement tracking and electrophysiology techniques have been described (Baraban S.C., et al., A large-scale mutagenesis screen to identify seizure-resistant zebrafish. Epilepsia 48, 1151-157 (2007); Baraban, S. C, Taylor M. R., Castro P. A., & Baier H., Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression Neuroscience 131, 759-768 (2005)). In preliminary experiments, HuC:GFP daniorerio was used in electrophysiology experiments to assess the position of the recording electrodes. Movement plots were generated for one fish per hole at 10 min recording intervals using the DanioVision system controlled by EthoVision XT software (Noldus Information Technology; Leesburg, VA). Seizures were scored as described (Baraban S. C, Taylor M. R., Castro P. A., & Baier H., Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience 131, 759-768 (2005)). Movement graphs were analyzed in terms of distance traveled (in mm) and average speed (in mm/s). Epileptiform events were analyzed in pClamp (Molecular Devices; Sunnyvale, CA) and were defined as

- 31 040818 membrane inclination up or down more than 2 times the initial background level, and they were classified as either similar to interictal (duration from 100 to 300 ms) or like ictal (duration from 1000 to 5000 ms). The frequency of impulses was determined by counting the number of epileptiform events per minute during the 10-min recording interval. The duration of the pulses was determined by measuring the interval from start to end for all events during the same interval.

Drugs were obtained from Sigma-Aldrich and dissolved in embryonic medium. Stock solutions were prepared in embryonic medium at a concentration of 1 mM and the pH was adjusted to ~7.5. Ganaxolone was a kind gift from BioCrea GmbH (Radebeul, Germany). Drug screening compounds were purchased from MicroSource Discovery Systems, Inc. (International Drug Collection; Gaylordsville, CT) and were provided as 10 mM solutions in DMSO. Test compounds were dissolved in embryonic medium and tested at concentrations ranging from 6.7 to 667 μM; final concentration of DMSO ~7%. The initial screening concentration of 667 µM was chosen to study behavior in free-swimming fish because it falls within the lower range of AED concentrations previously reported to be effective against PTZ-induced (10-20 mM) seizures in zebrafish larvae ( 0.1 to 25 mM) (Baraban S. C, Taylor M. R., Castro P. A., & Baier H., Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience 131, 759-768 (2005); Berghmans S., Hunt J., Roach A., & Goldsmith P., Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants Epilepsy Res. 75, 18-28 (2007); Afrikanova T., et al., Validation of the zebrafish pentylenetetrazol seizure model: locomotor versus electrographic responses to antiepileptic drugs PLoS One 8, e54166 (2013)), and was the most effective use of the small volume stock solution (250 µl) provided by MicroSource Discovery Syste ms, inc. A slightly higher concentration (1 mM) was chosen for the initial AED validation assays in FIG. 5 and 6 to account for any potential difficulties associated with diffusion through the agar. DMSO was evaluated for toxicity at dilutions from 0.01 to 100% using wild-type larvae (n=12 fish per concentration); DMSO at concentrations >25% was lethal.

In all drug screening studies, compounds were coded and experiments were blinded to the nature of the compound by investigators. The initial registration of seizure activity was performed for mutants in the medium for embryos; then a second graph was plotted after replacing the solution with the test compound. Each test compound classified as positive in the movement assay was visually confirmed alive based on movement in response to touch and a noticeable heartbeat. WT fish show little to no spontaneous swimming activity during these 10 min recording intervals (see FIG. 3B) and were not used in the drug development analysis.

Techniques for microarray analysis, quantitative PCR, and total preparation in situ hybridization have been described (Hortopan G. A., et al., Spontaneous seizures and altered gene expression in GABA signaling pathways in a mind bomb mutant zebrafish. J. Neurosci. 30, 13718-13728 (2010)).

Data are presented as means and SEM unless otherwise indicated. Pairwise statistical significance was determined using two-tailed unpaired Student's t-test, ANOVA, or Mann-Whitney rank sum test, as appropriate, unless otherwise indicated. Results were considered significant at P<0.05 unless otherwise indicated.

Example 2

Based on the previous discussion and some activity observed at 100 and 300 mg/kg doses in qualitative MES screening in mice, the inventors continued to quantify in MES/scMET/Tox mouse models to determine ED50/TD50. During the determination of TPE in the MES model, no activity was observed at an initial dose of 300 mg/kg. However, activity was observed at a dose of 500 mg/kg with 2/4 of the animals being protected after 0.25 min and 4/4 of the animals being protected after 30 min. No activity or toxicity (ability to grasp a rotating rod) was observed at any other dose or time point studied. No activity was observed in the scMET model. The data in the MES model show that there is significant activity/protection by ASP469016 in this model with an ED50<400 mg/kg.

Screening results for anticonvulsants IP quantification of mice

ASP ID: 469016 Screening ID: 1 Sponsor ID: 642 Sponsor Class:

Sponsor ASP/CM

Solvent code: MS Solvent preparation: M&P, TW

Animal weight: - g

Start Date: 06 May 2014 End Date: 09 May 2014

Link: 503:294,297.509:3,4

- 32 040818

ED50 value

Test Time (h) ED50 95% confidential interval Incline standard error p value MES 0.5 <400.0 - SCMET 0.5 >250.0 - TOX 0.5 >500.0 -

ED50, biological response

Test Time (h) Dose (mg/kg) Of death N/F C MES 0.50 350 0/8 MES 0.50 400 7/8 MES 0.50 500 4/4 SCMET 0.50 200 0/8 SCMET 0.50 250 0/4 TOX 0.50 500 0/4

Note: The presence of an asterisk (*) indicates that there are multiple comment codes.

Time to maximum effect

Time (watch) 0.25 0.5 10 2.0 4.0 6.0 8.0 24 h, 0 Test Death Dose N/F WITH N/F WITH N/F WITH N/F WITH N/F WITH N/F WITH N/F WITH N/F WITH N/ F WITH MES 300 0/4 0/4 0/4 0/4 0/4 / / / / MES 500 2/4 4/4 0/4 0/4 0/4 / / / / TOX 400 0/8 0/8 / / / / / / / TOX 500 0/4 0/4 0/4 / / / / / /

Note: N/F=number of animals active or toxic relative to number tested

C = comment code. The presence of an asterisk (*) indicates that there are multiple comment codes _____________________________________________

Comments for NIH Supplier comments The MES doses of 350 mg/kg and 400 mg/kg and the Scmet dose of 200 mg/kg were used for Lot Ori and all other doses were used for Lot A. Insufficient material to continue study

Example 3

ASP469016 was investigated in our original T31 screen (MES/scMET/Tox) at 30, 100 and 300 mg/kg. Data for each condition are presented as N/F, where N equals the number of animals protected and F equals the number of animals tested. For toxicity studies (TOX), N is equal to the number of animals showing toxicity effects and F is equal to the number of animals tested. The codes in column C refer to the comments of the technicians who performed the experiment, and they are defined in the comments section if necessary. Deaths have been noted. As shown in the 6 Hz (32 mA) model, only 1/4 of the animals were protected at 100 mg/kg for 30 min. In the MES-induced seizure model, only 1/4 of the animals were protected at doses of 100 and 300 mg/kg for 30 minutes. There was no toxicity (failure to grasp the rotating rod) or activity at any other dose or time point studied.

Anticonvulsant Screening Results - MES Mice and Identification at 6 Hz

ASP ID: 469016 Screening ID: 1 Sponsor ID: 642 Sponsor Class:

Sponsor ASP/CM

Diluent code: MS Preparation Pathway code: i.p. diluent: M&P, TW Animal weight: - g

Start date: February 11 End date: 11 Current (mA)

2014 February 2014

Link: 503:153

- 33 040818

Answer

Time watch) 0.5 2.0 0.25 1, oh 4.0 6.0 thirty 28.0 24 Test Dose Form Of death N/F with N/F WITH N/F WITH N/F WITH N/F with N/F WITH N/F with N/F WITH N/F WITH 6 Hz thirty 0/4 0/4 / / / / / / / 6 Hz 100 1/4 0/4 / / / / / / / 6 Hz 300 0/4 0/4 / / / / / / / MES thirty 0/4 0/4 / / / / / / / MES 100 1/4 0/4 / / / / / / / MES 300 1/4 0/4 / / / / / / / TOX thirty SUS 0/8 0/8 / / / / / / / TOX 100 SUS 0/8 0/8 / / / / / / / TOX 300 SUS 0/8 0/8 / / / / / / /

Note: N/F=number of animals active or toxic relative to number tested C=comment code.

The presence of an asterisk (*) indicates that there are multiple comment codes.

Example 4

Klemizol (149934-L6) was examined in the CEREP BioPrint Profile, which is a panel of 139 different in vitro receptor binding and enzyme assays. For the initial screening of BioPrint, a free compound concentration of 10 μM (1.0E-5 M) was used. Compound binding was calculated as % inhibition of binding of the radiolabeled ligand specific to each target. The enzyme inhibition effect of the compound was calculated as % inhibition of control enzyme activity. In each experiment, the respective reference compound was tested simultaneously with Clemisol (149934-L6) and the data compared to previously recorded values determined by CEREP. The experiment was accepted according to the CEREP validating standard procedure. Results showing inhibition (or stimulation for baseline assays) greater than 50% are considered to show significant effects of the test compounds. A summary of these results is given below:

Analysis 1,OE

5-ΗΤ2Α/Λ,) (agonist radioligand) 86%

5-HT2bN,) (agonist radioligand)82.5

5-HTi _A /hl (radioligand agonist)19.5

5-HTiB (h) (radioligand agonist) 4.4%

5-HTiB (h) (radioligand agonist)22.7

5-HT ₃ (h) (radioligand agonist) 9.8%

5-HT _4e (h) (radioligand agonist)GABA _A1 (h) (radioligand agonist)GABA _B( ib) (h) (radioligand agonist)BZD (central) (radioligand CBi (h) (radioligand agonist )17.9

CB ₂ (h) (agonist radioligand) 28.4

GABA-dependent C1 channel24.9

SK-Ca-channel GABA transporter Example 5.

Klemizol does not demonstrate antiepileptic activity through an antihistaminergic mechanism of action. 32 different antihistamine compounds, FIG. 10 were screened in the scn1Lab zebrafish assay and none of these compounds mimicked the antiepileptic effects of clemizol. Three of the compounds were toxic, consistent with clinical reports that antihistamines may exacerbate seizures in pediatric patients with epilepsy, five compounds exacerbated convulsive hanging.

Example 6

Applicants screened a Selleck Customized Library containing 62 drugs that act on the serotonin signaling pathways. These compounds were initially screened in the zebrafish locomotion assay and 15 compounds were identified as positive in the first pass locomotion assay (details of the assay can be found in Baraban et al. Nat. Comm. 2013 and Dinday and Baraban, eNeuro 2015) and can be seen in Fig. 11. These studies indicate that modulation of 5HT signaling, especially activation of postsynaptic 5HT receptors, has potential antiepileptic activity.

Conducted studies to re-examine the concentration-response of all compounds 5HT identified in the analysis of movement of the first pass. The results of trazodone (Desryl, Oleptro) are presented as a representative example. In addition to demonstrating robust inhibition of spontaneous convulsive behavior in the locomotion assay at concentrations between 100 and 750 μM (Fig. 12A), trazodone also effectively suppressed EEG activity in scn1Lab mutants (n=15) at concentrations between 250 and 500 μM (Fig. 12B). ) and in separate drug washout trials (n=12). Compounds with positive results include sumatriptan, naratriptan, rizatriptan, zolmitriptan, urapidil, BRL-54443 (3-(1-methylpiperidin-4-yl)-1H-indol-5-ol), lorcaserin, buspirone, ziprasidone, TCB-2 ( (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide), BRL-15572 (3-(4-(4-chlorophenyl)piperazin-1-yl)-1,1-diphenyl-2-propanol) , trazodone, BMY 7378 (8(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]dican-7,9-dione), atomoxetine and venlafaxine.

Additional Embodiments

Embodiment 1. A method of treating an epileptic disorder, the method comprising administering to an individual in need thereof a therapeutically effective amount of a 5HT receptor agonist or a pharmaceutically acceptable salt thereof.

Embodiment 2. The method of Embodiment 1 wherein the 5HT receptor agonist is a 5HT _2A receptor agonist or a 5HT _2B receptor agonist.

Embodiment 3. The method of Embodiment 2 wherein the 5HT receptor agonist is a dual 5HT _2A receptor and 5HT _2B receptor agonist.

Embodiment 4 The method of Embodiment 1 wherein the 5HT receptor agonist is not clemizole or fenfluramine.

Embodiment 5. The method of Embodiment 1 wherein the 5HT receptor agonist binds directly to the 5HT receptor.

Embodiment 6 The method of Embodiment 1 wherein the 5HT receptor agonist specifically activates the 5HT receptor.

Embodiment 7. The method of Embodiment 1 wherein the 5HT receptor agonist provides increased activity mediated by the 5HT _2A receptor or 5HT _2B receptor with equivalent or reduced activity mediated by the 5HT2C receptor.

Embodiment 8 The method of Embodiment 1 wherein the 5HT receptor agonist is not a serotonin reuptake inhibitor.

Embodiment 9. The method of Embodiment 1 wherein the 5HT receptor agonist does not significantly bind or modulate the activity of at least one of the 5HT1A, 5HT _1B , 5HT1D, 5HT2C, 5HT3, 5HT4, 5HT6, 5HT ₇ , NPY Y1, L-type Ca-channel, N-type Ca-channel, SK-Ca-channel, GABA-dependent Cl-channel, GABA transporter, GABA-A1 receptor, GABA-B1b receptor, Na-channel, 5HT transporter, CB1 receptor, CB2 receptor, BZD or ER estrogen alpha.

Embodiment 10. The method of Embodiment 1 wherein the 5HT receptor agonist is flibanserin, DOI HCl, norfenfluramine, or BW 723C86.

Embodiment 11. The method of Embodiment 1 wherein the 5HT receptor agonist is sumatriptan, naratriptap, rizatriptan, zolmitriptan, urapidil, BRL-54443 (3-(1methylpiperidin-4-yl)-1H-indol-5-ol), lorcaserin , Buspirone, Ziprasidone, TCB-2 ((4-bromo-3,6dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide), BRL-15572 (3-(4-(4-chlorophenyl)piperazin-1yl)-1,1-diphenyl -2-propanol), trazodone, BMY 7378 (8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8azaspiro[4.5]decan-7,9-dione), atomoxetine and venlafaxine.

Embodiment 12. The method of Embodiment 1 wherein the 5HT receptor agonist is trazodone or a pharmaceutically acceptable salt thereof. In embodiments, the pharmaceutically acceptable salt of trazadone is hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, propionate, tartrate, succinate, benzoate, glutamate, or a quaternary ammonium salt.

Embodiment 13. The method of Embodiment 1 wherein the epileptic disorder is Dravet, Lennox-Gastaut syndrome, infantile convulsions, or Otahara syndrome.

Embodiment 14. The method of Embodiment 13 wherein the epileptic disorder is Dravet's syndrome.

Embodiment 15. The method of Embodiment 1 wherein the epileptic disorder is pediatric epileptic disorder.

Embodiment 16. The method of Embodiment 1 wherein the subject has a cardiovascular disease.

Embodiment 17. The method of Embodiment 1 wherein the subject is treatment-resistant with a serotonin reuptake inhibitor.

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Embodiment 18. The method of Embodiment 1 wherein the subject is subject to side effects when the serotonin reuptake inhibitor is administered.

Embodiment 19. The method of Embodiment 17 or 18 wherein the serotonin reuptake inhibitor is fenfluramine.

Embodiment 20. The method of Embodiment 1 wherein the subject is on a ketogenic diet.

Embodiment 21. The method of Embodiment 1 wherein the 5HT receptor agonist inhibits seizure behavior or electrographic seizures in an individual with epilepsy, an individual with Alzheimer's disease, an individual with autism, or an individual with Parkinson's disease.

Embodiment 22. The method of Embodiment 1 wherein the 5HT receptor agonist reduces the occurrence of unprovoked seizures in the individual compared to no 5HT receptor agonist.

Embodiment 23. The method of Embodiment 1, wherein administration of the 5HT receptor agonist reduces or prevents myoclonic seizures or status epilepticus in the subject compared to no 5HT receptor agonist.

Embodiment 24. The method of Embodiment 1 wherein the 5HT receptor agonist is administered to the individual in an amount of from about 0.1 to about 1000 mg per kg of body weight.

Embodiment 25. The method of Embodiment 22, wherein the 5HT receptor agonist is administered to the individual at a daily dose of about 0.1 to about 1000 mg per kg of the individual's body weight.

Embodiment 26. The method of Embodiment 1 wherein the 5HT receptor agonist is co-administered with an antiepileptic drug (AED).

Embodiment 27. The method of Embodiment 1 wherein the 5HT receptor agonist is an adjuvant therapy for an antiepileptic drug (AED).

Embodiment 28. The method according to Embodiment 26 or 27 wherein the AED is acetazolamide, benzodiazepine, cannabadiols, carbamazepine, clobazam, clonezepam, eslicarbazepine acetate, ethosuximide, ettoin, felbamate, fenfluramine, fosphenytoin, gabapentin, ganaxolone, huperzine A, lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine, perampanel, piracetam, phenobarbital, phenytoin, potassium bromide, pregabalin, primidone, retigabine, rufinamide, valproic acid, sodium valproate, styripentol, tiagabine, topiramate, vigabatrin, or zonisamide.

Embodiment 29. The method of Embodiment 28 wherein the AED is valproic acid, sodium valproate, clonezepam, ethosuximide, felbamate, gabapentin, carbamazepine, oxcarbazepine, lamotrigine, levetiracetam, benzodiazepine, phenobarbital, pregabalin, primidone, tiagabine, topiramate, potassium bromide , phenytoin, styripentol, vigabatrin, or zonisamide.

Embodiment 30. The method of Embodiment 29 wherein the AED is valproic acid, sodium valproate, gabapentin, topiramate, carbamazepine, oxcarbazepine, or vigabatrin.

Embodiment 31. The method of Embodiment 26 wherein the AED is not fenfluramine or topiramate.

Embodiment 32. The method of Embodiment 26 wherein the AED is administered simultaneously or sequentially with the 5HT receptor agonist.

Embodiment 33. A method of treating an epileptic disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of a 5HT receptor agonist or a pharmaceutically acceptable salt thereof, wherein the subject has a cardiovascular disease, is refractory to treatment with a serotonin reuptake inhibitor, or is susceptible to side effects with the introduction of a serotonin reuptake inhibitor.

Embodiment 34. The method of Embodiment 33 wherein the 5HT receptor agonist is a 5HT2A receptor agonist or a 5HT _2B receptor agonist.

Embodiment 35. The method of Embodiment 32 wherein the 5HT receptor agonist is a dual 5HT2A receptor and 5HT _2B receptor agonist.

Embodiment 36. The method of Embodiment 33 wherein the 5HT receptor agonist is clemizol, a clemizol analog, or a pharmaceutically acceptable salt thereof.

Embodiment 37. The method of Embodiment 36 wherein the pharmaceutically acceptable salt is Clemizol HCl.

Embodiment 38. The method of Embodiment 36, wherein clemizol, a clemizol analog, or a pharmaceutically acceptable salt thereof forms part of a pharmaceutical composition.

Embodiment 39. The method of Embodiment 38, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

Embodiment 40. The method of Embodiment 38, wherein the pharmaceutical composition comprises a therapeutically effective amount of Clemizol, a Clemizol analog, or a pharmaceutically acceptable salt thereof.

Embodiment 41. The method of Embodiment 40 wherein the pharmaceutical

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Claims (4)

the composition is co-administered with an antiepileptic drug (AED). Embodiment 42. The method of Embodiment 41 wherein the pharmaceutical composition comprises clemizol, a clemizol analog, or a pharmaceutically acceptable salt thereof, and an AED. Embodiment 43. The method of Embodiment 33 wherein the receptor agonist 5HT is not a fenfluramine. Embodiment 44. The method of Embodiment 33 wherein the 5HT receptor agonist binds directly to the 5HT receptor. Embodiment 45. The method of Embodiment 33 wherein the 5HT receptor agonist specifically activates the 5HT receptor. Embodiment 46. The method of Embodiment 33 wherein the 5HT receptor agonist provides increased activity mediated by the 5HT _2A receptor or 5HT _2B receptor with equivalent or reduced activity mediated by the 5HT2C receptor. Embodiment 47. The method of Embodiment 33 wherein the 5HT receptor agonist is not a serotonin reuptake inhibitor. Embodiment 48. The method according to Embodiment 33, wherein the 5HT receptor agonist does not significantly bind or modulate the activity of at least one of the receptor 5HT1A, 5HT _1B , 5HT1D, 5HT _2C , 5HT ₃ , 5HT ₄ , 5HT6, 5HT ₇ . NPY Y1, L-type Ca-channel, N-type Ca-channel, SK-Ca-channel, GABA-dependent Cl-channel, GABA transporter, GABA-A1 receptor, GABA-B1b receptor, Na-channel, 5HT transporter, CB1 receptor , CB2 receptor, BZD, or ER estrogen alpha. Embodiment 49. The method of Embodiment 33 wherein the 5HT receptor agonist is sumatriptan, naratriptap, rizatriptan, zolmitriptan, urapidil, BRL-54443 (3-(1-methylpiperidin-4-yl)-1H-indol-5-ol) , lorcaserin, buspirone, ziprasidone, TSV-2 ((4-bromo-3,6dimethoxybenzocyclobuten-1 -yl)methylamine hydrobromide), BRL-15572 (3-(4-(4-chlorophenyl)piperazin-1 yl)-1, 1-diphenyl-2-propanol), trazodone, BMY 7378 (8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8azaspiro[4.5]decan-7,9-dione), atomoxetine and venlafaxine. Embodiment 50. The method of Embodiment 33 wherein the 5HT receptor agonist is trazodone or a pharmaceutically acceptable salt thereof. Embodiment 51. A method for modulating 5HT receptor activity comprising contacting the 5HT receptor with clemizol, a clemizol analog, or a pharmaceutically acceptable salt thereof. Embodiment 52. The method of Embodiment 51, wherein the modulation is an activation. Embodiment 53. The method of Embodiment 51 wherein the 5HT receptor is a 5HT2A receptor or a 5HT _2B receptor. Embodiment 54. A method of treating a disease or disorder caused by a deficiency of brain serotonin or the activity of one or more 5HT receptors, comprising administering to an individual in need thereof a therapeutically effective amount of clemizol, a clemizol analog, or a pharmaceutically acceptable salt thereof. Embodiment 55. The method of Embodiment 54 wherein the disease or disorder is not epilepsy. Embodiment 56. The method of Embodiment 54 wherein the disease or disorder is not Dravet's syndrome. Embodiment 57. The method of Embodiment 51 wherein the disease or disorder is selected from the group consisting of migraine, fragile X syndrome, Prader-Willi syndrome, schizophrenia, depression, Alzheimer's disease, autism, neuropathic pain, Parkinson's disease, irritable bowel syndrome, and dementia. Embodiment 58. The method of Embodiment 50 wherein the pharmaceutically acceptable is Clemizol HCl. CLAIM 1. A method of treating an epileptic disorder, said method comprising administering to an individual in need thereof a therapeutically effective amount of a 5HT receptor agonist or a pharmaceutically acceptable salt thereof, wherein said 5HT receptor agonist is trazodone or lorcaserin, wherein said epileptic disorder is Dravet's syndrome. 2. The method of claim 1 wherein said 5HT receptor agonist is a 5HT _2A receptor agonist or a 5HT _2B receptor agonist. 3. The method of claim 1 wherein said 5HT receptor agonist is trazodone or a pharmaceutically acceptable salt thereof. 4. The method of claim 1 wherein said 5HT receptor agonist is lorcaserin or a pharmaceutically acceptable salt thereof. -