CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

62/007,730, filed on June 4, 2014, the disclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The invention is directed to compositions of compounds and methods of using the compounds to treat, delay development of, palliate, and/or prevent epilepsy and/or epilepsy- related disorders. The compounds can modulate various neurological molecules,

neurological pathway and/or receptors, such as SCNl A, SCN8A, SCN9A, GABA receptors, and/or serotonin receptors.

BACKGROUND OF THE INVENTION

[0003] Epilepsy is a neurological condition which affects the nervous system. Epilepsy is also known as a seizure disorder. It is usually diagnosed after a person has had at least two seizures that were not caused by some known medical condition. About 65 million people in the world have epilepsy with about 2 million people in the US with epilepsy. The average incidence of epilepsy each year in the U. S is estimated at 150,000 or 48 for every 100,000 people (Epilepsy Foundation).

[0004] Seizures experienced as a result of Dravet Syndrome are intractable

(Panayiotopoulos, CP., "Dravet Syndrome" In: H. Cole & N. Lemmens, eds. A Clinical Guide to Epileptic Syndromes and their Treatment. S.l. Springer, pp. 283-287 (2010)). The best maintenance protocol for Dravet Syndrome remains under debate (Ceulemans, B. et al., "Successful use of fenfluramine as an add-on treatment for Dravet Syndrome," Epilepsia, pp. 1-9 (2012)). Other treatments that can be used includes valproate, benzodiazepines, melatonin, phenobarbital, ethosuximide, and bromides, all of which are temporarily beneficial (Ceulemans, et al., 2012) (Panayiotopoulos, 2010). Topiramate, stiripentol, zonisamide, and levetiracetam have also proven to be of some benefit (Ceulemans, et al., 2012 and Panayiotopoulos, 2010). Ketogenic diet has also been described as a way of addressing epilepsy (Panayiotopoulos, 2010). Other ways of addressing epilepsy included using combination antiepileptic drug (AED) maintenance therapy, avoidance of seizure aggravating drugs such as carbamazepine, lamotrigine, and phenytoin (Ceulemans, et al, 2012), and prevention of seizures by preventing hyperthermia and early treatment of infectious diseases (Ceulemans, et al, 2012 and Panayiotopoulos, 2010). Other treatments that have been published to help the management of Dravet Syndrome include verapamil, deep brain stimulation and vagus nerve stimulation (Ceulemans, et al., 2012).

[0005] However, all of these treatment approaches have their drawbacks in terms of efficacy, especially with respect to certain types of epilepsy, such as Dravet Syndrome.

Accordingly, new compositions and methods of treating and alleviating symptoms of epilepsy and other epilepsy-related conditions are needed.

[0006] All publications, references, patents and/or patent applications reference herein are hereby incorporation by reference in their entirety for all purposes.

SUMMARY OF THE INVENTION

[0007] The invention provides, inter alia, compositions and methods for treating, delaying development of, and/or preventing epilepsy and/or epilepsy-related disorders and palliating symptoms associated with epilepsy and/or epilepsy-related disorders. Accordingly, in one aspect, the invention provides for compositions comprising mifepristone for use in treating an individual having or suspected of having epilepsy and/or epilepsy-related disorders. In some embodiments, the composition comprises mifepristone in a therapeutically effective amount. In another aspect, the invention provides for compositions comprising bicalutamide for use in treating an individual having or suspected of having epilepsy and/or epilepsy-related disorders. In some embodiments, the composition comprises bicalutamide in a

therapeutically effective amount. In another aspect, the invention provides for compositions comprising progesterone for use in treating an individual having or suspected of having epilepsy and/or epilepsy-related disorders. In some embodiments, the composition comprises progesterone in a therapeutically effective amount. In another aspect, the invention provides for compositions comprising raloxifene for use in treating an individual having or suspected of having epilepsy and/or epilepsy-related disorders. In some embodiments, the composition is comprises raloxifene in a therapeutically effective amount. In any of the embodiments above and disclosed herein, the individual is a human. In any of the embodiments above and disclosed herein, the epilepsy and/or epilepsy-related disorders is selected from the group consisting of Dravet Syndrome, Lennox-Gastaut, generalized epilepsy with febrile seizure (GEFS+), severe myoclonic epilepsy of infancy (SMEI), epileptic encephalopathy, intractable epilepsy, absence epilepsy, temporal lobe epilepsy, frontal lobe epilepsy, occipital lobe epilepsy, parietal lobe epilepsy, partial seizures and general seizures. In some embodiments, the partial seizures are selected from the group consisting of simple partial seizures, complex partial seizures, aura, and secondarily generalized seizures. In some embodiments, the general seizures are selected from the group consisting of absence seizures (Petit mal), tonic seizures, clonic seizures, myoclonic seizures, atonic seizures, and tonic-clonic seizures (Grand mal).

[0008] In other aspects, the invention provides for methods of treating an individual with epilepsy and/or epilepsy-related disorders comprising administering to the individual a therapeutically effective amount of a composition comprising mifepristone. In other aspects, the invention provides for methods of treating an individual with epilepsy and/or epilepsy- related disorders comprising administering to the individual a therapeutically effective amount of a composition comprising bicalutamide. In other aspects, the invention provides for methods of treating an individual with epilepsy and/or epilepsy-related disorders comprising administering to the individual a therapeutically effective amount of a composition comprising progesterone. In other aspects, the invention provides for methods of treating an individual with epilepsy and/or epilepsy-related disorders comprising administering to the individual a therapeutically effective amount of a composition comprising raloxifene. In any of the embodiments above and disclosed herein, the epilepsy and/or epilepsy-related disorders is selected from the group consisting of Dravet Syndrome, Lennox-Gastaut, generalized epilepsy with febrile seizure (GEFS+), severe myoclonic epilepsy of infancy (SMEI), epileptic encephalopathy, intractable epilepsy, absence epilepsy, temporal lobe epilepsy, frontal lobe epilepsy, occipital lobe epilepsy, parietal lobe epilepsy, partial seizures and general seizures. In some embodiments, the partial seizures are selected from the group consisting of simple partial seizures, complex partial seizures, aura, and secondarily generalized seizures. In some embodiments, the general seizures are selected from the group consisting of absence seizures (Petit mal), tonic seizures, clonic seizures, myoclonic seizures, atonic seizures, and tonic-clonic seizures (Grand mal).

[0009] In other aspects, the invention provides for methods for modulating one or more neurological channel and/or receptor(s) selected from the group consisting of SCNl A, SCN8A, SCN9A, GABA receptors and serotonin receptors in an individual in need thereof, said method comprising administering an effective amount of mifepristone. In other aspects, the invention provides for methods for modulating one or more neurological channel and/or receptor(s) selected from the group consisting of SCNl A, SCN8A, SCN9A, GABA receptors and serotonin receptors in an individual in need thereof, said method comprising administering an effective amount of bicalutamide. In other aspects, the invention provides for methods for modulating one or more neurological channel and/or receptor(s) selected from the group consisting of SCNl A, SCN8A, SCN9A, GABA receptors and serotonin receptors in an individual in need thereof, said method comprising administering an effective amount of progesterone. In other aspects, the invention provides for methods for modulating one or more neurological channel and/or receptor(s) selected from the group consisting of SCNl A, SCN8A, SCN9A, GABA receptors and serotonin receptors in an individual in need thereof, said method comprising administering an effective amount of raloxifene. In any of the embodiments above and disclosed herein, the epilepsy and/or epilepsy-related disorders is selected from the group consisting of Dravet Syndrome, Lennox-Gastaut, generalized epilepsy with febrile seizure (GEFS+), severe myoclonic epilepsy of infancy (SMEI), epileptic encephalopathy, intractable epilepsy, absence epilepsy, temporal lobe epilepsy, frontal lobe epilepsy, occipital lobe epilepsy, parietal lobe epilepsy, partial seizures and general seizures. In some embodiments, the partial seizures are selected from the group consisting of simple partial seizures, complex partial seizures, aura, and secondarily generalized seizures. In some embodiments, the general seizures are selected from the group consisting of absence seizures (Petit mal), tonic seizures, clonic seizures, myoclonic seizures, atonic seizures, and tonic-clonic seizures (Grand mal).

[0010] In any of the embodiments above and disclosed herein, the neurological channel and/or receptor(s) is SCNl A. In any of the embodiments above and disclosed herein, the neurological channel and/or receptor(s) is SCN8A. In any of the embodiments above and disclosed herein, the neurological channel and/or receptor(s) is SCN9A. In any of the embodiments above and disclosed herein, the neurological channel and/or receptor(s) is a GABA receptor. In any of the embodiments above and disclosed herein, the neurological channel and/or receptor(s) is a serotonin receptor.

[0011] In other aspects, the invention provides for methods of treating a disease, disorder, symptom, or condition associated with one or more neurological channel and/or receptor(s) selected from the group consisting of SCNl A, SCN8A, SCN9A, GABA receptors and serotonin receptors in an individual, said method comprising administering to the individual an effective amount of mifepristone. In other aspects, the invention provides for methods of treating a disease, disorder, symptom, or condition associated with one or more neurological receptor(s) selected from the group consisting of SCNl A, SCN8A, SCN9A, GABA receptors and serotonin receptors in an individual, said method comprising administering to the individual an effective amount of bicalutamide. In other aspects, the invention provides for methods of treating a disease, disorder, symptom, or condition associated with one or more neurological receptor(s) selected from the group consisting of SCN1A, SCN8A, SCN9A, GABA receptors and serotonin receptors in an individual, said method comprising administering to the individual an effective amount of progesterone. In other aspects, the invention provides for methods of treating a disease, disorder, symptom, or condition associated with one or more neurological receptor(s) selected from the group consisting of SCN1A, SCN8A, SCN9A, GABA receptors and serotonin receptors in an individual, said method comprising administering to the individual an effective amount of raloxifene. In any of the embodiments above and disclosed herein, the epilepsy and/or epilepsy-related disorders is selected from the group consisting of Dravet Syndrome, Lennox-Gastaut, generalized epilepsy with febrile seizure (GEFS+), severe myoclonic epilepsy of infancy (SMEI), epileptic encephalopathy, intractable epilepsy, absence epilepsy, temporal lobe epilepsy, frontal lobe epilepsy, occipital lobe epilepsy, parietal lobe epilepsy, partial seizures and general seizures. In some embodiments, the partial seizures are selected from the group consisting of simple partial seizures, complex partial seizures, aura, and secondarily generalized seizures. In some embodiments, the general seizures are selected from the group consisting of absence seizures (Petit mal), tonic seizures, clonic seizures, myoclonic seizures, atonic seizures, and tonic-clonic seizures (Grand mal).

[0012] In other aspects, the invention provides for methods of palliating a disease state in an individual that is palliable by treatment with an agent capable of modulating sodium current, said method comprising administering to the individual an effective amount of mifepristone. In other aspects, the invention provides for methods of palliating a disease state in an individual that is palliable by treatment with an agent capable of modulating sodium current, said method comprising administering to the individual an effective amount of bicalutamide. In other aspects, the invention provides for methods of palliating a disease state in an individual that is palliable by treatment with an agent capable of modulating sodium current, said method comprising administering to the individual an effective amount of progesterone. In other aspects, the invention provides for methods of palliating a disease state in an individual that is palliable by treatment with an agent capable of modulating sodium current, said method comprising administering to the individual an effective amount of raloxifene. In any of the embodiments above and disclosed herein, the epilepsy and/or epilepsy-related disorders is selected from the group consisting of Dravet Syndrome, Lennox- Gastaut, generalized epilepsy with febrile seizure (GEFS+), severe myoclonic epilepsy of infancy (SMEI), epileptic encephalopathy, intractable epilepsy, absence epilepsy, temporal lobe epilepsy, frontal lobe epilepsy, occipital lobe epilepsy, parietal lobe epilepsy, partial seizures and general seizures. In some embodiments, the partial seizures are selected from the group consisting of simple partial seizures, complex partial seizures, aura, and secondarily generalized seizures. In some embodiments, the general seizures are selected from the group consisting of absence seizures (Petit mal), tonic seizures, clonic seizures, myoclonic seizures, atonic seizures, and tonic-clonic seizures (Grand mal).

SUMMARY OF THE DRAWINGS

[0013] Figure 1 shows a depiction of the predicted binding site of progesterone in SCNA1 A model as well a depiction of a similar binding site in mineralocorticoid receptor.

[0014] Figure 2 shows a schematic representation of the zebrafish tracking system.

[0015] Figure 3 depicts an example of low-resolution 96-well analysis (left), and high- resolution analysis, 16-well (right).

[0016] Figure 4 shows the zebrafish response to light and the response in darkness.

[0017] Figure 5 shows results for velocity amplitude, tail energy and absolute angular speed.

[0018] Figure 6 shows the predicted binding site of a drug in TRAP1.

[0019] Figure 7A shows activity plots showing locomotor activity of individual larvae over the course of a 10 minute recording session. Periods of movement are indicated in with brighter lines, periods of rest are indicated in black. Larvae were monitored using an automated tracking platform under either constant dark (top panels) or constant light (bottom panels). Figure 7B shows swim velocity in pixels per second for all larvae recorded under constant dark ("dark") and constant light ('light") conditions. "Didy" designates homozygous mutant scnllab ^s552 larvae; "Siblings" designates age-matched wild-type and heterozygous mutant siblings that were obtained from the same clutch.

[0020] Figure 8 A shows a diagram depicting experimental setup in which 500 millisecond light pulses are applied every 2 minutes during a 10 minute recording session. Light-induced seizure activity ("light input") is measured during the 5 second periods immediately following each light pulse. Unstimulated activity ("dark") is measured during the dark intervals between light pulses. Figure 8B shows an activity trace locomotor activity of single larvae in response to single 500 millisecond light pulses applied every 2 minutes (left) or dual 500 millisecond light pulses applied every 2 minutes (right). Figure 8C shows mean swim velocity in pixels per second for all larvae. Light-triggered seizure activity ("light input"; left) is measured in the 5 second intervals immediately following light stimuli. Unstimulated activity ("dark"; right) is measured during the dark intervals between light pulses. "Didy" designates homozygous mutant scnllabs552 larvae;

"Siblings" designates age-matched wild-type and heterozygous mutant siblings that were obtained from the same clutch. Figure 8D depicts a diagram showing experimental setup in which two consecutive 500 millisecond light pulses separated by 1 second are applied every 2 minutes during a 10 minute recording session. Bottom: Activity plots showing locomotor activity of individual scnllab ^s552 mutant larvae recorded in a 96-well plate over the course of a 10 minute recording session. Periods of movement are indicated in color, periods of rest are indicated in black. Larvae were monitored using an automated tracking platform and light pulses were applied at 2, 4, 6 and 8 minutes.

[0021] Figure 9 shows the location of the s552 and sal6474 point mutations in the Nav sodium channel alpha subunit. Transmembrane domains (dark gray: S1-S3 segments; lighter gray: S4 voltage-sensing segment; light gray: S5-S6, ion pore region). Circle, missense mutation (p.Metl208Arg); Square, truncation (p.Tyr462*).

[0022] Figure 10A shows Scnllab ^sal6474 homozygous mutant (bottom) and age-matched sibling control (top) larvae at 7 days post fertilization. Mutant larvae exhibit dark coloration due to dispersed melanosomes and fail to inflate their swim bladders. Figures 10B shows mean swim velocity in pixels per second for all larvae recorded under constant dark ("dark") and constant light ("light") conditions. Figure IOC shows mean swim velocity in pixels per second for all larvae in response to light pulses. Light-triggered seizure activity is measured in the 5 second intervals immediately following light stimuli, "sal 6474" designates homozygous mutant scnllab ^sal6474 larvae; "Siblings" designates age-matched wild-type and heterozygous mutant siblings that were obtained from the same clutch.

[0023] Figure 11 depicts mean swim velocity (pixels per second) in response to light pulses for scnllab ^s552 mutant larvae (left) and scnllab ^sal6474 mutant larvae (right) exposed to AUopregnanolone (1 μΜ), Progesterone (0.4 μΜ), Ganaxolone (1 μΜ), and Mifepristone (25 μΜ) at 7 days post fertilization. Light-triggered seizure activity was measured in the 5 second intervals immediately following light stimuli. Baseline ("bas.") recordings were acquired immediately prior to compound treatment. Subsequent recording sessions were performed at 45 minutes, 2 hours, and 4 hours post-treatment. All compounds were administered directly to the water in a final concentration of 1% DMSO. Larvae exposed to 1% DMSO only ("DMSO") served as negative controls. *<0.05, **<0.005, ***<0.0005, ****<0.0001. DETAILED DESCRIPTION

[0024] The invention provides, inter alia, compositions and methods for treating, delaying development of, and/or preventing epilepsy and/or epilepsy-related disorders and palliating symptoms associated with epilepsy and/or epilepsy-related disorders. The class of compounds disclosed herein have not previously been known or suggested to be helpful in addressing the problems of epilepsy (e.g., Dravet Syndrome) and/or epilepsy-related disorders. Mutations of the SCNl A gene are known to occur in a subset of individuals with Dravet Syndrome. Other channels that may be affected include SCN8A, SCN9A, GABA receptors, and/or serotonin receptors. Without being bound by theory, the compounds disclosed herein can modulate various neurological molecules and/or receptors, such as SCN1A, SCN8A, SCN9A, GABA receptors, and/or serotonin receptors. In some

embodiments, one or more of these neurological molecules and/or receptors are excited by the one or more of the compounds disclosed herein. In other embodiments, one or more of these neurological molecules and/or receptors are antagonized by the one or more of the compounds disclosed herein.

[0025] It is to be understood that the present invention is not limited to the particular embodiments, materials, and examples described herein, as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

Definitions

[0026] As used herein, "epilepsy" refers to a central nervous system disorder

(neurological disorder) in which the nerve cell activity in an individual's brain is disturbed, causing a seizure during which he/she experiences abnormal behavior, symptoms and sensations, including loss of consciousness. Seizure symptoms can vary. Some people with epilepsy simply stare blankly for a few seconds during a seizure, while others repeatedly twitch their arms or legs. In some embodiments, epilepsy does not include West Syndrom.

[0027] As used herein, an "individual" is a vertebrate, such as avian, preferably a mammal, such as a human. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, experimental animals, rodents (e.g., mice and rats) and pets.

[0028] As used herein, an "effective amount" or "therapeutically effective amount" is that amount sufficient to effect a desired biological effect, such as beneficial results, including clinical results. As such, an "effective amount" depends upon the context in which it is being applied. In the context of administering a composition that modulates a neurological molecule (e.g. sodium channel or neurological receptor), an effective amount of a compound is an amount sufficient to achieve such a modulation as compared to a control (e.g., a response obtained when no compound is administered). An effective amount can be administered in one or more administrations.

[0029] As used herein, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0030] As used herein, "palliating" a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened (or alleviated) and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder. In the context of epilepsy, palliation may occur upon modulation of one or more sodium channels (e.g., SCN1 A, SCN8A, SCN9A) or one or more neurological receptors (e.g., GABA receptors or serotonin receptors). Further, palliation does not necessarily occur by administration of one dose, but often occurs upon administration of a series of doses. Thus, an amount sufficient to palliate a response or disorder may be administered in one or more administrations.

[0031] As used herein, "inhibiting," "inhibition," "antagonizing" and its various noun and verbal permutations are used herein to describe the biological effects of the various compounds and classes of compounds on the receptors. It does not necessarily require 100% inhibition or antagonization. Partial inhibition or partial antagonization is encompassed within this definition. Any degree of inhibiting or antagonizing as compared to the relevant control (e.g., a degree observed when the compound is not used) would be encompassed in the definition.

[0032] As used herein, "modulate" can mean the excitation or the inhibition of varying degrees of the neurological molecules and/or neurological receptors described herein.

[0033] As used herein, "concurrent administration" includes overlapping in duration at least in part. For example, when two compounds (e.g., any of the compounds or class of compounds described herein that has bioactivity) are administered concurrently, their administration occurs within a certain desired time. The compounds' administration may begin and end on the same day. The administration of one compound can also precede the administration of a second compound by one or more days as long as both compounds are taken on the same day at least once. Similarly, the administration of one compound can extend beyond the administration of a second compound as long as both compounds are taken on the same day at least once. The bioactive compounds do not have to be taken at the same time each day to include concurrent administration.

[0034] As used herein, "sequential administration" includes that the administration of two compounds (e.g., the compounds described herein) do not occur on a same day.

[0035] As used herein, "intermittent administration includes the administration of a compound for a period of time (which can be considered a "first period of administration"), followed by a time during which the compound is not taken or is taken at a lower

maintenance dose (which can be considered "off-period") followed by a period during which the compound is administered again (which can be considered a "second period of administration"). Generally, during the second phase of administration, the dosage level of the compound will match that administered during the first period of administration but can be increased or decreased as medically necessary.

[0036] It must be noted that as used herein and in the appended embodiments, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a disorder" or "a compound" is a reference to one or more disorders or compounds and includes equivalents thereof known to those skilled in the art and so forth.

Compounds for Modulating Molecules and Receptors for Addressing Epilepsy and/or Epilepsy-Related Disorders

[0037] The invention provides for various compounds and classes of compounds that can be used to address different physiological parameters and/or symptoms of epilepsy and/or epilepsy-related disorders. Importantly, these compounds have not been previously taught nor suggested for the treatment, prevention, delaying development of, and/or alleviating symptoms of epilepsy and/or epilepsy-related disorders.

[0038] As further detailed in the examples, various compounds were identified using an in silico screening process for binding to different neurological targets (e.g., receptors) such as sodium channels such as SCN1A, SCN8A, SCN9A, as well as GABA receptors, and serotonin receptors. Representative compounds include mifepristone, bicalutamide, progesterone, and raloxifene (structure, CAS numbering and DrugBank ID shown in Example 1). Further screening of these compounds for relevant biological activity was confirmed in zebrafish disease models. The invention contemplates other classes of compounds and/or other compounds that share similar binding capabilities to the exemplary compounds listed below. As described herein, other compounds may be identified using in silico screening process.

Mifepristone

[0039] Mifepristone is a synthetic steroid compound with both antiprogesterone and antiglucocorticoid properties. It is also known as RU-486 or RU-38486. Mifepristone is a 19-nonsteroid, lacking the C19-methyl group of natural progesterone (P) and

glucocorticosteroids (G). The bioactive agent has been found to have a high affinity for both the progesterone receptor (PR) and the glucocorticosteroid receptor (GR).

[0040] Mifepristone is available commercially, is FDA-approved for terminating pregnancies and/or preventing pregnancies and is been marketed under tradenames

Mifegyne ^® (Exelgyn) and Mifeprex ^® (Danco Laboratories, Inc.). When used to address unwanted pregnancies, mifepristone is typically used with misoprostol. Mifepristone has also been approved as Korlym ^® (Corcept Therapeutics, Menlo Park, CA) to control high blood sugar levels (hyperglycemia) in adults with endogenous Cushing's syndrome. This drug was approved by the FDA for use in patients with endogenous Cushing's syndrome who have type 2 diabetes or glucose intolerance and are not candidates for surgery or who have not responded to prior surgery.

[0041] Importantly, mifepristone has not been described for use in addressing epilepsy and/or epilepsy-related disorders. Accordingly, the invention contemplates use of mifepristone, its related salts and forms, in a composition for use in treating, preventing, delaying development of and/or alleviating symptoms of epilepsy and/or epilepsy-related disorders. Various formulations of mifepristone are contemplated within the scope of the invention, including pharmaceutical formulation with the appropriate buffers, sterilization agents and other ingredients for administration to an individual in need of such treatment (e.g., patient). The formulations can be in different dosage amounts such that one or more dose is administered to the individual. For example, in one embodiment, any of about 0.1 μΜ, 0.2 μΜ, 0.3 μΜ, 0.4 μΜ, 0.5 μΜ, 0.6 μΜ, 0.7 μΜ, 0.8 μΜ, 0.9 μΜ, 1 μΜ, 1.1 μΜ, 1.2 μΜ, 1.3 μΜ, 1.4 μΜ, 1.5 μΜ, 1.6 μΜ, 1.7 μΜ, 1.8 μΜ, 1.9 μΜ, 2 μΜ, 2.1 μΜ, 2.2 μΜ, 2.3 μΜ, 2.4 μΜ, 2.5 μΜ, 2.6 μΜ, 2.7 μΜ, 2.8 μΜ, 2.9 μΜ, 3 μΜ, 3.5 μΜ, 4 μΜ, 4.5 μΜ, 5 μΜ, 5.5 μΜ, 6 μΜ, 6.5 μΜ, 7 μΜ, 7.5 μΜ, 8 μΜ, 8.5 μΜ, 9 μΜ, 9.5 μΜ, 10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ, 20 μΜ, 21 μΜ, 22 μΜ, 23 μΜ, 24 μΜ, 25 μΜ, 30 μΜ, 35 μΜ, 40 μΜ, 45 μΜ, 50 μΜ, 55 μΜ, 60 μΜ, 65 μΜ, 70 μΜ, 75 μΜ, 80 μΜ, 85 μΜ, 90 μΜ, 95 μΜ, or 100 μΜ or more mifepristone can be included in the formulations or methods contemplated by the present invention. The administration can be serial, parallel or concurrent (e.g., overlapping) regiment that is needed to achieve the desired medical benefit to the individual.

[0042] In other embodiments, use of mifepristone, its related salts and forms, in a composition for treating epilepsy and/or epilepsy-related disorders can reduce seizure activity by about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in an individual.

Bicalutamide

[0043] Bicalutamide (IUPAC name: N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(4- fluorophenyl)sulfonyl]-2-hydroxy-2-methylpropanamide) is an oral non-steroidal anti- androgen drug that has been approved by the FDA to treat stage D2 metastatic prostate cancer in combination with luteinizing hormone-releasing hormone (LHRH) analog.

Bicalutamide is marketed under the brand names Casodex ^® and Cosudex ^® (AstraZeneca) for prostate cancer treatment. Bicalutamide has also been used in clinical trial for ovarian cancer.

[0044] Importantly, bicalutamide has not been described for use in addressing epilepsy and/or epilepsy-related disorders. Accordingly, the invention contemplates use of bicalutamide, its related salts and forms, in a composition for use in treating, preventing, delaying development of and/or alleviating symptoms of epilepsy and/or epilepsy-related disorders. Various formulations of bicalutamide are contemplated within the scope of the invention, including pharmaceutical formulation with the appropriate buffers, sterilization agents and other ingredients for administration to an individual in need of such treatment (e.g., patient). The formulations can be in different dosage amounts such that one or more dose is administered to the individual. For example, in one embodiment, any of about 0.1 μΜ, 0.2 μΜ, 0.3 μΜ, 0.4 μΜ, 0.5 μΜ, 0.6 μΜ, 0.7 μΜ, 0.8 μΜ, 0.9 μΜ, 1 μΜ, 1.1 μΜ, 1.2 μΜ, 1.3 μΜ, 1.4 μΜ, 1.5 μΜ, 1.6 μΜ, 1.7 μΜ, 1.8 μΜ, 1.9 μΜ, 2 μΜ, 2.1 μΜ, 2.2 μΜ, 2.3 μΜ, 2.4 μΜ, 2.5 μΜ, 2.6 μΜ, 2.7 μΜ, 2.8 μΜ, 2.9 μΜ, 3 μΜ, 3.5 μΜ, 4 μΜ, 4.5 μΜ, 5 μΜ, 5.5 μΜ, 6 μΜ, 6.5 μΜ, 7 μΜ, 7.5 μΜ, 8 μΜ, 8.5 μΜ, 9 μΜ, 9.5 μΜ, 10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ, 20 μΜ, 21 μΜ, 22 μΜ, 23 μΜ, 24 μΜ, 25 μΜ, 30 μΜ, 35 μΜ, 40 μΜ, 45 μΜ, 50 μΜ, 55 μΜ, 60 μΜ, 65 μΜ, 70 μΜ, 75 μΜ, 80 μΜ, 85 μΜ, 90 μΜ, 95 μΜ, or 100 μΜ or more bicalutamide can be included in the formulations or methods contemplated by the present invention. The administration can be serial, parallel or concurrent (e.g., overlapping) regiment that is needed to achieve the desired medical benefit to the individual.

[0045] In other embodiments, use of bicalutamide, its related salts and forms, in a composition for treating epilepsy and/or epilepsy-related disorders can reduce seizure activity by about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in an individual.

Progesterone

[0046] Progesterone (IUPAC name: Pregn-4-ene-3,20-dione) is a C-21 steroid hormone involved in the female menstrual cycle, pregnancy (supports gestation) and embryogenesis of humans and other species. Progesterone was approved by the United States Food and Drug Administration as vaginal gel on July 31, 1997, an oral capsule on May 14, 1998 in an injection form on April 25, 2001 and as a vaginal insert on June 21, 2007. In Italy and Spain, Progesterone is sold under the trademark Progeffik ^® . Women have taken progesterone by mouth for inducing menstrual periods and treating abnormal uterine bleeding associated with hormonal imbalance, and severe symptoms of premenstrual syndrome (PMS). Progesterone is also used in combination with the hormone estrogen to "oppose estrogen" as part of hormone replacement therapy.

[0047] Progesterone is also used to ease withdrawal symptoms when certain drugs (benzodiazepines) are discontinued. Progesterone cream is sometimes used in hormone replacement therapy and for treating menopausal symptoms such as hot flashes. Topical progesterone is also used for treating or preventing certain allergies in which hormones play a role; and for treating bloating, breast tenderness, decreased sex drive, depression, fatigue, lumpy (fibrocystic) breasts, headaches, low blood sugar, increased blood clotting, infertility, irritability, memory loss, miscarriages, brittle bones (osteoporosis), bone loss in younger women, symptoms of PMS, thyroid problems, "foggy thinking," uterine cancer, uterine fibroids, water retention, weight gain, and vaginal irritation (vulval lichen sclerosis).

Progesterone gel is sometimes used inside the vagina to expand the cervix (cervical ripening), treat breast pain in women with noncancerous breast disease, and to prevent and treat abnormal thickening of the lining of the uterus (endometrial hyperplasia). Progesterone is also used intravaginally or by injection for treating infertility and symptoms of (PMS).

[0048] Importantly, progesterone has not been described for use in addressing epilepsy and/or epilepsy-related disorders. Accordingly, the invention contemplates use of progesterone, its related salts and forms, in a composition for use in treating, preventing, delaying development of and/or alleviating symptoms of epilepsy and/or epilepsy-related disorders. Various formulations of progesterone are contemplated within the scope of the invention, including pharmaceutical formulation with the appropriate buffers, sterilization agents and other ingredients for administration to an individual in need of such treatment (e.g., patient). The formulations can be in different dosage amounts such that one or more dose is administered to the individual. For example, in one embodiment, any of about 0.1 μΜ, 0.2 μΜ, 0.3 μΜ, 0.4 μΜ, 0.5 μΜ, 0.6 μΜ, 0.7 μΜ, 0.8 μΜ, 0.9 μΜ, 1 μΜ, 1.1 μΜ, 1.2 μΜ, 1.3 μΜ, 1.4 μΜ, 1.5 μΜ, 1.6 μΜ, 1.7 μΜ, 1.8 μΜ, 1.9 μΜ, 2 μΜ, 2.1 μΜ, 2.2 μΜ, 2.3 μΜ, 2.4 μΜ, 2.5 μΜ, 2.6 μΜ, 2.7 μΜ, 2.8 μΜ, 2.9 μΜ, 3 μΜ, 3.5 μΜ, 4 μΜ, 4.5 μΜ, 5 μΜ, 5.5 μΜ, 6 μΜ, 6.5 μΜ, 7 μΜ, 7.5 μΜ, 8 μΜ, 8.5 μΜ, 9 μΜ, 9.5 μΜ, 10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ, 20 μΜ, 21 μΜ, 22 μΜ, 23 μΜ, 24 μΜ, 25 μΜ, 30 μΜ, 35 μΜ, 40 μΜ, 45 μΜ, 50 μΜ, 55 μΜ, 60 μΜ, 65 μΜ, 70 μΜ, 75 μΜ, 80 μΜ, 85 μΜ, 90 μΜ, 95 μΜ, or 100 μΜ or more progesterone can be included in the formulations or methods contemplated by the present invention. The administration can be serial, parallel or concurrent (e.g., overlapping) regiment that is needed to achieve the desired medical benefit to the individual.

[0049] In other embodiments, use of progesterone, its related salts and forms, in a composition for treating epilepsy and/or epilepsy-related disorders can reduce seizure activity by about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in an individual.

Raloxifene

[0050] Raloxifene is an oral selective estrogen receptor modulator (SERM) that has estrogenic actions on bone and anti-estrogenic actions on the uterus and breast. It is used in the prevention of osteoporosis in postmenopausal women. Raloxifene is marketed as Evista ^® (Eli Lilly and Company). Raloxifene has been approved by the FDA for reducing the risk of invasive breast cancer in postmenopausal women with osteoporosis and in postmenopausal women at high risk for invasive breast cancer. Raloxifene is also indicated for the treatment and prevention of osteoporosis in postmenopausal women. [0051] Importantly, raloxifene has not been described for use in addressing epilepsy and/or epilepsy-related disorders. Accordingly, the invention contemplates use of raloxifene, its related salts and forms, in a composition for use in treating, preventing, delaying development of and/or alleviating symptoms of epilepsy and/or epilepsy-related disorders. Various formulations of raloxifene are contemplated within the scope of the invention, including pharmaceutical formulation with the appropriate buffers, sterilization agents and other ingredients for administration to an individual in need of such treatment (e.g., patient). The formulations can be in different dosage amounts such that one or more dose is administered to the individual. For example, in one embodiment, any of about 0.1 μΜ, 0.2 μΜ, 0.3 μΜ, 0.4 μΜ, 0.5 μΜ, 0.6 μΜ, 0.7 μΜ, 0.8 μΜ, 0.9 μΜ, 1 μΜ, 1.1 μΜ, 1.2 μΜ, 1.3 μΜ, 1.4 μΜ, 1.5 μΜ, 1.6 μΜ, 1.7 μΜ, 1.8 μΜ, 1.9 μΜ, 2 μΜ, 2.1 μΜ, 2.2 μΜ, 2.3 μΜ, 2.4 μΜ, 2.5 μΜ, 2.6 μΜ, 2.7 μΜ, 2.8 μΜ, 2.9 μΜ, 3 μΜ, 3.5 μΜ, 4 μΜ, 4.5 μΜ, 5 μΜ, 5.5 μΜ, 6 μΜ, 6.5 μΜ, 7 μΜ, 7.5 μΜ, 8 μΜ, 8.5 μΜ, 9 μΜ, 9.5 μΜ, 10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ, 20 μΜ, 21 μΜ, 22 μΜ, 23 μΜ, 24 μΜ, 25 μΜ, 30 μΜ, 35 μΜ, 40 μΜ, 45 μΜ, 50 μΜ, 55 μΜ, 60 μΜ, 65 μΜ, 70 μΜ, 75 μΜ, 80 μΜ, 85 μΜ, 90 μΜ, 95 μΜ, or 100 μΜ or more raloxifene can be included in the formulations or methods contemplated by the present invention. The administration can be serial, parallel or concurrent (e.g., overlapping) regiment that is needed to achieve the desired medical benefit to the individual.

[0052] In other embodiments, use of raloxifene, its related salts and forms, in a composition for treating epilepsy and/or epilepsy-related disorders can reduce seizure activity by about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in an individual.

Allopregnanolone

[0053] Allopregnanolone (3 -hydroxy-5a-pregnan-20-one) (ALLO) is an endogenous pregnane neurosteroid that is synthesized in vivo by 5a-reduction of progesterone [PMID 24001085, 21094889]. ALLO is a positive allosteric modulator of GABA _A Receptors that acts through binding sites that are distinct from the modulatory sites of benzodiazepine and barbiturates. Unlike progesterone, ALLO is not believed to have activity at nuclear steroid hormone receptors [PMID 17199022, 21094889]. Conversely, progesterone itself is not a modulator of GABAA Receptors but it can be metabolized to ALLO and other related neurosteroids that modulate GABA signaling. [0054] The invention contemplates use of ALLO, its related salts and forms, in a composition for use in treating, preventing, delaying development of and/or alleviating symptoms of epilepsy and/or epilepsy-related disorders. Various formulations of ALLO are contemplated within the scope of the invention, including pharmaceutical formulation with the appropriate buffers, sterilization agents and other ingredients for administration to an individual in need of such treatment (e.g., patient). The formulations can be in different dosage amounts such that one or more dose is administered to the individual. For example, in one embodiment, any of about 0.1 μΜ, 0.2 μΜ, 0.3 μΜ, 0.4 μΜ, 0.5 μΜ, 0.6 μΜ, 0.7 μΜ, 0.8 μΜ, 0.9 μΜ, 1 μΜ, 1.1 μΜ, 1.2 μΜ, 1.3 μΜ, 1.4 μΜ, 1.5 μΜ, 1.6 μΜ, 1.7 μΜ, 1.8 μΜ, 1.9 μΜ, 2 μΜ, 2.1 μΜ, 2.2 μΜ, 2.3 μΜ, 2.4 μΜ, 2.5 μΜ, 2.6 μΜ, 2.7 μΜ, 2.8 μΜ, 2.9 μΜ, 3 μΜ, 3.5 μΜ, 4 μΜ, 4.5 μΜ, 5 μΜ, 5.5 μΜ, 6 μΜ, 6.5 μΜ, 7 μΜ, 7.5 μΜ, 8 μΜ, 8.5 μΜ, 9 μΜ, 9.5 μΜ, 10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ, 20 μΜ, 21 μΜ, 22 μΜ, 23 μΜ, 24 μΜ, 25 μΜ, 30 μΜ, 35 μΜ, 40 μΜ, 45 μΜ, 50 μΜ, 55 μΜ, 60 μΜ, 65 μΜ, 70 μΜ, 75 μΜ, 80 μΜ, 85 μΜ, 90 μΜ, 95 μΜ, or 100 μΜ or more

ALLO can be included in the formulations or methods contemplated by the present invention. The administration can be serial, parallel or concurrent (e.g., overlapping) regiment that is needed to achieve the desired medical benefit to the individual.

[0055] In other embodiments, use of ALLO, its related salts and forms, in a composition for treating epilepsy and/or epilepsy-related disorders can reduce seizure activity by about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in an individual.

Ganaxolone

[0056] Ganaxolone (3a-hydroxy-3P-methyl-5a-pregnan-20-one) (GNX) is a 3P-methyl- substituted synthetic analog of ALLO that is orally active and has a more favorable pharmacokinetic-pharmacodynamic profile than its endogenous counterpart [PMID 9067315, 21094889]. GNX is a positive allosteric modulator of GABAA Receptors that act through binding sites that are distinct from the modulatory sites of benzodiazepine and barbiturates. Unlike progesterone, GNX is not believed to have activity at nuclear steroid hormone receptors [PMID 17199022, 21094889].

[0057] The invention contemplates use of GNX, its related salts and forms, in a composition for use in treating, preventing, delaying development of and/or alleviating symptoms of epilepsy and/or epilepsy-related disorders. Various formulations of GNX are contemplated within the scope of the invention, including pharmaceutical formulation with the appropriate buffers, sterilization agents and other ingredients for administration to an individual in need of such treatment (e.g., patient). The formulations can be in different dosage amounts such that one or more dose is administered to the individual. For example, in one embodiment, any of about 0.1 μΜ, 0.2 μΜ, 0.3 μΜ, 0.4 μΜ, 0.5 μΜ, 0.6 μΜ, 0.7 μΜ, 0.8 μΜ, 0.9 μΜ, 1 μΜ, 1.1 μΜ, 1.2 μΜ, 1.3 μΜ, 1.4 μΜ, 1.5 μΜ, 1.6 μΜ, 1.7 μΜ, 1.8 μΜ, 1.9 μΜ, 2 μΜ, 2.1 μΜ, 2.2 μΜ, 2.3 μΜ, 2.4 μΜ, 2.5 μΜ, 2.6 μΜ, 2.7 μΜ, 2.8 μΜ, 2.9 μΜ, 3 μΜ, 3.5 μΜ, 4 μΜ, 4.5 μΜ, 5 μΜ, 5.5 μΜ, 6 μΜ, 6.5 μΜ, 7 μΜ, 7.5 μΜ, 8 μΜ, 8.5 μΜ, 9 μΜ, 9.5 μΜ, 10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ, 20 μΜ, 21 μΜ, 22 μΜ, 23 μΜ, 24 μΜ, 25 μΜ, 30 μΜ, 35 μΜ, 40 μΜ, 45 μΜ, 50 μΜ, 55 μΜ, 60 μΜ, 65 μΜ, 70 μΜ, 75 μΜ, 80 μΜ, 85 μΜ, 90 μΜ, 95 μΜ, or 100 μΜ or more GNX can be included in the formulations or methods contemplated by the present invention. The administration can be serial, parallel or concurrent (e.g., overlapping) regiment that is needed to achieve the desired medical benefit to the individual.

[0058] In other embodiments, use of GNX, its related salts and forms, in a composition for treating epilepsy and/or epilepsy-related disorders can reduce seizure activity by about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in an individual.

Methods of Using Compounds for Addressing Epilepsy and/or Epilepsy-Related Conditions Populations of Individuals and/or Patients

[0059] The compounds described herein can be used to address epilepsy and/or epilepsy- related conditions in individual, including patients under physician's care. Non-limiting examples of epilepsy include Dravet Syndrome (DS), Lennox-Gastaut Syndrome (LGS), severe myoclonic epilepsy of infancy (SMEI), epileptic encephalopathy, intractable epilepsy, absence epilepsy, temporal lobe epilepsy, frontal lobe epilepsy, occipital lobe epilepsy, parietal lobe epilepsy, and generalized epilepsy with febrile seizure (GEFS+). Dravet Syndrome can be characterized by febrile and afebrile generalized and unilateral, clonic or tonic-clonic, seizures that can occur in the first year of life in an otherwise normal infant. Later, the association can be with myoclonus, atypical absences and partial seizures.

[0060] Epilepsy-related conditions can include partial seizures, which includes without limitation: simple partial seizures, complex partial seizures, aura, and secondarily generalized seizures. Epilepsy-related conditions can also include general seizures, which includes without limitation: absence seizures (Petit mal), tonic seizures, clonic seizures, myoclonic seizures, atonic seizures, and tonic-clonic seizures (Grand mal). [0061] The invention contemplates treating individuals with epilepsy and/or epilepsy- related conditions. In some embodiments, there are sub-populations of individuals to be treated who have not previously taken mifepristone, bicalutamide, progesterone, and/or raloxifene previously, for example, for addressing the diseases and/or indications set forth in the regulatory and/or marketing approval for those compounds. For example, for

mifepristone was approved by the FDA for addressing unwanted pregnancy, thus in one embodiment, a sub-population of individuals to be treated would not have previously received mifepristone for unwanted pregnancies. Similarly, other sub-populations of individuals to be treated bicalutamide, progesterone, and/or raloxifene would not have received those compounds for the indications listed in the regulatory and/or marketing approval.

[0062] In some embodiments, the individual is suspected of having epilepsy and/or epilepsy-related conditions. In other embodiments, the individual is diagnosed with epilepsy and/or epilepsy-related conditions.

[0063] The compounds of this invention can be used to treat individuals in need thereof with the monitoring and/or guidance of a physician, who can assess the symptoms and phenotypes of the individual to determine the medical condition. Compounds of this invention can be administered to the individual in a therapeutically effective amount.

[0064] The compounds described herein may be used alone or in conjunction with other compounds. They can be administered in various forms, such as, but not limited to oral dosage form, inhalational dosage form, parenteral injection dosage form, topical dosage form, and/or suppository dosage form.

Methods of Using Compounds for Modulating Neurological Pathways and/or Receptors

[0065] The compounds described herein may be used to modulate different neurological molecules, pathways and/or receptors. For example, sodium channels, which regulate the transportation of sodium into cells, can be affected by one of more of these compounds.

Sodium channels that are contemplated include, but are not limited to, can be SCN1A, SCN8A, or SCN9A. The compound can function as an agonist of an antagonist of the sodium channels. One of skill in the art can employ standard techniques for determining the agonist/antagonist effect on the sodium channels. Accordingly, in some embodiments, the compound(s) is an agonist or an antagonist of SCN1 A. In another embodiment, the compound(s) may be an agonist or antagonist of SCN8A. In another embodiment, the compound(s) may be an agonist or antagonist of SCN9A. [0066] The compounds identified in the manner described herein can also modulate other neurological pathways and/or receptors, such as GABA receptors or serotonin receptors. Accordingly, in some embodiments, the compound(s) may be an agonist or antagonist of GABA receptors. In other embodiments, the compound(s) may be an agonist or antagonist of serotonin receptors. One of skill in the art can employ standard techniques for determining the agonist/antagonist effect on these receptors.

[0067] The following examples are provided as illustrations of various embodiments of the invention but are not meant to limit the invention in any manner.

EXAMPLES

Example 1 Selection of Compounds Affecting Various Sodium Channel Receptors

[0068] In silico screenings of multiple compounds (FDA-approved and non-FDA- approved) were conducted to determine those compounds that would bind to SCN1 A ion channel. The binding affinities were predicted for potential efficacy, side effects and toxicity.

[0069] In vitro high throughput screening was then used in personalized in vitro models and/or single cell models to confirm binding affinity and activity on predicted molecular targets. Figure 1 shows a depiction of the predicted binding site of progesterone in SCNAl A model as well a depiction of a similar binding site in mineralocorticoid receptor.

[0070] From the selection process, four compounds (compound name, DrugBank ID, CAS# and structure shown below) were selected that could be an agonist and/or antagonist of SCN1A, SCN8A, GABA, and/or serotonin receptors.

Bicalutamide DB01128 90357-06-5

Progesterone DB00396 57-83-0

Raloxifene DB00481 84449-90-1

[0071] These compounds were further tested using Zebrafish disease model of a type of epilepsy, Dravet's Syndrome.

Example 2 Zebrafish Behavioral Screening Protocol

[0072] Zebrafish was used to create a disease model of Dravet's Syndrome, one type of epilepsy. The following protocol was used for this animal model: Let zebrafish embryos growing until 7dpf at 28C with circadian cycle of 12h L/12h D refreshing the E3 media every 2 days.

Load a 96 well plate with 1 larva per well in lOOuL of E3 media.

Let the larva set in dark for 5 min then acquire the 10 min video. This will represent the baseline.

Add lOOuL of the drug at the desired concentration in 1%DMS0/E3 media.

Place the plate in the incubator and record videos after 45min, 2h and 4h. Before acquiring the videos let the larva set in the dark for 5min.

Videos can be acquired in 12X8 field (low magnification) or 4X3 (high

magnification)

After the videos are recorded run the LiteTacking code.

E3 media

Per 1 liter

IX E3 50X E3

5.0 mM NaCl (58.44) 0.292g 14.6g

0.17mM KCl (74.55) 0.013g 0.65g

0.33mM CaCl (147.02) 0.044g 2.20g

0.33mM MgS04 (246.5) 0.081g 4.05g

[0073] Take 20 mis of 50X, q.s to 1 liter of dH20 for IX solution (for MWSU house distilled H20 Add 0.192 sodium bicarbonate to adjust pH to ~ 7.4). Optional: add 200 uls of 0.05% methylene blue to IX E3 as a fungicide. Multiwell plate: MultiScreen 96-well Transport Receiver Plate catalog number MATRNPS50.

Example 3 Behavioral Zebrafish Tracking System

[0074] The system that was designed provides a controlled environment for tracking locomotor activity and other behavioral readouts of zebrafish larvae in multi-well plates. As depicted in Figure 2, the outer enclosure shuts out all external light, while an internal IR light source allows behavior to be monitored in the dark. An internal white light source can be programmed to provide either continuous light or precisely timed light stimuli. The motorized zoom switches from a 96- to a 16-well field of view, allowing two different types of recording modalities: 1) a low resolution mode where it is possible to track the movement of all larvae in a 96-well plate simultaneously, and 2) a high resolution mode where subtle behavioral features can be detected and quantified in 16 wells at a time. The inventor has developed dedicated algorithms for acquisition and analysis using MATLAB. The code allows parameters such resolution, frame rate, duration of analysis, and light inputs to be customized for each experiment and enables automatic switching between low and high resolution modalities.

[0075] In the low-resolution modality, it is possible to automatically identify the positions of all larvae in each frame, allowing the determination of velocity, direction, and acceleration (normal and tangential). More complex analysis is possible in the high-resolution modality where morphological landmarks such as the eyes, the swim bladder, and position of the tail tip were precisely identified. These landmarks allow the derivation of additional metrics such as lateral vs. dorsal orientation, direction, angular speed, and tail-bending angle. Drugs were rapidly analyzed at multiple concentrations in a first-pass screen using the low-resolution modality. The positive hits were screened again at high-resolution to reduce false positives and to monitor a wider range of behaviors. Figure 3 depicts an example of low resolution 96-well analysis (left) and a high resolution analysis, 16 well (right).

Example 4 Screening of Progesterone and Mifepristone in Zebrafish Model

[0076] Progesterone and Mifepristone were screened in a Zebrafish Model as described above and herein. Figures 4 and 5 and Table 1 show the results from these experiments. Figure 4 shows the zebrafish response to light and the response in darkness. Figure 5 shows results for velocity amplitude, tail energy and absolute angular speed.

Table 1 Relative response with respect to DMSO normalized to baseline

Example 5 TRAP1 Binding

[0077] The procedures and protocols described herein was also used to predict binding site of a drug in Trapl (Figure 6) using the FINDSITE ^comb (H. Zhou and J. Skolnick.

FINDSITE ^comb : "A threading/structure -based, proteomic-scale virtual ligand screening approach" Journal of Chemical Information and Modeling 2013: 53(1): 230-240. PMCID: PMC355755.) Two patients with Chronic Fatigue Syndrome have been in a small scale trial for about 4 weeks. Both patients are doing well as measured as assessed by their increased mobility and energy.

Example 6 Effect on Sodium Channel Activity

[0078] The effects of progesterone and mifepristone on sodium channel activity are assessed in single ion channel recordings of SCN1 A mutants in frog oocytes using the standard protocols known in the art to a skilled practitioner. See, e.g., Dascal N., "The use of Xenopus oocytes for the study of ion channels" CRC Crit Rev Biochem. 22(4):317-87 (1987).

Example 7 Anti-seizure Activity

[0079] Progesterone and Mifepristone are screened for anti-seizure activity in mouse models of Dravet Syndrome using protocols known to one of skill in the art. See, e.g., "Sudden unexpected death in a mouse model of Dravet syndrome" Franck Kalume, et al J Clin Invest. Volume 123, Issue 4, pp. 1798-1808 (April 1, 2013).

Example 8 Testing of Compounds in Animal Models of SCN1 A with Mutations

[0080] Zebrafish have recently emerged as an important new non-rodent model for antiepileptic drug (AED) discovery [PMID 25710835]. Zebrafish larvae exposed to pentylenetetrazole (PTZ) and other convulsants exhibit elevated locomotor activity, seizurelike movements, and electrographic seizure activity. PTZ-induced convulsions can be readily monitored and quantified using automated tracking systems and are rescued many FDA approved AEDs [PMID 15730879, 17485198, 23342097, 25845493]. In addition to pharmacologically-induced seizures, antisense oligonucleotides have been used to knock down epilepsy associated genes in zebrafish embryos and several seizure-prone mutant lines have been characterized [PMID 20943912, 24002024, 25783594, 21692188, 23471908, 20819949].

[0081] A variety of childhood epilepsies have been associated with mutations in the SCN1A gene (MIM# 182389), which encodes the pore-forming alpha subunit of the Na _v l. l sodium channel and is widely expressed throughout the central nervous system. Disorders range from relatively mild forms like generalized epilepsy with febrile seizures plus (GEFS+; MIM #604233) to debilitating conditions such as Dravet syndrome (DS; also known as severe myoclonic epilepsy of infancy; MIM #607208) and intractable childhood epilepsy with generalized tonic-clonic seizures (ICE-GTC) [PMID 15880351, 19469841]. DS is the most common phenotype associated with SCN1 A mutations and is characterized by frequent febrile seizures that appear during the first year of life and are often refractory to treatment by standard anticonvulsants [PMID 17105460]. Over 1,200 SCN1A mutations have been identified to date and the most severe clinical phenotypes typically correlate with loss-of- function nonsense and frameshift mutations, or with missense mutations in critical residues of the pore region [PMID 19469841 , 25754450]. In addition to epilepsy, SCN1A variants have been linked to autism and rare cases of familial migraine, making it one of the most therapeutically relevant sodium channel genes [PMID 12610651, 23919895].

[0082] Progesterone and Mifepristone are screened in zebrafish models of Dravet

Syndrome with various mutations in the SCN1 A ion channel including frame shift mutations at various locations in the SCN1 A sequence, missense mutations in the pore and voltage sensing domains of SCN1 A, in the R931C mutation that likely locks the ion channel in the closed state.

[0083] Additionally, the zebrafish double indemnity (didy) s552 mutant was initially identified based on defects in the optokinetic response (OKR) assay and subsequently localized to the scnllab gene, which encodes a member of the voltage-gated sodium channel alpha family. The s552 mutation introduces a methionine (M) to an arginine (R) substitution at position 1208, which falls within the first transmembrane segment of domain III of the ion channel [PMID 16311625, 20484654]. Homozygous mutant larvae have a dark appearance due to dispersed melanosomes (which may be indicative of a neuroendocrine defect) and are not viable beyond -14 dpf [PMID 20484654]. In addition to OKR defects, scnllab" ⁵⁵² homozygous mutant larvae exhibit spontaneous electro graphic seizures and elevated swim activity beginning at ~4 dpf, making them a valuable animal model of DS and related epilepsies. Exposure of scnllab ^*552 mutant embryos to a variety of AEDs reveals a

pharmacological profile reminiscent of that associated with DS in humans [PMID 24002024]. Methods

[0084] At 7 dpf, homozygous mutants were identified based on the presence of dispersed melanosomes (PMID 20484654), and single larvae were distributed into individual wells of flat-bottomed 96-well microplates (MultiScreen 96-well Transport Receiver Plate, Millipore) in a volume of 100 μί, of E3 medium per well. Prior to application of test compounds, locomotor activity was monitored for 10 minutes using an automated video tracking system. 10 mM stocks of all test compounds were prepared in 100% dimethyl sulfoxide (DMSO). On the day of the experiment, 2x working stocks of each compound were prepared in E3 medium and the DMSO concentration was adjusted to 2%. 100 uL of the 2x working stock was added to each well immediately after acquisition of the baseline locomotor recording, resulting in the indicated screening concentrations (Table 2) and a final DMSO concentration of 1%. Additional locomotor activity recordings were acquired at 45 minutes, 2 hours, and 4 hours post-treatment.

Results

Light-induced seizures in scnllab mutants

[0085] Photosensitivity is reported in 30-40% of patients with DS and is typically characterized by isolated or recurrent massive myoclonic jerks and EEG showing generalized spike and polyspike waves [PMID 17105460, 21463274, 16302877]. This aspect of the DS phenotype has not been previously described in zebrafish Na _v l .1 mutants, so we first sought to determine if seizures can be triggered in scnllab ⁵⁵² larvae using simple visual stimuli. At 7 dpf, homozygous mutant larvae (distinguished by their dark pigmentation) [PMID

20484654], along with age -matched wild-type and heterozygous sibling controls, were transferred into 96-well plates and their locomotor activity was assessed under various conditions using an automated tracking platform. Previous studies have demonstrated that scnllab ⁵⁵² larvae exhibit increased swim velocities and spontaneous seizure-like behaviors [PMID 24002024]. Consistent with these findings, we observed significantly elevated locomotor activity in homozygous mutant larvae relative to wild-type siblings during 10 minute recording intervals carried out under either constant light (mean velocity of mutants=15±9 pixels s ^"1 , n=12; mean velocity of siblings=5±l pixels s ^"1 , n=12; p=0.002, Welch's t-test) or constant dark conditions (mean velocity of mutants=9±3 pixels s ^"1 , n=12; mean velocity of siblings=5±2 pixels s ^"1 , n=12; p=0.009), with the highest activity seen under constant light (Fig 7).

[0086] Photosensitivity in DS can be triggered by a variety of stimuli including intermittent light stimulation, bright light, and strong contrast between light and darkness [PMID 23093055]. We stimulated larvae by administering a brief (500ms) light pulse every 2 minutes in an otherwise dark environment over the course of a 10 minute recording session (Fig 8A). Mutant larvae consistently exhibited short rapid bursts of locomotor activity characterized by seizure-like movements during and immediately after each light pulse (Fig 8B). In contrast, wild-type siblings showed almost no increase in activity in response to light pulses. We quantified light-induced activity by measuring mean swim velocity during 5 second intervals immediately following the onset of each light pulse over the course of a 10 minute recording session (Fig 8C; mean velocity of mutants=50±19 pixels s ^"1 , n=12; mean velocity of siblings=15±7 pixels s ^"1 , n=12; p=3>< 10 ^"5 ). Locomotor activity during the two minute dark intervals between light pulses (excluding the 5 second periods following light stimuli) remained unvaried and was comparable to basal activity levels under continuous dark conditions for both mutants and siblings (Fig 8C). We observed that light-induced seizurelike behavior was further exacerbated by applying two consecutive light pulses in rapid succession (Fig 8B, C, D; 500ms light pulses separated by a 1 second dark interval; Fig 8B; mean velocity of mutants=96±24 pixels s ^"1 , n=12; mean velocity of siblings=19±7 pixels s ^"1 , n=12; p=l l0 ^"7 ).

[0087] To verify that photosensitivity is a general feature of scnllab loss-of-function mutations in zebrafish, rather than a unique phenotype associated with the s552 point mutation, we obtained a second mutant line that was generated as part of the Zebrafish Mutation Project (ZMP; PMID 23594742). The scnl lab ^8*16414 allele introduces a C to A mutation at position 1386 of the scnllab open reading frame, resulting in a premature stop codon at position 462 (p.Tyr462*; Fig 9). The mutation is located in the intracellular loop between domains I and II and presumably renders the ion channel nonfunctional.

Homozygous mutant sal 6474 larvae exhibit the same morphological phenotypes that are seen in scnllab ^s552 mutants [PMID 20484654], including failure to inflate their swim bladder and a dark appearance due to dispersed melanosomes (Fig 10A). Mutant larvae fail to thrive and begin to die at elevated rates relative to age-matched sibling controls beginning at approximately 13 dpf (survival of mutants at 14 dpf=32%, n=28; survival of siblings at 14 dpf=95%, n=39). Homozygous sal 6474 mutants also exhibit behavioral phenotypes consistent with those observed for the s552 allele, including elevated locomotor activity under constant light (mean velocity of mutants=2.55±2.33 pixels s ^"1 , n=8; mean velocity of siblings=0.34±0.18 pixels s ^"1 , n=8; p=0.03) and constant dark conditions (mean velocity of mutants=1.74±l . l 1, n=8; mean velocity of siblings=0.35±0.14, n=8; p=0.01). Light pulses consistently trigger sudden rapid bursts of seizure-like activity in sal 6474 mutants (mean velocity of mutants=16.76±5.84, n=8; mean velocity of siblings=3.04±1.67, n=8; p=2>< 10 ^~4 ). Taken together, these data show for the first time that photosensitivity is a general feature of scnllab mutations in zebrafish and establish an important new vertebrate genetic model for studying photosensitive epilepsies.

Screening for ntiepileptic activity in scnllab mutants

[0088] Mifepristone, Allopregnanolone, Ganaxolone, and Progesterone were tested for their ability to rescue light-induced seizure activity in s552 and sal 6474 mutant larvae.

Mifepristone (or RU-486), a synthetic C19 norsteroid with substitutions at positions CI 1 and C17, that acts as an antagonist of both progesterone and glucocorticoid receptors.

Allopregnanolone (3a-hydroxy-5a-pregnan-20-one) (ALLO) is an endogenous pregnane neurosteroid that is synthesized in vivo by 5 -reduction of progesterone [PMID 24001085, 21094889]. Ganaxolone (3 -hydroxy-3P-methyl-5a-pregnan-20-one) (GNX) is a 3P-methyl- substituted synthetic analog of ALLO that is orally active and has a more favorable pharmacokinetic-pharmacodynamic profile than its endogenous counterpart [PMID 9067315, 21094889]. Both ALLO and GNX are positive allosteric modulators of GABA _A Receptors that act through binding sites that are distinct from the modulatory sites of benzodiazepine and barbiturates. Unlike progesterone, ALLO and GNX are not believed to have activity at nuclear steroid hormone receptors [PMID 17199022, 21094889]. Conversely, progesterone itself is not a modulator of GABAA Receptors but it can be metabolized to ALLO and other related neurosteroids that modulate GABA signaling. Both ALLO and GNX exhibit potent anticonvulsant effects in diverse animal models [PMID 24001085] and appear to be less subject to anticonvulsant tolerance than benzodiazepines [PMID 23219031, 11082461]. Clinical trials have shown that ganaxolone reduces seizure frequency in adults with partial- onset seizures and children with refractory infantile spasms (West Syndrome) [PMID

23219031], although there are no reports to date of it being tested on patients with DS.

[0089] All compounds were tested on homozygous mutant scnllab ^s552 and scnllab ^sal6474 larvae in 96-well plates using the optimal light pulse parameters established above (i.e. two consecutive 500ms pulses separated by Is, administered every two minutes over a 10 minute recording session). Prior to compound application, an initial recording session was performed to establish the baseline locomotor activity level for each treatment group. Compounds were then applied directly to the wells at the indicated concentration and additional 10 minute recordings sessions were performed at 45 minutes, 2 hours, and 4 hours post-exposure. We evaluated the effect of compounds on light-induced seizures at all time points by calculating the mean swim velocity of each larvae during the 5 second periods following the onset of the light pulses. Velocities were normalized to baseline for each group. All four compounds caused a significant reduction in seizure activity at one or more of the assay time points relative to larvae that were treated with DMSO-only (Table 2; Figure 11). Table 2 Response of scnllab ^s mutant larvae to test compounds. "Seizure Activity" represents mean light-induced locomotor activity normalized to the baseline recording session for each treatment group.

Std Dev 32.30 15.59 16.19 16.16 t-test 3.15E-15 1.02E-14 3.96E-13

Ganaxolone 38398-32-2 1 μΜ Seizure Activity 100.00 39.13 46.71 41.08

Std Dev 34.28 18.15 22.85 21.82 t-test 2.18E-06 2.09E-05 4.47E-06

Mifepristone 84371-65-3 25 μΜ Seizure Activity 100.00 88.97 68.99 40.92

Std Dev 35.21 26.24 22.53 19.59 t-test 1.61E-01 1.05E-04 7.34E-11

Progesterone 57-83-0 0.4 μΜ Seizure Activity 100,00 31.15 27.86 38.33

Std Dev 31.73 25.38 18.19 26.55 t-test 1.52E-10 1.28E-11 4.97E-09

[0090] The effects of ALLO and GNX in our assay is consistent with their known mechanism of action as positive allosteric modulators of GABAA Receptors, with both compounds showing both anticonvulsive and sedative properties. The antiseizure effects of progesterone may occur through its conversion to ALLO, as has been shown in other animal models [PMID 14982969], although the time course of this effect is remarkably rapid, which light-induced seizure activity dropping to 26% of baseline within 45 minutes of application. Alternatively, in silico modeling predicts that these molecules directly interact with SCN1 A. Example 9 Testing of Compounds in Induced Pluripotent Cell Disease Models

[0091] Induced pluripotent stem cells are obtained from individuals with Dravet

Syndrome and used to generate a disease model for studying the effects of different compounds such as progesterone and mifepristone. Neuron derived from iPSCs derived from individuals with Dravet Syndrome are used to test these compounds to examine difference in seizures and response patterns to drugs in the iPSCs and the zebrafish.