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Home , Abdominal epilepsy, Eterobarb, Ilepcimide

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2013/188509 Al 19 December 2013 (19.12.2013) P O P C T

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every A61K 31/375 (2006.01) A61P 25/08 (2006.01) kind of national protection available): AE, AG, AL, AM, A61K 31/355 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (21) International Application Number: DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/US20 13/045355 HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KN, KP, KR, (22) International Filing Date: KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, 12 June 2013 (12.06.2013) MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, (25) Filing Language: English SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (26) Publication Language: English TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 61/660,207 15 June 2012 (15.06.2012) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, (71) Applicant: CREIGHTON UNIVERSITY [US/US]; 2500 UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, California Plaza, Omaha, NE 68178 (US). TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (72) Inventors: SIMEONE, Kristina; 2500 California Plaza, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, Omaha, NE 68178 (US). SIMEONE, Timothy; 2500 Cali TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, fornia Plaza, Omaha, NE 68178 (US). KM, ML, MR, NE, SN, TD, TG). (74) Agent: GRACE, Ryan, T.; Advent, LLP, 2425 South Published: 144th Street, Suite 202, Omaha, NE 68144 (US). THERAPY

BACKGROUND

[0001] is a neurological disorder defined by recurrent spontaneous . Approximately 1% of people in the US have epilepsy. Seizures are associated with cell toxicity, hyperexcitability and death. Anti-epileptic drugs act primarily on ion channels and receptors for calcium, sodium, potassium, glutamate and GABA. Current FDA approved anti-epileptic drugs that target these pathways fail to control seizures in 30% of patients.

SUMMARY

[0002] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key and/or essential features of the claimed subject matter. Also, this Summary is not intended to limit the scope of the claimed subject matter in any manner. Aspects of the disclosure pertain to methods and compositions for treating seizures.

BRIEF DESCRIPTION OF THE FIGURES

[0003] FIG. 1 depicts a graph showing the ontogeny of Kvl.l-/- seizures. On postnatal day (P)10, Kvl.l-/- mice do not exhibit seizures. Around P20, mice experience approximately 3-4 seizures daily. During their P30s, mice experience ~ 7 seizures per day.

[0004] FIG. 2 depicts a graph showing that seizure severity increases with age in Kvl .1-/- mice.

[0005] FIG. 3A depicts a schematic of mitochondrial redox and bar graph of impaired mitochondrial function in Kvl.l-/- mice. Polarometric methods monitor complex function and evaluate oxygen consumption via the coupling between transport of electrons thru complex I-IV and ATP synthesis at complex V in control wild-type (WT) and Kvl -Ι- brain tissue.

[0006] FIG. 3B shows that state III and state V respiratory functions are impaired (i.e. have reduced capacity) in epileptic Kvl.l-/-mice.

[0007] FIG. 3C shows that impaired respiratory function leads to unsuccessful transfer of electrons thru the electron transport chain and, hence, increased production of reactive oxygen species (ROS). n=5-6; * p < 0.05.

[0008] FIG. 4A depicts a schematic of fatty acid (FA, blue)-induced uncoupling.

[0009] FIG. 4B depicts a polarometric trace of functional uncoupling [i.e. fatty acid- induced respiration following blockade of complex V with oligomycin (state IV)] in wild-type (WT) mitochondria.

[0010] FIG 4C is a plot showing that Kvl.l-/- mitochondria have limited to no functional uncoupling capacity.

[0011] FIG. 5A depicts electrophysiological recordings showing inhibition of mitochondrial complex I of the electron transport chain creates a hyperexcitable environment in acute mouse hippocampal slices. Epilepsy is defined as a neuronal state of hyperexcitability and hypersynchrony. Extracellular recordings in the CA3 stratum radiatum reveals that a 30 min application of the mitochondrial complex I inhibitor rotenone (100 nM) increases the rate and amplitudes of spontaneous field potentials. The middle trace is an expansion of field potential marked by an asterisk in the top trace. Time-frequency analysis was performed on the signals to examine the component frequencies occurring during the field potentials. High frequency oscillations (HF05) in the 100-200 Hz bandwidth (called ripples) are normal and reflect small clusters of neurons firing action potentials synchronously. Rotenone application results in spectral disorganization and the generation of faster HFOs (called fast ripples) which reflects a desynchronization of neuronal firing. Fast ripples occur in humans and animals with epilepsy and are biomarkers for seizure-genic foci. [0012] FIG. 5B (Left) shows that increasing the potassium concentration in the artificial cerebral spinal fluid bath solution from 3mM to 8mM of a vehicle-treated hippocampal slice increases ripple occurrence, introduces fast ripples and causes regular seizure-like events. FIG. 5B (Right) shows that applying rotenone the intensity of ripples and fast ripples increases in the slices. Following application of high potassium, the seizure-like events are greater in amplitude and longer in duration, indicating greater seizure severity during mitochondrial dysfunction..

[0013] FIG. 6A depicts a trace and bar graphs showing that Ketogenic Diet ("KD")treatment reduces seizures and restores mitochondrial function. FIG. 6A shows a representative EEG trace of tonic-clonic seizures.

[0014] FIG. 6B shows that Kvl.l-/- mice naturally develop epilepsy with typical EEG and behavioral seizure manifestations. KD treatment significantly reduces daily Kvl.l-/- seizures (red bar) when compared to standard diet (SD) controls (black bar); n = 5-6, p<0.05.

[0015] FIG. 6C shows that KD treatment restores mitochondrial function, specifically enhances complex I driven state III respiratory capacity in Kvl.l-/- mice when compared to SD controls; n=5-6, p<0.05.

[0016] FIG. 7A depicts bar graphs, scatterplots and line graphs showing that targeting mitochondria with AATP is and antiepileptogenic. FIG. 7A is a double-plotted bar graph and scatterplot depicting the antiepileptogenic effects of AATP treatment. Young

Kvl -Ι- mice (-P22-P26) were continuously monitored for 48 hrs to establish a baseline seizure frequency and severity score for each animal. AATP treatment was then administered daily for 5 days, supplemented in their diet, and was followed by a 48 hr washout period (ascorbic acid, -570 mg/kg; sodium pyruvate, 100 mg/kg; tocopherol-succinate, -70 mg/kg). AATP diet treatment significantly reduced seizure frequency and severity when compared within groups (to pretreatment baseline scores) and between treated and age-matched controls. In addition, these effects persisted following discontinuation of treatment (washout).

[0017] FIGs 7B-C shows that AATP is anticonvulsant in severe . Mice underwent microneurosurgery and were implanted with subdural electroencephalographic (EEG) electrodes. Following a 5-7 day recovery, mice were pretreated with AATP (ascorbic acid, 250 mg/kg; sodium pyruvate, 500 mg/kg; tocopherol-succinate, 30 mg/kg, i.p. injection). Thirty min later, the proconvulsant kainic acid (10 mg/kg, i.p.) was administered to induce status epilepticus. Electroencephalographic and behavioral seizures were continuously monitored and scored for 3 hrs; *p < 0.05.

[0018] FIGs. 8A-C depict three plots showing that treating Kvl.l-/- mice with AATP completely restores mitochondrial function in Kvl.l-/- mice.

DETAILED DESCRIPTION

[0019] In one aspect, the disclosure comprises methods of treating seizures. Seizures are a transient symptom of abnormal excessive or synchronous neuronal activity in the brain. Seizures may manifest as a wild thrashing movement, a brief loss of awareness, an alteration in mental state, tonic or clonic movements, convulsions, a full body slump, and various other psychic symptoms such as deja vu or jamais vu. Recurrent, unprovoked seizures are medically known as epilepsy.

[0020] As more fully set forth herein impaired mitochondrial function is associated with seizure- genesis. This disclosure pertains to a composition that restores mitochondrial respiratory, antioxidant, and uncoupling functions in order to treat seizure. The composition may treat refractory seizures that are not treated by current seizure medicines and dietary regimens used to treat refractory seizure. In some instances, seizures may cause neuronal damage. As such, aspects of the disclosure also pertain to protection from neuronal damage by preventing seizures. (a) seizure [0021] This disclosure describes a method of treating seizure. Non limiting examples of seizures that may be treated by the composition may include seizures that occur in benign , , infantile spasms, juvenile , juvenile absence epilepsy, childhood absence epilepsy (pyknolepsy), hot water epilepsy, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Dravet syndrome, progressive myoclonus , reflex epilepsy, Rasmussen's syndrome, , limbic epilepsy, status epilepticus, abdominal epilepsy, massive bilateral myoclonus, catamenial epilepsy, Jacksonian seizure disorder, , photosensitive epilepsy, and epilepsy induced by chemoconvulsant. In nonhuman animals, chemoconvulsants may be kainite acid (KA), Pentylenetetrazole (PTZ), lithium pilocarpine, electroconvulsive shocks, vagal nerve stimulation, high frequency stimulation or kindling, and N-methyl-D-aspartate.

[0022] The method may treat seizures that occur in status epilepticus. Status epilepticus is a life-threatening condition in which the brain is in a state of persistent seizure lasting at least 1 hour, or recurrent seizures without regaining consciousness between seizures for at least 1 hour.

[0023] Treating seizures may comprise reducing seizure intensity, reducing seizure frequency, reducing epileptogenesis or a combination thereof. As used herein, the term "epileptogenesis" may be a gradual process by which a normal brain develops epilepsy resulting from acute brain insults such as traumatic brain injury, stroke, idiopathic ontogeny, or infection. In epileptogenesis, an array of events occur on molecular and cellular levels that cause neurons to fire in a disordered manner, resulting in seizures.

[0024] In some embodiments, treating seizure reduces seizure intensity. In other embodiments, treating seizure reduces seizure frequency. In yet other embodiments, treating seizures may comprise reducing epileptogenesis. In some embodiments, treating seizure reduces seizure intensity and epileptogenesis. In other embodiments, treating seizure reduces seizure frequency and epileptogenesis. In yet other embodiments, treating seizures reduces seizure frequency and seizure intensity. In other embodiments, treating seizures reduces seizure frequency, seizure intensity, and epileptogenesis.

[0025] Methods of measuring seizure intensity and frequency may include behavioral scoring, (EEG), electrocorticography (ECoG), electromyography (EMG), electrocardiography (EKG), fMRI, MRI or a combination thereof. In some embodiments, seizure intensity and frequency is measured using EEG. In other embodiments, seizure intensity and frequency is measured using behavioral scoring. Non limiting examples of behavioral scoring methods for measuring seizure intensity and frequency may include the Denver seizure score, the Racine scale, Luttjohann method, and the modified Racine scale. In some embodiments, seizure intensity and frequency is measured using the modified Racine scale for behavioral scoring as described in the examples. The modified Racine scale may be used to score seizure intensity as follows: 1, immobility; 2, forelimb and/ or tail extension; rigid posture; 3, repetitive movements; head bobbing; 4, rearing and falling; 5, continuous rearing and falling; 6, severe tonic clonic seizures

[0026] In one embodiment, a method treats seizure by restoring mitochondrial function in seizure-genic brain regions. As used herein, the term "seizure-genic brain region" may be any region of the brain where mitochondrial function may be impaired, leading to a seizure. For instance, seizures may originate from an epileptic focus where mitochondrial function is impaired, a small portion of the brain that serves as the irritant driving the epileptic response. Seizures may also originate from many independent foci (multifocal epilepsies) where mitochondrial function may be impaired, or from epileptic circuits that involve the whole brain or whole regions of the brain where mitochondrial function may be impaired. In some embodiments, the seizure-genic region of the brain is an epileptic focus in the brain where mitochondrial function may be impaired. In other embodiments, the seizure-genic region of the brain is from epileptic circuits that involve the whole brain or whole regions of the brain where mitochondrial function may be impaired.

[0027] A seizure-genic brain region may be any region of the brain where seizures may occur. Non limiting examples of a seizure-genic brain region may include the cortex, the hippocampus, the amygdala, the medulla, the pons, the thalamus, the cerebellum, the optic tectum, the pallium, the basal ganglia, the olfactory bulb, and the parahippocampal gyrus. In some embodiments, the seizure-genic brain region may be the hippocampus. In other embodiments, the seizure-genic brain region may be the cortex.

[0028] The method may be used to treat refractory seizures. As used herein, the term "refractory seizure" may be used to describe any seizures that do not come under control with established methods of controlling seizures. Non limiting examples of methods of controlling seizures may include medicines or diet changes. In some embodiments, the method of the invention may be used to treat refractory seizures that do not come under control with seizure medication. Non limiting examples of seizure medication may include such as ; Ameltolide; atolide; buramate; cinromide; citenamide; ; cyheptamide; dezinamide; dimethadione; divalproex sodium; eterobarb; ; ; hydrochloride; fluzinamide; fosphenyloin sodium; ; ilepcimide; ; magnesium sulfate; mephenyloin; mephobarbital; methetoin; methsuximide; milacemide hydrochloride; nabazenil; nafimidone hydrochloride; ; ; ; phenobarbital sodium; ; phenyloin; phenyloin sodium; ; ; ralitoline; remacemide hydrochloride; ropizine; sabeluzole; ; sulthiame; ; ; sodium; valproic acid; ; zoniclezole hydrochloride; and , and an antiepileptic such as felbanate; iamotrigine; ; tolgabide.

[0029] In other embodiments, the method may be used to treat refractory seizures that do not come under control with diet changes. Non- limiting examples of diet changes may include a ketogenic diet. The ketogenic diet is a high-fat, adequate-protein, low-carbohydrate diet used primarily to treat refractory epilepsy. Refractory seizures may not come under control with diet change such as a ketogenic diet because the subject may not be able to abide by the ketogenic diet. For instance the subject may not be able to abide by the ketogenic diet because of complicated administration regimens, palatability issues, and possible side effects due to high fat content. In some embodiments, the composition may be administered to a subject unable to abide by the ketogenic diet. [0030] Treating seizure using the method may reduce seizure frequency and intensity as measured by a seizure score. In some embodiments, seizure severity may be reduced by about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100%. In other embodiments, seizure severity may be reduced by about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or about 60%. In yet other embodiments, seizure severity may be reduced by about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or about 80%. In still other embodiments, seizure severity may be reduced by about 70, 7 1, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or about 100%.

[0031] In some embodiments, seizure intensity may be reduced by about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100%. In other embodiments, seizure intensity may be reduced by about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or about 60%. In yet other embodiments, seizure intensity may be reduced by about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or about 80%. In preferred embodiments, seizure intensity may be reduced by about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or about 100%.

(b) composition [0032] The method comprises administering a composition that restores mitochondrial function. The composition may comprise any compound that may restore mitochondrial function. Non limiting examples of compounds that may restore mitochondrial function may include ascorbic acid, vitamin E, pyruvate, ubiquinone, co-enzyme Q, or combinations thereof. Non limiting examples of combinations of compounds that may restore mitochondrial function may be as shown in Table A. Table A. Combinations Ascorbic acid Vitamin E Pyruvate Ubiquinone Co-enzyme O Ascorbic acid, Vitamin E Ascorbic acid, Pyruvate Ascorbic acid, Ubiquinone Ascorbic acid, Co-enzyme Q Vitamin E, Pyruvate Vitamin E, Ubiquinone Vitamin E, Co-enzyme Q Pyruvate, Ubiquinone Pyruvate, Co-enzyme Q Ubiquinone, Co-enzyme Q Ascorbic acid, Vitamin E, Pyruvate Ascorbic acid, Vitamin E, Ubiquinone Ascorbic acid, Vitamin E, Co-enzyme Q Ascorbic acid, Pyruvate, Ubiquinone Ascorbic acid, Pyruvate, Co-enzyme Q Ascorbic acid, Ubiquinone, Co-enzyme Q Vitamin E, Pyruvate, Ubiquinone Vitamin E, Pyruvate, Co-enzyme Q Vitamin E, Ubiquinone, Co-enzyme Q Pyruvate, Ubiquinone, Co-enzyme Q Ascorbic acid, Vitamin E, Pyruvate, Ubiquinone Ascorbic acid, Vitamin E, Pyruvate, Co-enzyme Q Ascorbic acid, Vitamin E, Ubiquinone, Co-enzyme Q Ascorbic acid, Pyruvate, Ubiquinone, Co-enzyme Q Vitamin E, Pyruvate, Ubiquinone, Co-enzyme Q Ascorbic acid, Vitamin E, Pyruvate, Ubiquinone, Co- enzyme Q

[0033] In some embodiments, the composition comprises ascorbic acid, vitamin E, and pyruvate. Ascorbic acid is a naturally occurring organic compound with antioxidant properties and is one form of vitamin C. Pyruvic acid is an organic acid, a ketone, as well as the simplest of the alpha-keto acids. In a cell, pyruvate is the output of glycolysis, the anaerobic metabolism of glucose. A group of compounds with Vitamin E activity may include tocopherols and tocotrienols. In some embodiments, the vitamin E of the composition may be a tocopherol. Non limiting examples of tocopherols that may be used in the composition may include a- tocopherol, [3-tocopherol, ytocopherol, and A-tocopherol. In some embodiments, the vitamin E of the composition may be a tocotrienol. Non limiting examples of tocotrienols that may be used in the composition may include a-tocotrienol, [3- tocotrienol, y-tocotrienol, and A- tocotrienol. In one embodiment, the vitamin E of the composition is a-tocopherol.

[0034] The dose of ascorbic acid, vitamin E, and pyruvate can vary depending on the body weight, sex, age and/or medical condition of the subject, the severity of the seizure, the method of administration, and the duration of the treatment, as well as the species of the subject.

[0035] When administered intravenously, the dose of ascorbic acid may be about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or about 300 mg/kg. In some embodiments, the intravenous dose of ascorbic acid may be about 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 2 11, 212, 213, 214, 215, 216, 217, 218, 219, or about 220 mg/kg. In other embodiments, the intravenous dose of ascorbic acid may be about 210, 2 11, 212, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, or about 230 mg/kg. In yet other embodiments, the intravenous dose of ascorbic acid may be about 220, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239 or about 240 mg/kg. In other embodiments, the intravenous dose of ascorbic acid may be about 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or about 250 mg/kg. In other embodiments, the intravenous dose of ascorbic acid may be about 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269 or about 270 mg/kg. In still other embodiments, the intravenous dose of ascorbic acid may be about 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, or about 280 mg/kg. In yet other embodiments, the intravenous dose of ascorbic acid may be about 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 or about 290 mg/kg. In additional embodiments, the intravenous dose of ascorbic acid may be about 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299 or about 300 mg/kg. In still other embodiments, the intravenous dose of ascorbic acid may be about 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259 or about 260 mg/kg. [0036] When administered intraorally, the dose of ascorbic acid may be about 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or about 700 mg/kg. In some embodiments, the intraoral dose of ascorbic acid may be about 500, 510, 520, 530, 540, 550, 560, 570, 580, 590 or about 600 mg/kg. In other embodiments, the intraoral dose of ascorbic acid may be about 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or about 700 mg/kg. In still other embodiments, the intraoral dose of ascorbic acid may be about 550, 560, 570, 580, 590, 600, 610, 620, 630, 640 or about 650 mg/kg.

[0037] When administered intravenously, the dose of vitamin E may be about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or about 40 mg/kg. In some embodiments, the intravenous dose of vitamin E may be about 20, 21, 22, 23, 24, or about 25 mg/kg. In other embodiments, the intravenous dose of vitamin E may be about 25, 26, 27, 28, 29, or about 30 mg/kg. In yet other embodiments, the intravenous dose of vitamin E may be about 30, 31, 32, 33, 34 or about 35 mg/kg. In other embodiments, the intravenous dose of vitamin may be about 35, 36, 37, 38, 39 or about 40 mg/kg. In still other embodiments, the intravenous dose of ascorbic acid may be about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or about 35 mg/kg.

[0038] When administered intraorally, the dose of vitamin E may be about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or about 100 mg/kg. In some embodiments, the intraoral dose of vitamin E may be about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or about 70 mg/kg. In other embodiments, the intraoral dose of vitamin E may be about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or about 80 mg/kg. In yet other embodiments, the intraoral dose of vitamin E may be about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or about 90 mg/kg. In other embodiments, the intraoral dose of vitamin E may be about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or about 90 mg/kg. In additional embodiments, the intraoral dose of vitamin E may be about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or about 100 mg/kg. In still other embodiments, the intraoral dose of vitamin E may be about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or about 85 mg/kg.

[0039] When administered intravenously, the dose of pyruvate may be about 450, 460, 470, 480, 490, 500, 510, 520, 530, 540 or about 550 mg/kg. In some embodiments, the intravenous dose of pyruvate may be about 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, or about 470 mg/kg. In other embodiments, the intravenous dose of pyruvate may be about 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, or about 480 mg/kg. In yet other embodiments, the intravenous dose of pyruvate may be about 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489 or about 490 mg/kg. In other embodiments, the intravenous dose of pyruvate may be about 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or about 500 mg/kg. In other embodiments, the intravenous dose of pyruvate may be about 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, or about 510 mg/kg. In still other embodiments, the intravenous dose of pyruvate may be about 500,

501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 5 11, 512, 513, 514, 515, 516, 517, 518, 519, or about 520 mg/kg. In yet other embodiments, the intravenous dose of pyruvate may be about 510, 5 11, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529 or about 530 mg/kg. In additional embodiments, the intravenous dose of pyruvate may be about 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539 or about 540 mg/kg. In still other embodiments, the intravenous dose of pyruvate may be about 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549 or about 550 mg/kg.

[0040] When administered intraorally, the dose of pyruvate may be about 1, 2, 3, 4, or about 5 g/kg. In some embodiments, the intraoral dose of pyruvate may be about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or about 5 mg/kg. In still other embodiments, the intraoral dose of pyruvate may be about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or about 3 mg/kg. [0041] In an exemplary embodiment, 250 mg/kg ascorbic acid, 500 mg/kg pyruvate, and 30 mg/kg tocopherol are administered intravenously. In another exemplary embodiment, 600 mg/kg ascorbic acid, 2.5 g/kg pyruvate, and 75 mg/kg tocopherol are administered orally.

[0042] Administering multiple doses of the composition per day may also be used as needed to provide the desired level of seizure therapy. For instance, one, two, three, four, or more doses of the composition may be administered per day. The composition may be administered before, during or after the seizures occur. In some embodiments, the composition may be administered before the seizures occur. In other embodiments, the composition may be administered during seizures. In yet other embodiments, the composition may be administered after the seizures occur.

(c) subject [0043] The method comprises administering a composition to a subject. Suitable subjects may include animals and humans. Non-limiting examples of suitable animals include companion animals such as cats, dogs, rodents, and horses; research animal such as mice, rats and other rodents; agricultural animals such as cows, cattle, pigs, goats, sheep, horses, deer, chickens and other fowl; zoo animals; and primates such as chimpanzees, monkeys, and gorillas. In one embodiment, the subject is a human. In another embodiment, the subject is a human with refractory seizure. In still another embodiment, the subject is a mouse. In yet another embodiment, the subject is the Kvl . 1 knockout mouse.

(d) administration [0044] The composition may be administered as oral, intravenous, intramuscular, subcutaneous, or parenteral routes. In some embodiments, the composition may be formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these compositions can be combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like. [0045] The composition may comprise a pharmaceutical carrier (or excipient). Such a carrier may be any solvent or solid material for encapsulation that is non-toxic. A carrier may give form or consistency, or act as a diluent. Suitable pharmaceutical carriers may include liquid carriers, such as normal saline and other non-toxic salts at or near physiological concentrations, and solid carriers not used for humans, such as talc or sucrose, or animal feed. Carriers may also include stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Carriers and excipients as well as formulations for parenteral and nonparenteral drug delivery are set forth in Remington's Pharmaceutical Sciences 19th Ed. Mack Publishing (1995).

[0046] For parenteral administration (including subcutaneous, intradermal, intravenous, intramuscular, and intraperitoneal), the composition may be an aqueous or an oil-based solution. Aqueous solutions may include a sterile diluent such as water, saline solution, a pharmaceutically acceptable polyol such as glycerol, propylene glycol, or other synthetic solvents; an antibacterial and/or antifungal agent such as benzyl , methyl paraben, , , thimerosal, and the like; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as etheylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and/or an agent for the adjustment of tonicity such as sodium chloride, dextrose, or a polyalcohol such as mannitol or sorbitol. The pH of the aqueous solution may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Oil-based solutions or suspensions may further comprise sesame, peanut, olive oil, or mineral oil.

[0047] In one embodiment, the composition may be administered orally. Non-limiting examples of oral formulations that may be used to administer the amino acid composition of the invention may be a nutritional formulation, a medical food, a medical beverage, in the form of a complete meal, part of a meal, as a food additive as a powder for dissolution, in the form of a pharmaceutical formulation such as in the form of a tablet, pill, sachet or capsule or by tube feeding such as by means of nasogastric, nasoduodenal, esophagostomy, gastrostomy, or jejunostomy tubes, or peripheral or total parenteral nutrition. In another embodiment, the compositions may be administered orally as a dietary supplement.

[0048] Compositions for oral administration generally contain inert excipients in addition to the amino acid ingredients of the composition. Oral preparations may be enclosed in gelatin capsules or compressed into tablets. Common excipients used in such preparations include pharmaceutically compatible fillers/diluents such as microcrystalline cellulose, hydroxypropyl methylcellulose, starch, lactose, sucrose, glucose, mannitol, sorbitol, dibasic calcium phosphate, or calcium carbonate; binding agents such as alginic acid, carboxymethylcellulose, microcrystalline cellulose, gelatin, gum tragacanth, or polyvinylpyrrolidone; disintegrating agents such as alginic acid, cellulose, starch, or polyvinylpyrrolidone; lubricants such as calcium stearate, magnesium stearate, talc, silica, or sodium stearyl fumarate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or citrus flavoring; coloring agents; and preservatives such as antioxidants (e.g., vitamin A, vitamin C, vitamin E, or retinyl palmitate), citric acid, or sodium citrate. Oral preparations may also be administered as aqueous suspensions, elixirs, or syrups. For these, the active ingredient may be combined with various sweetening or flavoring agents, coloring agents, and, if so desired, emulsifying and/or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof.

[0049] The compositions may be nutritionally complete, i.e. may include other vitamins, minerals, trace elements as well as , carbohydrate and fat and/or fatty acid sources so that they may be used as the sole source of nutrition supplying essentially all the required daily amounts of vitamins, minerals, carbohydrates, fat and/or fatty acids, proteins and the like.

(e) method of inducing seizure-like activity [0050] Other aspects of the disclosure include a method of inducing seizure-like activity. The method may be used to induce seizure-like activity in vitro by impairing mitochondrial function. In some embodiments, the method of inducing seizures comprises administering a composition that impairs mitochondrial function to a seizure-genic brain region, the hippocampus, in vitro. Non-limiting examples of compositions that may be used to impair mitochondrial function may include rotenone, As203, antimycin, cyanide, malonate, 2,4-dinitrophenol (DNP), carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP), and oligomycin. EXAMPLES

[0051] The following examples illustrate various iterations of the above disclosure.

Example 1. Bioenergetics are impaired in epilepsy. [0052] The Kvl.l knockout mouse naturally develops epilepsy with typical EEG and behavioral seizure manifestations. Seizure onset in the Kvl.l knockout mouse may occur during the third postnatal week, experiencing 7 seizures each day, with seizure frequency (FIG. 1) and severity (FIG. 2) continuing to increase with age. In these mice, it was found that three mitochondrial functions are impaired in seizure-genic brain regions. Mitochondria are organelles responsible for regulating cellular bioenergetic homeostasis. Due to its high- energy demand and metabolism, brain tissue, and particularly seizure-generating regions such as the hippocampus and cortex, is highly susceptible to impairment of mitochondrial function. Mutations to mitochondrial DNA and impaired mitochondrial respiratory capacity (i.e., energy failure) have been linked to several epilepsy syndromes; thus, deficiencies in cellular and network energy balance are associated with increased probability of hyperexcitable circuits. [0053] Oxidative phosphorylation (redox) occurs at mitochondrial respiratory complexes I-V located in the inner mitochondrial membrane (FIG. 3A). Mitochondrial complex I deficiency has been reported in the temporal lobe of epileptic patients. Functional mitochondrial impairments to proteins in the redox chain can lead to failed transfer of electrons, which then combine with molecular oxygen to form a series of reactive oxygen species (ROS) and nitrogen species. At suprathreshold levels, ROS damages DNA, oxidizes fatty acids in lipids and amino acids in proteins, damages mitochondrial, nuclear and lipid cellular membranes and can mediate mitochondrial permeability transition, which initiates cell injury and death cascades; collectively, interfering with normal bioenergetic homeostasis. In Kvl.l-/- mice, polarographic measurements indicate that complex I-driven state III and state V respiration are significantly reduced in isolated hippocampal and cortical mitochondria (FIG. 3B). This data suggests proteins comprising complex I and V subunits are reduced or damaged. Mitochondria provide a majority of cellular energy in the form of ATP. Reduced state III respiration may produce less ATP. ATP homeostasis helps maintain the neuronal membrane potential and thus is positioned to guard against hyperexcitability. [0054] First, it was discovered that isolated mitochondria from adult Kvl.l-/- mice generate significantly more ROS when compared to wild-type controls (FIG. 3C). It is also shown that ROS production increases during seizures and remains elevated for prolonged periods following seizure cessation. Higher constitutive oxidative stress experienced by the cell under normal conditions, and then exacerbated during seizure events (Kvl.l-/- mice experience ~7 seizures a day), can lead to damaged cellular membranes, proteins/ ion channels/ receptors, nucleotides (DNA R A) and further mitochondrial damage, thus perpetuating cellular bioenergetic imbalance.

[0055] Second, the testing found reduced functional uncoupling in Kvl.l-/- mice (FIGs. 4A-C). In the presence of free fatty acids, mitochondrial uncoupling proteins (UCPs, specifically UCP2 in the brain) translocate protons across the inner membrane back into the matrix (FIG. 4A) to uncouple or release the dependency of the redox chain on complex V (FIG. 4B). This allows for electrons to be successfully transferred, thus reducing ROS and the probability of permeability transition. It has been found that hippocampal mitochondria from rats with kainate-induced seizures have reduced functional uncoupling and enhanced ROS production. In addition, it has been found that acute application of fatty acids functionally uncouples control, but not Kvl.l-/- mitochondria (FIG. 4C). Because in these experiments, complex V is blocked by oligomycin (and therefore fatty-acid induced uncoupling is independent of the redox chain), this data indicates that Kvl -Ι- mitochondria have impaired or reduced expression of UCPs. Uncoupling capacity (i.e. enhanced ability to successfully transfer electrons) helps to withstand seizure- induced hypermetabolic states. Limited uncoupling capacity can result in a significant increase in ROS and cellular injury, thus interfering with bioenergetic homeostasis.

Example 2. Experimentally impairing mitochondria in control tissue promotes seizures. [0056] Impairing mitochondrial function alone is sufficient to promote seizures. In Kvl.l-/- hippocampal slices, pathological high frequency oscillations are prevalent throughout the in Kvl.l-/- hippocampal network. In addition, there is an increase in neurotransmitter release probabilities at specific hippocampal circuit synapses. To experimentally mimic the mitochondrial pathology seen in epilepsy, a drug that impairs mitochondrial function at respiratory complex I, rotenone, was applied to control tissue. It was found that acute application of rotenone is sufficient to promote seizures (FIG. 5A-B). Specifically, rotenone application promotes spectral disorganization and the generation of pathologic high frequency oscillations (called fast ripples) which reflects a desynchronization of neuronal firing. Fast ripples occur in humans and animals with epilepsy and are biomarkers for seizure-genic foci.

Example 3. Ketogenic diet and seizures. [0057] In patients with refractory seizures, the ketogenic diet (KD) is effective (specifically against intractable epilepsy and partial and generalized seizures), resulting in complete cessation of seizures in at least 7- 10% of patients and >50% reduction in seizure frequency in two-thirds of patients. Unfortunately, particularly in adult patients on the KD, noncompliance is high due to complicated administration regimens, palatability issues and the possibility of side effects due to the high fat content. The efficacy of the KD suggests that it targets a critical and novel anticonvulsant mechanism(s), which is effective against multiple epilepsy syndromes. The fact that seizure control gradually improves over the first few weeks of the KD, indicates that the anticonvulsant constituent of the KD does not merely act on a receptor or ion channel, but may require a longer temporal mechanism of action.

[0058] KD treatment reduces seizures of Kvl.l-/- mice by 75-100% (FIG. 6A and B). KD treatment is associated with increasing expression of at least 30 bioenergetic proteins, including mitochondrial complex I, 11 and V, and UCP2, as well as endogenous antioxidants. Two weeks of KD treatment fully restores state III respiration and reduces ROS production in Kvl.l-/- mitochondria to control values (FIG. 6B). In addition, it was found that KD treatment increases UCP expression and function in mitochondria isolated from control and Kvl.l-/- mice (FIG. 6C). Collectively, these data indicate that the KD promotes bioenergetic homeostasis.

Example 4. Restoration of bioenergetic homeostasis alone is anticonvulsant. [0059] Over ninety percent of the caloric intake in the KD is from fat. This high fat:low carbohydrate/protein ratio (3:1 or 4:1) of the KD forces fat catabolism as the primary energy source. Interestingly, fatty acids, specifically long chain polyunsaturated fatty acids, promote bioenergetic homeostasis. Specifically, they are used as metabolic fuels, signaling molecules, increase bioenergetic proteins and ATP production, and improve mitochondrial redox chain function and uncoupling functions. Seizures expend energy and deplete reserve stores, thus compromising cerebral energy balance. Therefore, targeting mechanisms responsible for maintaining energy homeostasis may provide novel anticonvulsant targets.

[0060] To that end, a treatment was designed to target specific components of the mitochondria to enhance mitochondrial bioenergetic functions called AATP. AATP comprises pyruvate, vitamin E, and vitamin C. The doses used are: ascorbic acid (vitamin C), 250 mg/kg; sodium pyruvate, 500 mg/kg; tocopherol-succinate (Vitamin E), 30 mg/kg. When administered orally, the doses used were: ascorbic acid (vitamin C), 600 mg/kg; sodium pyruvate, 2.5 g/kg; tocopherol-succinate (Vitamin E), about 75 mg/kg.

[0061] When treated with AATP, seizures induced by kainic acid 30 minutes after treatment with AATP are significant. In vitro, it was found that acute treatment that targets mitochondria is anticonvulsant in epileptic hippocampal slices. Daily treatment of AATP to idiopathic Kvl.l- /- model significantly reduced the onset of epilepsy, or epileptogenesis, seizure frequency and severity (FIG. 7A). Notably, after discontinuation of treatment (washout), seizures remained reduced when compared to age-matched controls. Furthermore, acute AATP treatment was significantly anticonvulsant during the severe state of status epilepticus (FIG. 7B and C). Furthermore, treating Kvl.l-/- mice with AATP completely restored mitochondrial function in

Kvl .1-1- mice in a manner that is similar to the effects of KD, (FIG. 8A-C).

[0062] Collectively, the data presented herein indicate that mitochondria impairment is sufficient to promote seizure-genesis and that collective restoration of mitochondrial respiratory, antioxidant and uncoupling functions can be an effective anticonvulsant strategy in epilepsy, particularly for refractory epilepsies. CLAIMS

1. A method of treating seizures, the method comprising administering a composition that restores mitochondrial function to a seizure-genic brain region.

2. The method of claim 1, wherein the composition comprises ascorbic acid, vitamin E, and pyruvate.

3. The method of claim 2, wherein the vitamin E is tocopherol.

4. The method of claim 1, wherein the composition is administered orally.

5. The method of claim 1, wherein the composition is administered intraperitoneally.

6. The method of claim 1, wherein the composition is administered intravenously.

7. The method of claim 1, wherein 50 mg/kg ascorbic acid, 500 mg/kg pyruvate, and 30 mg/kg tocopherol are administered intravenously.

8. The method of claim 1, wherein 600 mg/kg ascorbic acid, 2.5 g/kg pyruvate, and 75 mg/kg tocopherol are administered orally.

9. The method of claim 1, wherein treating seizure reduces seizure frequency.

10. The method of claim 1, wherein treating seizure reduces seizure intensity.

11. The method of claim 1, wherein treating seizure reduces epileptogenesis.

12. The method of claim 1, wherein the seizure is status epilepticus seizure.

13. The method of claim 1, wherein the seizure-genic brain region is the hippocampus or cortex.

14. The method of claim 1, wherein mitochondrial function is selected from the group consisting of respiratory, antioxidant, uncoupling functions of the mitochondria, and a combination thereof.

15. A method of treating a seizure, the method comprising administering a composition that restores mitochondrial function to a seizure-genic brain region, wherein the composition is administered to a subject suffering from seizure. 16. The method of claim 15, wherein seizure is a refractory seizure that does not respond to seizure medications.

17. The method of claim 16, wherein seizure medications comprises a ketogenic diet.

18. A method of treating a seizure, the method comprising administering a composition that restores mitochondrial function in a brain region, wherein the composition is administered to a subject unable or unwilling to abide by the ketogenic diet or any other dietary or pharmaceutical anticonvulsant treatment.

19. The method of claim 17, wherein the subject is unable to abide by the ketogenic diet because of complicated administration regimens, palatability issues, and possible side effects due to high fat content.

20. A method of inducing seizure-like activity, the method comprising administering a composition that impairs mitochondrial function to a seizure-genic brain region.

2 1. The method of claim 19, wherein seizure-like activity is induced by administering rotenone.

22. A composition, the composition comprising a combination of acorbic acid, pyruvate, and tocopherol, wherein the ratio of ascorbic acid to tocopherol is between about 1:6 and about 1:10, the ratio of ascorbic acid to pyruvate is between about 1:1.5 and about 1:4.5, and the ratio of tocopherol to pyruvate is between about 1:12 and about 1:40.

23. The composition of claim 22, wherein the composition is formulated for oral administration.

24. The composition of claim 22, wherein the composition is formulated for IV administration

25. The composition of claim 22, wherein the ratio of ascorbic acid to tocopherol is between about 1:7 and about 1:9.

26. The composition of claim 22, wherein the ratio of ascorbic acid to tocopherol is about 1:8.

27. The composition of claim 22, wherein the ratio of ascorbic acid to pyruvate is about 1:2. 28. The composition of claim 22, wherein the ratio of ascorbic acid to pyruvate is about 1:4.

29. The composition of claim 22, wherein the ratio of tocopherol to pyruvate is about 1:16.

30. The composition of claim 22, wherein the ratio of tocopherol to pyruvate is about 1:33.

A. CLASSIFICATION OF SUBJECT MATTER A61K 31/375(2006.01)i, A61K 31/355(2006.01)i, A61P 25/08(2006.01)i

According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) A61K 31/375; A61K 31/19; A61K 31/71 ; A61K 31/355; A61P 25/08

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Korean utility models and applications for utility models Japanese utility models and applications for utility models

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) eKOMPASS(KIPO internal) & Keywords: pyruvate, ascorbic acid, tocopherol, mitochondria, seizure, epilepsy

DOCUMENTS CONSIDERED TO BE RELEVANT

Category' Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

X WO 93-16690 Al (WARNER-LAMBERT COMPANY) 02 September 1993 22-30 See pages 27-30 , 46 and 47 ; and c l aims 1 , 4 , 6 and 7 .

SANTOS , I . M. S . e t a l . , Oxi dat ive stress in the hippocampus dur ing 22-30 exper iment a l s e izures can be amel i orated wi t h the ant i oxi dant ascorb i c a c i d " , Ox i dat ive Medi c ine and Ce l lul ar Longevi ty, 2009 , Vo l . 2 , I ssue 4 , pages 214-221 . See pages 214-216 and 218 .

BERBEL-GARCIA, A. e t a l . , Coenzyme Q 10 improves l act i c a c idos i s , 22-30 stroke l ike ep i sodes , and ep i l epsy i n a pat i ent wi t h MELAS (mi tochondr i a l myopathy, encephal opathy, l act i c a c i dos i s , and strokel ike ep i sodes) , Cl ini cal Neuropharmaco ogy, 2004, Vol . 27 , Issue 4 , pages 187-191 . See abstract .

BINDOKAS, V. P . e t a . , " Changes i n mi tochondr i a l funct ion resul t ing from 22-30 synapt i c act ivi t y i n the rat hippocampal s l i ce " , The Journal o f Neurosc ience , 1998 , Vo l . 18 , I ssue 12 , pages 4570-4587 . See abstract ; and pages 4570 and 4585 .

Further documents are listed in the continuation of Box C. See patent family annex.

Special categories of cited documents: later document published after the international filing date or priority document defining the general state of the art which is not considered date and not in conflict with the application but cited to understand to be of particular relevance the principle or theory underlying the invention earlier application or patent but published on or after the international document of particular relevance; the claimed invention cannot be filing date considered novel or cannot be considered to involve an inventive document which may throw doubts on priority claim(s) or which is step when the document is taken alone cited to establish the publication date of citation or other document of particular relevance; the claimed invention cannot be special reason (as specified) considered to involve an inventive step when the document is document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such combination means being obvious to a person skilled in the art document published prior to the international filing date but later document member of the same patent family than the priority date claimed Date of the actual completion of the international search Date of mailing of the international search report 26 September 2013 (26.09.2013) 26 September 2013 (26.09.2013) Name and mailing address of the ISA/KR Authorized officer * *» Korean Intellectual Property Office

1 9 Cheongsa-ro, Seo-gu, Daejeon Metropolitan City, CHOI Sung Hee ¾. 302-70 1, Republic of Korea * Facsimile No. +82-42-472-7140 Telephone No. +82-42-481-8740 Form PCT/ISA/210 (second sheet) (July 2009) C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

A SHOFFNER, J . M. et a l . , Myoc loni c ep i l epsy and ragged-red f iber di sease 22-30 (MERRF) i s assoc i ated wi t h a mi tochondr i a l DNA tRNA(Lys) mut at i on , Cel l , 1990 , Vo l . 61, pages 931-937 . See summary; and pages 931 and 935 .

Form PCT/ISA/210 (continuation of second sheet) (July 2009) Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)

This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:

Claims Nos.: 1- 1 because they relate to subject matter not required to be searched by this Authority, namely: Claims 1-21 pertain to methods for treatment of the human body by therapy, and thus relate to a subject matter which this International Searching Authority is not required, under Article 17(2)(a)(i) of the PCT and Rule 39.1(iv) of the Regulations under the PCT, to search.

Claims Nos.: □ because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically:

Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box No. Ill Observations where unity of invention is lacking (Continuation of item 3 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:

I IAs all required addtional search fees were timely paid by the applicant, this international search report covers all searchable claims.

I IAs all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment of any additional fee.

I IAs only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos.:

4. I INo required additional search fees were timely paid by the applicant. Consequently, this international search report restricted to the invention first mentioned in the claims; it is covered by claims Nos.:

Remark on Protest | | The additional search fees were accompanied by the applicant's protest and, where applicable, the payment of a protest fee. I IThe additional search fees were accompanied by the applicant's protest but the applicable protest fee was not paid within the time limit specified in the invitation. I INo protest accompanied the payment of additional search fees.

Form PCT/ISA/210 (continuation of first sheet (2)) (July 2009) Information on patent family members PCT/US2013/045355

Patent document Publication Patent family Publication cited in search report date member(s) date

WO 93-16690 Al 02/09/1993 AU 1992-12718 C 06/10/1992 AU 1995-28707 B2 21/01/1999 AU 1995-29080 B2 28/01/1999 AU 1995-30042 B2 21/01/1999 AU 1995-30045 B2 08/07/1999 AU 1996-56311 B2 09/12/1999 AU 1996-57885 B2 21/10/1999 AU 3315397 A 30/10/1997 AU 4275793 A 13/09/1993 AU 668084 B2 26/04/1996 CA 2104461 Al 02/09/1992 CA 2104461 C 30/07/2002 CA 2129732 Al 02/09/1993 CA 2218619 Al 28/11/1996 CA 2218619 C 28/08/2007 EP 0573465 Al 15/12/1993 EP 0573465 Bl 02/04/1997 EP 0627917 Al 14/12/1994 EP 0754052 Al 10/04/2002 EP 0754052 Bl 11/12/2002 EP 0759783 Al 20/02/2002 EP 0768877 Al 19/06/2002 EP 0773795 Al 09/02/2005 EP 0779820 Al 03/09/2003 EP 0783398 Al 16/07/1997 EP 0783398 Bl 09/01/2002 EP 0792163 Al 05/07/2000 EP 0792163 Bl 15/05/2002 EP 0796107 Al 02/05/2002 EP 0828514 Al 26/11/2003 EP 0828515 Al 27/12/2000 JP 09-511746 A 25/11/1997 JP 10-502344 A 03/03/1998 JP 10-502345 A 03/03/1998 JP 10-503200 A 24/03/1998 JP 10-505057 A 19/05/1998 JP 10-509451 A 14/09/1998 JP 2001-501576 A 06/02/2001 JP 2002-356421 A 13/12/2002 JP 2002-514937 A 21/05/2002 JP 2003-231632 A 19/08/2003 MX 9301014 A 01/09/1993 US 5602183 A 11/02/1997 US 5614561 A 25/03/1997 US 5633285 A 27/05/1997 US 5641814 A 24/06/1997 US 5646190 A 08/07/1997 us 5648380 A 15/07/1997 us 5652274 A 29/07/1997

Form PCT/ISA/210 (patent family annex) (July 2009) Information on patent family members PCT/US2013/045355

Patent document Publication Patent family Publication cited in search report date member(s) date

US 5658956 A 19/08/1997 us 5658957 A 19/08/1997 us 5663208 A 02/09/1997 us 5674912 A 07/10/1997 us 5692302 A 02/12/1997 us 5856364 A 05/01/1999 us 5863938 A 26/01/1999 us 5874479 A 23/02/1999 us 5981606 A 09/11/1999 o 92-15292 Al 17/09/1992 o 95-27501 Al 19/10/1995 wo 96-00572 Al 11/01/1996 wo 96-00584 Al 11/01/1996 wo 96-03149 Al 08/02/1996 wo 96-06640 Al 07/03/1996 wo 96-10474 Al 11/04/1996 wo 96-14868 Al 23/05/1996 wo 96-17624 Al 13/06/1996 wo 96-37227 Al 28/11/1996 wo 96-37228 Al 28/11/1996 wo 96-37229 Al 28/11/1996 wo 96-37230 Al 28/11/1996 ZA 9301297 A 27/06/1994

Form PCT/ISA/210 (patent family annex) (July 2009)