US009700541B2 (i2) United States Patent (io) Patent No.: US 9,700,541 B2 Simeone et al. (45) Date of Patent: Jul. 11, 2017 (54) SEIZURE THERAPY (56) References Cited (71) Applicant: Creighton University, Omaha, NE FOREIGN PATENT DOCUMENTS (US) WO 9316690 9/1993 WO WO 9316690 A1 * 9/1993 ............ A61K 8/361 (72) Inventors: Kristina A. Simeone, Omaha, NE (US); Timothy A. Simeone, Omaha, NE (US) OTHER PUBLICATIONS Itala Monica Sales Santos, Adriana Da Rocha Tome; Glaucio Barros (73) Assignee: Creighton University, Omaha, NE Saldanha, Paulo Michel Pinheiro Ferreira, Gardenia Carmem (US) Gadelha Militao, and Rivelilson Mendes De Freitas; “Oxidative stress in the hippocampus during experimental seizures can be ( * ) Notice: Subject to any disclaimer, the term of this ameliorated with the antioxidant ascorbic acid”; Oxidative Medi­ patent is extended or adjusted under 35 cine and Cellular Longevity; 2009; vol. 2, Issue 4; pp. 214-221. Angel Barbel-Garcia, Jose Ramon Barbera-Farre, Jesus Porta Etes- U.S.C. 154(b) by 0 days. sam, Antonio Martinez Salio, Ana Cabello, Eduardo Gutierrez- Rivas, and Yolanda Campos; “Coenzyme Q 10 Improves Lactic (21) Appi. No.: 14/405,816 Acidosis, Strokelike Episodes, and Epilepsy in a Patient with MELAS (Mitochondrial Myopathy, Encephalopathy, Lactic Acido­ sis, and Strokelike episodes)”, Clinical Neuropharmacology; 2004; (22) PCT Filed: Jun. 12, 2013 vol. 27, Issue 4; pp. 187-191. Vytautas P. Bindokas, Chong C. Lee, William F. Colmers, and (86) PCT No.: PCT/US2013/045355 Richard J. Miller; “Changes in Mitochondrial Function Resulting from Synaptic Activity in the Rat Hippocampal Slice”; 1998; vol. § 371 (c)(1), 18, Issue 12; pp. 4570-4587. (2) Date: Dec. 5, 2014 John M. Shoffner, Marie T. Lott, Angela M. S. Lezza, Peter Seibel, Scott W. Ballinger, and Douglas C. Wallace; “Myoclonic Epilepsy and Ragged-Red Fiber Disease (MERRF) is Associated with a (87) PCT Pub. No.: WO2013/188509 Mitochondrial DNA rRNAlys Mutation”; 1990; vol. 61; pp. 931 - 937. PCT Pub. Date: Dec. 19, 2013 * cited by examiner (65) Prior Publication Data Primary Examiner — Heidi Reese US 2015/0164857 Al Jun. 18, 2015 (74) Attorney, Agent, or Firm — Advent, LLP (57) ABSTRACT A method and a composition are utilized to affect mitochon­ Related U.S. Application Data drial functions on a seizure-genic brain region. In one (60) Provisional application No. 61/660,207, filed on Jun. aspect, the disclosure comprises methods of treating sei­ 15, 2012. zures. Seizures are a transient symptom of abnormal exces­ sive or synchronous neuronal activity in the brain. Seizures may manifest as a wild thrashing movement, a brief loss of (51) Int. Cl. awareness, an alteration in mental state, tonic or clonic A61K 31/355 (2006.01) movements, convulsions, a full body slump, and various A61K 31/375 (2006.01) other psychic symptoms such as déjà vu or jamais vu. A61K 31/19 (2006.01) Recurrent, unprovoked seizures are medically known as (52) U.S. Cl. epilepsy. In one implementation, the method of treating a CPC ........... A61K 31/355 (2013.01); A61K 31/19 seizure includes administering a composition that restores (2013.01); A61K 31/375 (2013.01) mitochondrial function to a seizure-genic brain region. In (58) Field of Classification Search some embodiments, the composition can include ascorbic None acid, vitamin E, and pyruvate. See application file for complete search history. 17 Claims, 12 Drawing Sheets U.S. Patent Jul. 11,2017 Sheet 1 of 12 US 9,700,541 B2 FIG. FIG. 1 Seizure Incidence Seizure U.S. Patent Jul. ll, 2017 Sheet 2 of 12 US 9,700,541 B2 FIG. FIG. 2 U.S. Patent In te r- Complex I Complex II Complex III Complex IV Complex V membrane 4H + 4H + 2H + H + Jul. Jul. ll, 2017 2017 Sheet 3 of3 12 12 FIG. 3A US 9,700,541 B2 U.S. Patent Jul. ll, 2017 Sheet 4 of 12 US 9,700,541 B2 ROS FIG. FIG. 3C % Control % State V FIG. FIG. 3B State II U.S. Patent Jul. 11, 2017 Sheet 5 of 12 US 9,700,541 B2 FIG. FIG. 4A o U.S. Patent Jul. ll, 2017 Sheet 6 of 12 US 9,700,541 B2 FIG. FIG. 4C Functional Uncoupling % State IV State % FIG. FIG. 4B WT WT Polarometric Trace State III U.S. Patent Jul. ll, 2017 Sheet 7 of 12 US 9,700,541 B2 m s m s FIG. FIG. 5A Baseline Rotenone U.S. Patent Vehicle Rotenone High Potassium Jul. ll, 2017 2017 Sheet 8 600 N 400 200 0 U 1 hr of 12 12 FIG. 5B US 9,700,541 B2 U.S. Patent Jul. ll, 2017 Sheet 9 of 12 US 9,700,541 B2 FIG. FIG. 6B FIG. 6C U.S. Patent Jul. ll, 2017 Sheet 10 of 12 US 9,700,541 B2 FIG. FIG. 7B r U.S. Patent Jul. 11,2017 Sheet 11 of 12 US 9,700,541 B2 Vehicle+KA AATP+KA * 'b -□ -B- FIG. FIG. 7C Time (hr) U.S. Patent Jul. 11,2017 Sheet 12 of 12 US 9,700,541 B2 State III respiration in hippocampus Stnte Ill resnirntinn in nnrtey US 9,700,541 B2 1 2 SEIZURE THERAPY exhibit seizures. Around P20, mice experience approxi­ mately 3-4 seizures daily. During their P30s, mice experi­ BACKGROUND ence -7 seizures per day. FIG. 2 depicts a graph showing that seizure severity Epilepsy is a neurological disorder defined by recurrent 5 increases with age in K v l.l-/- mice. spontaneous seizures. Approximately 1% of people in the FIG. 3A depicts a schematic of mitochondrial redox and US have epilepsy. Seizures are associated with cell toxicity, bar graph of impaired mitochondrial function in K v l.l-/- hyperexcitability and death. Anti-epileptic drugs act primar­ mice. Polarometric methods monitor complex function and ily on ion channels and receptors for calcium, sodium, evaluate oxygen consumption via the coupling between potassium, glutamate and GABA. Current FDA approved 10 transport of electrons thru complex I-IV and ATP synthesis anti-epileptic drugs that target these pathways fail to control at complex V in control wild-type (WT) and Kv 1.1-/- brain seizures in 30% of patients. tissue. FIG. 3B shows that state III and state V respiratory SUMMARY 15 functions are impaired (i.e. have reduced capacity) in epi­ leptic K v l.l-/- mice. In one aspect, the disclosure comprises methods of treat­ FIG. 3C shows that impaired respiratory function leads to ing seizures. Seizures are a transient symptom of abnormal unsuccessful transfer of electrons thru the electron transport excessive or synchronous neuronal activity in the brain. chain and, hence, increased production of reactive oxygen Seizures may manifest as a wild thrashing movement, a brief 20 species (ROS). n=5-6; * p<0.05. loss of awareness, an alteration in mental state, tonic or FIG. 4A depicts a schematic of fatty acid (FA, blue)- clonic movements, convulsions, a full body slump, and induced uncoupling. various other psychic symptoms such as déjà vu or jamais FIG. 4B depicts a polarometric trace of functional uncou­ vu. Recurrent, unprovoked seizures are medically known as pling [i.e. fatty acid-induced respiration following blockade epilepsy. 25 of complex V with oligomycin (state IV)] in wild-type (WT) In one implementation, the method of treating a seizure mitochondria. includes administering a composition that restores mito­ FIG. 4C is a plot showing that K v l.l-/- mitochondria chondrial function to a seizure-genic brain region. In another have limited to no functional uncoupling capacity. implementation, the method of treating a seizure includes FIG. 5A depicts electrophysiological recordings showing administering a composition that restores mitochondrial 30 inhibition of mitochondrial complex I of the electron trans­ function to a seizure-genic brain region, wherein the com­ port chain creates a hyperexcitable environment in acute position is administered to a subject suffering from seizure. mouse hippocampal slices. Epilepsy is defined as a neuronal In another implementation, the method of treating a seizure state of hyperexcitability and hypersynchrony. Extracellular includes administering a composition that restores mito­ recordings in the CA3 stratum radiatum reveals that a 30 min 35 application of the mitochondrial complex I inhibitor rote- chondrial function to a seizure-genic brain region, wherein none (100 nM) increases the rate and amplitudes of spon­ the composition is administered to a subject suffering from taneous field potentials. The middle trace is an expansion of seizure. In another implementation, the method of treating a field potential marked by an asterisk in the top trace. seizure includes administering a composition that restores Time-frequency analysis was performed on the signals to mitochondrial function in a brain region, wherein the com­ 40 examine the component frequencies occurring during the position is administered to a subject unable or unwilling to field potentials. High frequency oscillations (HF05) in the abide by a ketogenic diet or any other dietary or pharma­ 100-200 Hz bandwidth (called ripples) are normal and ceutical anticonvulsant treatment. In another implementa­ reflect small clusters of neurons firing action potentials tion, the method of treating a seizure includes administering synchronously. Rotenone application results in spectral dis­ a composition that impairs mitochondrial function to a 45 organization and the generation of faster HFOs (called fast seizure-genic brain region. In some embodiments, the com­ ripples) which reflects a desynchronization of neuronal position can include ascorbic acid, vitamin E, and pyruvate. firing. Fast ripples occur in humans and animals with In one specific embodiment, the composition includes a epilepsy and are biomarkers for seizure-genic foci. combination of acorbic acid, pyruvate, and tocopherol, FIG. 5B (Left) shows that increasing the potassium con­ wherein the ratio of ascorbic acid to tocopherol is between 50 centration in the artificial cerebral spinal fluid bath solution about 1:6 and about 1:10, the ratio of ascorbic acid to from 3 mM to 8 mM of a vehicle-treated hippocampal slice pyruvate is between about 1:1.5 and about 1:4.5, and the increases ripple occurrence, introduces fast ripples and ratio of tocopherol to pyruvate is between about 1:12 and causes regular seizure-like events.