US 20200215078A1 IN (19 ) United States (12 ) Patent Application Publication ( 10) Pub . No .: US 2020/0215078 A1 ROGAWSKI et al. (43 ) Pub . Date : Jul. 9 , 2020 (54 ) MITIGATION OF EPILEPTIC SEIZURES BY Publication Classification COMBINATION THERAPY USING (51 ) Int. Ci. BENZODIAZEPINES AND NEUROSTEROIDS A61K 31/57 (2006.01 ) A61K 45/06 (2006.01 ) ( 71) Applicant: The Regents of the University of A61P 25/08 ( 2006.01 ) California , Oakland , CA (US ) A61K 31/439 (2006.01 ) ( 72 ) Inventors : Michael A. ROGAWSKI, Sacramento , A61K 31/5513 ( 2006.01) CA (US ) ; Isaac N. PESSAH , Davis , A61K 31/5517 (2006.01 ) CA (US ) ; Zhengyu CAO , Woodland , (52 ) U.S. CI. CA (US ) ; Pamela J. LEIN , Davis , CA CPC A61K 31/57 ( 2013.01 ) ; A61K 45/06 (US ) (2013.01 ) ; A61K 31/5517 ( 2013.01 ) ; A61K 31/439 (2013.01 ) ; A61K 31/5513 ( 2013.01 ) ; ( 21) Appl. No .: 16 /544,480 A61P 25/08 (2018.01 ) Aug. 19 , 2019 ( 22 ) Filed : (57 ) ABSTRACT Related U.S. Application Data Provided are compositions comprising a benzodiazepine and (63 ) Continuation of application No. 15 /649,460 , filed on a neurosteroid , containing one or both of the benzodiazepine Jul. 13 , 2017 , now Pat . No. 10,426,786 , which is a and the neurosteroid in a subtherapeutic dose , and admin continuation of application No. 13 / 964,922 , filed on istration of such compositions for mitigation of an epileptic Aug. 12 , 2013 , now abandoned . seizure . Further provided are compositions comprising a (60 ) Provisional application No. 61 /798,094 , filed on Mar. benzodiazepine, a neurosteroid , and an NMDA blocker, and 15 , 2013 , provisional application No. 61/ 682,745 , administration of such compositions for mitigation of an filed on Aug. 13 , 2012 . epileptic seizure . Patent Application Publication Jul 9 ,? 2020 Sheet 1 of 25 US2020 / 0215078A1 Vehicle / TETS ???????? ????? ?? ???? conhecimentos están base Vehicle ?? ?? ??????????? ????? ?????????? ?????????? ???????? || ?????????? 3iN ??????? ||||| n 300 Phase 1 FIG . 1A Patent Application Publication Jul. 9 , 2020 Sheet 2 of 25 US 2020/0215078 A1 70 046 Log [ TETSJ M FIG . 1B FIG . 1C Patent Application Publication Jul. 9 , 2020 Sheet 3 of 25 US 2020/0215078 A1 V FIG . 1D T Phase Il response Washout Vehicle TETS ( 3 UM ) milione un Amenn 500AFU ad all 500 S FIG . 2 Patent Application Publication Jul 9 ,? 2020 Sheet 4 of 25 US2020 / 0215078A1 ??????????????? ????? ) 3 ?? M ( 100 ? M ( FIG . 3A Patent Application Publication Jul. 9 , 2020 Sheet 5 of 25 US 2020/0215078 A1 Phase 1 Fluo-4fluorescenceunits AUC)( dah FIG . 3B Phase II- Frequency Caktoscillations/10min FIG . 3C Patent Application Publication Jul. 9 , 2020 Sheet 6 of 25 US 2020/0215078 A1 Phase II- Amplitude Caktoscillationsamplitude (AF/F) . FIG . 3D Patent Application Publication Jul. 9 , 2020 Sheet 7 of 25 US 2020/0215078 A1 *** M TETS (0.23M ) WY w w 14 3 TETS (64M ) E wwwwwww FIG . 4A Spikefrequency(spikes/s) ??? 6 Basal 8 6 Log [ TETS ) M FIG . 4B Patent Application Publication Jul. 9 , 2020 Sheet 8 of 25 US 2020/0215078 A1 32 3.50 non potuit la tarde FIG . 5A 32 3.96 FIG . 5B Patent Application Publication Jul. 9 ,? 2020 Sheet 9 of 25 US2020 / 0215078A1 ????? ???? ?????? ??? ??????? ??????? ????????? * TETS (3M ) FIG . 6A Patent Application Publication Jul. 9 , 2020 Sheet 10 of 25 US 2020/0215078 A1 Fluo-4fluorescenceunits FIG . 6B Phase II- Frequency Ca2+oscillations/10min FIG . 6C Patent Application Publication Jul. 9 , 2020 Sheet 11 of 25 US 2020/0215078 A1 Phase 11- Amplitude Cal*oscillationsamplitude OF)/F( FIG . 6D Patent Application Publication Jul. 9 , 2020 Sheet 12 of 25 US 2020/0215078 A1 or DZP or TETS ????? ??? ????????? ??????? ??????? w TETS (3M ) DZP (0.03M ) will + TETS (3M ) ?????? ???? DZP (0.1M ) TETS (3uM ) will ull DZPDZP( (0.3M 0.34M ) whe * TET ( 3uM ) DZP (1M ) whanh * TETS ( 3uM ) FIG . 7A Patent Application Publication Jul. 9 , 2020 Sheet 13 of 25 US 2020/0215078 A1 Phase 1 ** Fluo-fluorescence4units Onv) DZP ( UM ) + TETS FIG . 7B Phase II- Frequency Caltoscillations/10min 0.3 DZP (UM ) + TETS FIG . 70 Patent Application Publication Jul. 9 , 2020 Sheet 14 of 25 US 2020/0215078 A1 Phase Il -Amplitude 38 Ca2+oscillationsamplitude (AF/F) ** 0.03 0.3 1 FIG . 7D Patent Application Publication Jul 9 ,? 2020 Sheet 15 of 25 US2020 / 0215078 Al www.lankan melewin | | | ???????? ???? ????? ???? || (11 / ?????? ????????? ??????? FIG . 8A Patent Application Publication Jul. 9 , 2020 Sheet 16 of 25 US 2020/0215078 A1 Phase I Fluo-fluorescence4units (AUC) AlloP (HMUM ) ) * TETESTETES FIG , 8B Phase Il- Frequency 30 20 oscillations/10minCalt IS 10 AlloP ( MOM )+ * TETES FIG . 8C Patent Application Publication Jul. 9 , 2020 Sheet 17 of 25 US 2020/0215078 A1 Phase Il -Amplitude Caltoscillationsamplitude la : 0.03 AlloP ( M ) * TETES FIG . 8D Patent Application Publication Jul 9 ,? 2020 Sheet 18 of 25 US2020 / 0215078 Al ??????? ?????? FIF ?????????? ?????????? FIG . 9A Patent Application Publication Jul. 9 , 2020 Sheet 19 of 25 US 2020/0215078 A1 Phasel . : . : . IN Fluo-4fluorescenceunits (AUC,%Control) 0 * DZP FIG . 9B Phase II- Frequency Caltoscillationsamplitude Control)(% 60 FIG . 9C Patent Application Publication Jul 9 ,? 2020 Sheet 20 of 25 US2020 / 0215078 Al Phase Il- Frequency ?? Ca2+oscillationsamplitude (%Control) TETS Allop DZP + AlloP + OZP FIG . 9D ?????? ?? ?? ??????? Veh ???????? TETS) 38 ( ???? ?????? ??????? TETS +A ???? ????????? TETS+ 0 ???? ???? ?? ????? ???? ?? ?? FIG . 10A Patent Application Publication Jul. 9 , 2020 Sheet 21 of 25 US 2020/0215078 A1 30 p < 0.01 p < 0.01 Ca*oscillations/10min TETS TETS + A TETS + DTETS + A + D FIG . 10B 1.2 p < 0.01 p< 0.01 , p< 0.01 Amplitude(AF/FO) TETS TETS +ATETS + DTETS + A + D FIG . 10C Patent Application Publication Jul. 9 , 2020 Sheet 22 of 25 US 2020/0215078 A1 TETS Average Time to Onset 36 43Suun 16 S T + TETS monic SoiZe 27 S + r. NO ...2 TETS + Diazepam TETS clonic semua diazepam ( 5 mg/ kg , ip ) . .* wit CARA .14* . **??????? ??????? FIG . 11 Patent Application Publication Jul. 9 , 2020 Sheet 23 of 25 US 2020/0215078 A1 FIG.12 Patent Application Publication Jul. 9 , 2020 Sheet 24 of 25 US 2020/0215078 A1 u 300 2.im FIG.13 Patent Application Publication Jul. 9 , 2020 Sheet 25 of 25 US 2020/0215078 A1 Inject mice Begin blood Mouse Blood Pressure DiastolicAverage BloodPressure 0.03 mg /kg DZP 0.03mg/ kg Allop 5mg/ kg DZP FIG . 14 US 2020/0215078 A1 Jul. 9. 2020 1 MITIGATION OF EPILEPTIC SEIZURES BY pentylenetetrazol. Limited cellular physiological studies and COMBINATION THERAPY USING results from [ SS ] t- butylbicyclophosphorothionate binding BENZODIAZEPINES AND NEUROSTEROIDS to brain membranes indicate that TETS inhibits GABA , receptors with an IC50 in the range of 1 uM (Squires et al , CROSS - REFERENCE TO RELATED 1983 ; Esser et al, 1991 ; Ratra et al. , 2001 ) and it is therefore APPLICATIONS comparable in potency to picrotoxin as an inhibitor of GABA , receptors (Squires et al. , 1983; Cole and Casida, [0001 ] This application is a continuation of U.S. applica 1986 , Ratra et al, 2001 ) . tion Ser. No. 13/ 964,922, filed on Aug. 12 , 2013 , which [ 0007 ] Cultured hippocampal neurons display synchro claims the benefit under 35 U.S.C. § 119 ( e ) of U.S. Provi nous spontaneous Ca2 + oscillations ( Tanaka et al, 1996 ) that sional Application No. 61 /682,745 filed on Aug. 13 , 2012 are driven by action potential- dependent synaptic transmis and U.S. Provisional Application No. 61/ 798,094 filed on sion Disruption of Ca2 + oscillations by environmental toxi Mar. 15 , 2013 , all of which are hereby incorporated herein cants has been reported (Soria -Mercado et al ., 2009 ; Cao et by reference in their entireties for all purposes . al , 2010 , Choi et al, 2010 , Percira et al. , 2010 , Cao et al ., STATEMENT OF GOVERNMENTAL SUPPORT 2011 ). Hippocampal neurons also exhibit spontaneous elec trical discharges as they form functional neuronal networks . [ 0002 ] This invention was made with Government support These discharges , as detected in extracellular recordings , under Grant Nos . AG032119 , NS072094 , and NS079202 consist of infrequent synchronized field potentials , mixed awarded by the National institutes of Health . The Govern with more frequent desynchronized random action poten ment has certain rights in this invention . tials (Cao et al. , 2012 ; Frega et al. , 2012 ) . Synchronous Ca2- oscillations and neuronal electrical firing co -occur FIELD ( Jimbo et al. , 1993) and are important in mediating neuronal [0003 ] Provided are compositions comprising a benzodi development and activity dependent dendritic growth (Way azepine and a neurosteroid , containing one or both of the man et al . , 2008 ) Genetic or environmental factors that benzodiazepine and the neurosteroid in a subtherapeutic interfere with neuronal transmission influence the overall dose , and administration of such compositions for mitigation neuronal networks activity (Kenet et al. , 2007, Meyer et al. , of an epileptic seizure . Further provided are compositions 2008 ; Shafer et al ., 2008 ; Frega et al. , 2012 ; Wayman et al. , comprising a benzodiazepine, a neurosteroid , and an NMDA 2012 ). For example picrotoxin , a GABA , receptor antago blocker, and administration of such compositions for miti nist, produces striking changes in network electric activity gation of an epileptic seizure . (Cao et al. , 2012 ; Frega et al. , 2012 ) . Diisopropylfluoro phosphate , an irreversible inhibitor of cholinesterase has BACKGROUND also been shown to elicit status epileptics in rats . Hippocam pal neurons