( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2020/0215078 A1

Home , Aptiganel, Azaspiracid, Bretazenil, Delucemine, Dexoxadrol, Dextrallorphan

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

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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 )

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 | | |

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????? ???? || (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

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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

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 dissociated from the brains of diisopropylfluo [0004 ] Tetramethylenedisulfotetramine ( TETS ), com monly called tetramine or TETS , is a highly toxic convulsant rophosphate exposed rats display significantly higher intra with a parenteral LD50 of 0.1-0.3 mg/ kg in mice or rats cellular Ca21 concentration which appears to be dependent ( Haskell and Voss, 1957 ; Voss et al. , 1961 ; Casida et al ., on the N -methy -D -aspartate receptors ( Deshpande et al. , 1976 ) . In adult humans, 7-10 mg is estimated as a lethal dose 2010 ). (Guan et al ., 1993 ) TETS was used as a rodenticide until [0008 ] In the present study, using rapid throughput assays banned worldwide in the early 1990's (Whitlow et al ., 2005 . we characterized the influence of TETS on the Ca21 dynam Banks et al. , 2012 ) . It is , however, still available illegally , ics and neuronal firing activity . Inasmuch as TETS induces and is responsible for accidental and intentional poisonings , changes in Ca2 : dynamics that are similar to those produced predominantly in China (Croddy , 2004, Wu and Sun , 2004 , by the GABA , receptor antagonists picrotoxin and bicuc Zhang et al. 2011 ), but also in other countries , including the ulline , our results support the view that TETS acts as a United States (Barrueto et al. , 2003 ) . Between 1991 and GABA , receptor antagonist. Using rapid throughput Ca2 . 2010 over 14,000 cases of TETS intoxication were reported measurement, we identified several agents that reduce or in China with 932 deaths (Li et al. , 2011) . Extreme toxicity, prevent the alterations in Ca2- dynamics induced by TETS , history of intentional mass poisonings, and the absence of a suggesting several treatment strategies for TETS - induced specific antidote raise concern that TETS is a potential seizures, including the GABA , receptor positive modulators chemical threat agent that could cause mass casualties if diazepam and allopregnanolone. In preliminary studies with released accidentally or intentionally (Whitlow et al. , 2005 ; mice , we confirmed that these two agents do inhibit TETS Jell and Yeung, 2010 ). induced clonic seizures and progression to tonic seizures and [0005 ] Mild to moderate poisoning with TETS leads to death supporting that measurement of Ca2 : dynamics is headache and dizziness whereas severe intoxication pro likely useful for identifying novel targeted interventions for duces status epilepticus and coma (Whitlow et al ., 2005 ; Li TETS poisoning . et al. , 2011 ) . Animal studies demonstrate that TETS is active as a convulsant when administered orally , parenterally and SUMMARY intraventricularly . Sublethal seizures are not associated with [0009 ] In one aspect, provided are compositions compris evidence of cellular injury or neurodegeneration although ing a benzodiazepine and a neurosteroid . In varying embodi there is delayed transient reactive astrocytosis and micro ments , the compositions comprise one or both of the ben glial activation (Zolkowska et al. , 2012 ) . zodiazepine and the neurosteroid in a subtherapeutic dose or [ 0006 ] The primary convulsant mechanism of TETS has amount. In varying embodiments , the compositions further been thought to relate to blockade of GABA , receptors and comprise a NMDA receptor antagonist. In some embodi the seizures induced in animals resemble those produced by ments , the composition is formulated for inhalational, intra other GABA , receptor antagonists including picrotoxin and nasal or intrapulmonary administration . In some embodi US 2020/0215078 A1 Jul. 9. 2020 2 ments , the composition is formulated for oral or tromethorphan , phencyclidine. CPPene ( 3 -[ ( R ) -2 -carboxyp transmucosal delivery . In varying embodiments , the com iperazin - 4 -yl )-prop - 2 - enyl- 1 - phosphonic acid ) , selfotel , position is formulated for parenteral delivery . In some amantadine dextrorphan , memantine, tiletamine , neramex embodiments , the parenteral delivery or administration is ane, cliprodil, remacemide, aptiganel , 1- Aminocyclopro via a route selected from the group consisting of inhala panecarboxylic acid ( ACPC ) , 7 -Chlorokynurenate , DCKA tional, intrapulmonary , intranasal , intramuscular, subcutane (5,7 - dichlorokynurenic acid ), kynurenic acid , CP - 101,606 ous, transmucosal and intravenous . In some embodiments , ( traxoprodil) , AZD6765 (lanicemine ) and GLYX -13 . In the composition is formulated for intramuscular delivery . In some embodiments , the NMDA receptor antagonist is dizo some embodiments , the benzodiazepine is an agonist of the cilpine (MK - 801) . In varying embodiments , the composition benzodiazepine recognition site on GABA , receptors and comprises a benzodiazepine and a neurosteroid formulated stimulates endogenous neurosteroid synthesis. In some in a cyclodextrin , e.g. , for intramuscular , intravenous and /or embodiments , the benzodiazepine is selected from the group subcutaneous administration . consisting of bretazenil, clonazepam , cloxazolam , clora [ 0010 ] In another aspect, provided are methods of pre zepate , diazepam , fludiazepam , flutoprazepam , lorazepam , venting or terminating a seizure in a subject in need thereof. midazolam , nimetazepam , nitrazepam , phenazepam , In varying embodiments , the methods comprise administra temazepam and clobazam . In some embodiments , the ben tion to the subject of an effective amount of a composition zodiazepine is selected from the group consisting of mida as described above and herein . Also provided are methods of zolam , lorazepam and diazepam . In some embodiments , the accelerating the termination or abortion of an impending neurosteroid is selected from the group consisting of allo seizure in a subject in need thereof. In varying embodiments , pregnanolone , allotetrahydrodeoxycorticosterone, ganax the methods comprise administration to the subject of an olone , alphaxolone , alphadolone, hydroxydione, minax effective amount of a composition as described above and olone, and Althesin . In some embodiments , the neurosteroid herein . In some embodiments , the composition is adminis is allopregnanolone. In some embodiments , the composition tered via inhalational or intrapulmonary administration . In comprises allopregnanolone and a benzodiazepine selected some embodiments , the composition is not heated prior to from the group consisting of midazolam , lorazepam and administration . In some embodiments , the composition is diazepam . In some embodiments , the neurosteroid is sus nebulized . In some embodiments , the nebulized particles are pended or dissolved in a cyclodextrin (e.g. , an a -cyclodex about 3 um or smaller. In some embodiments , the nebulized trin , a B -cyclodextrin or a y -cyclodextrin ). In varying particles are about 2-3 um . In some embodiments , the embodiments , the neurosteroid is suspended or dissolved in composition is delivered to the distal alveoli. In some a cyclodextrin selected from the group consisting of embodiments , the composition is administered orally . In hydroxypropyl- b -cyclodextrin , endotoxin controlled B - cy some embodiments, the composition is contained within a clodextrin sulfobutyl ethers , or cyclodextrin sodium salts soft gel capsule . In some embodiments , the composition is ( e.g. , CAPTISOL® ). In some embodiments , the neuroster administered parenterally . In some embodiments , the com oid is suspended or dissolved in an edible oil . In some position is administered via a parenteral route selected from embodiments , the edible oil comprises one or more veg the group consisting of inhalational, intrapulmonary , intra etable oils . In some embodiments , the vegetable oil is nasal, intramuscular, subcutaneous, transmucosal and intra selected from the group consisting of coconut oil, corn oil , venous. In some embodiments , the composition is admin cottonseed oil , olive oil, palm oil, peanut oil , rapeseed oil , istered transmucosally . In varying embodiments , themethod canola oil, safflower oil, sesame oil, soybean oil , sunflower comprises co - administering a benzodiazepine and a neuro oil , and mixtures thereof. In some embodiments , the edible steroid formulated in a cyclodextrin , e.g., intramuscularly , oil is canola oil . In some embodiments , the edible oil intravenously and /or subcutaneously . comprises one or more nut oils . In some embodiments , the [0011 ] In a related aspect , methods of preventing or ter nut oil is selected from the group consisting of almond oil , minating a seizure in a subject in need thereof, comprising cashew oil , hazelnut oil, macadamia oil, mongongo nut oil, administration to the subject of an effective amount of a pecan oil, pine nut oil, pistachio oil, walnut oil , and mixtures benzodiazepine and a neurosteroid . In varying embodi thereof. In some embodiments , the NMDA receptor antago ments, one or both of the benzodiazepine and the neuros nist is selected from the group consisting of dizocilpine teroid are administered in a subtherapeutic dose . Further are (MK -801 ) , meperidine, methadone , dextropropoxyphene , provided methods of accelerating the termination or abortion tramadol, ketobemidone , ketamine, dextromethorphan , of an impending seizure in a subject in need thereof. In phencyclidine , nitrous oxide ( N2O ) , AP5 ( R - 2 - amino - 5 varying embodiments , the methods comprise administration phosphonopentanoate ) , AP7 (2 -amino - 7 -phosphonohep to the subject of an effective amount of a benzodiazepine and tanoic acid ) , CPPene ( 3 - [ ( R )-2 -carboxypiperazin - 4 -yl ] a neurosteroid . In some embodiments , one or both of the prop - 2 -enyl - 1 -phosphonic acid ), selfotel, amantadine , benzodiazepine and the neurosteroid are administered in a dextrallorphan , dextrorphan , ethanol, eticyclidine, gacycli subtherapeutic dose . In some embodiments , the benzodiaz dine, ibogaine, magnesium , memantine , methoxetamine, epine and the neurosteroid are co - administered together rolicyclidine tenocyclidine ,methoxydine , tiletamine, xenon , and /or by the same route of administration . In some embodi neramexane , cliprodil , etoxadrol, dexoxadrol , WMS 2539 , ments , the benzodiazepine and the neurosteroid are co NEFA , remacemide, delucemine, 8A -PDHQ , aptiganel , administered separately and /or by different routes of admin HU -211 , rhynchophylline, 1 -Aminocyclopropanecarboxylic istration . In some embodiments , one or both of the acid (ACPC ), 7 -Chlorokynurenate , DCKA ( 5,7 -dichlo benzodiazepine and the neurosteroid are self- administered rokynurenic acid ) , kynurenic acid , lacosamide, CP - 101,606 by the subject . In some embodiments , one or both of the ( traxoprodil) , AZD6765 (lanicemine ) and GLYX - 13 . In benzodiazepine and the neurosteroid are administered via some embodiments , the NMDA receptor antagonist is inhalational or intrapulmonary administration . In some selected from the group consisting of ketamine , dex embodiments , one or both of the benzodiazepine and the US 2020/0215078 A1 Jul. 9. 2020 3 neurosteroid are not heated prior to administration . In some amexane , cliprodil, ramacemide, aptiganel, embodiments , one or both of the benzodiazepine and the 1 - Aminocyclopropanecarboxylic acid (ACPC ) . 7 - Chlo neurosteroid are nebulized . In some embodiments , the nebu rokynurenate. DCKA (5,7 - dichlorokynurenic acid ), lized particles are about 3 um or smaller . In some embodi kynurenic acid , CP - 101,606 ( traxoprodil ), AZD6765 ( lani ments , the nebulized particles are about 2-3 um . In some cemine ) and GLYX - 13 . In some embodiments , the subject is embodiments , one or both of the benzodiazepine and the experiencing aura . In some embodiments, the subject has neurosteroid are delivered to the distal alveoli . In some been warned of an impending seizure. In some embodi embodiments, one or both of the benzodiazepine and the ments , the subject is experiencing a seizure . In some neurosteroid are administered orally . In some embodiments , embodiments , the subject has status epilepticus, refractory one or both of the benzodiazepine and the neurosteroid are status epilepticus or super - refractory status epilepticus. In contained within a soft gel capsule . In some embodiments , some embodiments , the subject has myoclonic epilepsy . In one or both of the benzodiazepine and the neurosteroid are some embodiments , the subject suffers from seizure clusters . administered parenterally . In some embodiments , one or In some embodiments , the seizure is a tonic seizure . In some both of the benzodiazepine and the neurosteroid are admin embodiments , the seizure is a clonic seizure . In some istered via a parenteral route selected from the group con embodiments , the subject has been exposed to or is at risk sisting of inhalational , intrapulmonary , intranasal, intramus of being exposed to a nerve agent or a pesticide that can cular , subcutaneous, transmucosal and intravenous . In some cause seizures . In some embodiments , the subject has been embodiments , one or both of the benzodiazepine and the exposed to or is at risk of being exposed to tetramethylene neurosteroid are administered transmucosally . In varying disulfotetramine ( TETS ) . embodiments , the method comprises co -administering a [0013 ] In another aspect, further provided are kits com benzodiazepine and a neurosteroid formulated in a cyclo prising a benzodiazepine and a neurosteroid . In varying dextrin , e.g., intramuscularly , intravenously and /or subcuta embodiments , one or both of the benzodiazepine and the neously . neurosteroid are provided in unit dosage forms comprising [ 0012 ] With respect to further embodiments of the meth a subtherapeutic dose . In some embodiments , the kits further ods, in some embodiments , the benzodiazepine is selected comprise a NMDA receptor antagonist. In some embodi from the group consisting of bretazenil, clonazepam , clox ments , one or both of the benzodiazepine and the neuros azolam , clorazepate , diazepam , fludiazepam , flutoprazepam , teroid is formulated for inhalational, intranasal or intrapul lorazepam , midazolam , nimetazepam , nitrazepam , phenaze monary administration . In some embodiments , one or both pam , temazepam and clobazam . In some embodiments , the of the benzodiazepine and the neurosteroid is formulated for benzodiazepine is selected from the group consisting of oral or parenteral delivery . In some embodiments , one or midazolam , lorazepam , and diazepam . In some embodi both of the benzodiazepine and the neurosteroid are formu ments , the benzodiazepine is administered at a dose in the lated for a parenteral route selected from the group consist range of 0.3 ug/ kg to 3.0 ug/ kg . In varying embodiments , the ing of inhalational, intrapulmonary , intranasal, intramuscu benzodiazepine is administered at a dose that does not lar, subcutaneous , transmucosal and intravenous. In some decrease blood pressure . In some embodiments , the neuro embodiments , the benzodiazepine is an agonist of the ben steroid is selected from the group consisting of allopreg zodiazepine recognition site on GABA , receptors and nanolone , allotetrahydrodeoxycorticosterone, ganaxolone , stimulates endogenous neurosteroid synthesis . In some alphaxolone, alphadolone, hydroxydione , minaxolone, and embodiments , the benzodiazepine is selected from the group Althesin . In some embodiments , allopregnanolone is co consisting of bretazenil, clonazepam , cloxazolam , clora administered with a benzodiazepine selected from the group zepate, diazepam , fludiazepam , flutoprazepam , lorazepam , consisting ofmidazolam , lorazepam , and diazepam . In some midazolam , nimetazepam , nitrazepam , phenazepam , embodiments , the methods further comprise co - administra temazepam and clobazam . In some embodiments , the ben tion of an NMDA receptor antagonist . In some embodi zodiazepine is selected from the group consisting of mida ments , the NMDA receptor antagonist is selected from the zolam , lorazepam and diazepam . In some embodiments , the group consisting of dizocilpine (MK - 801) , meperidine, neurosteroid is selected from the group consisting of allo methadone , dextropropoxyphene, tramadol, ketobemidone, pregnanolone , allotetrahydrodeoxycorticosterone, ganax ketamine, dextromethorphan , phencyclidine , nitrous oxide olone, alphaxolone , alphadolone , hydroxydione , minax (N2O ), AP5 (R -2 -amino -5 -phosphonopentanoate ), AP7 olone , and Althesin . In some embodiments , the neurosteroid ( 2 -amino -7 -phosphonoheptanoic acid ) , CPPene (3 - [ (R )-2 is allopregnanolone . In some embodiments , the kit com carboxypiperazin - 4 -yl ) -prop - 2 - enyl- 1 -phosphonic acid ), prises allopregnanolone and a benzodiazepine selected from selfotel, amantadine , dextrallorphan , dextrorphan , ethanol, the group consisting of midazolam , lorazepam , and diaz eticyclidine, gacyclidine , ibogaine , magnesium , memantine , epam . In some embodiments, the neurosteroid is suspended methoxetamine , rolicyclidine tenocyclidine, methoxydine, or dissolved in a cyclodextrin ( e.g. , an a - cyclodextrin , a tiletamine , xenon , neramexane, eliprodil, etoxadrol, dexox B - cyclodextrin or a y -cyclodextrin ). In varying embodi adrol, WMS 2539 , NEFA , remacemide, delucemine , ments , the neurosteroid is suspended or dissolved in a 8A -PDHQ , aptiganel , HU -211 , rhynchophylline , 1 - Amino cyclodextrin selected from the group consisting ofhydroxy cyclopropanecarboxylic acid (ACPC ), 7 - Chlorokynurenate , propyl- L -cyclodextrin , endotoxin controlled ß -cyclodextrin DCKA (5,7 - dichlorokynurenic acid ) , kynurenic acid , lacos sulfobutyl ethers , or cyclodextrin sodium salts ( e.g. , CAP amide , CP - 101,606 ( traxoprodil ), AZD6765 ( lanicemine ) TISOL® ). In some embodiments , the neurosteroid is sus and GLYX - 13 . In some embodiments , the NMDA receptor pended or dissolved in an edible oil. In some embodiments , antagonist is selected from the group consisting ofketamine , the edible oil comprises one or more vegetable oils . In some dextromethorphan , phencyclidine , CPPene ( 3 - [ ( R ) -2 - car embodiments , the vegetable oil is selected from the group boxypiperazin -4 - yl) -prop - 2 -enyl - 1- phosphonic acid ) , selfo consisting of coconut oil, corn oil , cottonseed oil, olive oil , tel , amantadine, dextrorphan , memantine , tiletamine , ner palm oil, peanut oil, rapeseed oil , canola oil, safflower oil, US 2020/0215078 A1 Jul. 9. 2020 4

sesame oil , soybean oil, sunflower oil , and mixtures thereof. a vegetable oils . In some embodiments , the vegetable oil is In some embodiments , the edible oil is canola oil . In some selected from the group consisting of coconut oil, corn oil , embodiments , the edible oil comprises one or more nut oils . cottonseed oil, olive oil , palm oil, peanut oil , rapeseed oil, In some embodiments , the nut oil is selected from the group canola oil, safflower oil , sesame oil , soybean oil , sunflower consisting of almond oil , cashew oil, hazelnut oil, macada oil, and mixtures thereof. In some embodiments , the edible mia oil, mongongo nut oil, pecan oil , pine nut oil, pistachio oil is canola oil . In some embodiments , the edible oil oil , walnut oil , and mixtures thereof. In some embodiments , comprises one or more nut oils . In some embodiments , the the NMDA receptor antagonist is selected from the group nut oil is selected from the group consisting of almond oil, consisting of dizocilpine (MK -801 ) , meperidine, metha cashew oil, hazelnut oil, macadamia oil, mongongo nut oil , done , dextropropoxyphene, tramadol, ketobemidone , ket pecan oil, pine nut oil, pistachio oil, walnut oil, and mixtures amine, dextromethorphan , phencyclidine , nitrous oxide thereof in some embodiments , the NMDA receptor antago (N2O ) , AP5 ( R - 2 -amino - 5 -phosphonopentanoate ), AP7 nist is selected from the group consisting of dizocilpine (2 - amino - 7 -phosphonoheptanoic acid ), CPPene (3 -[ ( R ) -2 (MK - 801) , meperidine, methadone, dextropropoxyphene , carboxypiperazin - 4 - yl )-prop - 2 -enyl - 1 -phosphonic acid ), tramadol , ketobemidone, ketamine, dextromethorphan , selfotel, amantadine , dextrallorphan , dextrorphan , ethanol , phencyclidine , nitrous oxide (N2O ), AP5 ( R -2 -amino -5 eticyclidine, gacyclidine , ibogaine, magnesium , memantine , phosphonopentanoate ), AP7 ( 2 - amino -7 -phosphonohep methoxetamine , rolicyclidine , tenocyclidine, methoxydine, tanoic acid ), CPPene (3 - [( R ) -2 -carboxypiperazin - 4 - yl] tiletamine , xenon , neramexane , eliprodil, etoxadrol, dexox prop - 2 - enyl- 1 -phosphonic acid ) , selfotel, amantadine , adrol, WMS 2539 , NEFA , remamacemide , delucemine , dextrallorphan , dextrorphan , ethanol , eticyclidine, gacycli 8A -PDHQ , aptiganel , HU - 211 , rhynchophylline, 1 -Amino dine, ibogaine , magnesium , memantine , methoxetamine, cyclopropanecarboxylic acid ( ACPC ) , 7 -Chlorokynurenate , rolicyclidine tenocyclidine, methoxydine, tiletamine, xenon , DCKA ( 5,7 - dichlorokynurenic acid ) , kynurenic acid , lacos neramexane , cliprodil , etoxadrol, dexoxadrol, WMS 2539 , amide, CP - 101,606 (traxoprodil ) , AZD6765 ( lanicemine ) NEFA , remacemide, delucemine , 8A -PDHQ , aptiganel , and GLYX - 13 . In some embodiments , the NMDA receptor HU -211 , rhynchophylline , 1 - Aminocyclopropanecarboxylic antagonist is selected from the group consisting of ketamine , acid ( ACPC ) , 7 -Chlorokynurenate , DCKA (5,7 -dichlo dextromethorphan , phencyclidine . CPPene ( 3 - [ ( R ) -2 -car rokynurenic acid ), kynurenic acid , lacosamide, CP - 101,606 boxypiperazin - 4 - yl) -prop - 2 -enyl - 1 -phosphonic acid ), selfo ( traxoprodil) . AZD6765 (lanicemine ) and GLYX - 13 . In tel, amantadine, dextrorphan , memantine , tiletamine , ner some embodiments , the NMDA receptor antagonist is amexane , eliprodil , remacemide , aptiganel , selected from the group consisting of ketamine, dex 1 -Aminocyclopropanecarboxylic acid (ACPC ) , 7 - Chlo tromethorphan , phencyclidine, CPPene ( 3 - [ ( R ) -2 - carboxyp rokynurenate , DCKA ( 5,7 -dichlorokynurenic acid ), iperazin - 4 -yl ) -prop - 2 -enyl - 1 -phosphonic acid ), selfotel , kynurenic acid , CP - 101,606 ( traxoprodil) , AZD6765 (lani amantadine, dextrorphan , memantine, tiletamine, neramex cemine ) and GLYX - 13 . In some embodiments, the NMDA ane , eliprodil , remacemide, aptiganel, 1 - Aminocyclopro receptor antagonist is dizocilpine (MK - 801) . panecarboxylic acid (ACPC ), 7 -Chlorokynurenate , DCKA [0014 ] In another aspect , the invention provides compo ( 5,7 - dichlorokynurenic acid ) , kynurenic acid , CP - 101,606 sitions comprising a benzodiazepine , a neurosteroid and an (traxoprodil ), AZD6765 (lanicemine ) and GLYX -13 . NMDA receptor antagonist. In some embodiments , the [0015 ] In another aspect, the invention provides methods composition is formulated for inhalational or intrapulmo of preventing or terminating a seizure in a subject in need nary administration . In some embodiments , the composition thereof, comprising administration to the subject of an is formulated for oral or transmucosal delivery . In some effective amount of a composition as described above and embodiments , the benzodiazepine is an agonist of the ben herein . In another aspect , the invention provides methods of zodiazepine recognition site on GABA , receptors and accelerating the termination or abortion of an impending stimulates endogenous neurosteroid synthesis . In some seizure in a subject in need thereof, comprising administra embodiments , the benzodiazepine is selected from the group tion to the subject of an effective amount of a composition consisting of bretazenil, clonazepam , cloxazolam , clora as described above and herein . In a further aspect, the zepate, diazepam , fludiazepam , flutoprazepam , lorazepam , invention provides methods of preventing or terminating a midazolam , nimetazepam , nitrazepam , phenazepam , seizure in a subject in need thereof, comprising administra temazepam and clobazam . In some embodiments , the ben tion to the subject of an effective amount of a benzodiaz zodiazepine is midazolam . In some embodiments , the ben epine , a neurosteroid and an NMDA receptor antagonist . In zodiazepine is diazepam . In some embodiments , the neuro a further aspect, the invention provides methods of accel steroid is selected from the group consisting of erating the termination or abortion of an impending seizure allopregnanolone , allotetrahydrodeoxycorticosterone, in a subject in need thereof , comprising administration to the ganaxolone, alphaxolone, alphadolone, hydroxydione , subject of an effective amount of a benzodiazepine , a minaxolone , and Althesin . In some embodiments , the neu neurosteroid and an NMDA receptor antagonist . In some rosteroid is allopregnanolone. In some embodiments , the embodiments , the benzodiazepine , neurosteroid and NMDA neurosteroid is suspended or dissolved in a cyclodextrin receptor antagonist are co -administered together and /or by ( e.g. , an a - cyclodextrin , a ß -cyclodextrin or a y -cyclodex the same route of administration . In some embodiments , the trin ). In varying embodiments , the neurosteroid is suspended benzodiazepine , neurosteroid and NMDA receptor antago or dissolved in a cyclodextrin selected from the group nist are co - administered separately and /or by different routes consisting of hydroxypropyl- 3 -cyclodextrin , endotoxin con of administration . In some embodiments , the benzodiaz trolled B -cyclodextrin sulfobutyl ethers , or cyclodextrin epine is selected from the group consisting of bretazenil , sodium salts ( e.g. , CAPTISOL® ) . In some embodiments , clonazepam , cloxazolam , clorazepate, diazepam , fludiaz the neurosteroid is suspended or dissolved in an edible oil . epam , flutoprazepam , lorazepam ,midazolam , nimetazepam , In some embodiments , the edible oil comprises one or more nitrazepam , phenazepam , temazepam and clobazam . In US 2020/0215078 A1 Jul. 9 , 2020 5

some embodiments , the neurosteroid is selected from the antagonist ) that find use in the methods described herein group consisting of allopregnanolone, allotetrahydrodeoxy include , e.g. , oral (per os ( P.O.) ) administration , nasal or corticosterone, ganaxolone , alphaxolone , alphadolone , inhalation administration , administration as a suppository, hydroxydione, minaxolone , and Althesin . In some embodi topical contact, transdermal delivery (e.g. , via a transdermal ments , the NMDA receptor antagonist is selected from the patch ), intrathecal (IT ) administration , intravenous (“ iv ” ) group consisting of dizocilpine (MK - 801) , meperidine , administration , intraperitoneal ( “ ip ” ) administration , intra methadone , dextropropoxyphene, tramadol, ketobemidone, muscular (“ im ” ) administration , intralesional administra ketamine, dextromethorphan , phencyclidine , nitrous oxide tion , or subcutaneous (“ sc ” ) administration , or the implan (N2O ), AP5 (R -2 - amino - 5- phosphonopentanoate ), AP7 tation of a slow -release device e.g. , a mini- osmotic pump, a ( 2 -amino - 7 - phosphonoheptanoic acid ). CPPene ( 3 - [ ( R ) -2 depot formulation , etc., to a subject. Administration can be carboxypiperazin - 4 -yl ) -prop - 2 - enyl - 1 - phosphonic acid ), by any route including parenteral and transmucosal ( e.g. , selfotel, amantadine , dextrallorphan , dextrorphan , ethanol, oral, nasal, vaginal, rectal, or transdermal) . Parenteral eticyclidine, gacyclidine , ibogaine , magnesium , memantine , administration includes, e.g., intravenous, intramuscular , methoxetamine , rolicyclidine.tenocyclidine , methoxydine, intra - arterial, intradermal, subcutaneous , intraperitoneal, tiletamine, xenon , neramexane, eliprodil , etoxadrol, dexox intraventricular , iontophoretic and intracranial . Other modes adrol, WMS 2539, NEFA , remacemide , delucemine , of delivery include , but are not limited to , the use of 8A -PDHQ , aptiganel, HU - 211, rhynchophylline, 1 - Amino liposomal formulations , intravenous infusion , transdermal cyclopropanecarboxylic acid (ACPC ), 7 - Chlorokynurenate , patches , etc. DCKA ( 5,7 -dichlorokynurenic acid ), kynurenic acid , lacos [0017 ] The terms “ systemic administration ” and “ systemi amide, CP - 101,606 (traxoprodil ) , AZD6765 ( lanicemine ) cally administered ” refer to a method of administering a and GLYX - 13 . In some embodiments , the NMDA receptor compound or composition to a mammal so that the com antagonist is selected from the group consisting ofketamine , pound or composition is delivered to sites in the body , dextromethorphan , phencyclidine, CPPene ( 3 - [( R )-2 -car including the targeted site of pharmaceutical action , via the boxypiperazin - 4 - yl) -prop - 2 -enyl - 1 -phosphonic acid ) , selfo circulatory system . Systemic administration includes , but is tel , amantadine, dextrorphan , memantine , tiletamine , ner not limited to , oral, intranasal , rectal and parenteral ( e.g., amexane , eliprodil, remacemide, aptiganel, other than through the alimentary tract, such as intramus 1 - Aminocyclopropanecarboxylic acid (ACPC ), 7 - Chlo cular, intravenous, intra- arterial , transdermal and subcuta rokynurenate , DCKA (5,7 -dichlorokynurenic acid ) , neous ) administration . kynurenic acid , CP - 101,606 (traxoprodil ) , AZD6765 ( lani [0018 ] The term “ co -administration ” refers to the presence cemine ) and GLYX - 13. In one embodiment, the subject is of both active agents in the blood at the same time. Active experiencing aura . In one embodiment, the subject has been agents that are co -administered can be delivered concur warned of an impending seizure . In one embodiment, the rently ( i.e. , at the same time) or sequentially . subject is experiencing a seizure. In one embodiment, the [0019 ] The phrase " cause to be administered ” refers to the subject has status epilepticus , refractory status epilepticus or actions taken by a medical professional ( e.g., a physician ), super- refractory status epilepticus. In one embodiment, the or a person controlling medical care of a subject , that control subject has myoclonic epilepsy. In one embodiment, the and /or permit the administration of the agent( s )/ compound subject suffers from seizure clusters . In one embodiment, the ( s ) at issue to the subject. Causing to be administered can seizure is a tonic seizure . In one embodiment, the seizure is involve diagnosis and / or determination of an appropriate a clonic seizure . In one embodiment , the benzodiazepine is therapeutic or prophylactic regimen , and /or prescribing par self - administered by the subject. In one embodiment, the ticular agent( s )/ compounds for a subject. Such prescribing composition is administered via inhalational or intrapulmo can include, for example , drafting a prescription form , nary administration . In one embodiment, the composition is annotating a medical record , and the like . not heated prior to administration . In one embodiment, the [ 0020 ] The term “ effective amount” or “ pharmaceutically benzodiazepine is nebulized . In one embodiment, the nebu effective amount ” refer to the amount and /or dosage , and / or lized particles are about 3 um or smaller . In one embodi dosage regime of one or more compounds necessary to bring ment, the nebulized particles are about 2.3 um . In one about the desired result e.g. , an amount sufficient prevent, embodiment, the benzodiazepine is delivered to the distal abort or terminate a seizure . alveoli. In one embodiment, the benzodiazepine is admin [0021 ] “ Sub -therapeutic dose ” refers to a dose of a phar istered at a dose in the range of 0.3 ug /kg to 3.0 ug/ kg . In macologically active agent ( s) , either as an administered dose varying embodiments , the benzodiazepine is administered at of pharmacologically active agent , or actual level of phar a dose that does not decrease blood pressure . In one embodi macologically active agent in a subject that functionally is ment, the composition is administered orally . In one embodi insufficient to elicit the intended pharmacological effect in ment, the composition is contained within a soft gel capsule . itself ( e.g., to abort or prevent a seizure ), or that quantita In one embodiment, the composition is administered trans tively is less than the established therapeutic dose for that mucosally . In various embodiments , the subject may be at particular pharmacological agent ( e g , as published in a risk of exposure to or may have been exposed to tetrameth reference consulted by a person of skill, for example , doses ylenedisulfotetramine ( TETS ) . for a pharmacological agent published in the Physicians ' Desk Reference , 67th Ed , 2013 , Thomson Healthcare or Definitions Brunton , et al. , Goodman & Gilman's The Pharmacological [0016 ] As used herein , “ administering” refers to local and Basis of Therapeutics, 12th edition , 2010 , McGraw -Hill systemic administration , e.g. , including enteral , parenteral, Professional) . A “ sub -therapeutic dose ” can be defined in pulmonary, and topical/ transdermal administration . Routes relative terms (i.e. , as a percentage amount ( less than 100 % ) of administration for the agents ( e.g., one or more of a of the amount of pharmacologically active agent conven benzodiazepine , a neurosteroid and/ or an NMDA receptor tionally administered ) . For example , a sub - therapeutic dose US 2020/0215078 A1 Jul. 9. 2020 6

amount can be about 1 % to about 75 % of the amount of sigma receptors. Neurosteroids find use as sedatives for the pharmacologically active agent conventionally adminis purpose of general anaesthesia for carrying out surgical tered . In some embodiments , a sub - therapeutic dose can be procedures , and in the treatment of epilepsy and traumatic less than about 75 % , 50 % , 30 % , 25 % , 20 % , 10 % or less, brain injury . Illustrative neurosteroids include , e.g., allo than the amount of pharmacologically active agent conven pregnanolone, Ganaxolone , alphaxolone , alphadolone , tionally administered A sub -therapeutic dose amount can be hydroxydione , minaxolone, and Althesin ( a mixture of in the range of about 1 % to about 75 % of the amount of alphaxolone and alphadolone ). pharmacologically active agent known to elicit the intended pharmacological effect . In some embodiments , a sub - thera BRIEF DESCRIPTION OF THE DRAWINGS peutic dose can be less than about 75 % , 50 % , 30 % , 25 % , [0029 ] FIGS . 1A - D illustrate TETS - induced Ca2- dys 20 % , 10 % or less, than the amount of pharmacologically regulation in hippocampal neurons. ( A ) Representative active agent known to elicit the intended pharmacological traces showing how acute exposure to TETS (0.1-10 UM ) effect. influences Ca2 : fluctuations in hippocampal neurons 13-17 [0022 ] As used herein , the terms “ treating ” and “ treat DIV . Note that neurons exhibit spontaneous synchronous ment” refer to delaying the onset of, retarding or reversing Ca21 oscillations at this developmental stage indicative of the progress of, reducing the severity of, or alleviating or functional network connectivity. The effects of TETS were preventing either the disease or condition to which the term analyzed in the initial 5 min following addition (Phase I) and applies , or one or more symptoms of such disease or in the subsequent 10 min (Phase II ). In Phase I , the inte condition . grated intracellular Ca2 + level increased in a concentration [ 0023 ] The term “mitigating ” refers to reduction or elimi dependent fashion (B ) , and there was a plateau response at nation of one or more symptoms of that pathology or higher concentrations (3 , 10 uM ) that decayed slowly over disease, and /or a reduction in the rate or delay of onset or the 5 min period . In Phase II , there was a concentration severity of one or more symptoms of that pathology or dependent reduction in the frequency and an increase in the disease , and/ or the prevention of that pathology or disease . amplitude of the spontaneous synchronized Ca2- oscilla [ 0024 ] The terms " reduce, " " inhibit, " " relieve, " " allevi tions (C ,D ). The traces shown for Phase II are representative ate ” refer to the detectable decrease in the frequency, sever samples of the 10 min Phase II period . This experiment was ity and/ or duration of seizures . A reduction in the frequency , repeated three times with similar results . severity and /or duration of seizures can be measured by [0030 ] FIG . 2 illustrates reversal of TETS - induced Phase self -assessment ( e.g. , by reporting of the patient) or by a II effects after washout of TETS Traces show synchronized trained clinical observer . Determination of a reduction of the Ca2- oscillations that are reduced in frequency and frequency, severity and /or duration of seizures can be made increased in amplitude in the presence of TETS . The dotted by comparing patient status before and after treatment . red line is a representative trace before (“ Phase II response " ) [0025 ] As used herein , the phrase " consisting essentially and after TETS (" Washout” ) . The solid black line is a of ' refers to the genera or species of active pharmaceutical representative recording from a control experiment in which agents ( e.g., neurosteroid in combination with benzodiaz the culture was treated with vehicle and subjected to the epine, optionally in further combination with an NMDA same washout procedure. blocker) and excipient ( e.g., a cyclodextrin , an edible oil ) [0031 ] FIGS. 3A - D illustrate TETS , picrotoxin , and bicu included in a method or composition . In various embodi culline trigger similar neuronal Ca2 + dysregulation . ( A ) ments , other unmentioned or unrecited active ingredients Representative traces from experiments comparing the and inactive are expressly excluded . In various embodi effects of TETS ( 3 uM ) , picrotoxin (100 uM ) , and bicucul ments , additives ( e.g. , surfactants , acids (organic or fatty ) , line (100 UM ) on Ca2 + fluctuations . The three agents alcohols , esters , co - solvents , solubilizers , lipids, polymers , produce similar acute elevation of the integrated Ca2 + level glycols ) are expressly excluded . ( B ) with plateau responses in Phase I , and they decreased the [0026 ] The terms “ subject, ” “ individual, ” and “ patient” oscillator) frequency (C ) while increasing the amplitude of interchangeably refer to a mammal , preferably a human or Ca2 + transients in Phase II ( D ) . p < 0.01, inhibitors vs. a non -human primate , but also domesticated mammals ( e.g., control, data were pooled from three experiments performed canine or feline ), laboratory mammals ( e.g. , mouse , rat, at least in duplicate . rabbit , hamster , guinea pig ) and agricultural mammals ( e.g. , [0032 ] FIGS . 4A -B illustrate TETS -reversibly alters spon equine, bovine , porcine , ovine ) . In various embodiments , the taneous electrical discharges in hippocampal neurons. ( A ) subject can be a human ( e.g. , adult male , adult female , Representative raster plots of neuronal discharges before , adolescent male , adolescent female , male child , female during and after exposure to vehicle (DMSO ) (left panels ) or child ) under the care of a physician or other healthworker in TETS ( right panels) . Neuronal network activity was stable a hospital, psychiatric care facility , as an outpatient , or other for up to 60 min in the absence or presence of vehicle clinical context. In certain embodiments the subjectmay not control . TETS solutions of increasing concentration were be under the care or prescription of a physician or other added serially to the wells . After recording for 10 min , the healthworker. solution was removed and replaced by a higher concentra [0027 ] The term “ edible oil” refers to an oil that is tion or by vehicle (wash out) . TETS concentrations of 2 and digestible by a mammal . Preferred oils are edible or digest 6 UM caused a clustered burst discharge pattern and ible without inducing undesirable side effects . increased the overall discharge frequency ( B ) . This experi [0028 ] The term “ neuroactive steroid ” or “ neurosteroid ” ment was repeated three times each performed in duplicate refers to steroid compounds that rapidly alter neuronal with similar results . * , p < 0.05 , ** , p < 0.01, TETS vs. basal . excitability through interaction with neurotransmitter - gated [0033 ] FIGS. 5A - B illustrates TETS - induced a pattern of ion channels . Neurosteroids act as allosteric modulators of clustered electrical burst firing in hippocampal neuronal cell neurotransmitter receptors, such as GABAA, NMDA , and cultures at 14 days in vitro . Representative traces of neu US 2020/0215078 A1 Jul. 9 , 2020 7

ronal electrical firing from an MEA recording before ( A ) and (B ) and increases in Ca2 + transient amplitude ( C ). The first after ( B ) addition of TETS (6uM ) . The software only allows arrowhead indicates the addition of TETS or vehicle . The a display of 200 ms; the actual total period of clustered second arrowhead indicates the addition of vehicle or diaz bursts after TETS treatment often lasted up to 10 s (see FIG . epam or allopregnanolone or the combination . Each data 3A , right panel, 4th row ) . point represents Mean + SEM , n = 6 wells . [ 0034 ] FIGS. 6A - D illustrate MK - 801, but not nifedipine , [0039 ] FIG . 11 illustrates that high dose diazepam rescues partially mitigates TETS ( 3 uM ) -induced neuronal Ca2 + animals from TETS - induced tonic seizures and death . Rep dysregulation . ( A ) Representative traces illustrating effects resentative EEG recordings from mice administered TETS of pre- exposure to MK - 801 and nifedipine on TETS - in (0.15 mg/ kg , i.p. ) with and without pretreatment with diaz duced Ca2 + dysregulation . ( B ) Effects ofMK - 801 (MK ) and epam ( dose / route ). Time to seizure onset and seizure dura nifedipine (NIF ) on TETS - induced increase in integrated tion are expressed as the mean + S.E.M . ( n = x per treatment Ca2 + levels in Phase I. ( C , D ) Effects of MK - 801 and group ) . Administration of diazepam immediately following nifedipine on the TETS - induced synchronous Ca2 + transient the second clonic seizure prevented a fatal tonic seizure . oscillation frequency decrease (C ) and amplitude increase EEG recording in TETS -exposed animals rescued by diaz ( D ) in Phase II . ** , p < 0.01 , TETS vs. vehicle control, ## , epam indicated no additional seizure for up to 1 h post- TETS p < 0.01, MK -801 + TETS Vs TETS , n = 6 pooled from two exposure . experiments . [ 0040 ] FIG . 12. Adult male NIH Swiss mice were injected [0035 ] FIGS . 7 A - D Diazepam partially mitigates TETS with TETS ( 1.p.) . Two minutes following the second clonic induced neuronal Ca2 + dysregulation . ( A ) Representative seizure , mice were injected i.p. with diazepam ( in saline ) or traces illustrating effects of pre -exposure to increasing con allopregnanolone (Allop , in ß -cyclodextrin ) singly or in centrations of diazepam ( 0.03-1 uM ) on TETS - induced combination . Seizure time to onset, number and duration Ca2 + dysregulation . ( B ) Effect of diazepam (DZP ) on were monitored for 1 h post - TETS exposure . % Survival is TETS - induced increase in integrated Ca2 + levels in Phase I. at 24 h post TETS injection . Data presented as the ( C , D ) Effect of diazepam on the TETS - induced synchronous mean + SEM ( n = 6-8 per group ) . ** p < 0.05 as determined by Ca2 + transient oscillation frequency decrease (C ) and ampli one way ANOVA with Tukey's post hoc test . tude increase (D ) in Phase II . ** , p < 0.01, TETS vs. vehicle [0041 ] FIG . 13. Adult male NIH Swiss mice were injected control, # , p < 0.05, ## , p < 0.01, diazepam + TETS vs. TETS , i.p. with diazepam ( in saline ) or allopregnanolone ( Allop , in n = 6 pooled from two experiments . B -cyclodextrin ) singly or in combination 10 minutes prior to [0036 ] FIGS. 8A -D illustrate allopregnanolone partially i.p. injection of TETS . Seizure time to onset, duration and mitigates TETS - induced neuronal Ca2 + signaling dysregu number were monitored for 1 h post - TETS injection . % lation . ( A ) Representative traces illustrating effects of pre Survival is at 24 h post TETS injection . Data presented as exposure to increasing concentrations of allopregnanolone the mean + SEM ( n = 8 per group ). ** p < 0.01 as determined by (0.03-1 uM ) on TETS - induced Ca2 + dysregulation . ( B ) one way ANOVA with Tukey's post hoc test . Effect of allopregnanolone (AlloP ) on TETS - induced [0042 ] FIG . 14 illustrates the effect of benzodiazepine and increase in integrated Ca2 + levels in Phase I. (C , D ) Effect of neuro -steroid treatments on blood pressure . Adult male NIH allopregnanolone on the TETS - induced synchronous Ca2 + Swiss mice were i.p. injected with diazepam ( DZP ) or transient oscillation frequency decrease (C ) and amplitude allopregnanolone (AlloP ) alone or in combination . Blood increase (D ) in Phase II . ** , p < 0.01, TETS vs. vehicle pressure (BP ) was measured using a tail cuff CODA non control, # , p < 0.05 , ## , p < 0.01 , allopregnanolone + TETS Vs invasive blood measuring system from Kent Scientific . This TETS , n =6 pooled from two experiments . system utilizes volume pressure recording technology to [0037 ] FIGS . 9A - D illustrate low concentrations of allo detect changes that correspond to systolic and diastolic BP . pregnanolone and diazepam in combination act synergisti Diastolic BP is measured and systolic BP calculated . BP is cally to mitigate TETS - induced neuronal Ca2 + signaling measured for 6 days prior to testing to obtain baseline BP dysregulation . ( A ) Representative traces illustrating effect of and allow animals to acclimate to the chamber . Measure pre -exposure to allopregnanolone (0.1 uM ), diazepam (0.1 ments consisted of 20 cycles of 30 sec each with 10 sec delay uM ) or a combination of allopregnanolone ( 0.1 uM ) and between each measurement. Data presented as the diazepam (0.1 uM ) on TETS - induced Ca2 + dysregulation . mean + SEM (n = 6 per group ). ( C , D ) Effects of allopregnanolone or diazepam alone or the combination on TETS - induced synchronous Ca2 + transient DETAILED DESCRIPTION oscillation frequency decrease ( C ) and amplitude increase [0043 ] 1. Introduction ( D ) in phase II. ** , p < 0.01 , TETS vs. vehicle control, ## , [0044 ] Tetramethylenedisulfotetramine ( TETS ) is a potent p < 0.01, allopregnanolone/ diazepam + TETS vs TETS , n = 8 convulsant that is considered a chemical threat agent. We pooled from two experiments . characterized TETS as an activator of spontaneous Ca2 : [0038 ] FIGS. 10A - C illustrate that exposure of mouse oscillations and electrical burst discharges in mouse hip hippocampal neurons following TETS challenge with diaz pocampal neuronal cultures at 13-17 days in vitro using epam (0.1 uM ) and allopregnanolone (0.1 uM ) in combina FLIPR® Fluo - 4 fluorescence measurements and extracellu tion effectively mitigates TETS dysregulated Ca2 + dynam lar multielectrode array (MEA ) recording Acute exposure to ics . ( A ) Representative traces illustrating effects of post TETS ( 22 uM ) reversibly altered the pattern of spontaneous TETS treatment with diazepam or allopregnanolone or the neuronal discharges, producing clustered burst firing and an combination on TETS - induced Ca2 + dysregulation . Amelio overall increase in discharge frequency TETS also dramati ration of TETS - induced alterations in the Phase II response cally affected Ca2 + dynamics causing an immediate but ( see , Cao et al, Toxicological Sciences ( 2012 ) 130 : 362-372 ) transient elevation of neuronal intracellular Ca2 , followed by diazepam and allopregnanolone , either singly or in com by decreased frequency of Ca21 oscillations having greater bination , on the frequency of synchronous Ca2+ oscillation peak amplitudes . The effect on Ca21 dynamics was similar to US 2020/0215078 A1 Jul. 9 , 2020 8

that elicited by picrotoxin and bicuculline, supporting the remain unclear whether they arise from a portion of the brain view that TETS acts by inhibiting GABA , receptor function . or from more widespread circuits . The effect of TETS on Ca2l dynamics requires activation of [ 0049 ] Epilepsy syndromes are further divided by pre NMDA receptors, since the changes induced by TETS were sumptive cause idiopathic , symptomatic , and cryptogenic . prevented by MK -801 block of NMDA receptors , but not Idiopathic epilepsies are generally thought to arise from nifedipine block of L - type Ca2- channels. Pre - treatment genetic abnormalities that lead to alterations in brain excit with theGABA , receptor positive modulators diazepam and ability. Symptomatic epilepsies arise from the effects of an allopregnanolone partially mitigated TETS - induced changes epileptic lesion , whether that lesion is focal, such as a tumor, in Ca2- dynamics . Moreover , low , minimally effective con or a defect in metabolism causing widespread injury to the centrations of diazepam (0.1 uM ) and allopregnanolone (0.1 brain . Cryptogenic epilepsies involve a presumptive lesion UM ) , when administered together, were highly effective in that is otherwise difficult or impossible to uncover during suppressing TETS- induced alterations in Ca2: dynamics , evaluation . Forms of epilepsy are well characterized and suggesting that the combination of positive modulators reviewed , e.g. in Epilepsy: A Comprehensive Textbook synaptic and extrasynaptic GABA , receptors have therapeu ( 3 -volume set) , Engel , et al. , editors , 2nd Edition , 2007 , tic potential . These rapid throughput in vitro assays may Lippincott , Williams and Wilkins ; and The Treatment of assist in the identification of single agents or combinations Epilepsy : Principles and Practice , Wyllie , et al. , editors , 4th that have utility in the treatment of TETS intoxication . Edition , 2005, Lippincott, Williams and Wilkins; and Browne and Holmes, Handbook of Epilepsy , 4th Edition , [ 0045 ] 2. Conditions Amenable to Treatment 2008 , Lippincott , Williams and Wilkins. [0046 ] Co - administration of a benzodiazepine and a neu [0050 ] 3. Subjects Amenable to Treatment rosteroid . In varying embodiments , one or both of the [0051 ] In various embodiments , the patient may be expe benzodiazepine and the neurosteroid are administered in a riencing an electrographic or behavioral seizure or may be sub - therapeutic dose or amount finds use in the rapid ame experiencing a seizure aura , which itself is a localized lioration and /or termination of seizures. In various embodi seizure that may spread and become a full blown behavioral ments , the seizures may be due to an epileptic condition . seizure . For example , the subject may be experiencing aura Optionally , an NMDA receptor antagonist is also co - admin that alerts of the impending onset of a seizure or seizure istered . cluster. [0047 ] The term " epilepsy” refers to a chronic neurologi [0052 ] Alternatively , the subject may be using a seizure cal disorder characterized by recurrent unprovoked seizures . prediction device that alerts of the impending onset of a These seizures are transient signs and /or symptoms of seizure or seizure cluster . Implantable seizure prediction abnormal, excessive or synchronous neuronal activity in the devices are known in the art and described , e.g., in brain . There are over 40 different types of epilepsy , includ D'Alessandro , et al. , IEEE TRANSACTIONS ON BIO ing without limitation childhood absence epilepsy , juvenile MEDICAL ENGINEERING , VOL . 50. NO . 5. MAY 2003 , absence epilepsy , benign Rolandic epilepsy, clonic seizures , and U.S. Patent Publication Nos . 2010/0198098 , 2010 / complex partial seizures , frontal lobe epilepsy , febrile sei 0168603 , 2009/0062682 , and 2008/0243022 . zures , infantile spasms, juvenile myoclonic epilepsy, Len [ 0053 ] The subject may have a personal or familialhistory nox -Gastaut syndrome, Landau - Kleffner Syndrome, myo of any of the epileptic conditions described herein . The clonic seizures , mitochondrial disorders associated with subject may have been diagnosed as having any of the seizures, Lafora Disease , progressive myoclonic epilepsies, epileptic conditions described herein . In some embodiments , reflex epilepsy , and Rasmussen's syndrome. There are also the subject has or is at risk of suffering status epilepticus, numerous types of seizures including simple partial seizures , refractory status epilepticus or super- refractory status epi complex partial seizures , generalized seizures , secondarily lepticus. In some embodiments , the subject has or is at risk generalized seizures , temporal lobe seizures, tonic -clonic of suffering a myoclonic seizure or myoclonic epilepsy , e.g., seizures , tonic seizures , psychomotor seizures , limbic sei juvenile myoclonic epilepsy. The PTZ seizure model dem zures , status epilepticus, refractory status epilepticus or onstrated herein is predictive of utility and /or activity in super -refractory status epilepticus, abdominal seizures , aki counteracting myoclonic seizures or myoclonic epilepsy in netic seizures , autonomic seizures , massive bilateral myo humans. clonus, drop seizures , focal seizures, gelastic seizures , Jack [0054 ] In various embodiments , the subjectmay be at risk sonian march , motor seizures, multifocal seizures, neonatal of exposure to or may have been exposed to tetramethyl seizures, nocturnal seizures, photosensitive seizure, sensory enedisulfotetramine (TETS ). seizures, sylvan seizures, withdrawal seizures and visual [ 0055 ] In various embodiments , the subject may be at risk reflex seizures . of exposure to or may have been exposed to a nerve agent [0048 ] The most widespread classification of the epilep or a pesticide that can cause seizures . Illustrative nerve sies divides epilepsy syndromes by location or distribution agents that can cause seizures include, e.g., organophospho of seizures ( as revealed by the appearance of the seizures rus nerve agents , e.g. , tabun , sarin , soman , GF, VR and / or and by EEG ) and by cause . Syndromes are divided into VX . Illustrative pesticides that can cause seizures include , localization -related epilepsies, generalized epilepsies , or e.g., organophosphate pesticides ( e.g., Acephate (Orthene ) , epilepsies of unknown localization . Localization - related Azinphos- methyl (Gusathion , Guthion ), Bensulide (Be epilepsies , sometimes termed partial or focal epilepsies , tasan , Lescosan ), Bomyl ( Swat ) , Bromophos (Nexion ) , Bro arise from an epileptic focus, a small portion of the brain that mophos - ethyl (Nexagan ) , Cadusafos ( Apache, Ebufos , serves as the irritant driving the epileptic response . Gener Rugby) , Carbophenothion ( Trithion ), Chlorethoxyfos (For alized epilepsies, in contrast , arise from many independent tress ), Chlorfenvinphos ( Apachlor, Birlane ) , Chlormephos foci (multifocal epilepsies ) or from epileptic circuits that (Dotan ), Chlorphoxim (Baythion - C ) , Chlorpyrifos (Brodan , involve the whole brain . Epilepsies of unknown localization Dursban , Lorsban ), Chlorthiophos (Celathion ) , Coumaphos US 2020/0215078 A1 Jul. 9 , 2020 9

( Asuntol , Co -Ral ) , Crotoxyphos (Ciodrin , Cypona ), Crufo ion channels , specifically GABA , receptors Neuroactive mate (Ruelene ), Cyanofenphos (Surecide ), Cyanophos steroids have a wide range of applications from sedation to (Cyanox ), Cythioate (Cyflee , Proban ) , DEF (De - Green ), treatment of epilepsy and traumatic brain injury . Neuroac E - Z - Off D ) , Demeton (Systox ), Demeton - S -methyl (Dura tive steroids act as direct agonists and allosteric positive tox , Metasystoxl) , Dialifor ( Torak ) , Diazinon , Dichlorofen modulators of GABA , receptors. Several synthetic neuro active steroids have been used as sedatives for the purpose thion , ( VC - 13 Nemacide ), Dichlorvos ( DDVP , Vapona ), of general anaesthesia for carrying out surgical procedures . Dicrotophos (Bidrin ) , Dimefos (Hanane , Pestox XIV ) , Exemplary sedating neuroactive steroids include without Dimethoate (Cygon , DeFend ) , Dioxathion (Delnav ) , Disul limitation alphaxolone, alphadolone , hydroxydione and foton (Disyston ) , Ditalimfos, Edifenphos , Endothion , EPBP minaxolone. The neuroactive steroid ganaxolone finds use ( S - seven ), EPN , Ethion (Ethanox ), Ethoprop (Mocap ) , Ethyl for the treatment of epilepsy. In various embodiments , the parathion ( E605 , Parathion , thiophos ), Etrimfos (Ekamet ) , benzodiazepine or non -benzodiazepine benzodiazepine Famphur (Bash , Bo - Ana , Famfos ) , Fenamiphos (Nemacur ), receptor agonist is co - administered with an endogenously Fenitrothion (Accothion , Agrothion , Sumithion ) , Fenophos occurring neurosteroid or other neuroactive steroid . Illustra phon (Agritox , trichloronate ) , Fensulfothion (Dasanit ), Fen tive endogenous neuroactive steroids, e.g., allopregnanolone thion (Baytex , Entex , Tiguvon ), Fonofos (Dyfonate , and tetrahydrodeoxycorticosterone find use . In some N -2790 ), Formothion ( Anthio ), Fosthietan (Nem - A - Tak ) , embodiments , the neurosteroid is selected from the group Heptenophos (Hostaquick ), Hiometon (Ekatin ), Hosalone consisting of allopregnanolone , allotetrahydrodeoxycorti (Zolone ) , IBP (Kitazin ) , Iodofenphos (Nuvanol - N ) , Isazofos costerone , ganaxolone , alphaxolone, alphadolone , hydroxy (Brace , Miral , Triumph ), Isofenphos (Amaze , Oftanol) , dione, minaxolone, and Althesin . Isoxathion ( E -48 , Karphos ), Leptophos (Phosvel ) , Mala [0062 ] In various embodiments the neurosteroid is allo thion (Cythion ), Mephosfolan (Cytrolane ), Merphos ( Easy pregnanolone (ALP ) Allopregnanolone, also known as Off - D , Folex ), Methamidophos (Monitor ) , Methidathion 3a -hydroxy -5a - pregnan - 20 -one or 30,5c - tetrahydropro ( Supracide, Ultracide ), Methyl parathion (E601 . Penncap gesterone , IUPAC name 1- ( 3 - Hydroxy - 10,13 - dimethyl -2,3 , M ). Methyl trithion , Mevinphos (Duraphos , Phosdrin ), 4,5,6,7,8,9,11,12,14,15,16,17 - tetradecahydro - 1H -cyclo Mipafox ( Isopestox , Pestox XV ) , Monocrotophos ( Azo penta [ a ]phenanthren - 17 -yl ) ethanone , and referenced as drin ), Naled (Dibrome ) , Oxydemeton -methyl (Metasystox CAS number 516-54-1 , is a prototypic neurosteroid present in the blood and also the brain . It is a metabolite of R ), Oxydeprofos (Metasystox -S ) , Phencapton (G 28029 ), progesterone and modulator of GABA , receptors . While Phenthoate (Dimephenthoate , Phenthoate ) , Phorate (Ram allopregnanolone, like other GABA , receptor active neuro part, Thimet) , Phosalone ( Azofene , Zolone ) , Phosfolan (Cy steroids such as allotetrahydrodeoxycorticosterone (30,21 lan , Cyolane ), Phosmet ( Imidan , Prolate ) , Phosphamidon dihydroxy -5a - pregnan -20 - one ; THDOC ), positively modu ( Dimecron ), Phostebupirim (Aztec ) , Phoxim (Baythion ) , lates all GABA4 receptor isoforms, those isoforms Pirimiphos - ethyl (Primicid ) , Pirimiphos- methyl ( Actellic ) , containing 8- subunits exhibit greater magnitude potentiation Profenofos (Curacron ), Propetamphos (Safrotin ) , Propyl Allopregnanolone has pharmacological properties similar to thiopyrophosphate ( Aspon ), Prothoate (Fac ) , Pyrazophos other positive modulators of GABA , receptors , including ( Afugan , Curamil) , Pyridaphenthion (Ofunack ), Quinalphos anxiolytic and anticonvulsant activity . Allopregnanolone is (Bayrusil ) , Ronnel (Fenchlorphos , Korlan ), Schradan neuroprotective in many animal models of neurodegenera (OMPA ) , Sulfotep (Bladafum , Dithione , Thiotepp ) , Sulpro fos (Bolstar , Helothion ) , Temephos ( Abate , Abathion ), Ter tive conditions, including, e.g. , Alzheimer's disease (Wang bufos (Contraven , Counter ), Tetrachlorvinphos (Gardona , et al , Proc Natl Acad Sci USA . 2010 Apr. 6 ; 107 ( 14 ) :6498 Rabon ) , Tetraethyl pyrophosphate ( TEPP ) , Triazophos 503 ) , cerebral edema (Limmroth et al. , Br J Pharmacol. ( Hostathion ) , and Trichlorfon (Dipterex , Dylox , Neguvon , 1996 January ; 117 ( 1) : 99-104 ) and traumatic brain injury Proxol ). (He et al ., Restor Neurol Neurosci . 2004 ; 22 ( 1 ) : 19-31 ; and 10056 ] 4. Therapeutic Agents He, et al. , Exp Nerol. 2004 October; 189 ( 2 ) :404-12 ) , Mood [0057 ] Generally , the compositions and methods comprise disorders (Robichaud and Debonnel, Int J Neuropsychop co - administering a benzodiazepine and a neurosteroid . In harmacol. 2006 April ; 9 ( 2 ) : 191-200 ) , Niemann - Pick type C varying embodiments , one or both of the benzodiazepine disease (Griffin et al ., Nat Med . 2004 July , 10 ( 7 ) 704-11) and and the neurosteroid are co - administered at a sub - therapeu acts as an anticonvulsant against chemically induced sei tic dose or amount. Optionally , an NMDA receptor antago zures , including the pentylentetrazol (PTZ ) model (Kokate nist is co - administered . The agents can be co -administered et al ., J Pharmacol Exp Ther. 1994 September; 270 ( 3 ) concurrently or sequentially . The agents can be co -admin 1223-9 ) The chemical structure of allopregnanolone is istered via the same or different routes of administration . In depicted below in Formula I: various embodiments, the agents are co -administered in a single composition . [0058 ] a . Benzodiazepines [ 0059 ] Any benzodiazepine known in the art finds use in the present compositions and methods. Illustrative benzodi azepine that find use include without limitation bretazenil, clonazepam , cloxazolam , clorazepate , diazepam , fludiaz epam , flutoprazepam , lorazepam ,midazolam , nimetazepam , nitrazepam , phenazepam , temazepam and clobazam . In some embodiments , the benzodiazepine is midazolam . In some embodiments , the benzodiazepine is diazepam . 3 [ 0060 ] b . Neurosteroids HOITU ... [0061 ] The terms “ neuroactive steroid ” or “ neurosteroids" H interchangeably refer to steroids that rapidly alter neuronal excitability through interaction with neurotransmitter- gated US 2020/0215078 A1 Jul. 9. 2020 10

[ 0063 ] In various embodiments , the compositions com [0067 ] c . NMDA Receptor Antagonists prise a sulfate , salt , hemisuccinate , nitrosylated , derivative [0068 ] Illustrative NMDA receptor antagonists that find or congener of allopregnanolone. use include without limitation , e.g., dizocilpine (MK -801 ) , [0064 ] Delivery of other neurosteroids also can be meperidine , methadone , dextropropoxyphene, tramadol, enhanced by formulation in a cyclodextrin and /or in an ketobemidone , ketamine, dextromethorphan , phencyclidine , edible oil. Other neurosteroids that can be formulated in a nitrous oxide ( N , O ) , AP5 ( R - 2 -amino - 5 - phosphonopentano cyclodextrin and/ or in an edible oil, include without limita ate ), AP7 ( 2 - amino - 7 - phosphonoheptanoic acid ) , CPPene tion allotetrahydrodeoxycorticosterone (30,21 -dihydroxy ( 3 - [ ( R ) -2 - carboxypiperazin - 4 - yl] -prop - 2 -enyl - 1 - phospho 5a -pregnan - 20 -one ; THDOC ) , 3 0,21 - dihydroxy - 5b -preg nic acid ), selfotel , amantadine, dextrallorphan , dextrorphan , nan - 20 -one , pregnanolone (3a -hydroxy - 58 -pregnan - 20 ethanol, eticyclidine , gacyclidine , ibogaine, magnesium , one ) , Ganaxolone ( INN , also known as CCD - 1042 ; IUPAC memantine, methoxetamine, rolicyclidine.tenocyclidine , name ( 3a ,5a ) -3 - hydroxy - 5 -methylpregnan - 20 -one ; 1 - [ (3R , methoxydine, tiletamine , xenon , neramexane , eliprodil , 55,8R , 9S , 105,138,148,17S ) -3 -hydroxy -3,10,13 - trimethyl etoxadrol, dexoxadrol, WMS 2539 , NEFA , remacemide , 1,2,4,5,6,7,8,9,11,12,14,15,16,17 -tetradecahydrocyclopenta delucemine, 8A -PDHQ , aptiganel , 1 HU -211 , rhynchophyl [ a ]phenanthren - 17 -yllethanone ) , alphaxolone , alphadolone , line, 1 - Aminocyclopropanecarboxylic acid (ACPC ) , hydroxydione, minaxolone , and Althesin ( a mixture of 7 - Chlorokynurenate , DCKA ( 5,7 - dichlorokynurenic acid ), alphaxolone , alphadolone , tetrahydrodeoxycorticosterone , kynurenic acid , lacosamide , CP - 101,606 ( traxoprodil ) , pregnenolone, dehydroepiandrosterone (DHEA ), 7 - substi AZD6765 ( lanicemine ) and GLYX - 13 . In some embodi tuted benz [c ] indene - 3 -carbonitriles ( see , e.g., Hu, et al. , J ments , the NMDA receptor antagonist is selected from the Med Chem . ( 1993) 36 (24 ): 3956-67 ); 7- ( 2- hydroxyethyl ) group consisting of ketamine, dextromethorphan , phency benz [ e ] indene analogues (see , e.g., Han , et al ., J Med Chem . clidine , CPPene ( 3 - [ ( R )-2 - carboxypiperazin - 4 - yl ] -prop - 2 (1995 ) 38 (22 ) :4548-56 ); 3 alpha- hydroxy - 5 alpha -pregnan enyl- 1- phosphonic acid ), selfotel, amantadine , dextrorphan , 20 - one and 3 alpha -hydroxy - 5 beta - pregnan - 20 -one ana memantine , tiletamine, neramexane, cliprodil, remacemide , logues ( see. e.g., Han , et al. , J Med Chem . (1996 ) 39( 21 ) : aptiganel , 1 -Aminocyclopropanecarboxylic acid ( ACPC ), 4218-32 ) ; enantiomers of dehydroepiandrosterone sulfate , 7 - Chlorokynurenate , DCKA (5,7 -dichlorokynurenic acid ), pregnenolone sulfate , and ( 3alpha, 5beta ) -3 - hydroxypreg kynurenic acid , CP - 101,606 (traxoprodil ) , AZD6765 ( lani nan - 20 -one sulfate ( see , e.g. , Nilsson , et al ., J Med Chem . cemine ) and GLYX - 13 . In some embodiments , the NMDA ( 1998 ) 41 ( 14) : 2604-13) : 13,24 - cyclo -18,21 - dinorcholane receptor antagonist is dizocilpine (MK - 801) . analogues ( see , e.g., Jiang, et al , J Med Chem . (2003 ) [0069 ] 5. Formulation and Administration 46 ( 25) :5334-48 ): N -acylated 17a -aza -D -homosteroid ana [0070 ] In various embodiments , one or more of the ben logues ( see , e.g., Covey, et al, J Med Chem . ( 2000 ) 43 (17 ) : zodiazepines and one or more neurosteroids are formulated 3201-4 ) ; 5 beta -methyl - 3 -ketosteroid analogues (see , e.g., for intramuscular, intravenous, subcutaneous, intrapulmo Zeng , et al. , J Org Chem . ( 2000 ) 65 ( 7) : 2264-6 ); 18 -no nary and /or inhalational administration . In various embodi randrostan - 17 -one analogues (see , e.g. , Jiang , et al. , J Org ments , the benzodiazepines are formulated for delivery via Chem . (2000 ) 65 ( 11) : 3555-7 ) ; (3alpha , 5alpha )- and (3alpha , an inhaler . In various embodiments other routes of delivery, 5beta )-3 -hydroxypregnan - 20 -one analogs (see . e.g., Zeng , et described herein may be appropriate . Optionally a NMDA al ., J Med Chem . (2005 ) 48 ( 8 ) :3051-9 ) ; benz [ f ] indenes (see , receptor antagonist is included in the compositions and / or e.g. , Scaglione , et al ., J Med Chem . (2006 ) 49( 15 ): 4595 co -administration . 605 ) ; enantiomers of androgens (see , e.g., Katona , et al. , Eur [0071 ] Appropriate dosing will depend on the sire and J Med Chem . (2008 ) 43 ( 1) : 107-13 ), cyclopenta [ b ]phenan health of the patient and can be readily determined by a threnes and cyclopenta [b ]anthracenes (see , e.g. , Scaglione, trained clinician . Initial doses are low and then can be et al. , J Med Chem . (2008 ) 51 (5 ) 1309-18 ), 2beta -hydroxy incrementally increased until the desired therapeutic effect is gonane derivatives (see , e.g. , Wang , et al. , Tetrahedron achieved with little or no adverse side effects . Determination (2007 ) 63 (33 ) :7977-7984 ) , 16 -alphaxalone and corre of an effective amount for administration in a single dosage sponding 17- carbonitrile analogues ( see, e.g. , Bandyopad is well within the capability of those skilled in the art , hyaya, et al. , Bioorg Med Chem Lett. ( 2010 ) 20 ( 22 ) :6680-4 ) ; especially in light of the detailed disclosure provided herein A ( 16 ) and A ( 17 ( 20 ) analogues of A (16 )-alphaxalone (see , Generally , an efficacious or effective amount of the agents e.g. , Stastna , et al. , J Med Chem . (2011 ) 54 ( 11 ) :3926-34 ) ; ( e.g., one or more benzodiazepines and one or more neuro neurosteroid analogs developed by CoCensys (now Purdue steroids , optionally including a NMDA receptor antagonist ) Neuroscience ) (e.g. , CCD - 3693 , Co2-6749 (a.k.a. , GMA is determined by first administering a low dose or small 839 and WAY - 141839 ) ; neurosteroid analogs described in amount of the agent and then incrementally increasing the U.S. Pat. No. 7,781,421 and in PCT Patent Publications WO administered dose or dosages, adding a second or third 2008/157460 ; WO 1993/003732 ; WO 1993/018053; WO medication as needed , until a desired effect of is observed in 1994/027608 ; WO 1995/021617 ; WO 1996/016076 ; WO the treated subject with minimal or no toxic side effects . 1996/040043 , as well as salts , hemisuccinates, nitrosylated , Applicable methods for determining an appropriate dose and sulfates and derivatives thereof . dosing schedule for administration of a combination of [0065 ] In various embodiments , the steroid or neuroster agents of the present invention are described , for example , oid is not a sex hormone . In various embodiments, the in Goodman and Gilman's The Pharmacological Basis of steroid or neurosteroid is not progesterone . Therapeutics, 12th Edition , 2010, supra , in a Physicians' [ 0066 ] As appropriate , the steroid or neurosteroid (e.g. , Desk Reference (PDR ) , 67th Edition , 2013 ; in Remington : allopregnanolone ) may or may not be micronized . As appro The Science and Practice of Pharmacy, 21° Ed ., 2005 , priate , the steroid or neurosteroid ( e.g., allopregnanolone ) supra , and in Martindale : The Complete Drug Reference , may or may not be enclosed in microspheres in suspension Sweetman , 2005 , London : Pharmaceutical Press ., and in in the oil . Martindale, Martindale : The Extra Pharmacopoeia , 31st US 2020/0215078 A1 Jul. 9. 2020 11

Edition , 1996 , Amer Pharmaceutical Assn , each of which are Farr et al. , 1995 Thorax 50 :639-644 ) These systems are hereby incorporated herein by reference . therefore used mostly for local delivery of drugs to the [0072 ] In various embodiments , the agents ( e.g., one or pulmonary airways . In addition , metered doses inhalers may more benzodiazepines and one or more neurosteroids, not be able to deliver sufficient volumes of even a concen optionally including a NMDA receptor antagonist ) are nebu trated midazolam solution to produce the desired rapid lized . Methods and systems for intrapulmonary delivery of antiseizure effect. Accordingly , in various embodiments , a agents , e.g., benzodiazepines , are known in the art and find metered doses inhaler is not used for delivery of the ben use . Illustrative systems for aerosol delivery of benzodiaz zodiazepine , e.g., midazolam . In one embodiment a nebu epines by inhalation are described , e.g., in U.S. Pat . Nos . lization system with the capability of delivering < 5 um 5,497,763 ; 5,660,166 ; 7,060,255 ; and 7,540,286 ; and U.S. particles ( e.g., the PARILC Star, which has a high efficiency, Patent Publication Nos. 2003/0032638 ; and 2006/0052428 , 78 % respirable fraction 0.1-5 um , see , e.g. , pari.com ) is used each of which are hereby incorporated herein by reference in for intrapulmonary administration . Electronic nebulizers their entirety for all purposes . Preferably , the agents ( e.g. , which employ a vibrating mesh or aperture plate to generate one or more benzodiazepines and one or more neurosteroids , an aerosol with the required particle size can deliver suffi optionally including a NMDA receptor antagonist) are nebu cient quantities rapidly and find use (See , e.g. , Knoch and lized without the input of heat . Keller, 2005 Expert Opin Drug Deliv 2 : 377-390 ) . Also , [0073 ] For administration of the nebulized and / or aero custom -designed hand- held , electronic nebulizers can be solized agents ( e.g., one or more benzodiazepines and one or made and find use . more neurosteroids, optionally including a NMDA receptor [0076 ] Aerosolized delivery of the agents (e.g. , one or antagonist ), the size of the aerosol particulates can be within more benzodiazepines and one or more neurosteroids, a range appropriate for intrapulmonary delivery, particularly optionally including a NMDA receptor antagonist ) can allow delivery to the distal alveoli . In various embodiments , the for reduced dosing to achieve desired efficacy, e.g. , in aerosol particulates have a mass median aerodynamic diam comparison to intravenous or intranasal delivery . eter ( “MMAD ” ) of less than about 5 um , 4 um , 3 um , for [0077 ] In various embodiments, the agents ( e.g., one or example , ranging from about 1 um to about 3 um , e.g. , from more benzodiazepines and one or more neurosteroids, about 2 um to about 3 um , e.g. , ranging from about 0.01 um optionally including a NMDA receptor antagonist ) are dis to about 0.10 um . Aerosols characterized by a MMAD solved or suspended in a cyclodextrin . In varying embodi ranging from about 1 um to about 3 um can deposit on ments , the cyclodextrin is an a -cyclodextrin , a B -cyclodex alveoli walls through gravitational settling and can be trin or a y -cyclodextrin . In varying embodiments , the absorbed into the systemic circulation , while aerosols char cyclodextrin is selected from the group consisting of acterized by a MMAD ) ranging from about 0.01 um to 0.10 hydroxypropyl- L -cyclodextrin , endotoxin controlled B -cy um can also be deposited on the alveoli walls through clodextrin sulfobutyl ethers , or cyclodextrin sodium salts diffusion . Aerosols characterized by a MMAD ) ranging ( e.g., CAPTTISOL® ). Such formulations are useful for from about 0.15 um to about 1 um are generally exhaled . intramuscular, intravenous and /or subcutaneous administra Thus, in various embodiments , aerosol particulates can have tion . a MMAD ranging from 0.01 um to about 5 um , for example , [0078 ] In various embodiments , the agents ( e.g., one or ranging from about 0.05 um to about 3 um , for example , more benzodiazepines and one or more neurosteroids , ranging from about 1 um to about 3 um , for example , optionally including a NMDA receptor antagonist ) are dis ranging from about 0.01 um to about 0.1 um . The nebulized solved or suspended in an oil that is edible and / or digestible and / or aerosolized benzodiazepines can be delivered to the by the subject, e.g., without undesirable side effects . distal alveoli, allowing for rapid absorption and efficacy . [ 0079 ] In various embodiments , the edible oil comprises [0074 ] In various embodiments , the agents ( e.g., one or one or more vegetable oils . In various embodiments , the more benzodiazepines and one or more neurosteroids , vegetable oil is selected from the group consisting of coco optionally including a NMDA receptor antagonist ) are for nut oil, corn oil, cottonseed oil, olive oil, palm oil, peanut mulated in a solution comprising excipients suitable for oil , rapeseed oil , canola oil, safflower oil, sesame oil, soy aerosolized intrapulmonary delivery . The solution can com bean oil , sunflower oil , and mixtures thereof. prise one or more pharmaceutically acceptable carriers and / [0080 ] In some embodiments , the edible oil comprises one or excipients . Pharmaceutically acceptable refers to or more nut oils . In some embodiments , the nut oil is approved or approvable by a regulatory agency of the selected from the group consisting of almond oil , cashew oil, Federal or a state government or listed in the U.S Pharma hazelnut oil, macadamia oil, mongongo nut oil, pecan oil , copoeia or other generally recognized pharmacopoeia for pine nut oil , pistachio oil, walnut oil, and mixtures thereof. use in animals , and more particularly in humans. Preferably , [0081 ] In some embodiments , the edible oil does not the solution is buffered such that the solution is in a comprise castor oil. In some embodiments , the edible oil relatively neutral pH range , for example , a pH in the range does not comprise peanut oil . of about 4 to 8 , for example , a pH in the range of about 5-7 . [0082 ] Generally, the oils used in the present compositions In some embodiments , the benzodiazepine is formulated in are isolated from the source, e.g., plant, and used without a buffered solution , for example , phosphate -buffered saline . including further additives ( e.g. , surfactants , acids (organic [0075 ] In various embodiments , the agents (e.g. , one or or fatty ) , alcohols , esters , co - solvents , solubilizers , lipids, more benzodiazepines and one or more neurosteroids, polymers, glycols ) or processing . In various embodiments , optionally including a NMDA receptor antagonist ) are pre the oil vehicle further comprises a preservative ( e.g. , vitamin pared as a concentrated aqueous solution . Ordinary metered E ). dose liquid inhalers have poor efficiency for the delivery to [0083 ] The oil- agents ( e.g. , one or more benzodiazepines the deep lung because the particle size is not sufficiently and one or more neurosteroids, optionally including a small (Kim et al. 1985 Am Rev Resp Dis 132 : 137-142 ; and NMDA receptor antagonist ) compositions can be formulated US 2020/0215078 A1 Jul. 9 , 2020 12

for oral and / or transmucosal delivery using any method stored softgels , the greatest risk from microorganisms comes known in the art . In one embodiment, the oil -agents ( e.g. , from molds and yeasts . For this reason , preservatives can be one or more benzodiazepines and one or more neurosteroids, incorporated into the capsule shell. Suitable preservatives optionally including a NMDA receptor antagonist ) compo include alkyl esters of p -hydroxy benzoic acid such as sition is formulated in a capsule , e.g., for oral delivery. methyl, ethyl, propyl, butyl and heptyl esters (collectively [0084 ] a . Capsules known as “ parabens ” ) or combinations thereof. [ 0085 ] The capsule shells can be prepared using one or [ 0096 ] Flavorings can be used to mask unpleasant odors more film forming polymers . Suitable film forming poly and tastes of fill formulations. Suitable flavorings include mers include natural polymers , such as gelatin , and synthetic synthetic and natural flavorings . The use of flavorings can be film forming polymers , such asmodified celluloses. Suitable problematic due to the presence of aldehydes which can modified celluloses include , but are not limited to , hydroxy cross- link gelatin . As a result , buffering salts and acids can propyl methyl cellulose , methyl cellulose, hydroxypropyl be used in conjunction with flavorings that contain alde methyl cellulose acetate succinate , hydroxypropyl methyl hydes in order to inhibit cross - linking of the gelatin . cellulose phthalate , and cellulose acetate phthalate . Hard or [0097 ] b . Enteric Capsules soft capsules can be used to administer the hormone . Hard [0098 ] Alternatively, the liquid fills can be incorporated shell capsules are typically prepared by forming the two into an enteric capsule , wherein the enteric polymer is a capsule halves, filling one of the halves with the fill solution , component of the capsule shell, as described in WO 2004 / and then sealing the capsule halves together to form the 030658 to Banner Pharmacaps , Inc. The enteric capsule finished capsule . Soft gelatin capsules are typically prepared shell is prepared from a mass comprising a film - forming using a rotary die encapsulation process as described below . polymer, an acid - insoluble polymer which is present in an [ 0086 ] i . Gelatin Capsules amount making the capsule resistant to the acid within the [ 0087 ] Gelatin is the product of the partial hydrolysis of stomach , an aqueous solvent, and optionally , one or more collagen . Gelatin is classified as either Type A or Type B plasticizers and / or colorants . Other suitable shell additives gelatin . Type A gelatin is derived from the acid hydrolysis of including opacifiers , colorants , humectants , preservatives, collagen while Type B gelatin is derived from the alkaline flavorings , and buffering salts and acids may be added . hydrolysis of collagen . Traditionally , bovine bones and skins [0099 ] i. Film -Forming Polymers have been used as raw materials for manufacturing Type A [0100 ] Exemplary film - forming polymers can be of natu and Type B gelatin while porcine skins have been used ral or synthetic origin . Natural film - forming polymers extensively for manufacturing Type A gelatin . In general , include gelatin and gelatin - like polymers . Other suitable acid -processed gelatins form stronger gels than lime- pro natural film - forming polymers include shellac , alginates , cessed gelatins of the same average molecular weight. The pectin , and zeins. Synthetic film - forming polymers include capsules can be formulated as hard or soft gelatin capsules . hydroxypropyl methyl cellulose ,methyl cellulose, hydroxy [ 0088 ] ii. Non - Gelatin Capsules propyl methyl cellulose acetate succinate , hydroxypropyl [0089 ] Capsules can be prepared from non - gelatin mate methyl cellulose phthalate , cellulose acetate phthalate , and rials , such as carrageenan or modified celluloses. Carra acrylates such as poly (meth )acrylate . The weight ratio of geenan is a natural polysaccharide hydrocolloid , which is acid - insoluble polymer to film - forming polymer is from derived from seaweed . It includes a linear carbohydrate about 15 % to about 50 % In one embodiment, the film polymer of repeating sugar units , without a significant forming polymer is gelatin . degree of substitution or branching . Most , if not all, of the [0101 ] ii . Acid - Insoluble Polymers galactose units on a carrageenan molecule possess a sulfate [0102 ] Exemplary acid - insoluble polymers include cellu ester group . There are three main types of carrageenan : lose acetate phthalate , cellulose acetate butyrate , hydroxy kappa, iota and lambda ; although minor forms called mu and propyl methyl cellulose phthalate , algenic acid salts such as nu carrageenan also exist. sodium or potassium alginate , shellac , pectin , acrylic acid [0090 ] iii. Shell Additives methylacrylic acid copolymers (available under the trade [0091 ] Suitable shell additives include plasticizers , opaci name EUDRAGIT® from Rohm America Inc. , Piscataway , fiers , colorants , humectants , preservatives, flavorings, and N.J. as a powder or a 30 % aqueous dispersion ; or under the buffering salts and acids, and combinations thereof. tradename EASTACRYL® , from Eastman Chemical Co. [0092 ] Plasticizers are chemical agents added to gelatin to Kingsport . Tenn . , as a 30 % dispersion ) in one embodiment, make the material softer and more flexible . Suitable plasti the acid - insoluble polymer is EUDRAGIT® L100 , which is cizers include, but are not limited to , glycerin , sorbitol a methacrylic acid /methacrylic acid methyl ester copolymer . solutions which are mixtures of sorbitol and sorbitan , and The acid -insoluble polymer is present in an amount from other polyhydric alcohols such as propylene glycol and about 8 % to about 20 % by weight of the wet gelatin mass . maltitol or combinations thereof. The weight ratio of acid - insoluble polymer to film - forming [0093 ] Opacifiers are used to opacify the capsule shell polymer is from about 15 % to about 50 % . when the encapsulated active agents are light sensitive . [0103 ] iii . Aqueous Solvent Suitable opacifiers include titanium dioxide , zinc oxide , [0104 ] Hard and soft capsules are typically prepared from calcium carbonate and combinations thereof. solutions or suspensions of the film forming polymer and the [0094 ] Colorants can be used for marketing and product acid - insoluble polymer. Suitable solvents include water , identification / differentiation purposes . Suitable colorants aqueous solvents , and organic solvents . In one embodiment, include synthetic and natural dyes and combinations thereof. the solvent is water or an aqueous solvent. Exemplary [ 0095 ] Humectants can be used to suppress the water aqueous solvents include water or aqueous solutions of activity of the softgel . Suitable humectants include glycerin alkalis such as ammonia , sodium hydroxide , potassium and sorbitol , which are often components of the plasticizer hydroxide , ethylene diamine, hydroxylamine , tri- ethanol composition . Due to the low water activity of dried, properly amine , or hydroalcoholic solutions of the same. The alkali US 2020/0215078 A1 Jul. 9 , 2020 13 can be adjusted such that the final pH of the gelatin mass is [0118 ] h . Soft Capsules less than or equal to 9.0 , preferably less than or equal to 8.5 , [0119 ] Soft capsules are typically produced using a rotary more preferably less than or equal to 8.0 . In one embodi die encapsulation process . The gel mass is fed either by ment, the alkali is a volatile alkali such as ammonia or gravity or through positive displacement pumping to two ethylene diamine . Upon drying of the finished capsule , the heated ( 48-65 ° C.) metering devices . The metering devices water content of the capsule is from about 2 % to about 10 % control the flow of gel into cooled (10-18 ° C.) , rotating casting drums. Ribbons are formed as the cast gel masses set by weight of the capsule , preferably from about 4 % to about on contact with the surface of the drums. 8 % by weight of the capsule . [0120 ] The ribbons are fed through a series of guide rolls [0105 ] iv. Plasticizers and between injection wedges and the capsule - forming dies [ 0106 ] Exemplary plasticizers include glycerol, glycerin , A food - grade lubricant oil is applied onto the ribbons to sorbitol, polyethylene glycol, citric acid , citric acid esters reduce their tackiness and facilitate their transfer. Suitable such as triethylcitrate , polyalcohols with 3-6 carbons and lubricants include mineral oil , medium chain triglycerides, combinations thereof. The plasticizer to polymer ( film form and soybean oil. Fill formulations are fed into the encapsu ing polymer plus acid - insoluble polymer ) ratio is from about lation machine by gravity . In the preferred embodiment, the 10 % to about 50 % b of the polymer weight. soft capsules contain printing on the surface , optionally [ 0107 ] c . Methods ofManufacture identifying the encapsulated agent and / or dosage . [0108 ] i . Capsule Fill [0121 ] Upon drying of the finished capsule , the water content of the capsule is from about 2 % to about 10 % by [0109 ] The fill material is prepared by dissolving the weight of the capsule , preferably from about 4 % to about 8 % steroid or neurosteroid ( e.g., allopregnanolone ) in the carrier by weight of the capsule . containing a fatty acid solvent , such as oleic acid . The [0122 ] c . Enteric Capsules mixture of hormone and fatty acid may be heated to facilitate [0123 ] A method of making an enteric capsule shell is dissolution of the hormone. Upon cooling to room tempera described in WO 2004/030658 to Banner Pharmacaps, Inc. ture and encapsulation , the solution remains a liquid. The fill The enteric mass is typically manufactured by preparing an is typically deacrated prior to encapsulation in a soft gelatin aqueous solution comprising a film - forming , water soluble capsule . Additional excipients including, but not limited to , polymer and an acid - insoluble polymer and mixing the co - solvents , antioxidants may be added to the mixture of the solution with one or more appropriate plasticizers to form a hormone and fatty acid Again the mixture may be heated to gelatin mass . Alternatively , the enteric mass can be prepared facilitate dissolution of the excipients . The steroid or neu by using a ready -made aqueous dispersion of the acid rosteroid ( e.g., allopregnanolone) is fully dissolved in the insoluble polymer by adding alkaline materials such as carrier of the present invention and remains so upon storage. ammonium , sodium , or potassium hydroxides or other alka [0110 ] ii . Capsule Shell lis that will cause the acid - insoluble polymer to dissolve . [ 0111 ] a . Gelatin or Non -Gelatin Capsules The plasticizer- wetted , film - forming polymer can then be [0112 ] The main ingredients of the capsule shell are gela mixed with the solution of the acid - insoluble polymer. The tin (or a gelatin substitute for non -gelatin capsules ), plasti mass can also be prepared by dissolving the acid - insoluble cizer, and purified water . The primary difference between polymer or polymers in the form of salts of the above soft and hard capsules is the amount of plasticizer present in mentioned bases or alkalis directly in water and mixing the the capsule shell . solution with the plasticizer -wetted , film - forming polymer. [0113 ] Typical gel formulations contain ( w / w ) 40-50 % The mass is cast into films or ribbons using heat controlled gelatin , 20-30 % plasticizer, and 30-40 % purified water . drums or surfaces . The fill material is encapsulated in a soft Most of the water is subsequently lost during capsule drying. capsule using a rotary die . The capsules are dried under The ingredients are combined to form a molten gelatin mass controlled conditions of temperature and humidity . The final using either a cold melt or a hotmelt process . The prepared moisture content of the shell composition is from about 2 % gel masses are transferred to preheated , temperature -con to about 10 % by weightof the capsule shell , preferably from trolled , jacketed holding tanks where the gel mass is aged at about 4 % to about 8 % by weight by weight of the capsule 50-60 ° C. until used for encapsulation . shell . [0124 ] Alternatively , release of the agents ( e.g. , one or [ 0114 ] i. Cold Melt Process more benzodiazepines and one or more neurosteroids , [ 0115 ] The cold melt process involves mixing gelatin with optionally including a NMDA receptor antagonist) from the plasticizer and chilled water and then transferring the mix capsule can be modified by coating the capsule with one or ture to a jacket- heated tank . Typically , gelatin is added to the more modified release coatings, such as sustained release plasticizer at ambient temperature (18-22 ° C.) . Themixture coatings, delayed release coatings, and combinations is cooked ( 57-95 ° C.) under vacuum for 15-30 minutes to a thereof . homogeneous , deaerated gel mass. Additional shell addi [0125 ] The concentration of the agents (e.g. , one or more tives can be added to the gel mass at any point during the gel benzodiazepines and one or more neurosteroids, optionally manufacturing process or they may be incorporated into the including a NMDA receptor antagonist ) in the vehicle ( e.g., finished gel mass using a high torque mixer. cyclodextrin and /or edible oil ) is preferably in unit dosage [0116 ] ii. Hot Melt Process form . The term " unit dosage form ” , as used in the specifi [0117 ] The hot melt process involves adding , under mild cation , refers to physically discrete units suitable as unitary agitation , the gelatin to a preheated (60-80 ° C.) mixture of dosages for human subjects and animals , each unit contain plasticizer and water and stirring the blend until complete ing a predetermined quantity of active material calculated to melting is achieved . While the hot melt process is faster than produce the desired pharmaceutical effect in association the cold melt process , it is less accurately controlled and with the required pharmaceutical diluent, carrier or vehicle . more susceptible to foaming and dusting . The specifications for the novel unit dosage forms of this US 2020/0215078 A1 Jul. 9 , 2020 14

invention are dictated by and directly dependent on (a ) the subject can self -administer via the intrapulmonary route a unique characteristics of the active material and the particu dose of the benzodiazepine . If the benzodiazepine is admin lar effect to be achieved and ( b ) the limitations inherent in istered in an efficacious amount , the sensation of aura should the art of compounding such an active material for use in subside and /or the seizure prediction device should no humans and animals , as disclosed in detail in this specifi longer predict the imminent occurrence of an impending cation , these being features of the present invention . seizure . If the sensation of aura does not subside and / or the [0126 ] In various embodiments, the benzodiazepines are seizure prediction device continues to predict an impending administered at a dose that is less than about 10 % , 15 % , seizure , a second dose of benzodiazepine can be adminis 25 % , 50 % or 75 % of established doses for their adminis tered . tration for the prevention or mitigation of an epileptic [0131 ] In other embodiments , the efficacy is monitored by seizure . In some embodiments , the benzodiazepine is admin a caregiver. For example , in a subject experiencing the onset istered at a dose in the range of about 0.05 mg/ kg to about of a seizure or in situations where a seizure has commenced , 1.0 mg/ kg , for example , about 0.2 mg/ kg to about 0.8 mg/ kg , the subjectmay require intrapulmonary administration of the for example , about 0.05 mg /kg , 0.08 mg/ kg , 0.1 mg/ kg , 0.2 benzodiazepine by a caregiver. If the benzodiazepine is mg/ kg , 0.3 mg/ kg , 0.4 mg/kg , 0.5 mg/ kg , 0.6 mg/ kg , 0.7 administered in an efficacious amount, the seizure, along mg/ kg , 0.8 mg/ kg , 0.9 mg/ kg , or 1.0 mg/kg . In some with the subject's symptoms of the seizure , should rapidly embodiments the benzodiazepine is administered at a dose terminate or abort if the seizure does not terminate , a second in the range of about 10 ug /kg to about 80 ug/ kg , for dose of the benzodiazepine can be administered . example , about 20 ug/ kg to about 60 ug/ kg , for example , [0132 ] 7. Kits about 25 g /kg to about 50 g /kg , for example , about 10 ug /kg , [0133 ] The pharmaceutical compositions and neurosteroid 15 ug /kg , 20 ug /kg , 25 ug/ kg , 30 ug/ kg , 35 ug /kg , 40 ug /kg , and benzodiazepine combinations can be provided in a kit . 45 ug/ kg , 50 ug/ kg , 60 ug/ kg, 70 ug /kg , or 80 ug/ kg . In some In certain embodiments , a kit of the present invention embodiments , the benzodiazepine is administered at a dose comprises one or more benzodiazepines and one or more in the range of about 0.3 ug /kg to about 3.0 ug/ kg . In varying neurosteroids in separate formulations . In varying embodi embodiments , the benzodiazepine is administered at a dose ments , one or both of the benzodiazepine and the neuros that does not decrease blood pressure . When co - adminis teroid are provided in subtherapeutic doses or amounts in tered with one or more neurosteroids, the benzodiazepine certain embodiments , the kits comprise one or more benzo can be co -administered at a dose that is less than about 10 % , diazepines and one or more neurosteroids within the same 15 % , 259 % , 50 % or 75 % of the aforementioned doses or at formulation . In varying embodiments , one or both of the a dose that is less than about 10 % , 15 % , 25 % , 50 % or 75 % benzodiazepine and the neurosteroid are provided in sub of established doses for their administration for the preven therapeutic doses or amounts . In certain embodiments , the tion or mitigation of an epileptic seizure. When co -admin kits provide the one or more benzodiazepines and one or istered with one or more neurosteroids, the benzodiazepine more neurosteroids independently in uniform dosage for can be co -administered at a dose that is less than about 10 % , mulations throughout the course of treatment. In varying 15 % , 25 % , 50 % or 75 % of doses known to be efficacious via embodiments, one or both of the benzodiazepine and the a selected route of administration ( e.g. , oral , intramuscular, neurosteroid are provided in subtherapeutic doses or intravenous , subcutaneous and /or intrapulmonary) . amounts . In certain embodiments , the kits provide the one or [ 0127] In various embodiments , the compositions are for more benzodiazepines and one or more neurosteroids in mulated for administration of about 5 mg/ kg to about 50 graduated dosages over the course of treatment, either mg/kg of the steroid or neurosteroid ( e.g., allopreg increasing or decreasing , but usually increasing to an effi nanolone ) , e.g., about 5 mg/ kg , 10 mg/ kg , 15 mg/ kg , 20 cacious dosage level, according to the requirements of an mg/ kg , 25 mg/ kg , 30 mg/ kg , 35 mg/ kg , 40 mg/ kg , 45 mg/ kg , individual. In varying embodiments , one or both of the or 50 mg/ kg . When co -administered with one or more benzodiazepine and the neurosteroid are provided in sub benzodiazepines, the steroid or neurosteroid ( e.g. , allopreg therapeutic doses or amounts . nanolone) can be co -administered at a dose that is less than [0134 ] In some embodiments , the benzodiazepine is about 10 % , 15 % , 259 % , 50 % or 75 % of the aforementioned selected from the group consisting of bretazenil, clonaze doses or at a dose that is less than about 10 % , 15 % , 25 % , pam , cloxazolam , clorazepate , diazepam , fludiazepam , flu 50 % or 75 % of established doses for their administration for toprazepam , lorazepam , midazolam , nimetazepam , nitraze the prevention or mitigation of an epileptic seizure . When pam , phenazepam , temazepam and clobazam . In some co -administered with one or more benzodiazepines, the embodiments , the benzodiazepine is selected from the group neurosteroid can be co -administered at a dose that is less consisting of midazolam , lorazepam and diazepam . In some than about 10 % , 15 % , 25 % , 50 % or 75 % of doses known to embodiments , the neurosteroid is selected from the group be efficacious via a selected route of administration ( e.g. , consisting of allopregnanolone, allotetrahydrodeoxycorti oral, intramuscular , intravenous, subcutaneous and/ or intra costerone, ganaxolone , alphaxolone, alphadolone , hydroxy pulmonary ) . dione , minaxolone , and Althesin . In some embodiments , the (0128 ] 6. Monitoring Efficacy neurosteroid is allopregnanolone. In some embodiments , the [0129 ] Co -administration of a benzodiazepine and a neu kit comprises allopregnanolone and a benzodiazepine rosteroid (optionally with an NMDA receptor antagonist) to selected from the group consisting of midazolam , loraze a subject results in the prevention of the occurrence of an pam , and diazepam . impending seizure and / or the rapid termination or abortion [0135 ] In some embodiments , the kits further comprise a of a seizure in progress . NMDA receptor antagonist . In some embodiments , the [0130 ] In various embodiments , efficacy can be monitored NMDA receptor antagonist is selected from the group con by the subject . For example , in a subject experiencing aura sisting of dizocilpine (MK - 801 ), meperidine , methadone , or receiving a warning from a seizure prediction device, the dextropropoxyphene, tramadol, ketobemidone , ketamine, US 2020/0215078 A1 Jul. 9. 2020 15 dextromethorphan , phencyclidine , nitrous oxide (N , O ) , AP5 Example 1 (R -2 - amino -5 -phosphonopentanoate ), AP7 (2 -amino -7 phosphonoheptanoic acid ) , CPPene (3 -[ ( R ) -2 -carboxypiper Tetramethylenedisulfotetramine Alters Ca2 + azin -4 -yl ] -prop -2 -enyl - 1 -phosphonic acid ), selfotel, aman Dynamics in Cultured Hippocampal Neurons: tadine , dextrallorphan , dextrorphan , ethanol , eticyclidine , Mitigation by NMDA Blockade and GABAA gacyclidine , ibogaine, magnesium , memantine, methoxet Receptor Positive Modulation amine , rolicyclidine.tenocyclidine , methoxydine , tiletamine , xenon , neramexane, eliprodil, etoxadrol, dexoxadrol, WMS Materials and Methods 2539. NEFA , remacemide , delucemine, 8A -PDHQ , apti ganel , HU -211 , rhynchophylline , 1- Aminocyclopropanecar Materials boxylic acid (ACPC ), 7 -Chlorokynurenate , DCKA (5,7 [0138 ] Fetal bovine serum and soybean trypsin inhibitor dichlorokynurenic acid ), kynurenic acid , lacosamide, were obtained from Atlanta Biologicals (Norcross , Ga. ). CP- 101,606 ( traxoprodil ), AZD6765 (lanicemine ) and DNase , poly - L - lysine , cytosine arabinoside , ( - ) - 5 -methyl GLYX - 13 . In some embodiments , the NMDA receptor 10,11 - dihydro - 5H -dibenzo [ a , d ] cyclohepten - 5,10 - imine antagonist is selected from the group consisting of ketamine, maleate (MK -801 ) , Hydroxypropyl- ß -cyclodextran , and dextromethorphan , phencyclidine , CPPene ( 3 - [ ( R )-2 -car (3,5 - dimethyl 2,6 - dimethyl- 4- ( 2 -nitrophenyl ) -1,4 -dihydro boxypiperazin -4 - yl) -prop - 2 -enyl - 1- phosphonic acid ) , selfo pyridine -3,5 - dicarboxylate (nifedipine ) were from Sigma Aldrich (St. Louis , Mo.) . The Ca21 fluorescence dye Fluo - 4 , tel, amantadine , dextrorphan , memantine, tiletamine, ner Pluronic F - 127 and Neurobasal medium were purchased amexane , eliprodil, remacemide , aptiganel, from Life Technology (Grand Island , N.Y. ). Tetramethyl 1 - Aminocyclopropanecarboxylic acid (ACPC ), 7 - Chlo enedisulfotetramine ( TETS ) was synthesized as described rokynurenate , DCKA ( 5,7 -dichlorokynurenic acid ) , previously (Zolkowska et al ., 2012 ) . Diazepam was from kynurenic acid , CP - 101,606 ( traxoprodil) , AZD6765 (lani Western Medical Supply (Arcadia , Ca.) . Allopregnanolone cemine ) and GLYX - 13 . In some embodiments, the NMDA (3a -hydroxy -5a -pregnan - 20 -one ; > 99 % ) was provided by receptor antagonist is dizocilpine (MK - 801) . M A. Rogawski. [0136 ] In some embodiments , one or both of the benzo [0139 ] Primary Cultures of Hippocampal Neurons. diazepine and the neurosteroid is formulated for inhala [0140 ] Animals were treated humanely and with regard for tional , intranasal or intrapulmonary administration . In some alleviation of suffering according to protocols approved by the Institutional Animal Care and Use Committee of the embodiments , one or both of the benzodiazepine and the University of California , Davis . Hippocampal neuron cul neurosteroid is formulated for oral or parenteral delivery . In tures were dissociated from hippocampi dissected from some embodiments , one or both of the benzodiazepine and C57BL /6J mouse pups at postnatal day 0-1 and maintained the neurosteroid are formulated for a parenteral route in Neurobasal complete medium [Neurobasal medium selected from the group consisting of inhalational, intrapul supplemented with NS21, 0.5 mM L - glutamine, HEPES ] monary, intranasal, intramuscular , subcutaneous, transmu with 5 % fetal bovine serum . For Ca21 imaging studies using cosal and intravenous . In some embodiments , the benzodi FLIPR , dissociated hippocampal cells were plated onto azepine is an agonist of the benzodiazepine recognition site poly - L - lysine coated clear- bottom , black wall, 96 -well on GABA , receptors and stimulates endogenous neuroster imaging plate (BD , Franklin Lakes, N.J. , USA ) at a density oid synthesis . In some embodiments , the neurosteroid is of 0.8x105/ well . For microelectrode array (MEA ) experi suspended or dissolved in a cyclodextrin ( e.g., an a -cyclo ments , 120 ul of cell suspension at a density of 1.5x10 dextrin , B - cyclodextrin or a y - cyclodextrin ) . In varying cells /ml were added to a 12 -well Maestro plate (Axion embodiments , the neurosteroid is suspended or dissolved in Biosystems, Atlanta , Ga. ). After 2 h incubation , a volume of a cyclodextrin selected from the group consisting of 1.0 ml of serum - free Neurobasal complete medium was hydroxypropyl - ß - cyclodextrin , endotoxin controlled ß - cy added to each well . The medium was changed twice a week clodextrin sulfobutyl ethers , or cyclodextrin sodium salts by replacing half volume of culture medium with scrum - free ( e.g. , CAPTISOL® ). In some embodiments , the neuroster Neurobasal complete medium . The neurons were main oid is suspended or dissolved in an edible oil . In some tained at 37 ° C. with 5 % CO2 and 95 % humidity . embodiments , the edible oil comprises one or more veg [0141 ] Measurement of Synchronous Intracellular Ca2 etable oils . In some embodiments , the vegetable oil is Oscillations . selected from the group consisting of coconut oil, corn oil , [0142 ] Hippocampal neurons between 13-17 days in vitro cottonseed oil, olive oil , palm oil, peanut oil , rapeseed oil, (DIV ) were used to investigate how TETS alters synchro canola oil, safflower oil, sesame oil , soybean oil, sunflower nous Ca2: oscillations that normally occur in healthy neu oil , and mixtures thereof. In some embodiments , the edible rons at this developmental stage . This method permits oil is canola oil. In some embodiments , the edible oil simultaneous measurements of intracellular Ca2 . transients comprises one or more nut oils . In some embodiments , the in a 96 -well format as described as previously (Cao et al. , nut oil is selected from the group consisting of almond oil, 2010 ). Baseline recording were acquired in Locke's buffer cashew oil , hazelnut oil , macadamia oil, mongongo nut oil, ( 8.6 mM HEPES , 5.6 mM KC1, 154 mM NaCl, 5.6 mM pecan oil, pine nut oil, pistachio oil, walnut oil , and mixtures glucose , 1.0 mM MgCl2 , 2.3 mm CaCl , and 0.0001 mM thereof. glycine , pH 7.4 ) for 10 min followed by addition of TETS and /or pharmacological agents using a programmable EXAMPLES 96 - channel pipetting robotic system , and the intracellular Ca2 , was monitored for an additional 30 min . Unless [0137 ] The following examples are offered to illustrate , otherwise indicated , pharmacological interventions were but not to limit the claimed invention . introduced 10 min prior to TETS . TETS triggered an imme US 2020/0215078 A1 Jul. 9 , 2020 16 diate rise in [Ca2.Ji that was analyzed by quantifying the [0148 ] For comparison , we studied the influence of picro Area Under the Curve ( AUC ; in arbitrary fluorescence units ) toxin (PTX ; 100 uM ), a noncompetitive blocker of GABA , of the Fluo - 4 fluorescence units for a duration of 5 min receptor and bicuculline ( 100 uM ), a competitive antagonist following TETS addition . TETS also altered the frequency ofGABA , receptors on the Ca21 dynamics . Both antagonists and amplitude of neuronal synchronous Ca2 | oscillations, elicited similar Phase I and Phase II responses as TETS which were analyzed during the 10 min period after addition ( FIG . 3 ). of TETS . [0149 ] TETS Enhances Neuronal Electric Network Activ [0143 ] MEA Recording . ity in Primary Cultured Hippocampal Neurons . [0144 ] All MEA recordings were conducted at 37 ° C. in [0150 ] Extracellular recordings of electrical activity from culture medium without perfusion using a 12 -well Maestro multiple sites within the neuronal cultures at a high spatial system ( Axion BioSystems, Atlanta , Ga. ) . Each well con resolution provide a robust measure of network activity and tains 64 electrodes ( 30 um diameter ) in an 8x8 grid with connectivity ( Johnstone et al. , 2010 ) After recording the inter -electrode spacing of 200 um . Before recording basal basal electrical activity for 10 min , increasing concentra electrical activity , the cultures were equilibrated in freshly tions of TETS were serially introduced into the wells . The prepared , pre- warmed neurobasal complete medium for 1 h . recording was continued for 10 min at each TETS concen The 12 -well Maestro plates were loaded onto a temperature tration A control well was simultaneously recorded follow regulated headstage containing the recording amplifier and ing introduction of DMSO vehicle ( 0.01-0.1 % ) . Basal raw extracellular electrical signals were acquired using Axis recordings for up to 60 min showed that network firing software ( Axion BioSystems, Atlanta , Ga. ). Signals from the activity was stable in the absence or presence of DMSO amplifier were digitized at a rate of 25 KHZ , and filtered vehicle control (FIG . 4A , left panel ). Exposure to TETS using Butterworth Band -pass filter ( cutoff frequency of 300 concentrations of 2 uM and greater produced a dramatic Hz) . The Axis software was used to detect spontaneous change in discharge pattern . Events became more highly events that exceeded a threshold of six times of the noise . clustered (FIG . 4A , right panel and FIG . 5 ) and the duration Raster plot and spike rate analysis data were performed by of clustered bursts induced by 6 UM TETS can last up to 10 exporting the raw data to the NeuroExplorer software (ver s (FIG . 4A , right panel , 4th row ). There was an overall sion 4.0 , NEX Technologies , Littleton , Mass . ). increase in the discharge rate ( FIG . 4B . ) After washout of [0145 ] Data Analysis . Graphing and statistical analysis TETS , the neuronal network firing recovered to basal con were performed using GraphPad Prism software (Version ditions. 5.0 , GraphPad Software Inc., San Diego , Calif. ) . EC50 [0151 ] NMDA Receptors , but not L - type Ca2 : Channels values were determined by non - linear regression using a Are Required for TETS — Induced Ca2: Dysregulation . three -parameter logistic equation. Statistical significance [0152 ] We next examined the possible involvement of between different groups was calculated using Student's NMDA receptors and L - type Ca2 + channels in the effects of 1 -test or by an ANOVA and, where appropriate , a Dunnett's TETS on Ca2- dynamics . Preincubation of neuronal cul Multiple Comparison Test ; p values below 0.05 were con tures for 10 min with MK -801 ( 1 uM ) , an NMDA receptor sidered statistically significant. blocker , attenuated both Phase I and Phase II effects of TETS (FIG . 6B -D ) MK -801 slightly suppressed basal Ca2: oscil Results lations, which is consistent with an earlier report ( Tanaka et [0146 ] Effects of TETS on Ca2 · Oscillations in Primary al. , 1996 ) . By contrast, nifedipine ( 1 uM ) , which inhibits Cultured Hippocampal Neurons. L -type voltage activated Ca21 channels , was without effect [0147 ] Cultured hippocampal neurons ( 13-17 DIV ) on TETS - induced Phase I or Phase II Ca2 | responses (FIG . exhibit spontaneous synchronous Ca2- oscillations whose 6B - D ) . These results indicate that NMDA receptors but not frequency and amplitude can be quantitatively assessed in L -type Ca2 : channels are required for the effects of TETS on real time using FLIPR® ( FIG . 1A ). Addition of DMSO Ca2| fluctuations . vehicle had no significant effect on the properties of the [0153 ] Diazepam and Allopregnanolone Partially Mitigate synchronous Ca2 : oscillations during the 5 min Phase I TETS - Induced Ca2: Dysregulation . period or the 10 min Phase II period (FIG . 1A , top trace ). By [0154 ] We next determined if the GABA , receptor posi contrast , exposure of the neurons to TETS caused an imme tive modulators diazepam and allopregnanolone could pro diate increase in the amplitude of the oscillations and at tect against TETS - induced Ca21 dysregulation . FIG . 7A ( top higher concentrations ( 3 and 10 uM ) a sustained plateau trace ) demonstrates that the oscillatory activity of neurons response that decayed slowly over the 5 min Phase I period . exposed to vehicle remained stable over the entire recording The integrated Ca2- signal (area under the curve: AUC ) period . Introduction of diazepam ( 0.1 , 0.3 , or 1 uM ) caused during the Phase I period exhibited a concentration - depen an attenuation in the amplitude of basal spontaneous Ca2 + dent increase , with an EC50 value of 2.7 uM [ 95 % confi oscillations (FIG . 7A ) Pre- exposure to diazepam caused a dence interval (95 % CI) : 1.4-5.2 uM ] (FIG . 1B ) . During small concentration -dependent reduction of the Phase I Phase II , TETS caused a concentration - dependent decrease integrated rise in Ca2 + induced by TETS that reached in the frequency of the synchronous Ca2 + oscillations with statistical significance only at 1 uM (FIG . 7B ). Diazepam an EC50 value of 1.7 UM ( 95 % CI 0.69-4.12 uM ; FIG . 1C ) did not eliminate the Phase I plateau response (FIG . 7A ). Along with the reduction in frequency, TETS increased the Diazepam also caused a partial inhibition of the Phase II mean Ca2: oscillation amplitude with an EC50 value of 1.8 frequency and amplitude effects of TETS , with the effect on UM ( 95 % CI: 1.12-2.80 uM ; FIG . 1D ). TETS modestly amplitude reaching significance at 0.1 uM (FIG . 7C , D ). prolonged the mean duration of individual Ca2: transients [0155 ] As shown in FIG . 8 , allopregnanolone similarly compared to that measured from vehicle - exposed control attenuated the effects of TETS on Ca21 dysregulation . Allo neurons. TETS -induced phase II Ca21 responses (both fre pregnanolone (0.1-1 uM ) caused a concentration -dependent quency and amplitude ) were reversible (FIG . 2 ) suppression of basal spontaneous Ca2: fluctuations and it US 2020/0215078 A1 Jul. 9. 2020 17 partially attenuated the response in Phase I at 1 uM without neuronal Ca2- dynamics and electrical discharge activity eliminating the plateau in Ca21 levels (FIG . 8A , B ). Allo occur within the same concentration range , suggesting the pregnanolone at 0.3 and 1 uM also inhibited the Phase II two effects are mechanistically linked . TETS - induced effect of TETS on frequency and amplitude with a com changes on Ca2 : dynamics and on electrical discharges are pletely reversed Phase II effect on amplitude at 1 uM (FIG . similar to those observed with the GABA , receptor antago 8C ,D ). nists bicuculline or picrotoxin ( Arnold et al. , 2005 ; Cao et [0156 ] Low Concentrations of Diazepam and Allopreg al. , 2012 ) . Additionally , TETS modulation of Ca2 : dynamics nanolone in Combination Mitigate TETS - Induced Ca2 : Dys and spontaneous neuronal firing activity in a concentration regulation . dependent manner with EC50 values of approximately 1-2 [ 0157 ] We next evaluated the effect of a combination of UM which is consistentwith the affinity of TETS for GABA , diazepam and allopregnanolone, each at a low concentration receptors (Bowery et al. , 1975 ; Dray, 1975 , Roberts et al. , (0.1 uM ) that by itself has minimal effects on Phase I or 1981 ) . Collectively these data support the view that the Phase II Ca2: dysregulation . As shown in FIG . 9 , the GABA , receptor blocking activity of TETS is responsible combination they strongly mitigated both Phase I and Phase for the effects . Like picrotoxin , TETS is believed to be a II effects . In fact, the combination treatment was able to reversible inhibitor of GABA , receptors , which is also largely eliminate the plateau response obtained with acute consistent with the rapid reversibility of its effects in the TETS exposure (FIG . 9A ) , an effect not obtained with MEA assay . 10 - fold higher concentrations of diazepam (FIG . 7 ) or [0160 ] TETS -triggered alterations in electric firing and allopregnanolone ( FIG . 8 ) alone. synchronous Ca2 + oscillations appear to rely on spontane ous action potentials since they are prevented by tetrodo Discussion toxin block of Na + channels . The neuronal specificity of [0158 ] In the present study , we characterized the effects of TETS in producing both Phase I and Phase II Ca2 . responses TETS on hippocampal neurons in culture using MEA field in hippocampal cultures is also indicated by the observations potential recording and Fluo - 4 fluorescence measurements that addition of TETS up to 3 uM to the culture medium of of Ca2l dynamics in the neuronal network . Over time, skeletal myotubes alters neither basal Ca2. homeostasis nor hippocampal neurons in culture develop a rich network of electrically evoked Ca2: transients (i.e. , excitation -contrac processes and form numerous functional synaptic contacts tion coupling) . (Mennerick et al. , 1995 ; Arnold et al ., 2005) . Cultures that [0161 ] A key observation in the present study is that the have developed for 13-17 DIV as used in the present study alterations in Ca2l dynamics induced by TETS was largely are well organized and there is robust spontaneous electrical inhibited by MK - 801 demonstrating that NMDA receptors activity mediated by excitatory and inhibitory transmission are required . While direct activation of NMDA receptors by between neurons. Neurons within the cultures exhibit spon TETS is not excluded , activation of NMDA receptors by taneous action potentials and cultures of sufficient cell bath application ofNMDA increase the neuronal firing in a density may show synchronized bursting of neurons evenly distributed pattern which is not similar to the clus throughout the entire culture (Arnold et al. , 2005 ) . Excit tered bursts firng elicited by TETS or other GABA , recep atory synaptic transmission is mediated by functional glu tors blocker/ antagonist such as picrotoxin (Cao et al. , 2012 ) tamate receptors of the NMDA and AMPA types ( Abele et The NMDA receptor dependence for TETS response to the al. , 1990 ) . Importantly , the cultures contain GABAergic Ca2- is consistent with earlier evidences in vivo that the neurons, comprising approximately 10 percent of the neu NMDA receptor antagonist MK -801 inhibits picrotoxin or rons, that form robust inhibitory synaptic conne ns medi bicuculline - induced convulsion in mice (Obara , 1995 ; ated by GABA , receptors ( Jensen et al. , 1999 ; Jensen et al. , Czlonkowska et al. , 2000 ) and ex vivo that the NMDA 2000 ) . Inhibitory synaptic potentials in hippocampal cul antagonist 2 - APV suppresses picrotoxin - induced Ca2 . tures have physiological properties that are similar to those responses as well as the frequency and duration of the obtained in intact preparations ( Jensen et al. , 1999 ) The epileptiform discharges in hippocampal slice preparation GABAergic neurons impose tonic inhibition onto the net (Kohr and Heinemann , 1989 ) . How the suppression of work so that exposure of hippocampal cultures to GABA , GABA , receptors activity by TETS affects NMDA receptor receptor antagonists causes increased action potential firing , functions remains to be established . One possibility is that spontaneous rhythmic neuronal depolarizations , and burst the Phase I [Ca2 + ]i response may involve presynaptic ing . The rhythmic depolarizations and bursting is dependent glutamate transmission . In support , bicuculline - induced upon action potentials as it is eliminated by tetrodotoxin . [Ca2- ] i responses have been shown to involve synaptic but [ 0159 ] MEA recording allow the electrical activity of not- extra - synaptic NMDA receptor activation (Hardingham multiple neurons within the cultures to be monitored et al. , 2001 ; Hardingham et al. , 2002) . While the relationship whereas FLIPR® Fluo - 4 fluorescence measurements pro between the Ca21 signals in the rapid throughput FLIPR vide a dynamic assessment of aggregate intracellular Ca2| assay and epileptic activity remain to be determined , our levels (Cao et al ., 2010 ; Cao et al. , 2012 ). Using these observation that NMDA receptors are required for the assays , we found that TETS dramatically increases intrac TETS - induced changes in Ca2 : dynamics supports the con ellular Ca2. levels and alters Ca21 dynamics , initially caus cept that the effects on Ca21 are a surrogate for epileptic ing an transient increase on the intracellular Ca2 : concen activity and may be useful as a model for therapeutics tration ( [ Ca2- li ) followed by a decrease on the Ca2 : discovery. This is further supported by our demonstration oscillations having bigger amplitude. Assessment of ongo that GABA , receptors positive allosterical enhancer, diaz ing electric activity in the cultures with MEA recording epam or allopregnanolone partially suppress TETS - induced showed an overall increase in discharge frequency and a modulation of Ca2 + dynamics . change in the pattern of the discharges to clustering followed [0162 ] Consistent with the role of GABA , receptors in by periods of electrical silence. The actions of TETS on restraining bursting and altered Ca21 dynamics is our obser US 2020/0215078 A1 Jul. 9 , 2020 18 vation that the GABA , receptor positive modulators diaz a simple model for transcription and protein synthesis epam and allopregnanolone are able to protect against the dependent plasticity . The Journal of physiology 564 :3-19 . effects of TETS on Ca2 + dynamics. Allopregnanolone was [0167 ] Banks C N , Rogawski M A , Yang D and P. J. L more effective on mitigation of Phase I response induced by ( 2012) Tetramethylenedisulfotetramine. In : Encyclopedia TETS than diazepam . This is consistent with the fact that of Toxicology , 3rd edition , volume 2 (Edited by P Wex diazepam only acts on synaptic GABA , receptors , whereas ler ) . Elsevier , Oxford , UK . In Press . neurosteroids such as allopregnanolone can enhance both [0168 ] Barrueto F , Nelson L S , Hoffman R S , Heller MR , extrasynaptic synaptic GABA , receptors (Kokate et al ., Furdyna P M , Hoffman R J, Whitlow KS, Belson MG 1994 ; Lambert et al. , 2003 ; Reddy and Rogawski, 2012 ). and Henderson A K ( 2003 ) Poisoning by an illegally However , neither diazepam nor allopregnanolone alone was imported Chinese rodenticide containing tetramethylene fully effective , even at the highest concentrations tested ( 1 disulfotetramine — New York City , 2002 (Reprinted from UM ) . Unexpectedly , we found that the combination of diaz MMWR , vol 52 , pg 199-201, 2003 ). Jama- J Am Med epam and allopregnanolone , each at a threshold concentra Assoc 289: 2640-2642 . tion of 0.1 uM , was highly effective atprotecting against the [0169 ] Bowery N G , Brown D A and Collins J F ( 1975 ) effects of TETS on Ca2 + dynamics, causing a nearly com Tetramethylenedisulphotetramine: an inhibitor of gamma plete inhibition of the Phase I response, including the plateau aminobutyric acid induced depolarization of the isolated in Ca2- , as well as the Phase II changes. The combination superior cervical ganglion of the rat. British journal of of a benzodiazepine and a neurosteroid has not to our knowledge previously been studied in a simplified func pharmacology 53: 422-424 . tional system . It is well recognized that benzodiazepines [0170 ] Cao Z , Hulsizer S , Tassone F , Tang H T , Hagerman such as diazepam only act on synaptic GABA , receptors , RJ, RogawskiMA , Hagerman P J and Pessah I N ( 2012 ) whereas neurosteroids such as allopregnanolone preferen Clustered Burst Firing in FMRI Premutation Hippocam tially enhance extrasynaptic GABA4 receptors although pal Neurons Amelioration with Allopregnanolone. they also act on synaptic receptors as well (Kokate et al. , Human molecular genetics 21: 2923-2935 . 1994 ; Lambert et al. , 2003 ; Reddy and Rogawski, 2012 ). [0171 ] Cao Z , LePage K T , Frederick MO , Nicolaou KC Without being bound to theory , it appears that the combined and Murray TF ( 2010 ) Involvement of caspase activation action on synaptic and extrasynaptic receptors accounts for in azaspiracid - induced neurotoxicity in neocortical neu the unique potency of the drug combination . rons . Toxicological sciences: an official journal of the [0163 ] Alternatively , there may be an interaction at the Society of Toxicology 114 : 323-334 . level of individual GABA , receptors . The recognition sites [0172 ] Cao Z , Shafer T J and Murray T F ( 2011 ) Mecha for neurosteroids on GABA , receptors are distinct from nisms of pyrethroid insecticide - induced stimulation of those that recognize benzodiazepines and barbiturates calcium influx in neocortical neurons . The Journal of ( Johnston , 1996 ). It is conceivable , however, that allopreg pharmacology and experimental therapeutics 336 : 197 nanolone and diazepam could produce a synergistic 205 . enhancement of GABA , receptors in a similar fashion as the [ 0173 ] Casida J E , Eto M , Moscioni A D , Engel J L , synergism that occurs between barbiturates and benzodiaz Milbrath D S and Verkade J G ( 1976 ) Structure - Toxicity epines, where there is known to be allosteric coupling Relationships of 2,6,7 - Trioxabicyclo [ 2.2.2 ] -Octanes and ( DeLorey et al. , 1993 ) . Related Compounds. Toxicology and applied pharmacol [0164 ] In summary, we have developed rapid throughput ogy 36 : 261-279. methods to detect TETS - induced Ca21 dysregulation and [0174 ] Choi H , Pereira A R , Cao Z , Shuman C F, Engene altered electrical activity in cultured hippocampal neurons. N , Byrum T, Matainaho T, Murray T F , Mangoni A and We demonstrated that two GABA , receptors allosteric Gerwick W H (2010 ) The hoiamides , structurally intrigu modulators , allopregnanolone and diazepam , when intro ing neurotoxic lipopeptides from Papua New Guinea duced singly prior to TETS , mitigate TETS - induced Ca2 : marine cyanobacteria . Journal of natural products dysregulation , demonstrating that the in vitro methods 73 : 1411-1421. described here have translational value to identify new [0175 ] Cole L M and Casida J E ( 1986 ) Polychlorocy therapies and optimize combinatorial strategies for the pre cloalkane insecticide -induced convulsions in mice in rela vention of TETS poisoning . The basic approaches described tion to disruption of the GABA -regulated chloride iono here are of general utility for investigating chemically phore . 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[ 0208 ] Soria -Mercado I F , Pereira A , Cao Z , Murray TF Phase II response. Addition of vehicle (0.1 % DMSO ) was no and Gerwick W H ( 2009 ) Alotamide A , a novel neurop effect on the Ca21 dynamics over the recording period of 45 harmacological agent from the marine cyanobacterium min . However, a concentration of 3 uM of TETS produced Lyngbya bouillonii Organic letters 11: 4704-4707 . an acute Phase I and a prolonged Phase II effect, as previ [0209 ] Squires R F , Casida J F , Richardson M and Sae ously reported (Cao et al 2012 ) While addition of diazepam derup E (1983 ) [355 ] t- butylbicyclophosphorothionate ( 0.1 uM ) or allopregnanolone (0.1 uM ) singly was without binds with high affinity to brain - specific sites coupled to significant effect on TETS - induced decreased Ca2 + oscilla gamma- aminobutyric acid - A and ion recognition sites . tion frequency, diazepam (0.1 uM ) and allopregnanolone Molecular pharmacology 23: 326-336 . (0.1 uM ) in combination effectively recovered synchronous [0210 ] Tanaka T , Saito H and Matsuki N ( 1996 ) Intracel Ca2l oscillations characteristics comparable to those lular calcium oscillation in cultured rat hippocampal observed with vehicle - treated cultures . Although allopreg neurons: a model for glutamatergic neurotransmission . nanolone (0.1 uM ) alone decreased the TETS - induced Ca2: Japanese journal of pharmacology 70 :89-93 . oscillation amplitude -20 % ( p < 0.01 ) , the post - TETS treat [0211 ] Voss E , Haskell A R and Gartenbe L ( 1961) Reduc ment with diazepam (0.1 uM ) and allopregnanolone (0.1 tion of Tetramine Toxicity by Sedatives and Anticonvul uM ) in combination conferred much greater recovery of sants . J Pharm Sci 50 : 858-& . Ca2 - oscillation amplitude to that below vehicle control, and [ 0212 ] Wayman GA, Bose DD , Yang D , Lesiak A , Bruun occurred rapidly after the addition of diazepam and allo D , Impey S , Ledoux V , Pessah I N and Lein PJ (2012 ) pregnanolone (FIG . 10 ). These data clearly demonstrate that PCB 95 Modulates Calcium -Dependent Signaling Path diazepam in combination with a neurosteroid , such as allo way Responsible for Activity -Dependent Dendritic pregnanolone, act in a synergistic manner to mitigate the Growth . Environmental health perspectives. severity of seizures and prevent the lethality of seizurogenic [0213 ] Wayman GA , Lee YS, Tokumitsu H , Silva A J and agents After the seizures have already started . Soderling TR (2008 ) Calmodulin -kinases : modulators of neuronal development and plasticity Neuron 59: 914-931 . Example 3 [0214 ] Whitlow KS, Belson M , Barrueto F , Nelson L and Henderson A K ( 2005) Tetramethylenedisulfotetramine: In Vivo Assay Demonstrating the Therapeutic old agent and new terror. Annals of emergency medicine Efficacy of Combined Benzodiazepine and 45 : 609-613 . Neurosteroid in Mitigating TETS - Induced Seizures [ 0215 ] Wu Y Q and Sun C Y ( 2004 ) Poison control and Death services in China . Toxicology 198: 279-284 . [0219 ] When administered to adult male NIII Swiss mice [0216 ] Zhang Y , Su M and Tian D P (2011 ) Tetramine at lethal doses, TETS typically causes two clonic seizures poisoning A case report and review of the literature within the first 20 minutes after TETS injection with each Forensic science international 204 : e24-27 . seizure lasting approximately 30 to 45 seconds . These clonic [0217 ] Zolkowska D , Banks C N , Dhir A , Inceoglu B , seizures are followed by a tonic seizure that results in the Sanborn JR , McCoy MR , Bruun D A , Hammock B D , death of > 95 % of the TETS - intoxicated animals ( FIG . 11) . Lein P J and Rogawski M A ( 2012 ) Characterization of Mice can be rescued from TETS - induced death if they are seizures induced by acute and repeated exposure to administered a very high dose of diazepam (5 mg/ kg , i.p.) tetramethylenedisulfotetramine. The Journal of pharma immediately following the second clonic seizure (FIG . 11 ) . cology and experimental therapeutics 341: 435-446 . Administration of diazepam at 0.03 mg/ kg immediately following the second clonic seizure protected < 10 % of the Example 2 TETS - intoxicated animals from death (FIG . 12 ) . Pretreat ment with diazepam ( 0.1 mg/ kg 10 minutes before TETS Combination Treatment with a Benzodiazepine and injection ) protected < 30 % of the TETS - intoxicated animals a Neurosteroid Mitigates the Severity and Prevents from death (FIG . 13 ) Post -administration of the neurosteroid the Lethality of Seizures Even when Administering allopregnanolone at 0.03 mg/ kg was no more efficacious after Seizure has Started than low dose diazepam in protecting TETS - intoxicated [0218 ] In cultured hippocampal neurons , higher concen animals from death (FIG . 12 ) . Pretreatment with allopreg tration of TETS (> 3 uM ) produces an acute elevation of nanolone at 0.1 mg/ kg protected ~ 50 % of the TETS - intoxi intracellular Ca21 levels ( Phase I response ) and a prolonged cated animals ( FIG . 13 ) . When administered simultaneously , Ca2: response with increased Ca2 : oscillation amplitude and the subthreshold doses of diazepam and allopregnanolone decreased frequency of the Ca2 : oscillations (Phase II significantly increased survival of TETS - intoxicated ani response ). Both Phase I and Phase II response can be mals . When administered immediately after the second mitigated by the pretreatment of diazepam and allopreg clonic seizure, this benzodiazepine and neurosteroid com nanolone. More importantly , pre - treatment with a combina bination , 100 % of the TETS - intoxicated animals survived tion of low concentrations of diazepam and allopreg (FIG . 12 ). Used as a pretreatment, this combinatorial nanolone, which have minimal effect against TETS - induced therapy protected ~ 75 % of the TETS - intoxicated animals Ca2 + response, normalized the TETS Ca2 + response to the (FIG . 13 ). Importantly , the therapeutic combination of sub control level (Cao et al. , Toxicological Sciences , 130 : 362 threshold diazepam and allopregnanolone had no effect on 372 ). In this study, we examined whether post- treatment blood pressure , whereas the dose of diazepam required to ( after TETS triggers Phase I and Phase II Ca2 : responses ) of prevent TETS - induced tonic seizures and death when neurons with diazepam and /or allopregnanolone mitigate administered singly caused significant hypotension ( FIG . alterations triggered by TETS , which is more relevant to the 14 ). TETS poising . Since addition of TETS induces acute phase [0220 ] The significance of these in vivo data are 3 - fold : I response , we therefore only focused on the TETS - induced ( 1 ) these data confirm the predictive value of the in vitro US 2020/0215078 A1 Jul. 9. 2020 21 screening system ; ( 2 ) these data demonstrate that combina ate (Rampart , Thimet) , Phosalone ( Azofene , Zolone) , Phos torial therapy with a benzodiazepine and a neurosteroid , at folan (Cylan , Cyolane ), Phosmet (Imidan , Prolate) , Phos subthreshold doses that singly have no effect, is efficacious phamidon (Dimecron ), Phostebupirim (Aztec ) , Phoxim in preventing seizures and in the case of TETS, in preventing (Baythion ) , Pirimiphos - ethyl (Primicid ) , Pirimiphos -methyl death associated with tonic seizures ; and ( 3 ) the combina ( Actellic ) , Profenofos (Curacron ) , Propetamphos (Safrotin ) , torial therapy avoids an important off- target adverse effect Propyl thiopyrophosphate (Aspon ), Prothoate (Fac ), Pyra ( significant decrease in blood pressure ) associated with use zophos ( Afugan , Curamil) , Pyridaphenthion (Ofunack ), of diazepam when used singly at therapeutic doses. Quinalphos (Bayrusil ), Ronnel (Fenchlorphos , Korlan ) , [ 0221 ] It is understood that the examples and embodi Schradan (OMPA ), Sulfotep (Bladafum , Dithione , Thio ments described herein are for illustrative purposes only and tepp ) , Sulprofos (Bolstar , Helothion ), Temephos ( Abate , that various modifications or changes in light thereof will be Abathion ), Terbufos (Contraven , Counter ) , Tetrachlorvin suggested to persons skilled in the art and are to be included phos (Gardona , Rabon ), Tetraethyl pyrophosphate ( TEPP ) , within the spirit and purview of this application and scope of tetramethylenedisulfotetramine ( TETS ) , Triazophos (Hosta the appended claims. All publications, patents , and patent thion ) , and Trichlorfon (Dipterex , Dylox , Neguvon , Proxol) . applications cited herein are hereby incorporated by refer 132. The method of claim 129 , wherein the subject is at ence in their entirety for all purposes . risk of being exposed to tetramethylenedisulfotetramine 1-128 . ( canceled ) ( TETS ) . 129. A method of preventing or mitigating a seizure in a 133. The method of claim 129 , wherein the benzodiaz subject in need thereof, comprising administration to the epine and the allopregnanolone are co -administered together subject of an effective amount of a benzodiazepine and and / or by the same route of administration . allopregnanolone , wherein both of the benzodiazepine and 134. The method claim 129 , wherein the benzodiazepine the allopregnanolone are administered in a subtherapeutic and the allopregnanolone are co -administered separately dose , wherein the subject is at risk of exposure to a nerve and /or by different routes of administration . agent or a pesticide that can cause seizures . 135. The method claim 129 , wherein one or both of the 130. The method of claim 129 , wherein the subject is at benzodiazepine and the allopregnanolone are administered risk of being exposed to an organophosphorous nerve agent by the inhalational or intrapulmonary route of administra or pesticide . tion . 131. The method of claim 129 , wherein the subject is at 136. The method of claim 135 , wherein the benzodiaz risk of being exposed to tabun , sarin , soman , GF, VR , VX , epine and the allopregnanolone are not heated prior to Acephate (Orthene ), Azinphos -methyl (Gusathion , administration . Guthion ), Bensulide (Betasan , Lescosan ) , Bomyl (Swat ) , 137. The method of claim 135 or 136 , wherein one or both Bromophos (Nexion ), Bromophos -ethyl (Nexagan ) , of the benzodiazepine and allopregnanolone are aerosolized . Cadusafos ( Apache , Ebufos, Rugby ) , Carbophenothion (Tri 138. The method claim 137 , wherein one or both of the thion ), Chlorethoxyfos ( Fortress ) , Chlorfenvinphos benzodiazepine and the allopregnanolone aerosolized par ( Apachlor, Birlane ) , Chlormephos (Dotan ), Chlorphoxim ticulates having a mass median aerodynamic diameter rang ( Baythion -C ), Chlorpyrifos (Brodan , Dursban , Lorsban ), ing from about 1 um to about 3 um . Chlorthiophos ( Celathion ), Coumaphos ( Asuntol, Co -Ral ) , 139. The method of claim 129 , wherein the benzodiaz Crotoxyphos ( Ciodrin , Cypona ) , Crufomate (Ruelene ), Cya epine is administered at a dose in the range of 0.3 pg /kg to nofenphos ( Surecide ), Cyanophos (Cyanox ), Cythioate (Cy 3.0 pg /kg . flee , Proban ), DEF (De - Green ) , Demeton ( Systox ) , Deme 140. The method of claim 129 , wherein the allopreg ton - S -methyl (Duratox , Metasystoxl) , Dialifor ( Torak ), nanolone is administered at a dose in the range of about 5 Diazinon , Dichlorofenthion , (VC - 13 Nemacide ) , Dichlorvos mg/ kg to about 50 mg /kg . (DDVP , Vapona ), Dicrotophos (Bidrin ), Dimefos (Hanane , 141. The method of claim 129 , wherein the benzodiaz Pestox XIV ) , Dimethoate (Cygon , DeFend ), Dioxathion epine is selected from the group consisting of bretazenil , (Delnav ), Disulfoton (Disyston ), Ditalimfos, Edifenphos, clonazepam , cloxazolam , clorazepate , diazepam , fludiaz Endothion , EPBP ( S - seven ) , EPN , Ethion (Ethanox ) , Ethop epam , flutoprazepam , lorazepam ,midazolam , nimetazepam , rop (Mocap ) , Ethyl parathion ( E605, Parathion , thiophos) , nitrazepam , phenazepam , temazepam and clobazam . Etrimfos ( Ekamet ), Famphur (Bash , Bo - Ana, Famfos ), 142. The method of claim 129 , further comprising the Fenamiphos (Nemacur ) , Fenitrothion (Accothion , Agro co -administration of an NMDA receptor antagonist . thion , Sumithion ), Fenophosphon (Agritox , trichloronate ), 143. The method of claim 140 , wherein the NMDA Fensulfothion ( Dasanit ) , Fenthion (Baytex , Entex , Tiguvon ) , receptor antagonist is selected from the group consisting of Fonofos (Dyfonate , N -2790 ), Formothion (Anthio ), Fosthi dizocilpine (MK -801 ) , meperidine , methadone , dextro etan (Nem - A - Tak ), Heptenophos (Hostaquick ), Hiometon propoxyphene , tramadol, ketobemidone , ketamine, dex ( Ekatin ), Hosalone (Zolone ), IBP (Kitazin ), Iodofenphos tromethorphan, phencyclidine, nitrous oxide (N2O ), AP5 (Nuvanol - N ) , Isazofos (Brace , Miral , Triumph ), Isofenphos ( R - 2 - amino - 5 - phosphonopentanoate ), AP7 ( 2 -amino -7 (Amaze , Oftanol) , Isoxathion ( E -48 , Karphos ) , Leptophos phosphonoheptanoic acid ) , CPPene ( 3 - [ ( R ) -2 - carboxypiper (Phosvel ) , Malathion (Cythion ) , Mephosfolan ( Cytrolane) , azin -4 - yl )-prop - 2 -enyl - 1- phosphonic acid ), selfotel , aman Merphos (Easy Off - D , Folex ), Methamidophos (Monitor ), tadine , dextrallorphan , dextrorphan , ethanol, eticyclidine , Methidathion (Supracide , Ultracide) , Methyl parathion gacyclidine, ibogaine , magnesium , memantine , methoxet ( E601, Penncap - M ) , Methyl trithion , Mevinphos (Duraphos , amine, rolicyclidine.tenocyclidine ,methoxydine , tiletamine, Phosdrin ), Mipafox ( Isopestox , Pestox XV ), Monocroto xenon , neramexane , eliprodil , etoxadrol, dexoxadrol, WMS phos (Azodrin ), Naled (Dibrome ) , Oxydemeton -methyl 2539 , NEFA , remacemide, delucemine , 8A - PDHQ , apti (Metasystox - R ), Oxydeprofos (Metasystox - S ) , Phencapton ganel, HU -211 , rhynchophylline, 1 - Aminocyclopropanecar (G 28029 ), Phenthoate (Dimephenthoate , Phenthoate ), Phor boxylic acid ( ACPC ) , 7 -Chlorokynurenate , DCKA (5,7 US 2020/0215078 A1 Jul. 9 , 2020 22 dichlorokynurenic acid ), kynurenic acid , lacosamide , CP - 101,606 (traxoprodil ) , AZD6765 ( lanicemine ) and GLYX - 13 . 144. The method of claim 140 , wherein the benzodiaz epine, allopregnanolone and NMDA receptor antagonist are co -administered together and /or by the same route of admin istration . 145. The method of claim 140, wherein the benzodiaz epine , allopregnanolone and NMDA receptor antagonist are administered separately and /or by different routes of admin istration .