U.S. patent application number 13/964922 was filed with the patent office on 2014-02-20 for mitigation of epileptic seizures by combination therapy using benzodiazepines and neurosteroids.
This patent application is currently assigned to The Regents of the University of California. The applicant listed for this patent is The Regents of the University of California. Invention is credited to Zhengyu CAO, Pamela J. LEIN, Isaac N. PESSAH, Michael A. ROGAWSKI.
Application Number | 20140050789 13/964922 |
Document ID | / |
Family ID | 50100199 |
Filed Date | 2014-02-20 |
United States Patent
Application |
20140050789 |
Kind Code |
A1 |
ROGAWSKI; Michael A. ; et
al. |
February 20, 2014 |
MITIGATION OF EPILEPTIC SEIZURES BY COMBINATION THERAPY USING
BENZODIAZEPINES AND NEUROSTEROIDS
Abstract
Provided are compositions comprising a benzodiazepine and a
neurosteroid, containing one or both of the benzodiazepine and the
neurosteroid in a subtherapeutic dose, and administration of such
compositions for mitigation of an epileptic seizure. Further
provided are compositions comprising a benzodiazepine, a
neurosteroid, and an NMDA blocker, and administration of such
compositions for mitigation of an epileptic seizure.
Inventors: |
ROGAWSKI; Michael A.;
(Sacramento, CA) ; PESSAH; Isaac N.; (Davis,
CA) ; CAO; Zhengyu; (Woodland, CA) ; LEIN;
Pamela J.; (Davis, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
50100199 |
Appl. No.: |
13/964922 |
Filed: |
August 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61682745 |
Aug 13, 2012 |
|
|
|
61798094 |
Mar 15, 2013 |
|
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Current U.S.
Class: |
424/489 ;
514/171; 514/220; 514/221; 514/289 |
Current CPC
Class: |
A61K 31/439 20130101;
A61P 25/08 20180101; A61K 31/57 20130101; A61K 45/06 20130101; A61K
31/5513 20130101; A61K 31/5517 20130101; A61K 31/5517 20130101;
A61K 2300/00 20130101; A61K 31/5513 20130101; A61K 2300/00
20130101; A61K 31/439 20130101; A61K 2300/00 20130101; A61K 31/57
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/489 ;
514/171; 514/220; 514/221; 514/289 |
International
Class: |
A61K 31/57 20060101
A61K031/57; A61K 31/5513 20060101 A61K031/5513; A61K 31/439
20060101 A61K031/439; A61K 31/5517 20060101 A61K031/5517 |
Goverment Interests
STATEMENT OF GOVERNMENTAL SUPPORT
[0002] This invention was made with Government support under Grant
Nos. AG032119, NS072094, and NS079202 awarded by the National
Institutes of Health. The Government has certain rights in this
invention.
Claims
1. A composition comprising a benzodiazepine and a neurosteroid,
wherein the composition comprises one or both of the benzodiazepine
and the neurosteroid in a subtherapeutic dose.
2. The composition of claim 1, further comprising a NMDA receptor
antagonist.
3. The composition of claim 1, wherein the composition is
formulated for oral or transmucosal delivery or administration.
4. The composition of claim 1, wherein the composition is
formulated for parenteral delivery.
5. The composition of claim 4, wherein the parenteral delivery or
administration is via a route selected from the group consisting of
inhalational, intrapulmonary, intramuscular, subcutaneous,
transmucosal and intravenous.
6. The composition of claim 1, wherein the benzodiazepine is an
agonist of the benzodiazepine recognition site on GABA.sub.A
receptors and stimulates endogenous neurosteroid synthesis.
7. The composition of claim 1, wherein the benzodiazepine is
selected from the group consisting of bretazenil, clonazepam,
cloxazolam, clorazepate, diazepam, fludiazepam, flutoprazepam,
lorazepam, midazolam, nimetazepam, nitrazepam, phenazepam,
temazepam and clobazam.
8. The composition of claim 1, wherein the benzodiazepine is
selected from the group consisting of midazolam, lorazepam and
diazepam.
9. The composition of claim 1, wherein the neurosteroid is selected
from the group consisting of allopregnanolone,
allotetrahydrodeoxycorticosterone, ganaxolone, alphaxolone,
alphadolone, hydroxydione, minaxolone, and Althesin.
10. The composition of claim 1, wherein the neurosteroid is
allopregnanolone.
11. The composition of claim 1, wherein the composition comprises
allopregnanolone and a benzodiazepine selected from the group
consisting of midazolam, lorazepam and diazepam.
12-17. (canceled)
18. The composition of claim 2, wherein the NMDA receptor
antagonist is selected from the group consisting of dizocilpine
(MK-801), meperidine, methadone, dextropropoxyphene, tramadol,
ketobemidone, ketamine, dextromethorphan, phencyclidine, nitrous
oxide (N.sub.2O), AP5 (R-2-amino-5-phosphonopentanoate), AP7
(2-amino-7-phosphonoheptanoic acid), CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrallorphan, dextromethorphan,
dextrorphan, ethanol, eticyclidine, gacyclidine, ibogaine,
magnesium, memantine, methoxetamine, rolicyclidine, tenocyclidine,
methoxydine, tiletamine, xenon, neramexane, eliprodil, etoxadrol,
dexoxadrol, WMS 2539, NEFA, remacemide, delucemine, 8A-PDHQ,
aptiganel, HU-211, remacemide, rhynchophylline, 1
Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA
(5,7-dichlorokynurenic acid), kynurenic acid, and lacosamide.
19. The composition of claim 2, wherein the NMDA receptor
antagonist is dizocilpine (MK-801).
20. A method of preventing or terminating a seizure in a subject in
need thereof, comprising administration to the subject of an
effective amount of a composition of claim 1.
21. A method of accelerating the termination or abortion of an
impending seizure in a subject in need thereof, comprising
administration to the subject of an effective amount of a
composition of claim 1.
22-31. (canceled)
32. A method of preventing or terminating a seizure in a subject in
need thereof, comprising administration to the subject of an
effective amount of a benzodiazepine and a neurosteroid, wherein
one or both of the benzodiazepine and the neurosteroid are
administered in a subtherapeutic dose.
33. A method of accelerating the termination or abortion of an
impending seizure in a subject in need thereof, comprising
administration to the subject of an effective amount of a
benzodiazepine and a neurosteroid, wherein one or both of the
benzodiazepine and the neurosteroid are administered in a
subtherapeutic dose.
34-63. (canceled)
64. A kit comprising a benzodiazepine and a neurosteroid, wherein
one or both of the benzodiazepine and the neurosteroid are provided
in unit dosage forms comprising a subtherapeutic dose.
65-128. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Application No. 61/682,745 filed
on Aug. 13, 2012 and U.S. Provisional Application No. 61/798,094
filed on Mar. 15, 2013, both of which are hereby incorporated
herein by reference in their entirety for all purposes.
FIELD
[0003] Provided are compositions comprising a benzodiazepine and a
neurosteroid, containing one or both of the benzodiazepine and the
neurosteroid in a subtherapeutic dose, and administration of such
compositions for mitigation of an epileptic seizure. Further
provided are compositions comprising a benzodiazepine, a
neurosteroid, and an NMDA blocker, and administration of such
compositions for mitigation of an epileptic seizure.
BACKGROUND
[0004] Tetramethylenedisulfotetramine (TETS), commonly called
tetramine or TETS, is a highly toxic convulsant with a parenteral
LD50 of 0.1-0.3 mg/kg in mice or rats (Haskell and Voss, 1957; Voss
et al., 1961; Casida et al., 1976). In adult humans, 7-10 mg is
estimated as a lethal dose (Guan et al., 1993). TETS was used as a
rodenticide until banned worldwide in the early 1990's (Whitlow et
al., 2005; Banks et al., 2012). It is, however, still available
illegally, and is responsible for accidental and intentional
poisonings, predominantly in China (Croddy, 2004; Wu and Sun, 2004;
Zhang et al., 2011), but also in other countries, including the
United States (Barrueto et al., 2003). Between 1991 and 2010 over
14,000 cases of TETS intoxication were reported in China with 932
deaths (Li et al., 2011). Extreme toxicity, history of intentional
mass poisonings, and the absence of a specific antidote raise
concern that TETS is a potential chemical threat agent that could
cause mass casualties if released accidentally or intentionally
(Whitlow et al., 2005; Jett and Yeung, 2010).
[0005] Mild to moderate poisoning with TETS leads to headache and
dizziness whereas severe intoxication produces status epilepticus
and coma (Whitlow et al., 2005; Li et al., 2011). Animal studies
demonstrate that TETS is active as a convulsant when administered
orally, parenterally and intraventricularly. Sublethal seizures are
not associated with evidence of cellular injury or
neurodegeneration although there is delayed transient reactive
astrocytosis and microglial activation (Zolkowska et al.,
2012).
[0006] The primary convulsant mechanism of TETS has been thought to
relate to blockade of GABA.sub.A receptors and the seizures induced
in animals resemble those produced by other GABA.sub.A receptor
antagonists including picrotoxin and pentylenetetrazol. Limited
cellular physiological studies and results from
[.sup.35S]t-butylbicyclophosphorothionate binding to brain
membranes indicate that TETS inhibits GABA.sub.A receptors with an
1050 in the range of 1 .mu.M (Squires et al., 1983; Esser et al.,
1991; Ratra et al., 2001) and it is therefore comparable in potency
to picrotoxin as an inhibitor of GABA.sub.A receptors (Squires et
al., 1983; Cole and Casida, 1986; Ratra et al., 2001).
[0007] Cultured hippocampal neurons display synchronous spontaneous
Ca2+ oscillations (Tanaka et al., 1996) that are driven by action
potential-dependent synaptic transmission. Disruption of Ca2+
oscillations by environmental toxicants has been reported
(Soria-Mercado et al., 2009; Cao et al., 2010; Choi et al., 2010;
Pereira et al., 2010; Cao et al., 2011). Hippocampal neurons also
exhibit spontaneous electrical discharges as they form functional
neuronal networks. These discharges, as detected in extracellular
recordings, consist of infrequent synchronized field potentials,
mixed with more frequent desynchronized random action potentials
(Cao et al., 2012; Frega et al., 2012). Synchronous Ca2+
oscillations and neuronal electrical firing co-occur (Jimbo et al.,
1993) and are important in mediating neuronal development and
activity dependent dendritic growth (Wayman et al., 2008). Genetic
or environmental factors that interfere with neuronal transmission
influence the overall neuronal networks activity (Kenet et al.,
2007; Meyer et al., 2008; Shafer et al., 2008; Frega et al., 2012;
Wayman et al., 2012). For example picrotoxin, a GABA.sub.A receptor
antagonist, produces striking changes in network electric activity
(Cao et al., 2012; Frega et al., 2012). Diisopropylfluorophosphate,
an irreversible inhibitor of cholinesterase has also been shown to
elicit status epileptics in rats. Hippocampal neurons dissociated
from the brains of diisopropylfluorophosphate exposed rats display
significantly higher intracellular Ca2+ concentration which appears
to be dependent on the N-methyl-D-aspartate receptors (Deshpande et
al., 2010).
[0008] In the present study, using rapid throughput assays we
characterized the influence of TETS on the Ca2+ dynamics and
neuronal firing activity. Inasmuch as TETS induces changes in Ca2+
dynamics that are similar to those produced by the GABA.sub.A
receptor antagonists picrotoxin and bicuculline, our results
support the view that TETS acts as a GABA.sub.A receptor
antagonist. Using rapid throughput Ca2+ measurement, we identified
several agents that reduce or prevent the alterations in Ca2+
dynamics induced by TETS, suggesting several treatment strategies
for TETS-induced seizures, including the GABA.sub.A receptor
positive modulators diazepam and allopregnanolone. In preliminary
studies with mice, we confirmed that these two agents do inhibit
TETS-induced clonic seizures and progression to tonic seizures and
death supporting that measurement of Ca2+ dynamics is likely useful
for identifying novel targeted interventions for TETS
poisoning.
SUMMARY
[0009] In one aspect, provided are compositions comprising a
benzodiazepine and a neurosteroid. In varying embodiments, the
compositions comprise one or both of the benzodiazepine and the
neurosteroid in a subtherapeutic dose or amount. In varying
embodiments, the compositions further comprise a NMDA receptor
antagonist. In some embodiments, the composition is formulated for
inhalational, intranasal or intrapulmonary administration. In some
embodiments, the composition is formulated for oral or transmucosal
delivery. In varying embodiments, the composition is formulated for
parenteral delivery. In some embodiments, the parenteral delivery
or administration is via a route selected from the group consisting
of inhalational, intrapulmonary, intranasal, intramuscular,
subcutaneous, transmucosal and intravenous. In some embodiments,
the composition is formulated for intramuscular delivery. In some
embodiments, the benzodiazepine is an agonist of the benzodiazepine
recognition site on GABA.sub.A receptors and stimulates endogenous
neurosteroid synthesis. In some embodiments, the benzodiazepine is
selected from the group consisting of bretazenil, clonazepam,
cloxazolam, clorazepate, diazepam, fludiazepam, flutoprazepam,
lorazepam, midazolam, nimetazepam, nitrazepam, phenazepam,
temazepam and clobazam. In some embodiments, the benzodiazepine is
selected from the group consisting of midazolam, lorazepam and
diazepam. In some embodiments, the neurosteroid is selected from
the group consisting of allopregnanolone,
allotetrahydrodeoxycorticosterone, ganaxolone, alphaxolone,
alphadolone, hydroxydione, minaxolone, and Althesin. In some
embodiments, the neurosteroid is allopregnanolone. In some
embodiments, the composition comprises allopregnanolone and a
benzodiazepine selected from the group consisting of midazolam,
lorazepam and diazepam. In some embodiments, the neurosteroid is
suspended or dissolved in a cyclodextrin (e.g., an
.alpha.-cyclodextrin, a .beta.-cyclodextrin or a
.gamma.-cyclodextrin). In varying embodiments, the neurosteroid is
suspended or dissolved in a cyclodextrin selected from the group
consisting of hydroxypropyl-.beta.-cyclodextrin, endotoxin
controlled .beta.-cyclodextrin sulfobutyl ethers, or cyclodextrin
sodium salts (e.g., CAPTISOL.RTM.). In some embodiments, the
neurosteroid is suspended or dissolved in an edible oil. In some
embodiments, the edible oil comprises one or more vegetable oils.
In some embodiments, the vegetable oil is selected from the group
consisting of coconut oil, corn oil, cottonseed oil, olive oil,
palm oil, peanut oil, rapeseed oil, canola oil, safflower oil,
sesame oil, soybean oil, sunflower oil, and mixtures thereof. In
some embodiments, the edible oil is canola oil. In some
embodiments, the edible oil comprises one or more nut oils. In some
embodiments, the nut oil is selected from the group consisting of
almond oil, cashew oil, hazelnut oil, macadamia oil, mongongo nut
oil, pecan oil, pine nut oil, pistachio oil, walnut oil, and
mixtures thereof. In some embodiments, the NMDA receptor antagonist
is selected from the group consisting of dizocilpine (MK-801),
meperidine, methadone, dextropropoxyphene, tramadol, ketobemidone,
ketamine, dextromethorphan, phencyclidine, nitrous oxide
(N.sub.2O), AP5 (R-2-amino-5-phosphonopentanoate), AP7
(2-amino-7-phosphonoheptanoic acid), CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,
eticyclidine, gacyclidine, ibogaine, magnesium, memantine,
methoxetamine, rolicyclidine.tenocyclidine, methoxydine,
tiletamine, xenon, neramexane, eliprodil, etoxadrol, dexoxadrol,
WMS 2539, NEFA, remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211,
rhynchophylline, 1-Aminocyclopropanecarboxylic acid (ACPC),
7-Chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenic
acid, lacosamide, CP-101,606 (traxoprodil), AZD6765 (lanicemine)
and GLYX-13. In some embodiments, the NMDA receptor antagonist is
selected from the group consisting of ketamine, dextromethorphan,
phencyclidine, CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrorphan, memantine, tiletamine,
neramexane, eliprodil, remacemide, aptiganel,
1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA
(5,7-dichlorokynurenic acid), kynurenic acid, CP-101,606
(traxoprodil), AZD6765 (lanicemine) and GLYX-13. In some
embodiments, the NMDA receptor antagonist is dizocilpine (MK-801).
In varying embodiments, the composition comprises a benzodiazepine
and a neurosteroid formulated in a cyclodextrin, e.g., for
intramuscular, intravenous and/or subcutaneous administration.
[0010] In another aspect, provided are methods of preventing or
terminating a seizure in a subject in need thereof. In varying
embodiments, the methods comprise administration to the subject of
an effective amount of a composition as described above and herein.
Also provided are methods of accelerating the termination or
abortion of an impending seizure in a subject in need thereof. In
varying embodiments, the methods comprise administration to the
subject of an effective amount of a composition as described above
and herein. In some embodiments, the composition is administered
via inhalational or intrapulmonary administration. In some
embodiments, the composition is not heated prior to administration.
In some embodiments, the composition is nebulized. In some
embodiments, the nebulized particles are about 3 .mu.m or smaller.
In some embodiments, the nebulized particles are about 2-3 .mu.m.
In some embodiments, the composition is delivered to the distal
alveoli. In some embodiments, the composition is administered
orally. In some embodiments, the composition is contained within a
soft gel capsule. In some embodiments, the composition is
administered parenterally. In some embodiments, the composition is
administered via a parenteral route selected from the group
consisting of inhalational, intrapulmonary, intranasal,
intramuscular, subcutaneous, transmucosal and intravenous. In some
embodiments, the composition is administered transmucosally. In
varying embodiments, the method comprises co-administering a
benzodiazepine and a neurosteroid formulated in a cyclodextrin,
e.g., intramuscularly, intravenously and/or subcutaneously.
[0011] In a related aspect, methods of preventing or terminating a
seizure in a subject in need thereof, comprising administration to
the subject of an effective amount of a benzodiazepine and a
neurosteroid. In varying embodiments, one or both of the
benzodiazepine and the neurosteroid are administered in a
subtherapeutic dose. Further are provided methods of accelerating
the termination or abortion of an impending seizure in a subject in
need thereof. In varying embodiments, the methods comprise
administration to the subject of an effective amount of a
benzodiazepine and a neurosteroid. In some embodiments, one or both
of the benzodiazepine and the neurosteroid are administered in a
subtherapeutic dose. In some embodiments, the benzodiazepine and
the neurosteroid are co-administered together and/or by the same
route of administration. In some embodiments, the benzodiazepine
and the neurosteroid are co-administered separately and/or by
different routes of administration. In some embodiments, one or
both of the benzodiazepine and the neurosteroid are
self-administered by the subject. In some embodiments, one or both
of the benzodiazepine and the neurosteroid are administered via
inhalational or intrapulmonary administration. In some embodiments,
one or both of the benzodiazepine and the neurosteroid are not
heated prior to administration. In some embodiments, one or both of
the benzodiazepine and the neurosteroid are nebulized. In some
embodiments, the nebulized particles are about 3 .mu.m or smaller.
In some embodiments, the nebulized particles are about 2-3 .mu.m.
In some embodiments, one or both of the benzodiazepine and the
neurosteroid are delivered to the distal alveoli. In some
embodiments, one or both of the benzodiazepine and the neurosteroid
are administered orally. In some embodiments, one or both of the
benzodiazepine and the neurosteroid are contained within a soft gel
capsule. In some embodiments, one or both of the benzodiazepine and
the neurosteroid are administered parenterally. In some
embodiments, one or both of the benzodiazepine and the neurosteroid
are administered via a parenteral route selected from the group
consisting of inhalational, intrapulmonary, intranasal,
intramuscular, subcutaneous, transmucosal and intravenous. In some
embodiments, one or both of the benzodiazepine and the neurosteroid
are administered transmucosally. In varying embodiments, the method
comprises co-administering a benzodiazepine and a neurosteroid
formulated in a cyclodextrin, e.g., intramuscularly, intravenously
and/or subcutaneously.
[0012] With respect to further embodiments of the methods, in some
embodiments, the benzodiazepine is selected from the group
consisting of bretazenil, clonazepam, cloxazolam, clorazepate,
diazepam, fludiazepam, flutoprazepam, lorazepam, midazolam,
nimetazepam, nitrazepam, phenazepam, temazepam and clobazam. In
some embodiments, the benzodiazepine is selected from the group
consisting of midazolam, lorazepam, and diazepam. In some
embodiments, the benzodiazepine is administered at a dose in the
range of 0.3 .mu.g/kg to 3.0 .mu.g/kg. In varying embodiments, the
benzodiazepine is administered at a dose that does not decrease
blood pressure. In some embodiments, the neurosteroid is selected
from the group consisting of allopregnanolone,
allotetrahydrodeoxycorticosterone, ganaxolone, alphaxolone,
alphadolone, hydroxydione, minaxolone, and Althesin. In some
embodiments, allopregnanolone is co-administered with a
benzodiazepine selected from the group consisting of midazolam,
lorazepam, and diazepam. In some embodiments, the methods further
comprise co-administration of an NMDA receptor antagonist. In some
embodiments, the NMDA receptor antagonist is selected from the
group consisting of dizocilpine (MK-801), meperidine, methadone,
dextropropoxyphene, tramadol, ketobemidone, ketamine,
dextromethorphan, phencyclidine, nitrous oxide (N.sub.2O), AP5
(R-2-amino-5-phosphonopentanoate), AP7
(2-amino-7-phosphonoheptanoic acid), CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,
eticyclidine, gacyclidine, ibogaine, magnesium, memantine,
methoxetamine, rolicyclidine.tenocyclidine, methoxydine,
tiletamine, xenon, neramexane, eliprodil, etoxadrol, dexoxadrol,
WMS 2539, NEFA, remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211,
rhynchophylline, 1-Aminocyclopropanecarboxylic acid (ACPC),
7-Chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenic
acid, lacosamide, CP-101,606 (traxoprodil), AZD6765 (lanicemine)
and GLYX-13. In some embodiments, the NMDA receptor antagonist is
selected from the group consisting of ketamine, dextromethorphan,
phencyclidine, CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrorphan, memantine, tiletamine,
neramexane, eliprodil, remacemide, aptiganel,
1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA
(5,7-dichlorokynurenic acid), kynurenic acid, CP-101,606
(traxoprodil), AZD6765 (lanicemine) and GLYX-13. In some
embodiments, the subject is experiencing aura. In some embodiments,
the subject has been warned of an impending seizure. In some
embodiments, the subject is experiencing a seizure. In some
embodiments, the subject has status epilepticus, refractory status
epilepticus or super-refractory status epilepticus. In some
embodiments, the subject has myoclonic epilepsy. In some
embodiments, the subject suffers from seizure clusters. In some
embodiments, the seizure is a tonic seizure. In some embodiments,
the seizure is a clonic seizure. In some embodiments, the subject
has been exposed to or is at risk of being exposed to a nerve agent
or a pesticide that can cause seizures. In some embodiments, the
subject has been exposed to or is at risk of being exposed to
tetramethylenedisulfotetramine (TETS).
[0013] In another aspect, further provided are kits comprising a
benzodiazepine and a neurosteroid. In varying embodiments, one or
both of the benzodiazepine and the neurosteroid are provided in
unit dosage forms comprising a subtherapeutic dose. In some
embodiments, the kits further comprise a NMDA receptor antagonist.
In some embodiments, one or both of the benzodiazepine and the
neurosteroid is formulated for inhalational, intranasal or
intrapulmonary administration. In some embodiments, one or both of
the benzodiazepine and the neurosteroid is formulated for oral or
parenteral delivery. In some embodiments, one or both of the
benzodiazepine and the neurosteroid are formulated for a parenteral
route selected from the group consisting of inhalational,
intrapulmonary, intranasal, intramuscular, subcutaneous,
transmucosal and intravenous. In some embodiments, the
benzodiazepine is an agonist of the benzodiazepine recognition site
on GABA.sub.A receptors and stimulates endogenous neurosteroid
synthesis. In some embodiments, the benzodiazepine is selected from
the group consisting of bretazenil, clonazepam, cloxazolam,
clorazepate, diazepam, fludiazepam, flutoprazepam, lorazepam,
midazolam, nimetazepam, nitrazepam, phenazepam, temazepam and
clobazam. In some embodiments, the benzodiazepine is selected from
the group consisting of midazolam, lorazepam and diazepam. In some
embodiments, the neurosteroid is selected from the group consisting
of allopregnanolone, allotetrahydrodeoxycorticosterone, ganaxolone,
alphaxolone, alphadolone, hydroxydione, minaxolone, and Althesin.
In some embodiments, the neurosteroid is allopregnanolone. In some
embodiments, the kit comprises allopregnanolone and a
benzodiazepine selected from the group consisting of midazolam,
lorazepam, and diazepam. In some embodiments, the neurosteroid is
suspended or dissolved in a cyclodextrin (e.g., an
.alpha.-cyclodextrin, a .beta.-cyclodextrin or a
.gamma.-cyclodextrin). In varying embodiments, the neurosteroid is
suspended or dissolved in a cyclodextrin selected from the group
consisting of hydroxypropyl-.beta.-cyclodextrin, endotoxin
controlled .beta.-cyclodextrin sulfobutyl ethers, or cyclodextrin
sodium salts (e.g., CAPTISOL.RTM.). In some embodiments, the
neurosteroid is suspended or dissolved in an edible oil. In some
embodiments, the edible oil comprises one or more vegetable oils.
In some embodiments, the vegetable oil is selected from the group
consisting of coconut oil, corn oil, cottonseed oil, olive oil,
palm oil, peanut oil, rapeseed oil, canola oil, safflower oil,
sesame oil, soybean oil, sunflower oil, and mixtures thereof. In
some embodiments, the edible oil is canola oil. In some
embodiments, the edible oil comprises one or more nut oils. In some
embodiments, the nut oil is selected from the group consisting of
almond oil, cashew oil, hazelnut oil, macadamia oil, mongongo nut
oil, pecan oil, pine nut oil, pistachio oil, walnut oil, and
mixtures thereof. In some embodiments, the NMDA receptor antagonist
is selected from the group consisting of dizocilpine (MK-801),
meperidine, methadone, dextropropoxyphene, tramadol, ketobemidone,
ketamine, dextromethorphan, phencyclidine, nitrous oxide
(N.sub.2O), AP5 (R-2-amino-5-phosphonopentanoate), AP7
(2-amino-7-phosphonoheptanoic acid), CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,
eticyclidine, gacyclidine, ibogaine, magnesium, memantine,
methoxetamine, rolicyclidine.tenocyclidine, methoxydine,
tiletamine, xenon, neramexane, eliprodil, etoxadrol, dexoxadrol,
WMS 2539, NEFA, remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211,
rhynchophylline, 1-Aminocyclopropanecarboxylic acid (ACPC),
7-Chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenic
acid, lacosamide, CP-101,606 (traxoprodil), AZD6765 (lanicemine)
and GLYX-13. In some embodiments, the NMDA receptor antagonist is
selected from the group consisting of ketamine, dextromethorphan,
phencyclidine, CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrorphan, memantine, tiletamine,
neramexane, eliprodil, remacemide, aptiganel,
1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA
(5,7-dichlorokynurenic acid), kynurenic acid, CP-101,606
(traxoprodil), AZD6765 (lanicemine) and GLYX-13. In some
embodiments, the NMDA receptor antagonist is dizocilpine
(MK-801).
[0014] In another aspect, the invention provides compositions
comprising a benzodiazepine, a neurosteroid and an NMDA receptor
antagonist. In some embodiments, the composition is formulated for
inhalational or intrapulmonary administration. In some embodiments,
the composition is formulated for oral or transmucosal delivery. In
some embodiments, the benzodiazepine is an agonist of the
benzodiazepine recognition site on GABA.sub.A receptors and
stimulates endogenous neurosteroid synthesis. In some embodiments,
the benzodiazepine is selected from the group consisting of
bretazenil, clonazepam, cloxazolam, clorazepate, diazepam,
fludiazepam, flutoprazepam, lorazepam, midazolam, nimetazepam,
nitrazepam, phenazepam, temazepam and clobazam. In some
embodiments, the benzodiazepine is midazolam. In some embodiments,
the benzodiazepine is diazepam. In some embodiments, the
neurosteroid is selected from the group consisting of
allopregnanolone, allotetrahydrodeoxycorticosterone, ganaxolone,
alphaxolone, alphadolone, hydroxydione, minaxolone, and Althesin.
In some embodiments, the neurosteroid is allopregnanolone. In some
embodiments, the neurosteroid is suspended or dissolved in a
cyclodextrin (e.g., an .alpha.-cyclodextrin, a .beta.-cyclodextrin
or a .gamma.-cyclodextrin). In varying embodiments, the
neurosteroid is suspended or dissolved in a cyclodextrin selected
from the group consisting of hydroxypropyl-.beta.-cyclodextrin,
endotoxin controlled .beta.-cyclodextrin sulfobutyl ethers, or
cyclodextrin sodium salts (e.g., CAPTISOL.RTM.). In some
embodiments, the neurosteroid is suspended or dissolved in an
edible oil. In some embodiments, the edible oil comprises one or
more a vegetable oils. In some embodiments, the vegetable oil is
selected from the group consisting of coconut oil, corn oil,
cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil,
canola oil, safflower oil, sesame oil, soybean oil, sunflower oil,
and mixtures thereof. In some embodiments, the edible oil is canola
oil. In some embodiments, the edible oil comprises one or more nut
oils. In some embodiments, the nut oil is selected from the group
consisting of almond oil, cashew oil, hazelnut oil, macadamia oil,
mongongo nut oil, pecan oil, pine nut oil, pistachio oil, walnut
oil, and mixtures thereof. In some embodiments, the NMDA receptor
antagonist is selected from the group consisting of dizocilpine
(MK-801), meperidine, methadone, dextropropoxyphene, tramadol,
ketobemidone, ketamine, dextromethorphan, phencyclidine, nitrous
oxide (N.sub.2O), AP5 (R-2-amino-5-phosphonopentanoate), AP7
(2-amino-7-phosphonoheptanoic acid), CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,
eticyclidine, gacyclidine, ibogaine, magnesium, memantine,
methoxetamine, rolicyclidine.tenocyclidine, methoxydine,
tiletamine, xenon, neramexane, eliprodil, etoxadrol, dexoxadrol,
WMS 2539, NEFA, remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211,
rhynchophylline, 1-Aminocyclopropanecarboxylic acid (ACPC),
7-Chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenic
acid, lacosamide, CP-101,606 (traxoprodil), AZD6765 (lanicemine)
and GLYX-13. In some embodiments, the NMDA receptor antagonist is
selected from the group consisting of ketamine, dextromethorphan,
phencyclidine, CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrorphan, memantine, tiletamine,
neramexane, eliprodil, remacemide, aptiganel,
1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA
(5,7-dichlorokynurenic acid), kynurenic acid, CP-101,606
(traxoprodil), AZD6765 (lanicemine) and GLYX-13.
[0015] In another aspect, the invention provides methods of
preventing or terminating a seizure in a subject in need thereof,
comprising administration to the subject of an effective amount of
a composition as described above and herein. In another aspect, the
invention provides methods of accelerating the termination or
abortion of an impending seizure in a subject in need thereof,
comprising administration to the subject of an effective amount of
a composition as described above and herein. In a further aspect,
the invention provides methods of preventing or terminating a
seizure in a subject in need thereof, comprising administration to
the subject of an effective amount of a benzodiazepine, a
neurosteroid and an NMDA receptor antagonist. In a further aspect,
the invention provides methods of accelerating the termination or
abortion of an impending seizure in a subject in need thereof,
comprising administration to the subject of an effective amount of
a benzodiazepine, a neurosteroid and an NMDA receptor antagonist.
In some embodiments, the benzodiazepine, neurosteroid and NMDA
receptor antagonist are co-administered together and/or by the same
route of administration. In some embodiments, the benzodiazepine,
neurosteroid and NMDA receptor antagonist are co-administered
separately and/or by different routes of administration. In some
embodiments, the benzodiazepine is selected from the group
consisting of bretazenil, clonazepam, cloxazolam, clorazepate,
diazepam, fludiazepam, flutoprazepam, lorazepam, midazolam,
nimetazepam, nitrazepam, phenazepam, temazepam and clobazam. In
some embodiments, the neurosteroid is selected from the group
consisting of allopregnanolone, allotetrahydrodeoxycorticosterone,
ganaxolone, alphaxolone, alphadolone, hydroxydione, minaxolone, and
Althesin. In some embodiments, the NMDA receptor antagonist is
selected from the group consisting of dizocilpine (MK-801),
meperidine, methadone, dextropropoxyphene, tramadol, ketobemidone,
ketamine, dextromethorphan, phencyclidine, nitrous oxide
(N.sub.2O), AP5 (R-2-amino-5-phosphonopentanoate), AP7
(2-amino-7-phosphonoheptanoic acid), CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,
eticyclidine, gacyclidine, ibogaine, magnesium, memantine,
methoxetamine, rolicyclidine.tenocyclidine, methoxydine,
tiletamine, xenon, neramexane, eliprodil, etoxadrol, dexoxadrol,
WMS 2539, NEFA, remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211,
rhynchophylline, 1-Aminocyclopropanecarboxylic acid (ACPC),
7-Chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenic
acid, lacosamide, CP-101,606 (traxoprodil), AZD6765 (lanicemine)
and GLYX-13. In some embodiments, the NMDA receptor antagonist is
selected from the group consisting of ketamine, dextromethorphan,
phencyclidine, CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrorphan, memantine, tiletamine,
neramexane, eliprodil, remacemide, aptiganel,
1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA
(5,7-dichlorokynurenic acid), kynurenic acid, CP-101,606
(traxoprodil), AZD6765 (lanicemine) and GLYX-13. In one embodiment,
the subject is experiencing aura. In one embodiment, the subject
has been warned of an impending seizure. In one embodiment, the
subject is experiencing a seizure. In one embodiment, the subject
has status epilepticus, refractory status epilepticus or
super-refractory status epilepticus. In one embodiment, the subject
has myoclonic epilepsy. In one embodiment, the subject suffers from
seizure clusters. In one embodiment, the seizure is a tonic
seizure. In one embodiment, the seizure is a clonic seizure. In one
embodiment, the benzodiazepine is self-administered by the subject.
In one embodiment, the composition is administered via inhalational
or intrapulmonary administration. In one embodiment, the
composition is not heated prior to administration. In one
embodiment, the benzodiazepine is nebulized. In one embodiment, the
nebulized particles are about 3 .mu.m or smaller. In one
embodiment, the nebulized particles are about 2.3 .mu.m. In one
embodiment, the benzodiazepine is delivered to the distal alveoli.
In one embodiment, the benzodiazepine is administered at a dose in
the range of 0.3 .mu.g/kg to 3.0 .mu.g/kg. In varying embodiments,
the benzodiazepine is administered at a dose that does not decrease
blood pressure. In one embodiment, the composition is administered
orally. In one embodiment, the composition is contained within a
soft gel capsule. In one embodiment, the composition is
administered transmucosally. In various embodiments, the subject
may be at risk of exposure to or may have been exposed to
tetramethylenedisulfotetramine (TETS).
DEFINITIONS
[0016] As used herein, "administering" refers to local and systemic
administration, e.g., including enteral, parenteral, pulmonary, and
topical/transdermal administration. Routes of administration for
the agents (e.g., one or more of a benzodiazepine, a neurosteroid
and/or an NMDA receptor antagonist) that find use in the methods
described herein include, e.g., oral (per os (P.O.))
administration, nasal or inhalation administration, administration
as a suppository, topical contact, transdermal delivery (e.g., via
a transdermal patch), intrathecal (IT) administration, intravenous
("iv") administration, intraperitoneal ("ip") administration,
intramuscular ("im") administration, intralesional administration,
or subcutaneous ("sc") administration, or the implantation of a
slow-release device e.g., a mini-osmotic pump, a depot formulation,
etc., to a subject. Administration can be by any route including
parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or
transdermal). Parenteral administration includes, e.g.,
intravenous, intramuscular, intra-arterial, intradermal,
subcutaneous, intraperitoneal, intraventricular, ionophoretic and
intracranial. Other modes of delivery include, but are not limited
to, the use of liposomal formulations, intravenous infusion,
transdermal patches, etc.
[0017] The terms "systemic administration" and "systemically
administered" refer to a method of administering a compound or
composition to a mammal so that the compound or composition is
delivered to sites in the body, including the targeted site of
pharmaceutical action, via the circulatory system. Systemic
administration includes, but is not limited to, oral, intranasal,
rectal and parenteral (e.g., other than through the alimentary
tract, such as intramuscular, intravenous, intra-arterial,
transdermal and subcutaneous) administration.
[0018] The term "co-administration" refers to the presence of both
active agents in the blood at the same time. Active agents that are
co-administered can be delivered concurrently (i.e., at the same
time) or sequentially.
[0019] The phrase "cause to be administered" refers to the actions
taken by a medical professional (e.g., a physician), or a person
controlling medical care of a subject, that control and/or permit
the administration of the agent(s)/compound(s) at issue to the
subject. Causing to be administered can involve diagnosis and/or
determination of an appropriate therapeutic or prophylactic
regimen, and/or prescribing particular agent(s)/compounds for a
subject. Such prescribing can include, for example, drafting a
prescription form, annotating a medical record, and the like.
[0020] The term "effective amount" or "pharmaceutically effective
amount" refer to the amount and/or dosage, and/or dosage regime of
one or more compounds necessary to bring about the desired result
e.g., an amount sufficient prevent, abort or terminate a
seizure.
[0021] "Sub-therapeutic dose" refers to a dose of a
pharmacologically active agent(s), either as an administered dose
of pharmacologically active agent, or actual level of
pharmacologically active agent in a subject that functionally is
insufficient to elicit the intended pharmacological effect in
itself (e.g., to abort or prevent a seizure), or that
quantitatively is less than the established therapeutic dose for
that particular pharmacological agent (e.g., as published in a
reference consulted by a person of skill, for example, doses for a
pharmacological agent published in the Physicians' Desk Reference,
67th Ed., 2013, Thomson Healthcare or Brunton, et al., Goodman
& Gilman's The Pharmacological Basis of Therapeutics, 12th
edition, 2010, McGraw-Hill Professional). A "sub-therapeutic dose"
can be defined in relative terms (i.e., as a percentage amount
(less than 100%) of the amount of pharmacologically active agent
conventionally administered). For example, a sub-therapeutic dose
amount can be about 1% to about 75% of the amount of
pharmacologically active agent conventionally administered. In some
embodiments, a sub-therapeutic dose can be less than about 75%,
50%, 30%, 25%, 20%, 10% or less, than the amount of
pharmacologically active agent conventionally administered. A
sub-therapeutic dose amount can be in the range of about 1% to
about 75% of the amount of pharmacologically active agent known to
elicit the intended pharmacological effect. In some embodiments, a
sub-therapeutic dose can be less than about 75%, 50%, 30%, 25%,
20%, 10% or less, than the amount of pharmacologically active agent
known to elicit the intended pharmacological effect.
[0022] As used herein, the terms "treating" and "treatment" refer
to delaying the onset of, retarding or reversing the progress of,
reducing the severity of, or alleviating or preventing either the
disease or condition to which the term applies, or one or more
symptoms of such disease or condition.
[0023] The term "mitigating" refers to reduction or elimination of
one or more symptoms of that pathology or disease, and/or a
reduction in the rate or delay of onset or severity of one or more
symptoms of that pathology or disease, and/or the prevention of
that pathology or disease.
[0024] The terms "reduce," "inhibit," "relieve," "alleviate" refer
to the detectable decrease in the frequency, severity and/or
duration of seizures. A reduction in the frequency, severity and/or
duration of seizures can be measured by self-assessment (e.g., by
reporting of the patient) or by a trained clinical observer.
Determination of a reduction of the frequency, severity and/or
duration of seizures can be made by comparing patient status before
and after treatment.
[0025] As used herein, the phrase "consisting essentially of"
refers to the genera or species of active pharmaceutical agents
(e.g., neurosteroid in combination with benzodiazepine, optionally
in further combination with an NMDA blocker) and excipient (e.g., a
cyclodextrin, an edible oil) included in a method or composition.
In various embodiments, other unmentioned or unrecited active
ingredients and inactive are expressly excluded. In various
embodiments, additives (e.g., surfactants, acids (organic or
fatty), alcohols, esters, co-solvents, solubilizers, lipids,
polymers, glycols) are expressly excluded.
[0026] The terms "subject," "individual," and "patient"
interchangeably refer to a mammal, preferably a human or a
non-human primate, but also domesticated mammals (e.g., canine or
feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster,
guinea pig) and agricultural mammals (e.g., equine, bovine,
porcine, ovine). In various embodiments, the subject can be a human
(e.g., adult male, adult female, adolescent male, adolescent
female, male child, female child) under the care of a physician or
other healthworker in a hospital, psychiatric care facility, as an
outpatient, or other clinical context. In certain embodiments the
subject may not be under the care or prescription of a physician or
other healthworker.
[0027] The term "edible oil" refers to an oil that is digestible by
a mammal. Preferred oils are edible or digestible without inducing
undesirable side effects.
[0028] The term "neuroactive steroid" or "neurosteroid" refers to
steroid compounds that rapidly alter neuronal excitability through
interaction with neurotransmitter-gated ion channels. Neurosteroids
act as allosteric modulators of neurotransmitter receptors, such as
GABA.sub.A, NMDA, and sigma receptors. Neurosteroids find use as
sedatives for the purpose of general anaesthesia for carrying out
surgical procedures, and in the treatment of epilepsy and traumatic
brain injury. Illustrative neurosteroids include, e.g.,
allopregnanolone, Ganaxolone, alphaxolone, alphadolone,
hydroxydione, minaxolone, and Althesin (a mixture of alphaxolone
and alphadolone).
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIGS. 1A-D illustrate TETS-induced Ca2+ dysregulation in
hippocampal neurons. (A) Representative traces showing how acute
exposure to TETS (0.1-10 .mu.M) influences Ca2+ fluctuations in
hippocampal neurons 13-17 DIV. Note that neurons exhibit
spontaneous synchronous Ca2+ oscillations at this developmental
stage indicative of functional network connectivity. The effects of
TETS were analyzed in the initial 5 min following addition (Phase
I) and in the subsequent 10 min (Phase II). In Phase I, the
integrated intracellular Ca2+ level increased in a
concentration-dependent fashion (B), and there was a plateau
response at higher concentrations (3, 10 .mu.M) that decayed slowly
over the 5 min period. In Phase II, there was a
concentration-dependent reduction in the frequency and an increase
in the amplitude of the spontaneous synchronized Ca2+ oscillations
(C,D). The traces shown for Phase II are representative samples of
the 10 min Phase II period. This experiment was repeated three
times with similar results.
[0030] FIG. 2 illustrates reversal of TETS-induced Phase II effects
after washout of TETS. Traces show synchronized Ca2+ oscillations
that are reduced in frequency and increased in amplitude in the
presence of TETS. The dotted red line is a representative trace
before ("Phase II response") and after TETS ("Washout"). The solid
black line is a representative recording from a control experiment
in which the culture was treated with vehicle and subjected to the
same washout procedure.
[0031] FIGS. 3A-D illustrate TETS, picrotoxin, and bicuculline
trigger similar neuronal Ca2+ dysregulation. (A) Representative
traces from experiments comparing the effects of TETS (3 .mu.M),
picrotoxin (100 .mu.M), and bicuculline (100 .mu.M) on Ca2+
fluctuations. The three agents produce similar acute elevation of
the integrated Ca2+ level (B) with plateau responses in Phase I,
and they decreased the oscillatory frequency (C) while increasing
the amplitude of Ca2+ transients in Phase II (D). **, p<0.01,
inhibitors vs. control, data were pooled from three experiments
performed at least in duplicate.
[0032] FIGS. 4A-B illustrate TETS-reversibly alters spontaneous
electrical discharges in hippocampal neurons. (A) Representative
raster plots of neuronal discharges before, during and after
exposure to vehicle (DMSO) (left panels) or TETS (right panels).
Neuronal network activity was stable for up to 60 min in the
absence or presence of vehicle control. TETS solutions of
increasing concentration were added serially to the wells. After
recording for 10 min, the solution was removed and replaced by a
higher concentration or by vehicle (wash out). TETS concentrations
of 2 and 6 .mu.M caused a clustered burst discharge pattern and
increased the overall discharge frequency (B). This experiment was
repeated three times each performed in duplicate with similar
results. *, p<0.05, **, p<0.01, TETS vs. basal.
[0033] FIG. 5 illustrates TETS-induced a pattern of clustered
electrical burst firing in hippocampal neuronal cell cultures at 14
days in vitro. Representative traces of neuronal electrical firing
from an MEA recording before (A) and after (B) addition of TETS (6
.mu.M). The software only allows a display of 200 ms; the actual
total period of clustered bursts after TETS treatment often lasted
up to 10 s (see FIG. 3A, right panel, 4th row).
[0034] FIGS. 6A-D illustrate MK-801, but not nifedipine, partially
mitigates TETS (3 .mu.M)-induced neuronal Ca2+ dysregulation. (A)
Representative traces illustrating effects of pre-exposure to
MK-801 and nifedipine on TETS-induced Ca2+ dysregulation. (B)
Effects of MK-801 (MK) and nifedipine (NIF) on TETS-induced
increase in integrated Ca2+ levels in Phase I. (C,D) Effects of
MK-801 and nifedipine on the TETS-induced synchronous Ca2+
transient oscillation frequency decrease (C) and amplitude increase
(D) in Phase II. **, p<0.01, TETS vs. vehicle control, ##,
p<0.01, MK-801+ TETS vs TETS, n=6 pooled from two
experiments.
[0035] FIGS. 7A-D Diazepam partially mitigates TETS-induced
neuronal Ca2+ dysregulation. (A) Representative traces illustrating
effects of pre-exposure to increasing concentrations of diazepam
(0.03-1 .mu.M) on TETS-induced Ca2+ dysregulation. (B) Effect of
diazepam (DZP) on TETS-induced increase in integrated Ca2+ levels
in Phase I. (C,D) Effect of diazepam on the TETS-induced
synchronous Ca2+ transient oscillation frequency decrease (C) and
amplitude increase (D) in Phase II. **, p<0.01, TETS vs. vehicle
control, #, p<0.05, ##, p<0.01, diazepam+TETS vs. TETS, n=6
pooled from two experiments.
[0036] FIGS. 8A-D illustrate allopregnanolone partially mitigates
TETS-induced neuronal Ca2+ signaling dysregulation. (A)
Representative traces illustrating effects of pre-exposure to
increasing concentrations of allopregnanolone (0.03-1 .mu.M) on
TETS-induced Ca2+ dysregulation. (B) Effect of allopregnanolone
(AlloP) on TETS-induced increase in integrated Ca2+ levels in Phase
I. (C,D) Effect of allopregnanolone on the TETS-induced synchronous
Ca2+ transient oscillation frequency decrease (C) and amplitude
increase (D) in Phase II. **, p<0.01, TETS vs. vehicle control,
#, p<0.05, ##, p<0.01, allopregnanolone+TETS vs TETS, n=6
pooled from two experiments.
[0037] FIGS. 9A-D illustrate low concentrations of allopregnanolone
and diazepam in combination act synergistically to mitigate
TETS-induced neuronal Ca2+ signaling dysregulation. (A)
Representative traces illustrating effect of pre-exposure to
allopregnanolone (0.1 .mu.M), diazepam (0.1 .mu.M) or a combination
of allopregnanolone (0.1 .mu.M) and diazepam (0.1 .mu.M) on
TETS-induced Ca2+ dysregulation. (C,D) Effects of allopregnanolone
or diazepam alone or the combination on TETS-induced synchronous
Ca2+ transient oscillation frequency decrease (C) and amplitude
increase (D) in phase II. **, p<0.01, TETS vs. vehicle control,
##, p<0.01, allopregnanolone/diazepam+TETS vs TETS, n=8 pooled
from two experiments.
[0038] FIGS. 10A-C illustrate that exposure of mouse hippocampal
neurons following TETS challenge with diazepam (0.1 .mu.M) and
allopregnanolone (0.1 .mu.M) in combination effectively mitigates
TETS dysregulated Ca2+ dynamics. (A) Representative traces
illustrating effects of post-TETS treatment with diazepam or
allopregnanolone or the combination on TETS-induced Ca.sup.2+
dysregulation. Amelioration of TETS-induced alterations in the
Phase II response (see, Cao et al, Toxicological Sciences (2012)
130:362-372) by diazepam and allopregnanolone, either singly or in
combination, on the frequency of synchronous Ca.sup.2+ oscillation
(B) and increases in Ca.sup.2+ transient amplitude (C). The first
arrowhead indicates the addition of TETS or vehicle. The second
arrowhead indicates the addition of vehicle or diazepam or
allopregnanolone or the combination. Each data point represents
Mean.+-.SEM, n=6 wells.
[0039] FIG. 11 illustrates that high dose diazepam rescues animals
from TETS-induced tonic seizures and death. Representative EEG
recordings from mice administered TETS (0.15 mg/kg, i.p.) with and
without pretreatment with diazepam (dose/route). Time to seizure
onset and seizure duration are expressed as the mean.+-.S.E.M. (n=x
per treatment group). Administration of diazepam immediately
following the second clonic seizure prevented a fatal tonic
seizure. EEG recording in TETS-exposed animals rescued by diazepam
indicated no additional seizure for up to 1 h post-TETS
exposure.
[0040] FIG. 12. Adult male NIH Swiss mice were injected with TETS
(i.p.). Two minutes following the second clonic seizure, mice were
injected i.p. with diazepam (in saline) or allopregnanolone (AlloP,
in .beta.-cyclodextrin) singly or in combination. Seizure time to
onset, number and duration were monitored for 1 h post-TETS
exposure. % Survival is at 24 h post TETS injection. Data presented
as the mean.+-.SEM (n=6-8 per group). **p<0.05 as determined by
one way ANOVA with Tukey's post hoc test.
[0041] FIG. 13. Adult male NIH Swiss mice were injected i.p. with
diazepam (in saline) or allopregnanolone (AlloP, in
.beta.-cyclodextrin) singly or in combination 10 minutes prior to
i.p. injection of TETS. Seizure time to onset, duration and number
were monitored for 1 h post-TETS injection. % Survival is at 24 h
post TETS injection. Data presented as the mean.+-.SEM (n=8 per
group). **p<0.01 as determined by one way ANOVA with Tukey's
post hoc test.
[0042] FIG. 14 illustrates the effect of benzodiazepine and
neuro-steroid treatments on blood pressure. Adult male NIH Swiss
mice were i.p. injected with diazepam (DZP) or allopregnanolone
(AlloP) alone or in combination. Blood pressure (BP) was measured
using a tail cuff CODA non-invasive blood measuring system from
Kent Scientific. This system utilizes volume pressure recording
technology to detect changes that correspond to systolic and
diastolic BP. Diastolic BP is measured and systolic BP calculated.
BP is measured for 6 days prior to testing to obtain baseline BP
and allow animals to acclimate to the chamber. Measurements
consisted of 20 cycles of 30 sec each with 10 sec delay between
each measurement. Data presented as the mean.+-.SEM (n=6 per
group).
DETAILED DESCRIPTION
1. Introduction
[0043] Tetramethylenedisulfotetramine (TETS) is a potent convulsant
that is considered a chemical threat agent. We characterized TETS
as an activator of spontaneous Ca2+ oscillations and electrical
burst discharges in mouse hippocampal neuronal cultures at 13-17
days in vitro using FLIPR.RTM. Fluo-4 fluorescence measurements and
extracellular multielectrode array (MEA) recording. Acute exposure
to TETS (>2 .mu.M) reversibly altered the pattern of spontaneous
neuronal discharges, producing clustered burst firing and an
overall increase in discharge frequency. TETS also dramatically
affected Ca2+ dynamics causing an immediate but transient elevation
of neuronal intracellular Ca2+ followed by decreased frequency of
Ca2+ oscillations having greater peak amplitudes. The effect on
Ca2+ dynamics was similar to that elicited by picrotoxin and
bicuculline, supporting the view that TETS acts by inhibiting
GABA.sub.A receptor function. The effect of TETS on Ca2+ dynamics
requires activation of NMDA receptors, since the changes induced by
TETS were prevented by MK-801 block of NMDA receptors, but not
nifedipine block of L-type Ca2+ channels. Pre-treatment with the
GABA.sub.A receptor positive modulators diazepam and
allopregnanolone partially mitigated TETS-induced changes in Ca2+
dynamics. Moreover, low, minimally effective concentrations of
diazepam (0.1 .mu.M) and allopregnanolone (0.1 .mu.M), when
administered together, were highly effective in suppressing
TETS-induced alterations in Ca2+ dynamics, suggesting that the
combination of positive modulators synaptic and extrasynaptic
GABA.sub.A receptors have therapeutic potential. These rapid
throughput in vitro assays may assist in the identification of
single agents or combinations that have utility in the treatment of
TETS intoxication.
2. Conditions Amenable to Treatment
[0044] Co-administration of a benzodiazepine and a neurosteroid. In
varying embodiments, one or both of the benzodiazepine and the
neurosteroid are administered in a sub-therapeutic dose or amount
finds use in the rapid amelioration and/or termination of seizures.
In various embodiments, the seizures may be due to an epileptic
condition. Optionally, an NMDA receptor antagonist is also
co-administered.
[0045] The term "epilepsy" refers to a chronic neurological
disorder characterized by recurrent unprovoked seizures. These
seizures are transient signs and/or symptoms of abnormal, excessive
or synchronous neuronal activity in the brain. There are over 40
different types of epilepsy, including without limitation childhood
absence epilepsy, juvenile absence epilepsy, benign Rolandic
epilepsy, clonic seizures, complex partial seizures, frontal lobe
epilepsy, febrile seizures, infantile spasms, juvenile myoclonic
epilepsy, Lennox-Gastaut syndrome, Landau-Kleffner Syndrome,
myoclonic seizures, mitochondrial disorders associated with
seizures, Lafora Disease, progressive myoclonic epilepsies, reflex
epilepsy, and Rasmussen's syndrome. There are also numerous types
of seizures including simple partial seizures, complex partial
seizures, generalized seizures, secondarily generalized seizures,
temporal lobe seizures, tonic-clonic seizures, tonic seizures,
psychomotor seizures, limbic seizures, status epilepticus,
refractory status epilepticus or super-refractory status
epilepticus, abdominal seizures, akinetic seizures, autonomic
seizures, massive bilateral myoclonus, drop seizures, focal
seizures, gelastic seizures, Jacksonian march, motor seizures,
multifocal seizures, neonatal seizures, nocturnal seizures,
photosensitive seizure, sensory seizures, sylvan seizures,
withdrawal seizures and visual reflex seizures.
[0046] The most widespread classification of the epilepsies divides
epilepsy syndromes by location or distribution of seizures (as
revealed by the appearance of the seizures and by EEG) and by
cause. Syndromes are divided into localization-related epilepsies,
generalized epilepsies, or epilepsies of unknown localization.
Localization-related epilepsies, sometimes termed partial or focal
epilepsies, arise from an epileptic focus, a small portion of the
brain that serves as the irritant driving the epileptic response.
Generalized epilepsies, in contrast, arise from many independent
foci (multifocal epilepsies) or from epileptic circuits that
involve the whole brain. Epilepsies of unknown localization remain
unclear whether they arise from a portion of the brain or from more
widespread circuits.
[0047] Epilepsy syndromes are further divided by presumptive cause:
idiopathic, symptomatic, and cryptogenic. Idiopathic epilepsies are
generally thought to arise from genetic abnormalities that lead to
alterations in brain excitability. Symptomatic epilepsies arise
from the effects of an epileptic lesion, whether that lesion is
focal, such as a tumor, or a defect in metabolism causing
widespread injury to the brain. Cryptogenic epilepsies involve a
presumptive lesion that is otherwise difficult or impossible to
uncover during evaluation. Forms of epilepsy are well characterized
and reviewed, e.g., in Epilepsy: A Comprehensive Textbook (3-volume
set), Engel, et al., editors, 2nd Edition, 2007, Lippincott,
Williams and Wilkins; and The Treatment of Epilepsy: Principles and
Practice, Wyllie, et al., editors, 4th Edition, 2005, Lippincott,
Williams and Wilkins; and Browne and Holmes, Handbook of Epilepsy,
4th Edition, 2008, Lippincott, Williams and Wilkins.
3. Subjects Amenable to Treatment
[0048] In various embodiments, the patient may be experiencing an
electrographic or behavioral seizure or may be experiencing a
seizure aura, which itself is a localized seizure that may spread
and become a full blown behavioral seizure. For example, the
subject may be experiencing aura that alerts of the impending onset
of a seizure or seizure cluster.
[0049] Alternatively, the subject may be using a seizure prediction
device that alerts of the impending onset of a seizure or seizure
cluster. Implantable seizure prediction devices are known in the
art and described, e.g., in D'Alessandro, et al., IEEE TRANSACTIONS
ON BIOMEDICAL ENGINEERING, VOL. 50, NO. 5, MAY 2003, and U.S.
Patent Publication Nos. 2010/0198098, 2010/0168603, 2009/0062682,
and 2008/0243022.
[0050] The subject may have a personal or familial history of any
of the epileptic conditions described herein. The subject may have
been diagnosed as having any of the epileptic conditions described
herein. In some embodiments, the subject has or is at risk of
suffering status epilepticus, refractory status epilepticus or
super-refractory status epilepticus. In some embodiments, the
subject has or is at risk of suffering a myoclonic seizure or
myoclonic epilepsy, e.g., juvenile myoclonic epilepsy. The PTZ
seizure model demonstrated herein is predictive of utility and/or
activity in counteracting myoclonic seizures or myoclonic epilepsy
in humans.
[0051] In various embodiments, the subject may be at risk of
exposure to or may have been exposed to
tetramethylenedisulfotetramine (TETS).
[0052] In various embodiments, the subject may be at risk of
exposure to or may have been exposed to a nerve agent or a
pesticide that can cause seizures. Illustrative nerve agents that
can cause seizures include, e.g., organophosphorus nerve agents,
e.g., tabun, sarin, soman, GF, VR and/or VX. Illustrative
pesticides that can cause seizures include, e.g., organophosphate
pesticides (e.g., Acephate (Orthene), Azinphos-methyl (Gusathion,
Guthion), Bensulide (Betasan, Lescosan), Bomyl (Swat), Bromophos
(Nexion), Bromophos-ethyl (Nexagan), Cadusafos (Apache, Ebufos,
Rugby), Carbophenothion (Trithion), Chlorethoxyfos (Fortress),
Chlorfenvinphos (Apachlor, Birlane), Chlormephos (Dotan),
Chlorphoxim (Baythion-C), Chlorpyrifos (Brodan, Dursban, Lorsban),
Chlorthiophos (Celathion), Coumaphos (Asuntol, Co-Ral), Crotoxyphos
(Ciodrin, Cypona), Crufomate (Ruelene), Cyanofenphos (Surecide),
Cyanophos (Cyanox), Cythioate (Cyflee, Proban), DEF (De-Green),
E-Z-Off D), Demeton (Systox), Demeton-5-methyl (Duratox,
Metasystoxl), Dialifor (Torak), Diazinon, Dichlorofenthion, (VC-13
Nemacide), Dichlorvos (DDVP, Vapona), Dicrotophos (Bidrin), Dimefos
(Hanane, Pestox XIV), Dimethoate (Cygon, DeFend), Dioxathion
(Delnav), Disulfoton (Disyston), Ditalimfos, Edifenphos, Endothion,
EPBP (S-seven), EPN, Ethion (Ethanox), Ethoprop (Mocap), Ethyl
parathion (E605, Parathion, thiophos), Etrimfos (Ekamet), Famphur
(Bash, Bo-Ana, Famfos), Fenamiphos (Nemacur), Fenitrothion
(Accothion, Agrothion, Sumithion), Fenophosphon (Agritox,
trichloronate), Fensulfothion (Dasanit), Fenthion (Baytex, Entex,
Tiguvon), Fonofos (Dyfonate, N-2790), Formothion (Anthio),
Fosthietan (Nem-A-Tak), Heptenophos (Hostaquick), Hiometon
(Ekatin), Hosalone (Zolone), IBP (Kitazin), Iodofenphos
(Nuvanol-N), Isazofos (Brace, Miral, Triumph), Isofenphos (Amaze,
Oftanol), Isoxathion (E-48, Karphos), Leptophos (Phosvel),
Malathion (Cythion), Mephosfolan (Cytrolane), Merphos (Easy Off-D,
Folex), Methamidophos (Monitor), Methidathion (Supracide,
Ultracide), Methyl parathion (E601, Penncap-M), Methyl trithion,
Mevinphos (Duraphos, Phosdrin), Mipafox (Isopestox, Pestox XV),
Monocrotophos (Azodrin), Naled (Dibrome), Oxydemeton-methyl
(Metasystox-R), Oxydeprofos (Metasystox-S), Phencapton (G 28029),
Phenthoate (Dimephenthoate, Phenthoate), Phorate (Rampart, Thimet),
Phosalone (Azofene, Zolone), Phosfolan (Cylan, Cyolane), Phosmet
(Imidan, Prolate), Phosphamidon (Dimecron), Phostebupirim (Aztec),
Phoxim (Baythion), Pirimiphos-ethyl (Primicid), Pirimiphos-methyl
(Actellic), Profenofos (Curacron), Propetamphos (Safrotin), Propyl
thiopyrophosphate (Aspon), Prothoate (Fac), Pyrazophos (Afugan,
Curamil), Pyridaphenthion (Ofunack), Quinalphos (Bayrusil), Ronnel
(Fenchlorphos, Korlan), Schradan (OMPA), Sulfotep (Bladafum,
Dithione, Thiotepp), Sulprofos (Bolstar, Helothion), Temephos
(Abate, Abathion), Terbufos (Contraven, Counter), Tetrachlorvinphos
(Gardona, Rabon), Tetraethyl pyrophosphate (TEPP), Triazophos
(Hostathion), and Trichlorfon (Dipterex, Dylox, Neguvon,
Proxol).
4. Therapeutic Agents
[0053] Generally, the compositions and methods comprise
co-administering a benzodiazepine and a neurosteroid. In varying
embodiments, one or both of the benzodiazepine and the neurosteroid
are co-administered at a sub-therapeutic dose or amount.
Optionally, an NMDA receptor antagonist is co-administered. The
agents can be co-administered concurrently or sequentially. The
agents can be co-administered via the same or different routes of
administration. In various embodiments, the agents are
co-administered in a single composition.
[0054] a. Benzodiazepines
[0055] Any benzodiazepine known in the art finds use in the present
compositions and methods. Illustrative benzodiazepines that find
use include without limitation bretazenil, clonazepam, cloxazolam,
clorazepate, diazepam, fludiazepam, flutoprazepam, lorazepam,
midazolam, nimetazepam, nitrazepam, phenazepam, temazepam and
clobazam. In some embodiments, the benzodiazepine is midazolam. In
some embodiments, the benzodiazepine is diazepam.
[0056] b. Neurosteroids
[0057] The terms "neuroactive steroid" or "neurosteroids"
interchangeably refer to steroids that rapidly alter neuronal
excitability through interaction with neurotransmitter-gated ion
channels, specifically GABA.sub.A receptors. Neuroactive steroids
have a wide range of applications from sedation to treatment of
epilepsy and traumatic brain injury. Neuroactive steroids act as
direct agonists and allosteric positive modulators of GABA.sub.A
receptors. Several synthetic neuroactive steroids have been used as
sedatives for the purpose of general anaesthesia for carrying out
surgical procedures. Exemplary sedating neuroactive steroids
include without limitation alphaxolone, alphadolone, hydroxydione
and minaxolone. The neuroactive steroid ganaxolone finds use for
the treatment of epilepsy. In various embodiments, the
benzodiazepine or non-benzodiazepine benzodiazepine receptor
agonist is co-administered with an endogenously occurring
neurosteroid or other neuroactive steroid. Illustrative endogenous
neuroactive steroids, e.g., allopregnanolone and
tetrahydrodeoxycorticosterone find use. In some embodiments, the
neurosteroid is selected from the group consisting of
allopregnanolone, allotetrahydrodeoxycorticosterone, ganaxolone,
alphaxolone, alphadolone, hydroxydione, minaxolone, and
Althesin.
[0058] In various embodiments the neurosteroid is allopregnanolone
(ALP). Allopregnanolone, also known as
3.alpha.-hydroxy-5.alpha.-pregnan-20-one or
3.alpha.,5.alpha.-tetrahydroprogesterone, IUPAC name
1-(3-Hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahy-
dro-1H-cyclopenta[a]phenanthren-17-yl)ethanone, and referenced as
CAS number 516-54-1, is a prototypic neurosteroid present in the
blood and also the brain. It is a metabolite of progesterone and
modulator of GABA.sub.A receptors. While allopregnanolone, like
other GABA.sub.A receptor active neurosteroids such as
allotetrahydrodeoxycorticosterone
(3.alpha.,21-dihydroxy-5.alpha.-pregnan-20-one; THDOC), positively
modulates all GABA.sub.A receptor isoforms, those isoforms
containing .delta.-subunits exhibit greater magnitude potentiation.
Allopregnanolone has pharmacological properties similar to other
positive modulators of GABA.sub.A receptors, including anxiolytic
and anticonvulsant activity. Allopregnanolone is neuroprotective in
many animal models of neurodegenerative conditions, including,
e.g., Alzheimer's disease (Wang et al., Proc Natl Acad Sci USA.
2010 Apr. 6; 107(14):6498-503), cerebral edema (Limmroth et al., Br
J Pharmacol. 1996 January; 117(1):99-104) and traumatic brain
injury (He et al., Reston Neurol Neurosci. 2004; 22(1):19-31; and
He, et al., Exp Neurol. 2004 October; 189(2):404-12), Mood
disorders (Robichaud and Debonnel, Int J Neuropsychopharmacol. 2006
April; 9(2):191-200), Niemann-Pick type C disease (Griffin et al.,
Nat Med. 2004 July; 10(7):704-11) and acts as an anticonvulsant
against chemically induced seizures, including the
pentylenetetrazol (PTZ) model (Kokate et al., J Pharmacol Exp Ther.
1994 September; 270(3):1223-9). The chemical structure of
allopregnanolone is depicted below in Formula I:
##STR00001##
[0059] In various embodiments, the compositions comprise a sulfate,
salt, hemisuccinate, nitrosylated, derivative or congener of
allopregnanolone.
[0060] Delivery of other neurosteroids also can be enhanced by
formulation in a cyclodextrin and/or in an edible oil. Other
neurosteroids that can be formulated in a cyclodextrin and/or in an
edible oil, include without limitation
allotetrahydrodeoxycorticosterone
(3.alpha.,21-dihydroxy-5.alpha.-pregnan-20-one; THDOC),
3.alpha.,21-dihydroxy-5b-pregnan-20-one, pregnanolone
(3.alpha.-hydroxy-5.beta.-pregnan-20-one), Ganaxolone (INN, also
known as CCD-1042; IUPAC name
(3.alpha.,5.alpha.)-3-hydroxy-5-methylpregnan-20-one;
1-[(3R,5S,8R,9S,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethyl-1,2,4,5,6,7,-
8,9,11,12,14,15,16,17-tetradecahydrocyclopenta[a]phenanthren-17-yl]ethanon-
e), alphaxolone, alphadolone, hydroxydione, minaxolone, and
Althesin (a mixture of alphaxolone, alphadolone,
tetrahydrodeoxycorticosterone, pregnenolone, dehydroepiandrosterone
(DHEA), 7-substituted benz[e]indene-3-carbonitriles (see, e.g., Hu,
et al., J Med Chem. (1993) 36(24):3956-67);
7-(2-hydroxyethyl)benz[e]indene analogues (see, e.g., Han, et al.,
J Med Chem. (1995) 38(22):4548-56); 3 alpha-hydroxy-5
alpha-pregnan-20-one and 3 alpha-hydroxy-5 beta-pregnan-20-one
analogues (see, e.g., Han, et al., J Med Chem. (1996)
39(21):4218-32); enantiomers of dehydroepiandrosterone sulfate,
pregnenolone sulfate, and (3alpha,5beta)-3-hydroxypregnan-20-one
sulfate (see, e.g., Nilsson, et al., J Med Chem. (1998)
41(14):2604-13); 13,24-cyclo-18,21-dinorcholane analogues (see,
e.g., Jiang, et al., J Med Chem. (2003) 46(25):5334-48); N-acylated
17a-aza-D-homosteroid analogues (see, e.g., Covey, et al., J Med
Chem. (2000) 43(17):3201-4); 5 beta-methyl-3-ketosteroid analogues
(see, e.g., Zeng, et al., J Org Chem. (2000) 65(7):2264-6);
18-norandrostan-17-one analogues (see, e.g., Jiang, et al., J Org
Chem. (2000) 65(11):3555-7); (3alpha,5alpha)- and
(3alpha,5beta)-3-hydroxypregnan-20-one analogs (see, e.g., Zeng, et
al., J Med Chem. (2005) 48(8):3051-9); benz[f]indenes (see, e.g.,
Scaglione, et al., J Med Chem. (2006) 49(15):4595-605); enantiomers
of androgens (see, e.g., Katona, et al., Eur J Med Chem. (2008)
43(1):107-13); cyclopenta[b]phenanthrenes and
cyclopenta[b]anthracenes (see, e.g., Scaglione, et al., J Med Chem.
(2008) 51(5):1309-18); 2beta-hydroxygonane derivatives (see, e.g.,
Wang, et al., Tetrahedron (2007) 63(33):7977-7984);
.DELTA.16-alphaxalone and corresponding 17-carbonitrile analogues
(see, e.g., Bandyopadhyaya, et al., Bioorg Med Chem Lett. (2010)
20(22):6680-4); .DELTA.(16) and .DELTA.(17(20)) analogues of
.DELTA.(16)-alphaxalone (see, e.g., Stastna, et al., J Med Chem.
(2011) 54(11):3926-34); neurosteroid analogs developed by CoCensys
(now Purdue Neuroscience) (e.g., CCD-3693, Co2-6749 (a.k.a.,
GMA-839 and WAY-141839); neurosteroid analogs described in U.S.
Pat. No. 7,781,421 and in PCT Patent Publications WO 2008/157460;
WO 1993/003732; WO 1993/018053; WO 1994/027608; WO 1995/021617; WO
1996/016076; WO 1996/040043, as well as salts, hemisuccinates,
nitrosylated, sulfates and derivatives thereof.
[0061] In various embodiments, the steroid or neurosteroid is not a
sex hormone. In various embodiments, the steroid or neurosteroid is
not progesterone.
[0062] As appropriate, the steroid or neurosteroid (e.g.,
allopregnanolone) may or may not be micronized. As appropriate, the
steroid or neurosteroid (e.g., allopregnanolone) may or may not be
enclosed in microspheres in suspension in the oil.
[0063] c. NMDA Receptor Antagonists
[0064] Illustrative NMDA receptor antagonists that find use include
without limitation, e.g., dizocilpine (MK-801), meperidine,
methadone, dextropropoxyphene, tramadol, ketobemidone, ketamine,
dextromethorphan, phencyclidine, nitrous oxide (N.sub.2O), AP5
(R-2-amino-5-phosphonopentanoate), AP7
(2-amino-7-phosphonoheptanoic acid), CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,
eticyclidine, gacyclidine, ibogaine, magnesium, memantine,
methoxetamine, rolicyclidine.tenocyclidine, methoxydine,
tiletamine, xenon, neramexane, eliprodil, etoxadrol, dexoxadrol,
WMS 2539, NEFA, remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211,
rhynchophylline, 1-Aminocyclopropanecarboxylic acid (ACPC),
7-Chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenic
acid, lacosamide, CP-101,606 (traxoprodil), AZD6765 (lanicemine)
and GLYX-13. In some embodiments, the NMDA receptor antagonist is
selected from the group consisting of ketamine, dextromethorphan,
phencyclidine, CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrorphan, memantine, tiletamine,
neramexane, eliprodil, remacemide, aptiganel,
1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA
(5,7-dichlorokynurenic acid), kynurenic acid, CP-101,606
(traxoprodil), AZD6765 (lanicemine) and GLYX-13. In some
embodiments, the NMDA receptor antagonist is dizocilpine
(MK-801).
5. Formulation and Administration
[0065] In various embodiments, one or more of the benzodiazepines
and one or more neurosteroids are formulated for intramuscular,
intravenous, subcutaneous, intrapulmonary and/or inhalational
administration. In various embodiments, the benzodiazepines are
formulated for delivery via an inhaler. In various embodiments
other routes of delivery, described herein may be appropriate.
Optionally a NMDA receptor antagonist is included in the
compositions and/or co-administration.
[0066] Appropriate dosing will depend on the size and health of the
patient and can be readily determined by a trained clinician.
Initial doses are low and then can be incrementally increased until
the desired therapeutic effect is achieved with little or no
adverse side effects. Determination of an effective amount for
administration in a single dosage is well within the capability of
those skilled in the art, especially in light of the detailed
disclosure provided herein. Generally, an efficacious or effective
amount of the agents (e.g., one or more benzodiazepines and one or
more neurosteroids, optionally including a NMDA receptor
antagonist) is determined by first administering a low dose or
small amount of the agent and then incrementally increasing the
administered dose or dosages, adding a second or third medication
as needed, until a desired effect of is observed in the treated
subject with minimal or no toxic side effects. Applicable methods
for determining an appropriate dose and dosing schedule for
administration of a combination of agents of the present invention
are described, for example, in Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 12th Edition, 2010, supra;
in a Physicians' Desk Reference (PDR), 67.sup.th Edition, 2013; in
Remington: The Science and Practice of Pharmacy, 21.sup.st Ed.,
2005, supra; and in Martindale: The Complete Drug Reference,
Sweetman, 2005, London: Pharmaceutical Press., and in Martindale,
Martindale: The Extra Pharmacopoeia, 31st Edition., 1996, Amer
Pharmaceutical Assn, each of which are hereby incorporated herein
by reference.
[0067] In various embodiments, the agents (e.g., one or more
benzodiazepines and one or more neurosteroids, optionally including
a NMDA receptor antagonist) are nebulized. Methods and systems for
intrapulmonary delivery of agents, e.g., benzodiazepines, are known
in the art and find use. Illustrative systems for aerosol delivery
of benzodiazepines by inhalation are described, e.g., in U.S. Pat.
Nos. 5,497,763; 5,660,166; 7,060,255; and 7,540,286; and U.S.
Patent Publication Nos. 2003/0032638; and 2006/0052428, each of
which are hereby incorporated herein by reference in their entirety
for all purposes. Preferably, the agents (e.g., one or more
benzodiazepines and one or more neurosteroids, optionally including
a NMDA receptor antagonist) are nebulized without the input of
heat.
[0068] For administration of the nebulized and/or aerosolized
agents (e.g., one or more benzodiazepines and one or more
neurosteroids, optionally including a NMDA receptor antagonist),
the size of the aerosol particulates can be within a range
appropriate for intrapulmonary delivery, particularly delivery to
the distal alveoli. In various embodiments, the aerosol
particulates have a mass median aerodynamic diameter ("MMAD") of
less than about 5 .mu.m, 4 .mu.m, 3 .mu.m, for example, ranging
from about 1 .mu.m to about 3 .mu.m, e.g., from about 2 .mu.m to
about 3 .mu.m, e.g., ranging from about 0.01 .mu.m to about 0.10
.mu.m. Aerosols characterized by a MMAD ranging from about 1 .mu.m
to about 3 .mu.m can deposit on alveoli walls through gravitational
settling and can be absorbed into the systemic circulation, while
aerosols characterized by a MMAD ranging from about 0.01 .mu.m to
0.10 .mu.m can also be deposited on the alveoli walls through
diffusion. Aerosols characterized by a MMAD ranging from about 0.15
.mu.m to about 1 .mu.m are generally exhaled. Thus, in various
embodiments, aerosol particulates can have a MMAD ranging from 0.01
.mu.m to about 5 .mu.m, for example, ranging from about 0.05 .mu.m
to about 3 .mu.m, for example, ranging from about 1 .mu.m to about
3 .mu.m, for example, ranging from about 0.01 .mu.m to about 0.1
.mu.m. The nebulized and/or aerosolized benzodiazepines can be
delivered to the distal alveoli, allowing for rapid absorption and
efficacy.
[0069] In various embodiments, the agents (e.g., one or more
benzodiazepines and one or more neurosteroids, optionally including
a NMDA receptor antagonist) are formulated in a solution comprising
excipients suitable for aerosolized intrapulmonary delivery. The
solution can comprise one or more pharmaceutically acceptable
carriers and/or excipients. Pharmaceutically acceptable refers to
approved or approvable by a regulatory agency of the Federal or a
state government or listed in the U.S Pharmacopoeia or other
generally recognized pharmacopoeia for use in animals, and more
particularly in humans. Preferably, the solution is buffered such
that the solution is in a relatively neutral pH range, for example,
a pH in the range of about 4 to 8, for example, a pH in the range
of about 5-7. In some embodiments, the benzodiazepine is formulated
in a buffered solution, for example, phosphate-buffered saline.
[0070] In various embodiments, the agents (e.g., one or more
benzodiazepines and one or more neurosteroids, optionally including
a NMDA receptor antagonist) are prepared as a concentrated aqueous
solution. Ordinary metered dose liquid inhalers have poor
efficiency for the delivery to the deep lung because the particle
size is not sufficiently small (Kim et al., 1985 Am Rev Resp Dis
132:137-142; and Fan et al., 1995 Thorax 50:639-644). These systems
are therefore used mostly for local delivery of drugs to the
pulmonary airways. In addition, metered doses inhalers may not be
able to deliver sufficient volumes of even a concentrated midazolam
solution to produce the desired rapid antiseizure effect.
Accordingly, in various embodiments, a metered doses inhaler is not
used for delivery of the benzodiazepine, e.g., midazolam. In one
embodiment a nebulization system with the capability of delivering
<5 .mu.m particles (e.g., the PARI LC Star, which has a high
efficiency, 78% respirable fraction 0.1-5 .mu.m. see, e.g.,
pari.com) is used for intrapulmonary administration. Electronic
nebulizers which employ a vibrating mesh or aperture plate to
generate an aerosol with the required particle size can deliver
sufficient quantities rapidly and find use (See, e.g., Knoch and
Keller, 2005 Expert Opin Drug Deliv 2: 377-390). Also,
custom-designed hand-held, electronic nebulizers can be made and
find use.
[0071] Aerosolized delivery of the agents (e.g., one or more
benzodiazepines and one or more neurosteroids, optionally including
a NMDA receptor antagonist) can allow for reduced dosing to achieve
desired efficacy, e.g., in comparison to intravenous or intranasal
delivery.
[0072] In various embodiments, the agents (e.g., one or more
benzodiazepines and one or more neurosteroids, optionally including
a NMDA receptor antagonist) are dissolved or suspended in a
cyclodextrin. In varying embodiments, the cyclodextrin is an
.alpha.-cyclodextrin, a .beta.-cyclodextrin or a
.gamma.-cyclodextrin. In varying embodiments, the cyclodextrin is
selected from the group consisting of
hydroxypropyl-.beta.-cyclodextrin, endotoxin controlled
.beta.-cyclodextrin sulfobutyl ethers, or cyclodextrin sodium salts
(e.g., CAPTISOL.RTM.). Such formulations are useful for
intramuscular, intravenous and/or subcutaneous administration.
[0073] In various embodiments, the agents (e.g., one or more
benzodiazepines and one or more neurosteroids, optionally including
a NMDA receptor antagonist) are dissolved or suspended in an oil
that is edible and/or digestible by the subject, e.g., without
undesirable side effects.
[0074] In various embodiments, the edible oil comprises one or more
vegetable oils. In various embodiments, the vegetable oil is
selected from the group consisting of coconut oil, corn oil,
cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil,
canola oil, safflower oil, sesame oil, soybean oil, sunflower oil,
and mixtures thereof.
[0075] In some embodiments, the edible oil comprises one or more
nut oils. In some embodiments, the nut oil is selected from the
group consisting of almond oil, cashew oil, hazelnut oil, macadamia
oil, mongongo nut oil, pecan oil, pine nut oil, pistachio oil,
walnut oil, and mixtures thereof.
[0076] In some embodiments, the edible oil does not comprise castor
oil. In some embodiments, the edible oil does not comprise peanut
oil.
[0077] Generally, the oils used in the present compositions are
isolated from the source, e.g., plant, and used without including
further additives (e.g., surfactants, acids (organic or fatty),
alcohols, esters, co-solvents, solubilizers, lipids, polymers,
glycols) or processing. In various embodiments, the oil vehicle
further comprises a preservative (e.g., vitamin E).
[0078] The oil-agents (e.g., one or more benzodiazepines and one or
more neurosteroids, optionally including a NMDA receptor
antagonist) compositions can be formulated for oral and/or
transmucosal delivery using any method known in the art. In one
embodiment, the oil-agents (e.g., one or more benzodiazepines and
one or more neurosteroids, optionally including a NMDA receptor
antagonist) composition is formulated in a capsule, e.g., for oral
delivery.
[0079] a. Capsules
[0080] The capsule shells can be prepared using one or more film
forming polymers.
[0081] Suitable film forming polymers include natural polymers,
such as gelatin, and synthetic film forming polymers, such as
modified celluloses. Suitable modified celluloses include, but are
not limited to, hydroxypropyl methyl cellulose, methyl cellulose,
hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl
methyl cellulose phthalate, and cellulose acetate phthalate. Hard
or soft capsules can be used to administer the hormone. Hard shell
capsules are typically prepared by forming the two capsule halves,
filling one of the halves with the fill solution, and then sealing
the capsule halves together to form the finished capsule. Soft
gelatin capsules are typically prepared using a rotary die
encapsulation process as described below.
[0082] i. Gelatin Capsules
[0083] Gelatin is the product of the partial hydrolysis of
collagen. Gelatin is classified as either Type A or Type B gelatin.
Type A gelatin is derived from the acid hydrolysis of collagen
while Type B gelatin is derived from the alkaline hydrolysis of
collagen. Traditionally, bovine bones and skins have been used as
raw materials for manufacturing Type A and Type B gelatin while
porcine skins have been used extensively for manufacturing Type A
gelatin. In general, acid-processed gelatins form stronger gels
than lime-processed gelatins of the same average molecular weight.
The capsules can be formulated as hard or soft gelatin
capsules.
[0084] ii. Non-Gelatin Capsules
[0085] Capsules can be prepared from non-gelatin materials, such as
carrageenan or modified celluloses. Carrageenan is a natural
polysaccharide hydrocolloid, which is derived from seaweed. It
includes a linear carbohydrate polymer of repeating sugar units,
without a significant degree of substitution or branching. Most, if
not all, of the galactose units on a carrageenan molecule possess a
sulfate ester group. There are three main types of carrageenan:
kappa, iota and lambda; although minor forms called mu and nu
carrageenan also exist.
[0086] iii. Shell Additives
[0087] Suitable shell additives include plasticizers, opacifiers,
colorants, humectants, preservatives, flavorings, and buffering
salts and acids, and combinations thereof.
[0088] Plasticizers are chemical agents added to gelatin to make
the material softer and more flexible. Suitable plasticizers
include, but are not limited to, glycerin, sorbitol solutions which
are mixtures of sorbitol and sorbitan, and other polyhydric
alcohols such as propylene glycol and maltitol or combinations
thereof.
[0089] Opacifiers are used to opacify the capsule shell when the
encapsulated active agents are light sensitive. Suitable opacifiers
include titanium dioxide, zinc oxide, calcium carbonate and
combinations thereof.
[0090] Colorants can be used for marketing and product
identification/differentiation purposes. Suitable colorants include
synthetic and natural dyes and combinations thereof.
[0091] Humectants can be used to suppress the water activity of the
softgel. Suitable humectants include glycerin and sorbitol, which
are often components of the plasticizer composition. Due to the low
water activity of dried, properly stored softgels, the greatest
risk from microorganisms comes from molds and yeasts. For this
reason, preservatives can be incorporated into the capsule shell.
Suitable preservatives include alkyl esters of p-hydroxy benzoic
acid such as methyl, ethyl, propyl, butyl and heptyl esters
(collectively known as "parabens") or combinations thereof.
[0092] Flavorings can be used to mask unpleasant odors and tastes
of fill formulations. Suitable flavorings include synthetic and
natural flavorings. The use of flavorings can be problematic due to
the presence of aldehydes which can cross-link gelatin. As a
result, buffering salts and acids can be used in conjunction with
flavorings that contain aldehydes in order to inhibit cross-linking
of the gelatin.
[0093] b. Enteric Capsules
[0094] Alternatively, the liquid fills can be incorporated into an
enteric capsule, wherein the enteric polymer is a component of the
capsule shell, as described in WO 2004/030658 to Banner Pharmacaps,
Inc. The enteric capsule shell is prepared from a mass comprising a
film-forming polymer, an acid-insoluble polymer which is present in
an amount making the capsule resistant to the acid within the
stomach, an aqueous solvent, and optionally, one or more
plasticizers and/or colorants. Other suitable shell additives
including opacifiers, colorants, humectants, preservatives,
flavorings, and buffering salts and acids may be added.
[0095] i. Film-Forming Polymers
[0096] Exemplary film-forming polymers can be of natural or
synthetic origin. Natural film-forming polymers include gelatin and
gelatin-like polymers. Other suitable natural film-forming polymers
include shellac, alginates, pectin, and zeins. Synthetic
film-forming polymers include hydroxypropyl methyl cellulose,
methyl cellulose, hydroxypropyl methyl cellulose acetate succinate,
hydroxypropyl methyl cellulose phthalate, cellulose acetate
phthalate, and acrylates such as poly (meth)acrylate. The weight
ratio of acid-insoluble polymer to film-forming polymer is from
about 15% to about 50%. In one embodiment, the film forming polymer
is gelatin.
[0097] ii. Acid-Insoluble Polymers
[0098] Exemplary acid-insoluble polymers include cellulose acetate
phthalate, cellulose acetate butyrate, hydroxypropyl methyl
cellulose phthalate, algenic acid salts such as sodium or potassium
alginate, shellac, pectin, acrylic acid-methylacrylic acid
copolymers (available under the tradename EUDRAGIT.RTM. from Rohm
America Inc., Piscataway, N.J. as a powder or a 30% aqueous
dispersion; or under the tradename EASTACRYL.RTM., from Eastman
Chemical Co., Kingsport, Tenn., as a 30% dispersion). In one
embodiment, the acid-insoluble polymer is EUDRAGIT.RTM. L100, which
is a methacrylic acid/methacrylic acid methyl ester copolymer. The
acid-insoluble polymer is present in an amount from about 8% to
about 20% by weight of the wet gelatin mass. The weight ratio of
acid-insoluble polymer to film-forming polymer is from about 15% to
about 50%.
[0099] iii. Aqueous Solvent
[0100] Hard and soft capsules are typically prepared from solutions
or suspensions of the film forming polymer and the acid-insoluble
polymer. Suitable solvents include water, aqueous solvents, and
organic solvents. In one embodiment, the solvent is water or an
aqueous solvent. Exemplary aqueous solvents include water or
aqueous solutions of alkalis such as ammonia, sodium hydroxide,
potassium hydroxide, ethylene diamine, hydroxylamine, tri-ethanol
amine, or hydroalcoholic solutions of the same. The alkali can be
adjusted such that the final pH of the gelatin mass is less than or
equal to 9.0, preferably less than or equal to 8.5, more preferably
less than or equal to 8.0. In one embodiment, the alkali is a
volatile alkali such as ammonia or ethylene diamine. Upon drying of
the finished capsule, the water content of the capsule is from
about 2% to about 10% by weight of the capsule, preferably from
about 4% to about 8% by weight of the capsule.
[0101] iv. Plasticizers
[0102] Exemplary plasticizers include glycerol, glycerin, sorbitol,
polyethylene glycol, citric acid, citric acid esters such as
triethylcitrate, polyalcohols with 3-6 carbons and combinations
thereof. The plasticizer to polymer (film forming polymer plus
acid-insoluble polymer) ratio is from about 10% to about 50% of the
polymer weight.
[0103] c. Methods of Manufacture
[0104] i. Capsule Fill
[0105] The fill material is prepared by dissolving the steroid or
neurosteroid (e.g., allopregnanolone) in the carrier containing a
fatty acid solvent, such as oleic acid. The mixture of hormone and
fatty acid may be heated to facilitate dissolution of the hormone.
Upon cooling to room temperature and encapsulation, the solution
remains a liquid. The fill is typically deaerated prior to
encapsulation in a soft gelatin capsule. Additional excipients
including, but not limited to, co-solvents, antioxidants may be
added to the mixture of the hormone and fatty acid. Again the
mixture may be heated to facilitate dissolution of the excipients.
The steroid or neurosteroid (e.g., allopregnanolone) is fully
dissolved in the carrier of the present invention and remains so
upon storage.
[0106] ii. Capsule Shell
[0107] a. Gelatin or Non-Gelatin Capsules
[0108] The main ingredients of the capsule shell are gelatin (or a
gelatin substitute for non-gelatin capsules), plasticizer, and
purified water. The primary difference between soft and hard
capsules is the amount of plasticizer present in the capsule
shell.
[0109] Typical gel formulations contain (w/w) 40-50% gelatin,
20-30% plasticizer, and 30-40% purified water. Most of the water is
subsequently lost during capsule drying. The ingredients are
combined to form a molten gelatin mass using either a cold melt or
a hot melt process. The prepared gel masses are transferred to
preheated, temperature-controlled, jacketed holding tanks where the
gel mass is aged at 50-60.degree. C. until used for
encapsulation.
[0110] i. Cold Melt Process
[0111] The cold melt process involves mixing gelatin with
plasticizer and chilled water and then transferring the mixture to
a jacket-heated tank. Typically, gelatin is added to the
plasticizer at ambient temperature (18-22.degree. C.). The mixture
is cooked (57-95.degree. C.) under vacuum for 15-30 minutes to a
homogeneous, deaerated gel mass. Additional shell additives can be
added to the gel mass at any point during the gel manufacturing
process or they may be incorporated into the finished gel mass
using a high torque mixer.
[0112] ii. Hot Melt Process
[0113] The hot melt process involves adding, under mild agitation,
the gelatin to a preheated (60-80.degree. C.) mixture of
plasticizer and water and stirring the blend until complete melting
is achieved. While the hot melt process is faster than the cold
melt process, it is less accurately controlled and more susceptible
to foaming and dusting.
[0114] b. Soft Capsules
[0115] Soft capsules are typically produced using a rotary die
encapsulation process. The gel mass is fed either by gravity or
through positive displacement pumping to two heated (48-65.degree.
C.) metering devices. The metering devices control the flow of gel
into cooled (10-18.degree. C.), rotating casting drums. Ribbons are
formed as the cast gel masses set on contact with the surface of
the drums.
[0116] The ribbons are fed through a series of guide rolls and
between injection wedges and the capsule-forming dies. A food-grade
lubricant oil is applied onto the ribbons to reduce their tackiness
and facilitate their transfer. Suitable lubricants include mineral
oil, medium chain triglycerides, and soybean oil. Fill formulations
are fed into the encapsulation machine by gravity. In the preferred
embodiment, the soft capsules contain printing on the surface,
optionally identifying the encapsulated agent and/or dosage.
[0117] Upon drying of the finished capsule, the water content of
the capsule is from about 2% to about 10% by weight of the capsule,
preferably from about 4% to about 8% by weight of the capsule.
[0118] c. Enteric Capsules
[0119] A method of making an enteric capsule shell is described in
WO 2004/030658 to Banner Pharmacaps, Inc. The enteric mass is
typically manufactured by preparing an aqueous solution comprising
a film-forming, water soluble polymer and an acid-insoluble polymer
and mixing the solution with one or more appropriate plasticizers
to form a gelatin mass. Alternatively, the enteric mass can be
prepared by using a ready-made aqueous dispersion of the
acid-insoluble polymer by adding alkaline materials such as
ammonium, sodium, or potassium hydroxides or other alkalis that
will cause the acid-insoluble polymer to dissolve. The
plasticizer-wetted, film-forming polymer can then be mixed with the
solution of the acid-insoluble polymer. The mass can also be
prepared by dissolving the acid-insoluble polymer or polymers in
the form of salts of the above-mentioned bases or alkalis directly
in water and mixing the solution with the plasticizer-wetted,
film-forming polymer. The mass is cast into films or ribbons using
heat controlled drums or surfaces. The fill material is
encapsulated in a soft capsule using a rotary die. The capsules are
dried under controlled conditions of temperature and humidity. The
final moisture content of the shell composition is from about 2% to
about 10% by weight of the capsule shell, preferably from about 4%
to about 8% by weight by weight of the capsule shell.
[0120] Alternatively, release of the agents (e.g., one or more
benzodiazepines and one or more neurosteroids, optionally including
a NMDA receptor antagonist) from the capsule can be modified by
coating the capsule with one or more modified release coatings,
such as sustained release coatings, delayed release coatings, and
combinations thereof.
[0121] The concentration of the agents (e.g., one or more
benzodiazepines and one or more neurosteroids, optionally including
a NMDA receptor antagonist) in the vehicle (e.g., cyclodextrin
and/or edible oil) is preferably in unit dosage form. The term
"unit dosage form", as used in the specification, refers to
physically discrete units suitable as unitary dosages for human
subjects and animals, each unit containing a predetermined quantity
of active material calculated to produce the desired pharmaceutical
effect in association with the required pharmaceutical diluent,
carrier or vehicle. The specifications for the novel unit dosage
forms of this invention are dictated by and directly dependent on
(a) the unique characteristics of the active material and the
particular effect to be achieved and (b) the limitations inherent
in the art of compounding such an active material for use in humans
and animals, as disclosed in detail in this specification, these
being features of the present invention.
[0122] In various embodiments, the benzodiazepines are administered
at a dose that is less than about 10%, 15%, 25%, 50% or 75% of
established doses for their administration for the prevention or
mitigation of an epileptic seizure. In some embodiments, the
benzodiazepine is administered at a dose in the range of about 0.05
mg/kg to about 1.0 mg/kg, for example, about 0.2 mg/kg to about 0.8
mg/kg, for example, about 0.05 mg/kg, 0.08 mg/kg, 0.1 mg/kg, 0.2
mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8
mg/kg, 0.9 mg/kg, or 1.0 mg/kg. In some embodiments the
benzodiazepine is administered at a dose in the range of about 10
.mu.g/kg to about 80 .mu.g/kg, for example, about 20 .mu.g/kg to
about 60 .mu.g/kg, for example, about 25 .mu.g/kg to about 50
.mu.g/kg, for example, about 10 .mu.g/kg, 15 .mu.g/kg, 20 .mu.g/kg,
25 .mu.g/kg, 30 .mu.g/kg, 35 .mu.g/kg, 40 .mu.g/kg, 45 .mu.g/kg, 50
.mu.g/kg, 60 .mu.g/kg, 70 .mu.g/kg, or 80 .mu.g/kg. In some
embodiments, the benzodiazepine is administered at a dose in the
range of about 0.3 .mu.g/kg to about 3.0 .mu.g/kg. In varying
embodiments, the benzodiazepine is administered at a dose that does
not decrease blood pressure. When co-administered with one or more
neurosteroids, the benzodiazepine can be co-administered at a dose
that is less than about 10%, 15%, 25%, 50% or 75% of the
aforementioned doses or at a dose that is less than about 10%, 15%,
25%, 50% or 75% of established doses for their administration for
the prevention or mitigation of an epileptic seizure. When
co-administered with one or more neurosteroids, the benzodiazepine
can be co-administered at a dose that is less than about 10%, 15%,
25%, 50% or 75% of doses known to be efficacious via a selected
route of administration (e.g., oral, intramuscular, intravenous,
subcutaneous and/or intrapulmonary).
[0123] In various embodiments, the compositions are formulated for
administration of about 5 mg/kg to about 50 mg/kg of the steroid or
neurosteroid (e.g., allopregnanolone), e.g., about 5 mg/kg, 10
mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg,
45 mg/kg, or 50 mg/kg. When co-administered with one or more
benzodiazepines, the steroid or neurosteroid (e.g.,
allopregnanolone) can be co-administered at a dose that is less
than about 10%, 15%, 25%, 50% or 75% of the aforementioned doses or
at a dose that is less than about 10%, 15%, 25%, 50% or 75% of
established doses for their administration for the prevention or
mitigation of an epileptic seizure. When co-administered with one
or more benzodiazepines, the neurosteroid can be co-administered at
a dose that is less than about 10%, 15%, 25%, 50% or 75% of doses
known to be efficacious via a selected route of administration
(e.g., oral, intramuscular, intravenous, subcutaneous and/or
intrapulmonary).
6. Monitoring Efficacy
[0124] Co-administration of a benzodiazepine and a neurosteroid
(optionally with an NMDA receptor antagonist) to a subject results
in the prevention of the occurrence of an impending seizure and/or
the rapid termination or abortion of a seizure in progress.
[0125] In various embodiments, efficacy can be monitored by the
subject. For example, in a subject experiencing aura or receiving a
warning from a seizure prediction device, the subject can
self-administer via the intrapulmonary route a dose of the
benzodiazepine. If the benzodiazepine is administered in an
efficacious amount, the sensation of aura should subside and/or the
seizure prediction device should no longer predict the imminent
occurrence of an impending seizure. If the sensation of aura does
not subside and/or the seizure prediction device continues to
predict an impending seizure, a second dose of benzodiazepine can
be administered.
[0126] In other embodiments, the efficacy is monitored by a
caregiver. For example, in a subject experiencing the onset of a
seizure or in situations where a seizure has commenced, the subject
may require intrapulmonary administration of the benzodiazepine by
a caregiver. If the benzodiazepine is administered in an
efficacious amount, the seizure, along with the subject's symptoms
of the seizure, should rapidly terminate or abort. If the seizure
does not terminate, a second dose of the benzodiazepine can be
administered.
7. Kits
[0127] The pharmaceutical compositions and neurosteroid and
benzodiazepine combinations can be provided in a kit. In certain
embodiments, a kit of the present invention comprises one or more
benzodiazepines and one or more neurosteroids in separate
formulations. In varying embodiments, one or both of the
benzodiazepine and the neurosteroid are provided in subtherapeutic
doses or amounts. In certain embodiments, the kits comprise one or
more benzodiazepines and one or more neurosteroids within the same
formulation. In varying embodiments, one or both of the
benzodiazepine and the neurosteroid are provided in subtherapeutic
doses or amounts. In certain embodiments, the kits provide the one
or more benzodiazepines and one or more neurosteroids independently
in uniform dosage formulations throughout the course of treatment.
In varying embodiments, one or both of the benzodiazepine and the
neurosteroid are provided in subtherapeutic doses or amounts. In
certain embodiments, the kits provide the one or more
benzodiazepines and one or more neurosteroids in graduated dosages
over the course of treatment, either increasing or decreasing, but
usually increasing to an efficacious dosage level, according to the
requirements of an individual. In varying embodiments, one or both
of the benzodiazepine and the neurosteroid are provided in
subtherapeutic doses or amounts.
[0128] In some embodiments, the benzodiazepine is selected from the
group consisting of bretazenil, clonazepam, cloxazolam,
clorazepate, diazepam, fludiazepam, flutoprazepam, lorazepam,
midazolam, nimetazepam, nitrazepam, phenazepam, temazepam and
clobazam. In some embodiments, the benzodiazepine is selected from
the group consisting of midazolam, lorazepam and diazepam. In some
embodiments, the neurosteroid is selected from the group consisting
of allopregnanolone, allotetrahydrodeoxycorticosterone, ganaxolone,
alphaxolone, alphadolone, hydroxydione, minaxolone, and Althesin.
In some embodiments, the neurosteroid is allopregnanolone. In some
embodiments, the kit comprises allopregnanolone and a
benzodiazepine selected from the group consisting of midazolam,
lorazepam, and diazepam.
[0129] In some embodiments, the kits further comprise a NMDA
receptor antagonist. In some embodiments, the NMDA receptor
antagonist is selected from the group consisting of dizocilpine
(MK-801), meperidine, methadone, dextropropoxyphene, tramadol,
ketobemidone, ketamine, dextromethorphan, phencyclidine, nitrous
oxide (N.sub.2O), AP5 (R-2-amino-5-phosphonopentanoate), AP7
(2-amino-7-phosphonoheptanoic acid), CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,
eticyclidine, gacyclidine, ibogaine, magnesium, memantine,
methoxetamine, rolicyclidine.tenocyclidine, methoxydine,
tiletamine, xenon, neramexane, eliprodil, etoxadrol, dexoxadrol,
WMS 2539, NEFA, remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211,
rhynchophylline, 1-Aminocyclopropanecarboxylic acid (ACPC),
7-Chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenic
acid, lacosamide, CP-101,606 (traxoprodil), AZD6765 (lanicemine)
and GLYX-13. In some embodiments, the NMDA receptor antagonist is
selected from the group consisting of ketamine, dextromethorphan,
phencyclidine, CPPene
(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),
selfotel, amantadine, dextrorphan, memantine, tiletamine,
neramexane, eliprodil, remacemide, aptiganel,
1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA
(5,7-dichlorokynurenic acid), kynurenic acid, CP-101,606
(traxoprodil), AZD6765 (lanicemine) and GLYX-13. In some
embodiments, the NMDA receptor antagonist is dizocilpine
(MK-801).
[0130] In some embodiments, one or both of the benzodiazepine and
the neurosteroid is formulated for inhalational, intranasal or
intrapulmonary administration. In some embodiments, one or both of
the benzodiazepine and the neurosteroid is formulated for oral or
parenteral delivery. In some embodiments, one or both of the
benzodiazepine and the neurosteroid are formulated for a parenteral
route selected from the group consisting of inhalational,
intrapulmonary, intranasal, intramuscular, subcutaneous,
transmucosal and intravenous. In some embodiments, the
benzodiazepine is an agonist of the benzodiazepine recognition site
on GABA.sub.A receptors and stimulates endogenous neurosteroid
synthesis. In some embodiments, the neurosteroid is suspended or
dissolved in a cyclodextrin (e.g., an .alpha.-cyclodextrin, a
.beta.-cyclodextrin or a .gamma.-cyclodextrin). In varying
embodiments, the neurosteroid is suspended or dissolved in a
cyclodextrin selected from the group consisting of
hydroxypropyl-.beta.-cyclodextrin, endotoxin controlled
.beta.-cyclodextrin sulfobutyl ethers, or cyclodextrin sodium salts
(e.g., CAPTISOL.RTM.). In some embodiments, the neurosteroid is
suspended or dissolved in an edible oil. In some embodiments, the
edible oil comprises one or more vegetable oils. In some
embodiments, the vegetable oil is selected from the group
consisting of coconut oil, corn oil, cottonseed oil, olive oil,
palm oil, peanut oil, rapeseed oil, canola oil, safflower oil,
sesame oil, soybean oil, sunflower oil, and mixtures thereof. In
some embodiments, the edible oil is canola oil. In some
embodiments, the edible oil comprises one or more nut oils. In some
embodiments, the nut oil is selected from the group consisting of
almond oil, cashew oil, hazelnut oil, macadamia oil, mongongo nut
oil, pecan oil, pine nut oil, pistachio oil, walnut oil, and
mixtures thereof.
EXAMPLES
[0131] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
Tetramethylenedisulfotetramine Alters Ca2+ Dynamics in Cultured
Hippocampal Neurons: Mitigation by NMDA Blockade and GABA.sub.A
Receptor Positive Modulation
Materials and Methods
Materials
[0132] Fetal bovine serum and soybean trypsin inhibitor were
obtained from Atlanta Biologicals (Norcross, Ga.). DNase,
poly-L-lysine, cytosine arabinoside,
(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine
maleate (MK-801), Hydroxypropyl-.beta.-cyclodextran, and
(3,5-dimethyl
2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate
(nifedipine) were from Sigma-Aldrich (St. Louis, Mo.). The Ca2+
fluorescence dye Fluo-4, Pluronic F-127 and Neurobasal medium were
purchased from Life Technology (Grand Island, N.Y.).
Tetramethylenedisulfotetramine (TETS) was synthesized as described
previously (Zolkowska et al., 2012). Diazepam was from Western
Medical Supply (Arcadia, Calif.). Allopregnanolone
(3.alpha.-hydroxy-5.alpha.-pregnan-20-one; >99%) was provided by
M. A. Rogawski.
[0133] Primary Cultures of Hippocampal Neurons.
[0134] Animals were treated humanely and with regard for
alleviation of suffering according to protocols approved by the
Institutional Animal Care and Use Committee of the University of
California, Davis. Hippocampal neuron cultures were dissociated
from hippocampi dissected from C57BL/6J mouse pups at postnatal day
0-1 and maintained in Neurobasal complete medium [Neurobasal medium
supplemented with NS21, 0.5 mM L-glutamine, HEPES] with 5% fetal
bovine serum. For Ca2+ imaging studies using FLIPR, dissociated
hippocampal cells were plated onto poly-L-lysine coated
clear-bottom, black wall, 96-well imaging plate (BD, Franklin
Lakes, N.J., USA) at a density of 0.8.times.10.sup.5/well. For
microelectrode array (MEA) experiments, 120 .mu.l of cell
suspension at a density of 1.5.times.10.sup.6 cells/ml were added
to a 12-well Maestro plate (Axion BioSystems, Atlanta, Ga.). After
2 h incubation, a volume of 1.0 ml of serum-free Neurobasal
complete medium was added to each well. The medium was changed
twice a week by replacing half volume of culture medium with
serum-free Neurobasal complete medium. The neurons were maintained
at 37.degree. C. with 5% CO2 and 95% humidity.
[0135] Measurement of Synchronous Intracellular Ca2+
Oscillations.
[0136] Hippocampal neurons between 13-17 days in vitro (DIV) were
used to investigate how TETS alters synchronous Ca2+ oscillations
that normally occur in healthy neurons at this developmental stage.
This method permits simultaneous measurements of intracellular Ca2+
transients in a 96-well format as described as previously (Cao et
al., 2010). Baseline recording were acquired in Locke's buffer (8.6
mM HEPES, 5.6 mM KCl, 154 mM NaCl, 5.6 mM glucose, 1.0 mM
MgCl.sub.2, 2.3 mM CaCl.sub.2, and 0.0001 mM glycine, pH 7.4) for
10 min followed by addition of TETS and/or pharmacological agents
using a programmable 96-channel pipetting robotic system, and the
intracellular Ca2+ was monitored for an additional 30 min. Unless
otherwise indicated, pharmacological interventions were introduced
10 min prior to TETS. TETS triggered an immediate rise in [Ca2+]i
that was analyzed by quantifying the Area Under the Curve (AUC; in
arbitrary fluorescence units) of the Fluo-4 fluorescence units for
a duration of 5 min following TETS addition. TETS also altered the
frequency and amplitude of neuronal synchronous Ca2+ oscillations,
which were analyzed during the 10 min period after addition of
TETS.
[0137] MEA Recording.
[0138] All MEA recordings were conducted at 37.degree. C. in
culture medium without perfusion using a 12-well Maestro system
(Axion BioSystems, Atlanta, Ga.). Each well contains 64 electrodes
(30 .mu.m diameter) in an 8.times.8 grid with inter-electrode
spacing of 200 .mu.m. Before recording basal electrical activity,
the cultures were equilibrated in freshly prepared, pre-warmed
neurobasal complete medium for 1 h. The 12-well Maestro plates were
loaded onto a temperature regulated headstage containing the
recording amplifier and raw extracellular electrical signals were
acquired using Axis software (Axion BioSystems, Atlanta, Ga.).
Signals from the amplifier were digitized at a rate of 25 KHz, and
filtered using Butterworth Band-pass filter (cutoff frequency of
300 Hz). The Axis software was used to detect spontaneous events
that exceeded a threshold of six times of the noise. Raster plot
and spike rate analysis data were performed by exporting the raw
data to the NeuroExplorer software (version 4.0, NEX Technologies,
Littleton, Mass.).
[0139] Data Analysis. Graphing and statistical analysis were
performed using GraphPad Prism software (Version 5.0, GraphPad
Software Inc., San Diego, Calif.). EC50 values were determined by
non-linear regression using a three-parameter logistic equation.
Statistical significance between different groups was calculated
using Student's t-test or by an ANOVA and, where appropriate, a
Dunnett's Multiple Comparison Test; p values below 0.05 were
considered statistically significant.
Results
[0140] Effects of TETS on Ca2+ Oscillations in Primary Cultured
Hippocampal Neurons
[0141] Cultured hippocampal neurons (13-17 DIV) exhibit spontaneous
synchronous Ca2+ oscillations whose frequency and amplitude can be
quantitatively assessed in real time using FLIPR.RTM. (FIG. 1A).
Addition of DMSO vehicle had no significant effect on the
properties of the synchronous Ca2+ oscillations during the 5 min
Phase I period or the 10 min Phase II period (FIG. 1A, top trace).
By contrast, exposure of the neurons to TETS caused an immediate
increase in the amplitude of the oscillations and at higher
concentrations (3 and 10 .mu.M) a sustained plateau response that
decayed slowly over the 5 min Phase I period. The integrated Ca2+
signal (area under the curve; AUC) during the Phase I period
exhibited a concentration-dependent increase, with an EC50 value of
2.7 .mu.M [95% confidence interval (95% CI): 1.4-5.2 .mu.M] (FIG.
1B). During Phase II, TETS caused a concentration-dependent
decrease in the frequency of the synchronous Ca2+ oscillations with
an EC50 value of 1.7 .mu.M (95% CI: 0.69-4.12 .mu.M; FIG. 1C).
Along with the reduction in frequency, TETS increased the mean Ca2+
oscillation amplitude with an EC50 value of 1.8 .mu.M (95% CI:
1.12-2.80 .mu.M; FIG. 1D). TETS modestly prolonged the mean
duration of individual Ca2+ transients compared to that measured
from vehicle-exposed control neurons. TETS-induced phase II Ca2+
responses (both frequency and amplitude) were reversible (FIG.
2)
[0142] For comparison, we studied the influence of picrotoxin (PTX;
100 .mu.M), a noncompetitive blocker of GABA.sub.A receptor and
bicuculline (100 .mu.M), a competitive antagonist of GABA.sub.A
receptors on the Ca2+ dynamics. Both antagonists elicited similar
Phase I and Phase II responses as TETS (FIG. 3).
[0143] TETS Enhances Neuronal Electric Network Activity in Primary
Cultured Hippocampal Neurons.
[0144] Extracellular recordings of electrical activity from
multiple sites within the neuronal cultures at a high spatial
resolution provide a robust measure of network activity and
connectivity (Johnstone et al., 2010). After recording the basal
electrical activity for 10 min, increasing concentrations of TETS
were serially introduced into the wells. The recording was
continued for 10 min at each TETS concentration. A control well was
simultaneously recorded following introduction of DMSO vehicle
(0.01-0.1%). Basal recordings for up to 60 min showed that network
firing activity was stable in the absence or presence of DMSO
vehicle control (FIG. 4A, left panel). Exposure to TETS
concentrations of 2 .mu.M and greater produced a dramatic change in
discharge pattern. Events became more highly clustered (FIG. 4A,
right panel and FIG. 5) and the duration of clustered bursts
induced by 6 .mu.M TETS can last up to 10 s (FIG. 4A, right panel,
4th row). There was an overall increase in the discharge rate (FIG.
4B,). After washout of TETS, the neuronal network firing recovered
to basal conditions.
[0145] NMDA Receptors, but not L-Type Ca2+ Channels are Required
for TETS-Induced Ca2+ Dysregulation.
[0146] We next examined the possible involvement of NMDA receptors
and L-type Ca2+ channels in the effects of TETS on Ca2+ dynamics.
Preincubation of neuronal cultures for 10 min with MK-801 (1
.mu.M), an NMDA receptor blocker, attenuated both Phase I and Phase
II effects of TETS (FIG. 6B-D). MK-801 slightly suppressed basal
Ca2+ oscillations, which is consistent with an earlier report
(Tanaka et al., 1996). By contrast, nifedipine (1 .mu.M), which
inhibits L-type voltage activated Ca2+ channels, was without effect
on TETS-induced Phase I or Phase II Ca2+ responses (FIG. 6B-D).
These results indicate that NMDA receptors but not L-type Ca2+
channels are required for the effects of TETS on Ca2+
fluctuations.
[0147] Diazepam and Allopregnanolone Partially Mitigate
TETS-Induced Ca2+Dysregulation.
[0148] We next determined if the GABA.sub.A receptor positive
modulators diazepam and allopregnanolone could protect against
TETS-induced Ca2+ dysregulation. FIG. 7A (top trace) demonstrates
that the oscillatory activity of neurons exposed to vehicle
remained stable over the entire recording period. Introduction of
diazepam (0.1, 0.3, or 1 .mu.M) caused an attenuation in the
amplitude of basal spontaneous Ca2+ oscillations (FIG. 7A).
Pre-exposure to diazepam caused a small concentration-dependent
reduction of the Phase I integrated rise in Ca2+ induced by TETS
that reached statistical significance only at 1 .mu.M (FIG. 7B).
Diazepam did not eliminate the Phase I plateau response (FIG. 7A).
Diazepam also caused a partial inhibition of the Phase II frequency
and amplitude effects of TETS, with the effect on amplitude
reaching significance at 0.1 .mu.M (FIG. 7C, D).
[0149] As shown in FIG. 8, allopregnanolone similarly attenuated
the effects of TETS on Ca2+ dysregulation. Allopregnanolone (0.1-1
.mu.M) caused a concentration-dependent suppression of basal
spontaneous Ca2+ fluctuations and it partially attenuated the
response in Phase I at 1 .mu.M without eliminating the plateau in
Ca2+ levels (FIG. 8A, B). Allopregnanolone at 0.3 and 1 .mu.M also
inhibited the Phase II effect of TETS on frequency and amplitude
with a completely reversed Phase II effect on amplitude at 1 .mu.M
(FIG. 8C,D).
[0150] Low Concentrations of Diazepam and Allopregnanolone in
Combination Mitigate TETS-Induced Ca2+ Dysregulation.
[0151] We next evaluated the effect of a combination of diazepam
and allopregnanolone, each at a low concentration (0.1 .mu.M) that
by itself has minimal effects on Phase I or Phase II Ca2+
dysregulation. As shown in FIG. 9, the combination they strongly
mitigated both Phase I and Phase II effects. In fact, the
combination treatment was able to largely eliminate the plateau
response obtained with acute TETS exposure (FIG. 9A), an effect not
obtained with 10-fold higher concentrations of diazepam (FIG. 7) or
allopregnanolone (FIG. 8) alone.
Discussion
[0152] In the present study, we characterized the effects of TETS
on hippocampal neurons in culture using MEA field potential
recording and Fluo-4 fluorescence measurements of Ca2+ dynamics in
the neuronal network. Over time, hippocampal neurons in culture
develop a rich network of processes and form numerous functional
synaptic contacts (Mennerick et al., 1995; Arnold et al., 2005).
Cultures that have developed for 13-17 DIV as used in the present
study are well organized and there is robust spontaneous electrical
activity mediated by excitatory and inhibitory transmission between
neurons. Neurons within the cultures exhibit spontaneous action
potentials and cultures of sufficient cell density may show
synchronized bursting of neurons throughout the entire culture
(Arnold et al., 2005). Excitatory synaptic transmission is mediated
by functional glutamate receptors of the NMDA and AMPA types (Abele
et al., 1990). Importantly, the cultures contain GABAergic neurons,
comprising approximately 10 percent of the neurons, that form
robust inhibitory synaptic connections mediated by GABA.sub.A
receptors (Jensen et al., 1999; Jensen et al., 2000). Inhibitory
synaptic potentials in hippocampal cultures have physiological
properties that are similar to those obtained in intact
preparations (Jensen et al., 1999). The GABAergic neurons impose
tonic inhibition onto the network so that exposure of hippocampal
cultures to GABA.sub.A receptor antagonists causes increased action
potential firing, spontaneous rhythmic neuronal depolarizations,
and bursting. The rhythmic depolarizations and bursting is
dependent upon action potentials as it is eliminated by
tetrodotoxin.
[0153] MEA recording allow the electrical activity of multiple
neurons within the cultures to be monitored whereas FLIPR.RTM.
Fluo-4 fluorescence measurements provide a dynamic assessment of
aggregate intracellular Ca2+ levels (Cao et al., 2010; Cao et al.,
2012). Using these assays, we found that TETS dramatically
increases intracellular Ca2+ levels and alters Ca2+ dynamics,
initially causing an transient increase on the intracellular Ca2+
concentration ([Ca2+]i) followed by a decrease on the Ca2+
oscillations having bigger amplitude. Assessment of ongoing
electric activity in the cultures with MEA recording showed an
overall increase in discharge frequency and a change in the pattern
of the discharges to clustering followed by periods of electrical
silence. The actions of TETS on neuronal Ca2+ dynamics and
electrical discharge activity occur within the same concentration
range, suggesting the two effects are mechanistically linked.
TETS-induced changes on Ca2+ dynamics and on electrical discharges
are similar to those observed with the GABA.sub.A receptor
antagonists bicuculline or picrotoxin (Arnold et al., 2005; Cao et
al., 2012). Additionally, TETS modulation of Ca2+ dynamics and
spontaneous neuronal firing activity in a concentration-dependent
manner with EC50 values of approximately 1-2 .mu.M which is
consistent with the affinity of TETS for GABA.sub.A receptors
(Bowery et al., 1975; Dray, 1975; Roberts et al., 1981).
Collectively these data support the view that the GABA.sub.A
receptor blocking activity of TETS is responsible for the effects.
Like picrotoxin, TETS is believed to be a reversible inhibitor of
GABA.sub.A receptors, which is also consistent with the rapid
reversibility of its effects in the MEA assay.
[0154] TETS-triggered alterations in electric firing and
synchronous Ca2+ oscillations appear to rely on spontaneous action
potentials since they are prevented by tetrodotoxin block of Na+
channels. The neuronal specificity of TETS in producing both Phase
I and Phase II Ca2+ responses in hippocampal cultures is also
indicated by the observations that addition of TETS up to 3 .mu.M
to the culture medium of skeletal myotubes alters neither basal
Ca2+ homeostasis nor electrically evoked Ca2+ transients (i.e.,
excitation-contraction coupling).
[0155] A key observation in the present study is that the
alterations in Ca2+ dynamics induced by TETS was largely inhibited
by MK-801 demonstrating that NMDA receptors are required. While
direct activation of NMDA receptors by TETS is not excluded,
activation of NMDA receptors by bath application of NMDA increase
the neuronal firing in a evenly distributed pattern which is not
similar to the clustered bursts firing elicited by TETS or other
GABA.sub.A receptors blocker/antagonist such as picrotoxin (Cao et
al., 2012). The NMDA receptor dependence for TETS response to the
Ca2+ is consistent with earlier evidences in vivo that the NMDA
receptor antagonist MK-801 inhibits picrotoxin or
bicuculline-induced convulsion in mice (Obara, 1995; Czlonkowska et
al., 2000) and ex vivo that the NMDA antagonist 2-APV suppresses
picrotoxin-induced Ca2+ responses as well as the frequency and
duration of the epileptiform discharges in hippocampal slice
preparation (Kohr and Heinemann, 1989). How the suppression of
GABA.sub.A receptors activity by TETS affects NMDA receptor
functions remains to be established. One possibility is that the
Phase I [Ca2+]i response may involve presynaptic glutamate
transmission. In support, bicuculline-induced [Ca2+]i responses
have been shown to involve synaptic but not-extra-synaptic NMDA
receptor activation (Hardingham et al., 2001; Hardingham et al.,
2002). While the relationship between the Ca2+ signals in the rapid
throughput FLIPR assay and epileptic activity remain to be
determined, our observation that NMDA receptors are required for
the TETS-induced changes in Ca2+ dynamics supports the concept that
the effects on Ca2+ are a surrogate for epileptic activity and may
be useful as a model for therapeutics discovery. This is further
supported by our demonstration that GABA.sub.A receptors positive
allosterical enhancer, diazepam or allopregnanolone partially
suppress TETS-induced modulation of Ca2+ dynamics.
[0156] Consistent with the role of GABA.sub.A receptors in
restraining bursting and altered Ca2+ dynamics is our observation
that the GABA.sub.A receptor positive modulators diazepam and
allopregnanolone are able to protect against the effects of TETS on
Ca2+ dynamics. Allopregnanolone was more effective on mitigation of
Phase I response induced by TETS than diazepam. This is consistent
with the fact that diazepam only acts on synaptic GABA.sub.A
receptors, whereas neurosteroids such as allopregnanolone can
enhance both extrasynaptic synaptic GABA.sub.A receptors (Kokate et
al., 1994; Lambert et al., 2003; Reddy and Rogawski, 2012).
However, neither diazepam nor allopregnanolone alone was fully
effective, even at the highest concentrations tested (1 .mu.M).
Unexpectedly, we found that the combination of diazepam and
allopregnanolone, each at a threshold concentration of 0.1 .mu.M,
was highly effective at protecting against the effects of TETS on
Ca2+ dynamics, causing a nearly complete inhibition of the Phase I
response, including the plateau in Ca2+, as well as the Phase II
changes. The combination of a benzodiazepine and a neurosteroid has
not to our knowledge previously been studied in a simplified
functional system. It is well recognized that benzodiazepines such
as diazepam only act on synaptic GABA.sub.A receptors, whereas
neurosteroids such as allopregnanolone preferentially enhance
extrasynaptic GABA.sub.A receptors although they also act on
synaptic receptors as well (Kokate et al., 1994; Lambert et al.,
2003; Reddy and Rogawski, 2012). Without being bound to theory, it
appears that the combined action on synaptic and extrasynaptic
receptors accounts for the unique potency of the drug
combination.
[0157] Alternatively, there may be an interaction at the level of
individual GABA.sub.A receptors. The recognition sites for
neurosteroids on GABA.sub.A receptors are distinct from those that
recognize benzodiazepines and barbiturates (Johnston, 1996). It is
conceivable, however, that allopregnanolone and diazepam could
produce a synergistic enhancement of GABA.sub.A receptors in a
similar fashion as the synergism that occurs between barbiturates
and benzodiazepines, where there is known to be allosteric coupling
(DeLorey et al., 1993).
[0158] In summary, we have developed rapid throughput methods to
detect TETS-induced Ca2+ dysregulation and altered electrical
activity in cultured hippocampal neurons. We demonstrated that two
GABA.sub.A receptors allosteric modulators, allopregnanolone and
diazepam, when introduced singly prior to TETS, mitigate
TETS-induced Ca2+ dysregulation, demonstrating that the in vitro
methods described here have translational value to identify new
therapies and optimize combinatorial strategies for the prevention
of TETS poisoning. The basic approaches described here are of
general utility for investigating chemically diverse threat agents
that elicit changes in the electrical behavior or Ca2+ dynamics of
in vitro neuronal networks. These rapid throughput approaches are
useful for identifying novel targeted interventions and for
optimizing therapeutic strategies involving drug combinations.
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Example 2
Combination Treatment with a Benzodiazepine and a Neurosteroid
Mitigates the Severity and Prevents the Lethality of Seizures Even
when Administering after Seizure has Started
[0212] In cultured hippocampal neurons, higher concentration of
TETS (>3 .mu.M) produces an acute elevation of intracellular
Ca2+ levels (Phase I response) and a prolonged Ca2+ response with
increased Ca2+ oscillation amplitude and decreased frequency of the
Ca2+ oscillations (Phase II response). Both Phase I and Phase II
response can be mitigated by the pretreatment of diazepam and
allopregnanolone. More importantly, pre-treatment with a
combination of low concentrations of diazepam and allopregnanolone,
which have minimal effect against TETS-induced Ca2+ response,
normalized the TETS Ca2+ response to the control level (Cao et al.,
Toxicological Sciences, 130: 362-372). In this study, we examined
whether post-treatment (after TETS triggers Phase I and Phase II
Ca2+ responses) of neurons with diazepam and/or allopregnanolone
mitigate alterations triggered by TETS, which is more relevant to
the TETS poising. Since addition of TETS induces acute phase I
response, we therefore only focused on the TETS-induced Phase II
response. Addition of vehicle (0.1% DMSO) was no effect on the Ca2+
dynamics over the recording period of 45 min. However, a
concentration of 3 .mu.M of TETS produced an acute Phase I and a
prolonged Phase II effect, as previously reported (Cao et al 2012).
While addition of diazepam (0.1 .mu.M) or allopregnanolone (0.1
.mu.M) singly was without significant effect on TETS-induced
decreased Ca2+ oscillation frequency, diazepam (0.1 .mu.M) and
allopregnanolone (0.1 .mu.M) in combination effectively recovered
synchronous Ca2+ oscillations characteristics comparable to those
observed with vehicle-treated cultures. Although allopregnanolone
(0.1 .mu.M) alone decreased the TETS-induced Ca2+ oscillation
amplitude .about.20% (p<0.01), the post-TETS treatment with
diazepam (0.1 .mu.M) and allopregnanolone (0.1 .mu.M) in
combination conferred much greater recovery of Ca2+ oscillation
amplitude to that below vehicle control, and occurred rapidly after
the addition of diazepam and allopregnanolone (FIG. 10). These data
clearly demonstrate that diazepam in combination with a
neurosteroid, such as allopregnanolone, act in a synergistic manner
to mitigate the severity of seizures and prevent the lethality of
seizurogenic agents AFTER the seizures have already started.
Example 3
In Vivo Assay Demonstrating the Therapeutic Efficacy of Combined
Benzodiazepine and Neurosteroid in Mitigating TETS-Induced Seizures
and Death
[0213] When administered to adult male NIH Swiss mice at lethal
doses, TETS typically causes two clonic seizures within the first
20 minutes after TETS injection with each seizure lasting
approximately 30 to 45 seconds. These clonic seizures are followed
by a tonic seizure that results in the death of >95% of the
TETS-intoxicated animals (FIG. 11). Mice can be rescued from
TETS-induced death if they are administered a very high dose of
diazepam (5 mg/kg, i.p.) immediately following the second clonic
seizure (FIG. 11). Administration of diazepam at 0.03 mg/kg
immediately following the second clonic seizure protected <10%
of the TETS-intoxicated animals from death (FIG. 12). Pretreatment
with diazepam (0.1 mg/kg 10 minutes before TETS injection)
protected <30% of the TETS-intoxicated animals from death (FIG.
13). Post-administration of the neurosteroid allopregnanolone at
0.03 mg/kg was no more efficacious than low dose diazepam in
protecting TETS-intoxicated animals from death (FIG. 12).
Pretreatment with allopregnanolone at 0.1 mg/kg protected
.about.50% of the TETS-intoxicated animals (FIG. 13). When
administered simultaneously, the subthreshold doses of diazepam and
allopregnanolone significantly increased survival of
TETS-intoxicated animals. When administered immediately after the
second clonic seizure, this benzodiazepine and neurosteroid
combination, 100% of the TETS-intoxicated animals survived (FIG.
12). Used as a pretreatment, this combinatorial therapy protected
.about.75% of the TETS-intoxicated animals (FIG. 13). Importantly,
the therapeutic combination of subthreshold diazepam and
allopregnanolone had no effect on blood pressure, whereas the dose
of diazepam required to prevent TETS-induced tonic seizures and
death when administered singly caused significant hypotension (FIG.
14).
[0214] The significance of these in vivo data are 3-fold: (1) these
data confirm the predictive value of the in vitro screening system;
(2) these data demonstrate that combinatorial therapy with a
benzodiazepine and a neurosteroid, at subthreshold doses that
singly have no effect, is efficacious in preventing seizures and in
the case of TETS, in preventing death associated with tonic
seizures; and (3) the combinatorial therapy avoids an important
off-target adverse effect (significant decrease in blood pressure)
associated with use of diazepam when used singly at therapeutic
doses.
[0215] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
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