U.S. patent application number 17/014220 was filed with the patent office on 2021-07-29 for compositions and methods for treating refractory seizures.
The applicant listed for this patent is THE JOHNS HOPKINS UNIVERSITY, KENNEDY KRIEGER INSTITUTE, INC.. Invention is credited to Shilpa D. Kadam.
Application Number | 20210228518 17/014220 |
Document ID | / |
Family ID | 1000005523289 |
Filed Date | 2021-07-29 |
United States Patent
Application |
20210228518 |
Kind Code |
A1 |
Kadam; Shilpa D. |
July 29, 2021 |
COMPOSITIONS AND METHODS FOR TREATING REFRACTORY SEIZURES
Abstract
The present invention relates to the field of seizures. More
specifically, the present invention provides compositions and
methods for treating refractory seizures in neonates. In one
embodiment, the method comprises the steps of (a) administering to
the patient an amount of a KCC2 agonist and/or trkB antagonist
effective to restore KCC2 expression to normal physiological
levels; and (b) administering to the patient an effective amount of
an anti-seizure medication.
Inventors: |
Kadam; Shilpa D.;
(Pikesville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE JOHNS HOPKINS UNIVERSITY
KENNEDY KRIEGER INSTITUTE, INC. |
Baltimore
Baltimore |
MD
MD |
US
US |
|
|
Family ID: |
1000005523289 |
Appl. No.: |
17/014220 |
Filed: |
September 8, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16734801 |
Jan 6, 2020 |
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17014220 |
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15504141 |
Feb 15, 2017 |
10525024 |
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PCT/US2015/045170 |
Aug 14, 2015 |
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16734801 |
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62037654 |
Aug 15, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01K 2207/30 20130101;
A61K 45/06 20130101; A61K 31/196 20130101; A61K 31/55 20130101;
A01K 2267/0356 20130101; A61K 31/553 20130101; A01K 2207/20
20130101; A61K 31/4166 20130101; A61K 31/4015 20130101; A61K 31/501
20130101; A61K 31/515 20130101 |
International
Class: |
A61K 31/196 20060101
A61K031/196; A61K 31/4166 20060101 A61K031/4166; A61K 31/515
20060101 A61K031/515; A61K 31/501 20060101 A61K031/501; A61K
31/4015 20060101 A61K031/4015; A61K 31/55 20060101 A61K031/55; A61K
31/553 20060101 A61K031/553; A61K 45/06 20060101 A61K045/06 |
Goverment Interests
STATEMENT OF GOVERNMENTAL INTEREST
[0002] This invention was made with government support under grant
no. R21HD073105, awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method for treating refractory seizures in a neonatal patient
comprising the steps of: a. administering to the patient an amount
of a KCC2 agonist and/or trkB antagonist effective to restore KCC2
expression to normal physiological levels; and b. administering to
the patient an effective amount of an anti-seizure medication.
2. The method of claim 1, wherein the seizure is an
ischemia-related seizure.
3. The method of claim 1, wherein the KCC2 agonist comprises
N-ethylmaleimide (NEM), the chloride channel inhibitor
5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), CLP257, or
CLP290.
4. The method of claim 1, wherein the trkB antagonist comprises
ANA-12, N-T19, K252a or cyclotraxin-B.
5. The method of claim 1, wherein the KCC2 agonist is CLP290.
6. The method of claim 1, wherein the trkB antagonist is
ANA-12.
7. The method of claim 1, wherein the anti-seizure medication is
phenobarbital or phenytoin.
8. A method for treating refractory seizures in a neonatal patient
comprising the steps of: a. administering to the patient an amount
of ANA-12 and/or CLP290 effective to restore KCC2 expression to
normal physiological levels; and b. administering to the patient an
effective amount of an anti-seizure medication.
9. The method of claim 8, wherein the seizure is an
ischemia-related seizure.
10. The method of claim 8, wherein the anti-seizure medication is
phenobarbital or phenytoin.
11. A method for treating refractory seizures in a neonatal patient
comprising the steps of: a. administering to the patient an amount
of ANA-12 and/or CLP290 effective to restore KCC2 expression to
normal physiological levels; and b. administering to the patient an
effective amount of phenobarbital.
12. A method for treating refractory seizures in a patient
comprising the steps of: a. administering to the patient an
effective amount of an agent that increases KCC2 expression back to
normal levels, wherein the seizure downregulates KCC2 expression;
and b. administering to the patient an effective amount of an
anti-seizure medication.
13. The method of claim 12, wherein the patient is a neonate.
14. The method of claim 13, wherein the neonate is administered a
transient dose of an anti-seizure medication.
15. A method for treating ischemia-related seizures in a neonatal
patient comprising the steps of: a. administering a KCC2 agonist
and/or a trkB antagonist in an amount(s) effective to restore KCC2
expression back to normal physiological levels; and b.
administering a transient dose of an anti-seizure medication in an
amount effective to stop the seizures.
16-19. (canceled)
20. The method of claim 5, wherein the effective amount of the
anti-seizure medication or phenobarbital is reduced due to
anti-seizure activity of CLP290.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 16/734,801, filed Jan. 6, 2020, which is a
Continuation of U.S. patent application Ser. No. 15/504,141 filed
Feb. 15, 2017, now U.S. Pat. No. 10,525,024, issued Jan. 7, 2020,
which is a 35 U.S.C. .sctn. 371 U.S. national entry of
International Application PCT/US2015/045170, having an
international filing date of Aug. 14, 2015, which claims the
benefit of U.S. Provisional Application No. 62/037,654, filed Aug.
15, 2014, the content of each of the aforementioned applications is
herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to the field of seizures. More
specifically, the present invention provides compositions and
methods for treating refractory seizures in neonates.
BACKGROUND OF THE INVENTION
[0004] Detected in 1 to 3.5 per 1000 newborns, neonatal convulsions
refer to the seizures occurring within the first 28 days of life.
Neonatal seizures, if poorly managed, can result in severe
neurodevelopmental outcomes that threaten cognition, motor
function, and even life. The associated pathologies include, but
are not limited to hypoxic-ischemic encephalopathy (HIE), stroke,
intracranial hemorrhage, brain malformation, infarction, prenatal
and neonatal infections. However, HIE has been the most prevalent
cause of neonatal seizures, and the HIE-associated seizures pose a
great challenge to its current therapy because those have higher
propensity of showing a stubborn refractoriness to conventional
antiepileptic drugs (AEDs) provided as a first-line therapy in
clinics.
[0005] The refractoriness in neonatal seizures can be mainly
attributed to a neuronal chloride gradient that does not generate
hyperpolarization as much as the one established in the adult
nervous system. When the ion channels located at neuronal membrane
open, the net ion influx or efflux is determined by both electrical
and chemical gradient: an electrical gradient of a certain
threshold voltage that a neuron wants to maintain and a chemical
gradient that is determined by the net concentration of ions at
extracellular and intracellular environment. The mature nervous
system maintains a relatively low intracellular chloride
concentration such that an opening of chloride channels results in
an influx of negatively charged chloride ions which ultimately
renders a post-synaptic inhibition in central nervous system (CNS).
In contrast, the immature nervous system has a relatively higher
intracellular chloride concentration that results in less
hyperpolarization or even depolarization in some cases. Hence, the
HIE-associated neonatal seizures are not efficaciously modulated by
conventional anti-convulsants, phenobarbital and phenytoin, that
target GABA.sub.A receptors to open the chloride channels to induce
neuronal hyperpolarization and halting seizures eventually. The
difference in the chloride gradient also contributes to the
neonatal hyperexcitability that leads to a higher seizure
susceptibility observed in seizing neonates, especially in the
first 2 days in the neonatal period.
[0006] The depolarizing chloride gradient has been shown to play a
critical role in neurodevelopment such as neuronal migration,
proliferation, and maturation. The critical switch of neuronal
chloride gradient from depolarizing to hyperpolarizing occurs
within neonatal period, and cation chloride co-transporters (CCCs)
are one of the pivotal players that drive the neuronal chloride
gradient toward its adult level. In early developmental stage,
NKCC1 pumps in chloride ions into a neuron to build up a high
intracellular chloride concentration which results in a
depolarizing gradient necessary for the associated
neurodevelopment. In the later development, KCC2 pumps out chloride
ions to lower the intracellular chloride concentration which
renders a hyperpolarizing gradient when the chloride channels are
opened. KCC2 expression is neuron-specific.sup.15 whereas NKCC1
expression is relatively ubiquitous, therefore the ratio of these
two CCCs are critical for the efficacy of anti-convulsants that
depend on chloride ion influx. The well-studied developmental
profile of CCCs in human and rodents suggests: 1) the high
expression level of NKCC1 during early development decreases
throughout neonatal period, and stabilizes at the lowest level
after neonatal period, 2) the lower expression of KCC2 during early
development gradually increases throughout neonatal period, and
stabilizes at the highest level by the end of adolescent stage.
Thus, in seizing adults with fully matured KCC2 expression in
mature CNS, the delivery of conventional GABA.sub.A-modulating AEDs
drives an ideal hyperpolarization driven by the chloride influx
upon channel opening. However, in seizing neonates with lower KCC2
expression in a developing CNS, refractoriness to traditional AEDs
is often observed. Importantly, KCC has a caudal-rostral expression
pattern that the establishment of hyperpolarizing chloride gradient
starts at the spinal cord, and reaches the brain at last. This
relates to a neonate-specific phenomenon of electroclinical
dissociation where a neonate undergoes electrographic seizures
without behavioral manifestation.
[0007] Designing and investigating an efficient therapy for
treating refractory neonatal seizures is challenging because there
are intrinsic difficulties in dissociating the harmful effects of
hypoxic-ischemia and seizures. Many animal models have been
proposed to examine the refractory neonatal seizures that mimic HIE
condition such as in vitro chemo-convulsive, in vivo hypoxic, and
ischemic model. Ischemic models, with the highest clinical
relevance among many HIE models, have provided a crucial insight on
the role of CCCs in designing an optimal pharmacotherapy for
refractory seizures in neonates. Recent animal studies have focused
on targeting NKCC1 using Bumetanide, a potent NKCC1 blocker, to
control refractory seizures. Under ischemia, an acute upregulation
of NKCC1 expression occurs such that an increased chloride influx
causes hyperexcitability and more refractoriness to conventional
AEDs. Prevention of chloride influx by blocking NKCC1 with
Bumetanide may establish a neuronal environment that enables
GABA.sub.A modulating AEDs to act efficaciously upon an ischemic
insult. However, more studies are needed to ensure: 1) the safety
of Bumetanide in neonates as an adjunct therapy, and 2) the true
additional efficacy of Bumetanide on refractory seizures. Indeed,
chronic delivery of bumetanide may induce an overwhelming diuresis
in neonates with HIE suffering other pathophysiological
complications such as energy failure and altered homeostasis.
Accordingly, new methods for treating neonatal seizures are
needed.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIGS. 1I and 1IIA-1IID. Effect of ANA12+PB on the total
seizure burden at P7 and P10. FIG. 1I. Schematics of the
experimental design. Experimental paradigm for the acute ligation
surgery and synchronous video-EEG recordings with the time-course
of drug administrations and the durations for each procedure.
Arrowheads indicate time-points of drug administration after
carotid-ligation and brain-harvesting for 24 h WB data. FIG.
1IIA-1IID. Total time spent seizing on EEG was quantitated as
seizure burden at P7 and P10 over a 3 h period, and was represented
by bar graphs (black bar=baseline, gray=2nd h, and white=3rd h)
FIGS. 1IIA and 1IIC) vehicle (5% DMSO) and ANA12-alone treatment
groups. The data from FIGS. 1IIA and 1IIC were pooled to form a
ligate-control group represented in FIGS. 1IIC and 1IID. FIGS. 1IIC
and 1IID. ligate control vs. ANA12+PB+BTN. Within-group comparison
was done using repeated measures ANOVA (i.e., *=p<0.05;
**=p<0.01; ***=p<0.001). Brackets (@) denote significant
between-group comparisons at each hour (independent sample t-tests;
p<0.05).
[0009] FIG. 2A-2B. ANA12 rescued PB-resistance at P7 and improved
PB-efficacy at P10. The effect of ANA12 on PB-efficacy was
evaluated as percent PB seizure suppression over baseline: % PB
seizure suppression =100*[Post-PB seizure burden/baseline seizure
burden]. FIG. 2A. PB by itself failed to subdue ischemic seizures
at P7. ANA12+PB significantly subdued ischemic seizures by 62%
(One-way ANOVA; ***=p<0.001). FIG. 2B. PB alone subdued seizures
by 68% at P10. ANA12+PB improved this efficacy to 85%. This
improved PB-efficacy however was not statistically significant when
compared to the efficacy of PB alone. The sample size for saline+PB
group was n=13 and n=11 for P7 and P10 respectively; n for ligate
control and treated group was same as listed in FIG. 1 (see Table
2). Sz: Seizure.
[0010] FIG. 3A-3D. The anti-seizure efficacy of ANA12+PB was not
different by sex. The efficacy of ANA12+PB on total seizure burden
was evaluated by sex at P7 and P10. FIGS. 3A and 3C. Males at both
ages of P7 and P10 responded significantly to ANA12+PB. However, at
P10, BTN administration following PB weakened the statistical
significance of seizure suppression achieved by PB. FIGS. 3B and
3D. ANA12+PB efficacy was significant in females at both P7 and
P10. Additionally, a significant temporal increase in the total
seizure burden over time-course of 3 h period was detected in P7
females, but was absent in P7 males. At P7, BTN aggravated
PB-subdued seizures in females such that PB-efficacy was lost.
ANA12+ PB efficacy was not significantly different between males
and females. (Repeated measures ANOVA; *=p<0.05; **=p<0.01;
***=p<0.001).
[0011] FIG. 4A-4B. Frequency distribution of baseline seizure
burdens shows age-dependent susceptibility. FIGS. 4A and 4B.
Frequency histogram of baseline seizure burdens was plotted,
separated by age and sex. Both sexes displayed higher seizure
susceptibility at P7 compared to P10: sample size P7 (n=50) and P10
(n=39). The between-group comparison of baseline seizure burdens by
sex was not statistically different at either P7 or P10.
[0012] FIG. 5A-5D. ANA12+PB rescued KCC2 expression but had no
effect on NKCC1 expression at 24 h. Bar graphs represent mean
expression of KCC2 and NKCC1 in ipsilateral hemisphere normalized
to the contralateral at 24 h after ischemia. .beta.-actin was used
as an internal control. The protein bands for both co-transporters
were: 1) normalized to the level of actin for the same sample, 2)
followed by normalization of ipsilateral co-transporter expression
levels to contralateral hemisphere of the same pup FIGS. 5A and 5C.
The post-ischemic degradation of KCC2 expression (.about.20%) was
prevented in the ANA12+PB+BTN treated group at P7 and P10. FIGS. 5B
and 5D. Post-ischemic expression levels of NKCC1 in the treated
group were not significantly different from ligate controls
(One-way ANOVA; *=p<0.05; ***=p<0.001).
[0013] FIG. 6A-6H. Significant correlation between rescue of KCC2
degradation at 24 h and seizure suppression was detected. The
percent change of KCC2 (or NKCC1) expression of an ipsilateral
hemisphere [% change=100*(ipsi-contra/contra KCC2 expression)] was
correlated to the percent seizure suppression [% baseline seizure
burden=100*(baseline seizure burden-post-PB seizure
burden)/baseline seizure burden)]. The color gradient (blue or red)
applied to the background represents the data distribution in the
positive (blue) or negative (red) direction of KCC2 expression
after ligation/treatment. The dotted gray lines within each scatter
plot represent the mean values for percent seizure suppression in
each group (x axis). FIGS. 6A and 6C. The ligated control group at
both ages of P7 and P10 showed the post-ischemic degradation of
KCC2 expression evident by predominant distribution of dots on the
red gradient. FIGS. 6B and 6D. The post-ischemic KCC2 expression of
the treated group was rescued at both ages of P7 and P10, as seen
in the positive-shift of the dots towards the blue gradient (red
arrows). The correlation between the percent KCC2 expression and
percent seizure suppression (black arrows) was significant at P10
(Spearman's test; p=0.04) but not at P7. When P7 data were binned
by baseline seizure burden (<1200 sec vs. >1200 sec), the
correlation became significant for the pups that seized <1200
sec (p=0.02; data not shown). FIGS. 6E and 6G. The ligated control
group at both ages of P7 and P10 showed inconsistent and
non-significant changes in the post-ischemic NKCC1a expression.
FIGS. 6F and 6H. NKCC1 expression levels of post ANA12+PB+BTN
treatment did not show any significant correlations with
efficacious PB seizure suppression.
[0014] FIG. 7A-7C. Representative electrographic seizure.
Representative trace of a raw EEG recording of an ictal event
associated with tonic-clonic convulsive behavior on video lasting
90 seconds. FIG. 7A. Expanded time scale traces for time-points in
A. Arrowheads denote the start and end of the ictal event. FIG.
7B-7C. Raw EEG trace in A filtered by high and low frequency band
pass.
[0015] FIG. 8. Representative time-compressed plot (60.times.3=180
min) of continuous EEG traces recorded from P7 and P10 pups by
ligate control vs. treated group. Timeline of 3 h EEG recording
(top) and the representative compressed raw EEG traces for same
duration are depicted for ligate control vs. treated group at each
age. The timepoints of drug administrations (IP) are represented
below each 1 h time-slot. All traces were selected from female pups
that represented the mean total seizure burdens for each treatment
group. ANA12+PB administration significantly suppressed seizures at
P7 and P10, and BTN aggravated the PB-subdued seizures at P7 in
females.
[0016] FIG. 9A-9H. Ictal events and ictal duration of
electrographic seizures quantitated for different treatment groups.
The number of ictal events and mean ictal durations were
quantitated. FIG. 9A-9B. ANA12-alone group showed a significant
temporal increase in the number of ictal events at P7. However,
pair-wise comparisons for the ictal events and duration of vehicle
vs. ANA-alone group for each hour did not detect a statistical
significance. FIG. 9C-9D. Ictal durations were not significantly
different between vehicle and ANA-alone treated groups. FIG. 9E-9F.
Ligate control group at P7 showed a significant temporal increase
in the number of ictal events. The number of ictal events in
treated group significantly dropped post-PB at both ages of P7 and
P10. However, BTN administration significantly increased the number
of ictal events at P10. FIG. 9G-9H. Ictal durations for ligate
control and the treated group were not significantly different.
(Repeated measures ANOVA; *=p<0.05 **=p<0.01;
***=p<0.001).
[0017] FIG. 10A-10H. Ictal events and ictal duration of EEG
seizures quantitated for treatment groups by sex. The quantitation
of ictal events and ictal durations for treated group was further
separated by sex. FIG. 10A-10B. ANA12+PB significantly suppressed
the number of ictal events in males at both P7 and P10 compared to
ligate controls. FIG. 10C-10D. Ictal durations for ligate control
and the treated group were not significantly different in males.
FIG. 10E-10F. ANA12+PB significantly suppressed the number of ictal
events in females at both P7 and P10 compared to ligate controls.
FIG. 10G-10H. Ictal durations were not different by age or
treatment in females. Overall, the ictal events and durations were
not significantly different between male and female pups at either
age tested. (Repeated measures ANOVA; *=p<0.05 **=p<0.01;
***=p<0.001).
[0018] FIG. 11A-11D. ANA12+PB rescued KCC2 expression but had no
effect on NKCC1 expression at 3 h. Brains harvested at 3 h
post-ischemia underwent WB analyses to quantitate expression levels
of KCC2 and NKCC1, similar to 24 h WB analyses (FIG. 5). An
additional group of saline-only treatment group was included. FIGS.
11A and 11C. ANA12+PB+BTN treated group showed that the
post-ischemic degradation of KCC2 expression was rescued at both
ages of P7 and P10. The statistical significance for the rescue in
KCC2 expression was only present at P7 at the 3 h time-point
(One-way ANOVA; *=p<0.05; ***=p<0.001). FIGS. 11B and 11D.
NKCC1a expression remained similar to saline controls regardless of
the treatment paradigm, replicating the 24 h WB data.
[0019] FIG. 12A-12C. FIG. 12A. Representative video-frame from a
ligated pup having an ischemic tonic-clonic seizure. FIGS. 12B and
12C. Schematics of the time-line and experimental design.
[0020] FIG. 13A-13I. Age dependent seizure burden and PB-efficacy.
FIG. 13A-13C. Representative electrographic traces of ischemic
seizures recorded with sub-dermal scalp electrodes at P7, P10, and
P12 and their associated behavioral grades on video. Arrowheads
show the start and end of ictal events. FIG. 13D-13F. EEG seizure
burden, mean number of ictal events and ictal durations in
ligated-controls that received saline injections at each hour after
ligation. Seizure burden after ischemia at baseline recording was
highest at P7, and was significantly more severe than at P10
(p=0.01) and P12 (p=0.03): pairwise t-test FIG. 13G-13I.
Electrographic seizure burden, mean number of ictal events and
ictal durations in ligated-treated mice that received PB (1 h
post-ligation) and BTN (2 h post-ligation; adjunct to PB). PB (25
mg/kg; IP) was inefficacious as an anti-seizure agent at P7. At the
same loading dose, PB was signify cantly efficacious as an
anti-seizure agent at P10 and P12. BTN as an adjunct failed to
improve PB-efficacy at any age tested, and significantly blunted
PB-efficacy at P10. PB-efficacy at P10 and P12 (FIG. 13G) was due
to significant reduction in mean number of ictal events (FIG. 13H).
Ictal durations were not significantly different at any age tested
(FIG. 13I).
[0021] FIG. 14A-14B. Behavioral correlates of EEG seizures and
PB-efficacy. Electrographic seizures (grade 0-2) occurred at all
ages tested, and responded well to PB treatment at P10 and P12.
This response was not significant at P7 (repeated measures ANOVA).
Likewise, the convulsive seizures (grades 3-6) also showed
significant PB-efficacy at P10 and P12 only with significant BTN
aggravation of the convulsive seizures at P10 (repeated measures
ANOVA). gray *=P10, #=P12, represent significant p-values for
pairwise t-tests. Therefore, PB failed to block either
electrographic or convulsive seizures at P7.
[0022] FIG. 15A-15C. Ictal events vs. PB-efficacy. Correlations of
the number of ictal events before treatment (i.e.; during baseline
EEG recording) to the number of ictal events post-PB treatment at
P7, P10 and P12. Brackets in FIGS. 15B and 15C show the difference
in the PB-efficacy at P10 vs. P12. Post-PB seizure suppression at
P12 was consistently significant and uniform regardless of baseline
seizure seventies (p=0.09). In contrast, at P10 PB-efficacy was
significantly dependent on the baseline seizure severity (i.e.,
better efficacy with lower baseline seizure loads compared to
higher baseline seizure loads). Additional correlations run for
baseline vs. post-PB seizure burdens showed similar results (see
brackets in FIG. 15B); low baseline seizure burdens and PB efficacy
were not significantly correlated (for baseline seizure burden
<=250 sec; r=0.35, p=0.39; high baseline seizure burdens showed
significant positive correlations to their post-PB seizure burdens
(for baseline seizure burden>250 sec; r=0.61, p=0.03). This may
indicate the potential role of the number of ictal events that have
occurred before treatment on the efficacy of anti-seizure agents at
P10 (p=0.001).
[0023] FIG. 16A-16B. Age-dependent stroke injury. Severity of the
ischemic injury evaluated at P18 in ligated-treated mice.
Histopathological analyses were performed on a series of coronal
brain sections harvested at P18 for all the ages investigated.
Post-ischemic P7 brains were significantly less vulnerable to
necrotic infract injury compared to P10 and P12. Both hemispheric
and hippocampal atrophies associated with stroke injury were
significantly higher at P10 and P12. The stroke severities between
P10 and P12 were not significantly different.
[0024] FIG. 17A-17C. Developmental profile of KCC2 expression. FIG.
17A. IHC for KCC2 in cortex as a function of postnatal age in naive
brains respectively (Scale bar=250 um) showed increased neuronal
expression with age. FIG. 17B. Western blot quantitation of KCC2
expression as a function of postnatal age in naive brains (n=3 for
each age). Bar graphs show the co-transporter expression normalized
to actin expression of the same brains. FIG. 17C. A significant
sex-dependent lag of KCC2 expression was detected at P7 in naive
males compared to age-matched naive females (p<0.05), and this
dimorphism was not significant at older ages [M=male F=female (n=2
each at every age)]. Analyses for NKCC1 in the same brain samples
are not shown.
[0025] FIG. 18A-18B. Seizure severity by age and sex. Baseline
seizure burdens pooled for ligated-control and ligated-treated pups
showed an age-dependent susceptibility for ischemic seizures that
was significant in males but not in females.
[0026] FIG. 19A-19D. Post-ischemic KCC2 downregulation. Western
blot quantification of post-ligation expression of KCC2 in P7, P10,
and P12 ligated pups at acute and sub-acute time-points after
ischemia (n=3 each) in ipsi- and contralateral (i.e.; injured and
uninjured hemispheres respectively) hemispheres; FIG. 19A. At P7,
the significant downregulation of KCC2 detected within 6-8 h of
ligation (p=0.004) showed a complete recovery by 96 h. FIG. 19B. At
P10, KCC2 downregulation showed the same trend as at P7 at 48 h.
FIG. 19C. P12F pups showed a significant downregulation of KCC2 at
24 h (p<0.02). FIG. 19D. Scatter plot of KCC2 expression levels,
normalized to actin and shown as the percent of levels in their
respective contralateral uninjured hemispheres. All acute
time-points (6-48 h) were pooled from all three age groups (n=13).
Data show that ischemia in the CD1 mice results in an acute KCC2
downregulation in the ischemic injured hemispheres with
approximately 45.35% reduction in mean expression (pairwise t-test,
p=0.0002; contralateral control is 100%, which is represented as a
dotted gray line as a ratio of 1).
[0027] FIG. 20. Data using CLP290 (n=4) in an experimental paradigm
similar to ANA-12 indicating that the KCC2 agonist can act
independently as an anti-seizure agent and with PB to completely
block all ischemic seizures at a 20 mg/kg dose in the model when
compared to the vehicle injection (HPCD). Bottom trace in each
panel shows the seizure frequency as scored by electrographic
seizure activity on raw EEG in blue. Middle trace shows gamma
frequency seizure burst activity on 3 h EEG and top blue trace
shows low frequency activity due to movement artifacts during
ischemia induced status. This finding indicates that as a KCC2
agonist CLP290 also works efficiently to not only reverse
PB-resistant seizures but act as an anti-seizure agent by itself.
Arrow heads indicate time of 1 hourly drug injections from start of
the 3 h recording.
DETAILED DESCRIPTION OF THE INVENTION
[0028] It is understood that the present invention is not limited
to the particular methods and components, etc., described herein,
as these may vary. It is also to be understood that the terminology
used herein is used for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention. It must be noted that as used herein and in the
appended claims, the singular forms "a," "an," and "the" include
the plural reference unless the context clearly dictates otherwise.
Thus, for example, a reference to a "protein" is a reference to one
or more proteins, and includes equivalents thereof known to those
skilled in the art and so forth.
[0029] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Specific
methods, devices, and materials are described, although any methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the present invention.
[0030] All publications cited herein are hereby incorporated by
reference including all journal articles, books, manuals, published
patent applications, and issued patents. In addition, the meaning
of certain terms and phrases employed in the specification,
examples, and appended claims are provided. The definitions are not
meant to be limiting in nature and serve to provide a clearer
understanding of certain aspects of the present invention.
[0031] The present invention is based, at least in part, on the
discovery that refractory seizures can be treated using KCC2
agonists and/or trkB antagonists. More specifically, in particular
embodiments, the present invention provides novel pharmaceutical
interventions that enhance KCC2 function by using a KCC2 agonist
and/or small molecule Trk-B receptor antagonists as adjunct
therapies to convert phenobarbital (PB)-resistant seizures into
phenobarbital responsive seizures in neonates. Both drugs act
through very different mechanisms but achieve the same goal. They
help enhance KCC2 activity in injured neonatal brains and thus,
improve the outflow of chloride from neurons.
[0032] As described herein, the present inventors have shown that,
unlike in adult brains, KCC2 expression in the neonatal brain is
much lower and undergoes further down-regulation following an
ischemic insult, which is one of the most common causes of neonatal
seizures. Because it is the KCC2 activity on neuronal cell
membranes that help reduce the intracellular Cl concentrations; the
present inventors hypothesized that enhancing its activity in an
injured/seizing neonatal brain will act as an effective adjunct
treatment to convert PB-resistant seizures into PB-responsive
seizures. The present inventors have shown that a Trk-B antagonist
(ANA-12), when administered systemically to block the action of
excessive BDNF release following ischemic injury in immature brains
in conditions like ischemia, hypoxia, infection and inflammation,
prevents the disruption of the normally increasing KCC2 expression
profile and makes the refractory seizures responsive to PB. The
present inventors have also achieved similar results with a KCC2
agonist, CLP290.
[0033] This discovery provides a novel approach to treat and
prevent the emergence of refractory seizures in both neonates and
adults. Additionally, by correcting the developmental dysfunction
of ischemia related KCC2 downregulation, the present invention may
prevent the emergence of long-term neurological morbidities in the
form of ADHD, learning disabilities, autism spectrum disorders and
psychiatric disorders associated with a history a neonatal CNS
injury.
[0034] It has long been known that blocking Trk-B receptors may be
able to prevent some deleterious downstream effects in neurological
diseases (especially in pain research). However, the old generation
of Trk-B receptors antagonists was incapable of crossing the blood
brain barrier (BBB) and, therefore, clinical applicability remained
elusive. The current generation of small molecule antagonists have
been shown to be able to cross the BBB and therefore systemic
application during periods of acute CNS injury are now feasible and
are a novel approach to treat conditions where KCC2 down-regulation
plays a role in neurological pathogenesis. The present inventors
have identified a novel mechanism of seizure refractoriness in
neonatal ischemic seizures that involves the reduction and delay in
the developmental profile of KCC2 up-regulation that is important
to developing brains. This acute down-regulation and severe
attenuation of KCC2 function may underlie both the acute symptoms
but also the appearance of long-term morbidity due to the
dysfunctional period in early brain development. The novel approach
of enhancing KCC2 function using KCC2 agonists and/or Trk-B
receptor antagonists would be a short, acute systemic (i.e.,
non-CNS-invasive) intervention that would help better treat the
acute symptoms where current line of treatments are found
non-efficacious but more importantly prevent future morbidities
involving the brain by preventing disruption of early developmental
processes.
[0035] Examples of KCC2 agonists include, but are not limited to,
N-ethylmaleimide (NEM), the chloride channel inhibitor
5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), CLP257, CLP290
and analogs, functional derivatives and prodrugs thereof. Examples
of KCC2 agonists are described in WO2009/114950, WO2009/097695 and
WO2010132999.
[0036] Specific examples of trkB antagonists include, but are not
limited to, ANA-12 (Cazorla et al., 121(5) J. CLIN. INVEST. 1846-57
(2011)); N-T19 (Cazorla et al. (2011)); K252a; and cyclotraxin-B
(CTX-B) (Constandil et al., 13(6) J. PAIN 579-89 (2012)).
[0037] As used herein, the term "modulate" indicates the ability to
control or influence directly or indirectly, and by way of
non-limiting examples, can alternatively mean inhibit or stimulate,
agonize or antagonize, hinder or promote, and strengthen or weaken.
Thus, the term "modulator" refers to an agent that modulates KCC2
and/or trkB. Modulators may be organic or inorganic, small to large
molecular weight individual compounds, mixtures and combinatorial
libraries of inhibitors, agonists, antagonists, and biopolymers
such as peptides, nucleic acids, or oligonucleotides. A modulator
may be a natural product or a naturally-occurring small molecule
organic compound. In particular, a modulator may be a carbohydrate;
monosaccharide; oligosaccharide; polysaccharide; amino acid;
peptide; oligopeptide; polypeptide; protein; receptor; nucleic
acid; nucleoside; nucleotide; oligonucleotide; polynucleotide
including DNA and DNA fragments, RNA and RNA fragments and the
like; lipid; retinoid; steroid; glycopeptides; glycoprotein;
proteoglycan and the like; and synthetic analogues or derivatives
thereof, including peptidomimetics, small molecule organic
compounds and the like, and mixtures thereof. A modulator
identified according to the invention is preferably useful in the
treatment of a disease disclosed herein.
[0038] As used herein, an "antagonist" is a type of modulator and
the term refers to an agent that can, directly or indirectly,
block, suppress or reduce one or more functions or biological
activities of the target. An "agonist" is a type of modulator and
refers to an agent that, directly or indirectly, can activate,
stimulate or increase one or more functions or biological
activities of the target.
[0039] As used herein, the term "antibody" is used in reference to
any immunoglobulin molecule that reacts with a specific antigen. It
is intended that the term encompass any immunoglobulin (e.g., IgG,
IgM, IgA, IgE, IgD, etc.) obtained from any source (e.g., humans,
rodents, non-human primates, caprines, bovines, equines, ovines,
etc.). Specific types/examples of antibodies include polyclonal,
monoclonal, humanized, chimeric, human, or otherwise-human-suitable
antibodies. "Antibodies" also includes any fragment or derivative
of any of the herein described antibodies.
[0040] The terms "specifically binds to," "specific for," and
related grammatical variants refer to that binding which occurs
between such paired species as antibody/antigen, enzyme/substrate,
receptor/agonist, and lectin/carbohydrate which may be mediated by
covalent or non-covalent interactions or a combination of covalent
and non-covalent interactions. When the interaction of the two
species produces a non-covalently bound complex, the binding which
occurs is typically electrostatic, hydrogen-bonding, or the result
of lipophilic interactions. Accordingly, "specific binding" occurs
between a paired species where there is interaction between the two
which produces a bound complex having the characteristics of an
antibody/antigen or enzyme/substrate interaction. In particular,
the specific binding is characterized by the binding of one member
of a pair to a particular species and to no other species within
the family of compounds to which the corresponding member of the
binding member belongs. Thus, for example, an antibody typically
binds to a single epitope and to no other epitope within the family
of proteins. In some embodiments, specific binding between an
antigen and an antibody will have a binding affinity of at least
10.sup.-6 M. In other embodiments, the antigen and antibody will
bind with affinities of at least 10.sup.-7 M, 10.sup.-8 M to
10.sup.-9 M, 10.sup.-10 M, 10.sup.-11 M, or 10.sup.-12 M.
[0041] Optional" or "optionally" means that the subsequently
described event or circumstance can or cannot occur, and that the
description includes instances where the event or circumstance
occurs and instances where it does not.
[0042] The terms "subject," "individual," or "patient" are used
interchangeably herein, and refer to a mammal, particularly, a
human. In certain embodiments, the patient is an adult and/or a
neonate. In particular embodiments, the patient suffers from
seizures.
[0043] As used herein, the term "effective," means adequate to
accomplish a desired, expected, or intended result. More
particularly, a "therapeutically effective amount" as provided
herein refers to an amount of a KCC2 and/or trkB modulator of the
present invention, either alone or in combination with another
therapeutic agent, necessary to provide the desired therapeutic
effect, e.g., an amount that is effective to prevent, alleviate, or
ameliorate symptoms of a condition. In a specific embodiment, the
term "therapeutically effective amount" as provided herein refers
to an amount of a KCC2 agonist and/or trkB antagonist, necessary to
provide the desired therapeutic effect, e.g., an amount that is
effective to prevent, alleviate, or ameliorate symptoms of
seizures, e.g., refractory seizures. As would be appreciated by one
of ordinary skill in the art, the exact amount required will vary
from subject to subject, depending on age, general condition of the
subject, the severity of the condition being treated, the
particular compound and/or composition administered, and the like.
An appropriate "therapeutically effective amount" in any individual
case can be determined by one of ordinary skill in the art by
reference to the pertinent texts and literature and/or by using
routine experimentation.
[0044] As used herein, the terms "treatment," "treating," and the
like, refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease, condition or symptom thereof and/or
may be therapeutic in terms of a partial or complete cure for a
disease or condition and/or adverse effect attributable to the
disease or condition. "Treatment," as used herein, covers any
treatment of a disease or condition in a subject, particularly in a
human, and includes: (a) preventing the disease or condition from
occurring in a subject which may be predisposed to the disease or
condition but has not yet been diagnosed as having it; (b)
inhibiting the disease or condition, i.e., arresting its
development; and (c) relieving the disease or condition, e.g.,
causing regression of the disease or condition, e.g., to completely
or partially remove symptoms of the disease or condition.
[0045] A "trkB antagonist" refers to an agent that is able to
block, suppress or reduce (including significantly) trkB biological
activity, including downstream pathways mediated by trkB signaling,
such as binding of trkB to BDNF or NT-4/5 and/or elicitation of a
cellular response to BDNF or NT-4/5. The term "antagonist" implies
no specific mechanism of biological action whatsoever, and is
deemed to expressly include and encompass all possible
pharmacological, physiological, and biochemical interactions with
trkB whether direct or indirect, or whether interacting with BDNF,
NT-4/5, trkB, or through another mechanism, and its consequences
which can be achieved by a variety of different, and chemically
divergent, compositions. Exemplary trkB antagonists include, but
are not limited to, an anti-BDNF antibody, an anti-NT-4/5 antibody,
a BDNF or an NT-4/5 inhibitory compound, a BDNF or an NT-4/5
structural analog, a dominant-negative mutation of a trkB receptor
that binds BDNF and/or NT-4/5, a trkB immunoadhesin, an anti-trkB
antibody, and a trkB inhibitory compound. For purpose of the
present invention, it will be explicitly understood that the term
"antagonist" encompass all the previously identified terms, titles,
and functional states and characteristics whereby the trkB receptor
itself, a trkB biological activity or the consequences of the
biological activity, are substantially nullified, decreased, or
neutralized in any meaningful degree.
[0046] "Biological activity" of trkB receptor generally refers to
the ability to bind BDNF and NT-4/5 and/or activate trkB receptor
signaling pathways. Without limitation, a biological activity
includes any one or more of the following: the ability to bind its
ligand BDNF and/or NT-4/5; the ability to dimerize and/or
autophosphorylate after the ligand binding; the ability to activate
the trkB signaling pathway; the ability to promote cell
differentiation, proliferation, survival, growth and other changes
in cell physiology, including (in the case of neurons, including
peripheral and central neuron) change in neuronal morphology,
synaptogenesis, synaptic function, neurotransmitter and/or
neuropeptide release and regeneration following damage.
[0047] Accordingly, in one aspect, the present invention provides
compositions and methods for treating refractory seizures in a
neonatal patient. In one embodiment, the method comprises the steps
of (a) administering to the patient an effective amount of a KCC2
agonist and/or trkB antagonist; and (b) administering to the
patient an effective amount of an anti-seizure medication. In
certain embodiments, the amount of a KCC2 agonist and/or trkB
antagonist is effective to restore KCC2 expression to normal
physiological levels. In particular embodiments, the seizure is an
ischemia-related seizure. The KCC2 agonist can be, but is not
limited to, N-ethylmaleimide (NEM), the chloride channel inhibitor
5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), CLP257, or
CLP290. In a specific embodiment, the KCC2 agonist is CLP290. The
trkB antagonist can be, but is not limited to, ANA-12, N-T19, K252a
or cyclotraxin-B. In a specific embodiment, the trkB antagonist is
ANA-12. In particular embodiments, the anti-seizure medication is
phenobarbital or phenytoin.
[0048] The present invention also provides a method for treating
refractory seizures in a neonatal patient comprising the steps of
(a) administering to the patient an amount of ANA-12 and/or CLP290
effective to restore KCC2 expression to normal physiological
levels; and (b) administering to the patient an effective amount of
an anti-seizure medication. In a specific embodiment, the seizure
is an ischemia-related seizure. In other embodiments, the
anti-seizure medication is phenobarbital (PB) or phenytoin.
[0049] In particular embodiments, the present invention provides
methods and compositions for reducing the loading dose of PB
required as first line treatment for neonatal seizures. The first
loading dose is often ineffective in >50% of cases. Higher and
repeated doses of PB can be detrimental for immature brains both
acutely and in the lob-term with associated cell death. In a
further embodiment, a method for treating refractory seizures in a
neonatal patient comprises the steps of (a) administering to the
patient an amount of ANA-12 and/or CLP290 effective to restore KCC2
expression to normal physiological levels; and (b) administering to
the patient an effective amount of phenobarbital. In methods
comprising the use of CLP290 or any KCC2 agonist or trkB antagonist
that shows some anti-seizure effects by itself, the effective
amount of the anti-seizure medication or phenobarbital is reduced
due to anti-seizure activity of CLP290.
[0050] In yet another embodiment, a method for treating refractory
seizures in a patient comprises the steps of (a) administering to
the patient an effective amount of an agent that increases KCC2
expression back to normal levels, wherein the seizure downregulates
KCC2 expression; and (b) administering to the patient an effective
amount of an anti-seizure medication. In particular embodiments,
the patient is a neonate. In certain embodiments, the neonate is
administered a transient dose of an anti-seizure medication. The
present invention also provides a method for treating
ischemia-related seizures in a neonatal patient comprising the
steps of (a) administering a KCC2 agonist and/or a trkB antagonist
in an amount(s) effective to restore KCC2 expression back to normal
physiological levels; and (b) administering a transient dose of an
anti-seizure medication in an amount effective to stop the
seizures.
[0051] The compositions of the present invention can comprise an
effective amount of a KCC2 agonist and an anti-seizure medication.
In other embodiments, the compositions can comprise an effective
amount of a trkB antagonist and an anti-seizure medication. In
certain embodiments, the compositions can comprise an effective
amount of a KCC2 agonist, a trkB antagonist and an anti-seizure
medication. In a specific embodiment, a composition comprises
ANA-12 and PB. In another embodiment, a composition comprises
CLP290 and PB. In further embodiments, a composition comprises
ANA-12, CLP290 and PB.
[0052] Typically, a loading dose of PB for a neonate comprises
15-25 mg/kg and additional doses of 5-10 mg/kg can be given if
seizures are not controlled. The maximum loading dose is typically
about 40 mg/kg. The effective amount of administered PB can be
reduced by combining with a KCC2 agonist and/or trkB antagonist
(co-administered or PB following KCC2 agonist and/or trkB
antagonist administration) (particularly in embodiments in which
CLP290 is used). The compositions of the present invention can
comprise a lower amount of PB than is typically used. For example
the dose of PB can comprise about 5-90% less than the typical dose
for neonates including, but not limited to, 5%, 6%, 7%, 8%, 9%,
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,
23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,
36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,
49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% less than a typical PB
dose. The dose of PB can comprise about 5-90%, 10-85%, 15-75%,
20-70%, 25-65%, 30-60%, 35-55%, 40-50% less than the typical PB
dose. In specific embodiments, a composition can comprise an amount
of PB equivalent to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20 mg/kg. In other embodiments,
phenytoin can be used and the effective amount reduced accordingly
as recited above.
[0053] In another aspect, the present invention provides
compositions and methods for making a rodent ischemic seizure
model. In one embodiment, a method for making a rodent ischemic
seizure model comprises the steps of (a) anesthetizing the rodent;
(b) permanently double ligating the right common carotid artery;
and (c) closing the outer skin of the rodent. In particular
embodiments, the rodent is a mouse. In a more specific embodiment,
the mouse is a CD1 mouse. The rodent can be any species/strains
that can be manipulated using the present methods to provide an
ischemia only seizure background, as opposed to other strains that
require ischemia and a 90 minute low oxygen hypoxia to generate a
similar insult. In certain embodiments, the present invention
provides a rodent ischemic seizure model produced by the methods
described herein.
[0054] Without further elaboration, it is believed that one skilled
in the art, using the preceding description, can utilize the
present invention to the fullest extent. The following examples are
illustrative only, and not limiting of the remainder of the
disclosure in any way whatsoever.
EXAMPLES
[0055] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices,
and/or methods described and claimed herein are made and evaluated,
and are intended to be purely illustrative and are not intended to
limit the scope of what the inventors regard as their invention.
Efforts have been made to ensure accuracy with respect to numbers
(e.g., amounts, temperature, etc.) but some errors and deviations
should be accounted for herein. Unless indicated otherwise, parts
are parts by weight, temperature is in degrees Celsius or is at
ambient temperature, and pressure is at or near atmospheric. There
are numerous variations and combinations of reaction conditions,
e.g., component concentrations, desired solvents, solvent mixtures,
temperatures, pressures and other reaction ranges and conditions
that can be used to optimize the product purity and yield obtained
from the described process. Only reasonable and routine
experimentation will be required to optimize such process
conditions.
Example 1
Acute Trk-.beta. Inhibition Rescues Phenobarbital-Resistant
Seizures in a Mouse Model of Neonatal Ischemia
[0056] Neonatal seizures are commonly associated with
hypoxic-ischemic encephalopathy (HIE). Phenobarbital
(PB)-resistance is common and poses a serious challenge in clinical
management. Using a newly characterized neonatal mouse model of
ischemic seizures, this study investigated a novel strategy to
rescue PB-resistance. A small-molecule TrkB antagonist, ANA12, used
to selectively and transiently block post-ischemic BDNF-TrkB
signaling in vivo determined whether rescuing TrkB-mediated
post-ischemic degradation of KCC2 rescued PB-resistant seizures.
The anti-seizure efficacy of ANA12+PB was quantitated by; 1)
electrographic seizure burden using acute continuous video-EEGs and
2) post-treatment expression levels of KCC2 and NKCC1 using western
blot analysis in postnatal day 7 and 10 (P7, P10) CD1 pups with
unilateral carotid ligation. ANA12 significantly rescued
PB-resistant seizures at P7, and improved PB-efficacy at P10.
Single dose of ANA12+PB prevented the post-ischemic degradation of
KCC2 up to 24 h. As anticipated, ANA12 by itself had no
anti-seizure properties and was unable to prevent KCC2 degradation
at 24 h without follow-on PB. This indicates that unsubdued
seizures can independently lead to KCC2 degradation by non-TrkB
dependent pathways. This study, for the first time, reports the
potential therapeutic value of KCC2 modulation for the management
of PB-resistant seizures in neonates.
Introduction
[0057] The neonatal period is a critical window for the increased
occurrence of seizures, with a prevalence of 1 to 3.5 per 1000
newborns. Neonatal seizures, acquired from diverse pathologies, are
associated with severe morbidity and mortality. Hypoxic-ischemic
encephalopathy (HIE) accounts for 50-60% of the cases for neonatal
seizures. HIE-associated seizures in neonates are known for their
resistance to the conventional 1st-line anti-seizure drugs. GABAA
agonists like phenobarbital (PB) and benzodiazepines, which are
consistently efficacious in adults, fail as anti-seizure agents in
neonates. Viable alternative strategies in overcoming these hurdles
are currently lacking.
[0058] The pharmaco-resistance of 1st-line GABAA agonists in
immature brains has been largely attributed to the developmental
physiology of [C1-]i that is 20-40 mM higher in immature neurons
than in mature neurons. The age-dependent upregulation of K+Cl-
co-transporter (KCC2), operating as a Cl- extruder in an
electroneutral manner, has been shown to play a critical role in
the shift of GABAergic signaling from depolarizing to
hyperpolarizing. The expression of KCC2, which is predominantly
neuronal, increases exponentially with conceptional age, especially
perinatally, starting in the second half of gestation in humans to
reach the significantly higher and stable adult levels. Similar
developmental profiles for KCC2 have been noted in mice from the
age of P3 to P15 where P7 is considered as term. Recent findings in
both pre-clinical and human studies highlighting the important role
of KCC2 in excitotoxicity have helped shape our novel strategy of
modulating KCC2 to improve the efficacy of GABAA agonists in
neonatal seizures.
[0059] The activation of BDNF-TrkB signaling has been associated
with KCC2 downregulation in diverse pathological environments
associated with excitotoxicity. Despite this known causal-effect
relationship of BDNF-TrkB activation associated with degradation of
KCC2, the novel strategy of preventing post-ischemic degradation of
KCC2 by using a TrkB antagonist has never been tested in vivo. This
may have primarily been due to the inability of old-generation TrkB
antagonists to cross blood brain barrier and remain proteolytically
stable. The identification of a small-molecule TrkB antagonist,
ANA12, that can selectively block BDNF-TrkB binding directly at the
extracellular domain of TrkB receptor in a non-competitive manner,
allowed to test the following hypotheses: 1. Blocking BDNF-TrkB
pathway after the onset of ischemia will prevent the post-ischemic
degradation of KCC2; 2. If KCC2 degradation following ischemia
results in the emergence of PB-resistant seizures, preventing KCC2
degradation will rescue the emergence of PB-resistance by
maintaining KCC2 expression and function. This study investigated
the following "proof-of-concept" question: Can the age-dependent
PB-resistance for P7 seizures in a mouse model of neonatal ischemic
seizures be reversed by a small-molecule TrkB antagonist? Does
adding a NKCC1 antagonist, BTN, help improve this rescue?
Materials and Methods
[0060] Study approval. All experimental procedures were conducted
in compliance with guidelines by the Committee on the Ethics of
Animal Experiments, Johns Hopkins University (Permit Number:
A3272-01) and all protocols were approved by the Animal Care and
Use of Committee (IACUC) of Johns Hopkins. All litters of CD1 mice
with dams were purchased from Charles River Laboratories Inc.
(Wilmington, Mass.). Newly born litters of pups were delivered at
postnatal 3 days old (P3) and were allowed to acclimate. Food and
water were provided ad libitum. Equal numbers of male and female
pups were introduced into the study. The number of pups and litters
used in this study are listed in Table 2.
TABLE-US-00001 TABLE 2 Sample sizes for sham and ligated pups for
each age group for EEG and histology experiments. Age P7 P10 P12
Sum EEG Sham 5 4 3 12 Ligated Untreated 9 8 9 26 Ligated PB + BTN
20 16 18 54 Sum 34 28 30 92 P18 Histology: CV stain 15 15 19 49
[0061] Surgical procedure for ischemic insult and sub-dermal EEG
electrode implantation. The surgical protocol was similar to the
previously published work (Kang et al., 9 FRONTIERS CELLULAR
NEUROSCI. Art. 173 (2015); Kadam et al., 18 EPILEPSY BEHAV. 344-57
(2010)). At P7 or P10, animals were subjected to permanent
unilateral ligation of right common carotid artery using 6-0
surgisilk (Fine Science Tools, BC Canada) under isoflurane
anesthesia. The outer skin was closed with 6-0 monofilament nylon
(Covidien, MA), and lidocaine was applied as an additional local
anesthetic. Under continued anesthesia, animals were then implanted
with 3 sub-dermal EEG scalp electrodes: 1 reference, 1 ground, and
1 recording overlying the parietal cortex. Wire electrodes made for
use in humans (IVES EEG; Model #SWE-L25-MA, USA) were implanted
sub-dermally and fixed in position with adhesive. Pups were then
allowed to recover from anesthesia which took a couple of minutes.
Finally, animals were tethered to a preamplifier by connecting
sub-dermal electrodes within a recording chamber for 3 h of
video-EEG, maintained at 36.degree. C. with isothermal pads. At the
end of the recording session, the animals were returned to the dam
after removal of the sub-dermal electrodes. The average duration of
anesthesia for both ligation and electrode implantation in this
study added up to 16.18.+-.4.37 min. There is a known mortality
rate of .about.10-20% associated with the surgical procedure of
carotid-ligation and severe seizures in the model. The mortality
rates for the pups 24 h after surgery were n/n=9/45 (20%) pups at
P7 (6 males and 3 females) and n/n=7/52 (13%) pups at P10 (3 males
and 4 females), and were not significantly different, by age nor by
sex in this study (p=0.42 and p=0.57 respectively; Fisher's exact
test, two-tailed). Mortality rates following the surgical procedure
were also not significantly different by treatment (ligated control
vs. treated group; p=0.26).
[0062] Experimenal paradigm. Following unilateral permanent
carotid-ligation, vehicle [5% dimethyl sulfoxide (DMSO)], ANA12
(2.5 mg/kg; N-[2-[[(Hexahydro-2-oxo-1H-azepin-3-yl)
amino]carbonyl]phenyl]benzo[b]thiophene-2-carboxamide), or 0.9%
sodium chloride (Saline) was injected intraperitoneally (IP). The
general outline of the experimental paradigm is depicted in FIG.
1I. Pups were randomly assigned to three different treatment
regimens over the 3 h acute and continuous EEG recording period: 1)
vehicle+saline+saline, 2) ANA12+saline+saline, and 3) ANA12+PB+BTN.
Injection of 1st drug occurred immediately after ligation, and the
subsequent injections followed every hour (i.e.; FIG. 1IIB and
1IID). Each color in the bar graphs (FIGS. 1 & 3) represents
the parameters (i.e.; total EEG seizure burdens) quantitated for
the 60 min duration of EEG recordings during each hour: 1st h
(black bars), 2nd h (gray bars), and 3rd h (white bars).
[0063] The vehicle, 5% DMSO (Cat. No. 472301) was made in phosphate
buffered saline (v/v; pH 7.4). ANA12 (2.5 mg/kg) was dissolved in
the vehicle. ANA12 was stored at -20.degree. C. in aliquots ready
for use (Sigma-Aldrich; Cat. No. SML0209). PB was (25 mg/kg:
Sigma-Aldrich; Cat. No. P5178) dissolved in phosphate buffered
saline (made on the day of experiment) injection followed 1 h after
the vehicle or ANA12 injection, and BTN injection [0.1-0.2 mg/kg
dissolved in 100% alcohol; aliquoted and stored at -20.degree. C.;
protocol similar to Example 2 below (Kang et al. (2015)] followed 1
h after PB injection.
[0064] In vivo synchronous video-EEG recording and analyses. EEG
recording was acquired using Sirenia Acquisition software (v 1.6.4)
with synchronous video capture (Pinnacle Technology Inc. KS, USA).
Data acquisition was done with sampling rates of 400 Hz that had a
pre-amplifier gain of 100 and the filters of 1 Hz high-pass and 60
Hz low-pass to remove ambient noise. The data were scored by
binning the raw EEG trace in 10 sec epochs. Similar to Example 2
(Kang et al., 2015), seizures were defined as electrographic ictal
events that consisted of rhythmic spikes of high amplitude, diffuse
peak frequency of .gtoreq.7-8 Hz (i.e.; peak frequency detected by
automated spectral power analysis) lasting .gtoreq.6 seconds. Short
duration burst activity lasting <6 seconds (brief runs of
epileptiform discharge) was not included for seizure burden
calculations in this study.
[0065] Western blot at 3 h and 24 h post-ligation. All animals were
anesthetized with chloral hydrate (90 mg/ml; IP) before being
transcardially perfused. The whole brains of P7 and P10 pups were
harvested at either 3 h or 24 h post-ligation, and were frozen as
ipsilateral and contralateral hemisphere. The brains were stored in
-80.degree. C. until further use. Homogenized whole brain lysates
were suspended in cell lysis buffer with 10% protease/phosphatase
inhibitor cocktail. Total protein concentrations were quantified
through Bradford Assay (Bio-Rad) at 570 nm wavelength, and the
samples were diluted for 50 ug of protein at 20 ul of loading
volume for gel electrophoresis.
[0066] Samples were run on 4-20% gradient 1.5 mm 15 wells SDS gels
(Invitrogen) for 100-120 min with 130V, and were transferred onto
nitrocellulose membranes for overnight wet-transfer for minimum of
18 hours at 30V. After the transfer, the nitrocellulose membranes
underwent 1 h blocking step in odyssey buffer, before overnight
incubation in primary antibodies: rabbit .alpha.-KCC2 (1:1000,
Millipore; Cat. No. 07-432); rabbit .alpha.-NKCC1 (1:500,
Millipore, Cat. No. AB3560P); mouse .alpha.-actin (1:10000, LI-COR
Biosciences, Cat. No. 926-42214). On the next day, nitrocellulose
membranes were washed with TBS containing tween detergent, and then
were incubated in chemiluminescence secondaries for 1 h (1:5000 for
both goat .alpha.-mouse 680LT and goat .alpha.-rabbit 800CW, LI-COR
Biosciences). Chemiluminescent protein bands were analyzed using
the Odyssey infrared imaging system 2.1 (LI-COR Biosciences). The
optical density of each protein sample was normalized to the actin
bands run on each lane for internal control. The expression levels
of the proteins of interest in ipsilateral hemispheres were
normalized to the same in contralateral hemispheres for each pup.
The human brain was recently shown to express two splice variants
of NKCC1 (a and b) and the NKCC1 probe used in this study and all
previous studies referenced here can only detect the NKCC1a isoform
because the targeted epitope site of this probe overlaps with exon
21 that is spliced in the dominant isoform NKCC1b. A reliable
pan-NKCC1 probe is not currently available.
[0067] A smaller sample of naive age-matched controls (n=8, 4 each
at P8 and P11, equal sexes) was run with the 24 h WB data (brains
harvested at P8 and P11, i.e.; 24 h after P7 and P10 ligations).
KCC2 and NKCC1 expression in the contralateral uninjured
hemispheres from the ligate control pups (n=8, 4 each at P8 and
P11, equal sexes) was not significantly different from their
age-matched naive controls (p=0.49 and p=0.23; KCC2 and NKCC1
respectively). This pilot established that contralateral
hemispheres were ideal controls for normalization of KCC2 and NKCC1
expression in ipsilateral injured hemispheres for this study
similar to previously reported conclusions in another ischemia
model.
[0068] Statistics. Group means of total seizure burden, number of
ictal events, and ictal duration within each treatment group were
compared using repeated measures ANOVA with Bonferroni's post-hoc
correlations. Differences with p<0.05 were considered
statistically significant (repeated measures ANOVA; *=p<0.05
**=p<0.01; ***=p<0.001). The assumption of sphericity for
data was confirmed by Mauchly's test, similar to Example 2 (Kang et
al., 2015). Pairwise t-tests for group means were also reported.
Independent sample t-tests were used to make comparisons between
groups: i.e.; seizure burdens for P7 vs. P10. Western blot
quantifications were compared among treatment groups using One-way
ANOVAs. Correlation analyses were performed using non-parametric
comparisons (Spearman's test, two-tail). Error bars depicted in
figures represent mean.+-.SEM.
TABLE-US-00002 TABLE 1 Sample sizes of ligated pups in each
treatment- and age-group for EEG and WB experiments. EEG Western
Blot ANA12 ANA12 + PB ANA12 ANA12 + P7 Vehicle alone PB + BTN alone
P7 Vehicle alone PB + BTN Male 4 4 11 4 Male 3 4 7 Female 3 5 10 4
Female 2 4 9 Total 7 9 21 8 Total 5 8 16 ANA12 ANA12 + PB ANA12
ANA12 + P10 Vehicle alone PB + BTN alone P10 Vehicle alone PB + BTN
Male 4 5 9 7 Male 4 3 7 Female 4 6 12 5 Female 4 6 7 Total 8 11 21
12 Total 8 9 14
Results
[0069] Small-molecule TrkB antagonist ANA12, rescued PB-resistance
of seizures at P7 and improved PB-efficacy at P10. The effect of an
acute single post-ischemic dose of ANA12 on PB-resistant seizures
was evaluated at P7 and P10. PB administration at 1 h following
ischemic insult in the ANA12 treated group significantly reduced
the seizure burden at P7 and P10 (FIGS. 1B & D, FIG. 8:
repeated measures ANOVA). ANA12+PB significantly suppressed
seizures by 62% in P7 ischemic pups and by 85% in P10 ischemic pups
(FIGS. 2A & B, FIG. 8: One-way ANOVA). In the absence of ANA12,
PB failed to suppress seizures at P7, replicating the hallmark of
the age-dependent PB-resistance in the ischemic model (Example 2,
Kang et al., 2015). Similar to previous findings in the model, PB
by itself suppressed seizures by 68% (FIG. 2B, p=0.001) in P10
pups. ANA12+PB improved PB-efficacy to from 68% to 85%, however
this improvement was not significant (p=0.08). The seizure burdens
quantitated here were additionally evaluated by the number of ictal
events and the average ictal durations that constituted each
seizure burden. The data showed that the anti-seizure efficacy of
ANA12+PB at both ages was driven by a significant reduction in the
number of ictal events (FIGS. 9E & F). The mean ictal durations
did not change from baseline to the post-PB hour (FIGS. 9C-D and
G-H). Therefore, acute TrkB inhibition significantly rescued
PB-resistance at P7 and improved PB-efficacy at P10 by
significantly reducing the number of ictal events post-treatment.
Similar to Example 2 (Kang et al., 2015), PB-efficacy had no effect
on ictal durations.
[0070] The between-group comparison of vehicle vs. ANA12-alone
group did not differ significantly for any of the parameters of
baseline seizure burden, the number of ictal events, and mean ictal
duration at either P7 or P10 (FIGS. 1A & C). Additionally,
Fisher's exact test comparing post-treatment mortality rate did not
show a statistical significance (p=0.58 for P7, 1.00 for P10).
Based on these observations, the data for vehicle and ANA12-alone
group were pooled to form the "ligate control" group (n=15 and 19
for P7 and P10 respectively).
[0071] The efficacy of ANA12-alone as an anti-seizure agent was
evaluated. As expected, ANA12-alone without PB administration did
not have any anti-seizure effect at either P7 or P10. The baseline
seizure burdens of ANA12-alone group did not differ significantly
from that of vehicle group at either age (i.e., P7 p=1.00 and P10
p=0.63; One-way ANOVA). Additionally, ANA12 did not suppress the
baseline seizure burden prior to PB administration in the treated
groups at either age (FIGS. 1B & D). Therefore, an acute single
dose of ANA12, in the absence of subsequent PB administration,
failed to suppress ischemic seizures.
[0072] To evaluate whether vehicle or ANA12-alone had any effect on
seizure susceptibility, the latency to onset of ischemic seizures
following unilateral carotid ligation was evaluated in each pup.
Neither vehicle nor ANA12-alone significantly altered the seizure
onset latency. The effect of post-ligation drugs (saline, vehicle,
and ANA12) on the latency to the 1st-detected seizure were not
statistically different at either age tested: P7; saline
(321.7.+-.58.8 sec), vehicle (321.7.+-.171.3 sec), ANA12
(202.3.+-.38.8 sec), P10; saline (150.73 .+-.40.4 sec), vehicle
(164.4.+-.44.4 sec), ANA12 (245.8.+-.39.8 sec; One-way ANOVA: P7,
p=0.28; P10, p=0.30 respectively). The latency to seizures in each
treatment group did not significantly differ by age either. In
summary, vehicle and ANA12 did not have any significant effects on
age-dependent seizure susceptibility when evaluated by seizure
onset latency in the model.
[0073] The effect of adding an adjunct NKCC1 antagonist, BTN, was
also evaluated at the 3rd h of post-ischemia recording. BTN,
delivered 1 h after PB, failed to act as an efficacious
anti-seizure adjunct at either P7 or P10, similar to Example 2
(Kang et al., 2015). BTN did not reduce either the total seizure
burden or the number of ictal events (FIGS. 1B & D, white bars;
FIG. 8) at P7. BTN administration significantly blunted PB-efficacy
by increasing the seizure burden in P10 pups (FIG. 1D; white bar
p=0.01, FIG. 8). This aggravation occurred due to an increase in
the number of ictal events following BTN administration (FIG. 9F;
white bar p=0.02). Therefore, the blockage of NKCC1 after the
successful rescue of PB-resistance did not help enhance
PB-efficacy; instead, it resulted in an age- and sex-dependent
aggravation of PB-subdued seizures, replicating previous
observations in the model (Example 2, Kang et al., 2015).
[0074] ANA12 significantly reversed PB-resistance at P7 and
improved PB-efficacy at P10 in both sexes. The developmental
expression profile of chloride co-transporters has been reported to
be sexually dimorphic. To evaluate the role of biological sex in
the efficacy of ANA12, the data presented in FIG. 1B and D were
evaluated by sex. PB-efficacy was significant for both sexes at P7
and P10 (FIG. 3A-D, FIG. 10A-B and E-F; p<0.001 for all). In P7
pups, BTN significantly blunted ANA12+PB efficacy only in females
(FIG. 3B). At P10, BTN blunted ANA12+PB anti-seizure efficacy in
males but not in females (FIG. 10B; white bars). In summary,
PB-efficacy was not different by sex and age. However, the
BTN-associated aggravation of ischemic seizures was
sex-dependent.
[0075] Age- and sex-dependent seizure susceptibility to ischemic
seizures was unaltered by TrkB antagonist. The developmental
regulation of chloride gradients is one of the known mechanisms
that makes immature neurons hyper-excitable and more susceptible to
ischemic seizures compared to mature neurons. The ligate control
group displayed graded increase in the total seizure burden over
the 3 h window of EEG recording (FIG. 1B), which was statistically
significant at P7 but not at P10. Temporal increase of seizure
burden during acute post-ischemic window is one of the
characteristic features of the model (Kang et al., 2015). ANA12,
administered after the onset of ischemia did not alter the
significant age-dependent seizure susceptibility previously
documented in this model (Example 2, Kang et al., 2015). Total
seizure burden for ligate control group (i.e.; over 3 h duration
post-ischemia) was significantly higher in P7 (213%) pups compared
to P10 pups (p=0.01), thus replicating the characteristic feature
of the model (FIGS. 1A & C).
[0076] The ligate control and treated groups did not differ in
their baseline seizure burdens (i.e.; black bars FIGS. 1B & D)
at P7 or P10. Additionally, similar to the characteristics
previously noted for the model, a significantly higher seizure
susceptibility was observed in P7 male pups compared to P10 male
pups (p=0.001).This age-dependent susceptibility was not
significant (p=0.76) between P7 and P10 females (FIGS. 4A & B).
Therefore, the age- and sex-dependent seizure susceptibility to
ischemic seizures established in the model was not altered by
ANA12.
[0077] ANA12 followed by PB rescues the ischemic downregulation of
KCC2 expression at both P7 and P10. Approximately .about.20%
downregulation of KCC2 expression in the ipsilateral ischemic
hemisphere compared to the contralateral hemisphere was previously
documented in the model when evaluated at 24 h post-ischemia
(Example 2, Kang et al., 2015). The effect of TrkB inhibition on
post-ischemic KCC2 degradation was evaluated. Quantification of
KCC2 expression at 24 h after an ischemic insult indicated that the
post-ischemic degradation of KCC2 expression was significantly
prevented in the treated group at both P7 and P10 (FIGS. 5A &
C). At P7, both vehicle and ANA12-alone group exhibited KCC2
degradation that was approximately 15-18% lower compared to the
contralateral hemisphere; which was statistically significant
between ANA12-alone and the treated group (FIG. 5A; p<0.05). The
expression levels of KCC2 in the PB-resistance rescued group was
.about.110%, and therefore similar to the contralateral hemisphere.
At P10, both vehicle and ANA12-alone group displayed KCC2
degradation, while the treated group showed significant rescue.
However, the vehicle group showed .about.30% KCC2 degradation
whereas ANA12-alone group showed .about.12% KCC2 degradation,
compared to the contralateral hemisphere. The KCC2 downregulation
was only significant between the vehicle group and the treated
group (FIG. 5C). The ligate control group representing the vehicle
and ANA12-alone data pooled together similar to the seizure burden
data in FIGS. 1B & D, showed significant post-ischemic
degradation of KCC2 expression (16.2% and 20.0% for P7 and P10
respectively) when compared to the ANA12+PB treated group (p=0.04
and 0.002; P7 and P10 respectively). NKCC1 expression was also
quantitated in the same manner as it was done for KCC2, and no
statistically significant differences were detected amongst
treatment groups (FIGS. 5B & D). Additionally, the overall
percent NKCC1 change in the treated group was also not
significantly different by age (P7 vs. P10; One-way ANOVA p=0.28).
In summary, a single acute dose of ANA12, in the presence of
follow-on PB, significantly prevented the post-ischemic degradation
of KCC2 expression even at 24 h post-ischemia, and did not
significantly alter NKCC1 expression.
[0078] KCC2 expression levels were also evaluated acutely at 3 h
post-ischemia. An additional group of saline treatment was included
in this experiment, to establish the KCC2 expression in untreated
ischemic pups. The results were similar to the findings for the 24
h data. At P7, all groups except the ANA12+PB treated group
underwent approximately 15-20% downregulation of KCC2 expression
(FIG. 11A). A similar 20% loss of KCC2 surface expression has been
shown to decrease KCC2 function by 50%. The KCC2 expression levels
for saline group and ANA12-alone group significantly differed from
the treated group, but the vehicle group did not (One-way ANOVA;
p<0.05). At P10, the vehicle and ANA12-alone groups also
underwent degradation of KCC2, however no statistical significance
was detected when compared to the ANA12+PB treated group (FIG.
11C). Similar to the 24 h results, NKCC1 expression among groups
was not statistically different (FIGS. 11B & D).
[0079] Since severe seizures can independently degrade KCC2 in a
non-TrkB dependent pathway, a pilot experiment to evaluate the
efficacy of ANA12 to rescue PB-resistance after the occurrence of
status-like seizures was additionally conducted. In contrast to
experimental paradigm described in FIG. 1, ANA12 was administered 1
h after PB in P7 seizing pups (i.e.; 2 h after repetitive ischemic
seizures, PB+ANA12). The post-PB administration of ANA12 protocol,
failed to subdue the PB-resistant seizures at P7 (p=1.00; n=3).
Additionally, this treatment protocol also failed to rescue
post-ischemic KCC2 degradation when evaluated at 24 h (p=1.00; n=5)
similar to the vehicle-treated pups. These data indicate that the
TrkB-pathway-mediated KCC2 degradation (.about.20%) starts early
after ischemia and is irreversible by administration of ANA-12
after emergence of PB-resistance.
[0080] Correlation of PB-efficacy and KCC2 expression. Correlations
between seizure suppression and KCC2 degradation were evaluated to
determine whether the PB-efficacy was driven by the prevention of
KCC2 degradation. P10 pups showed a significant correlation between
efficacious seizure suppression and rescue of KCC2 expression at
P10 (FIG. 6D; p=0.04, r=0.6). This correlation was not significant
at P7 (FIG. 6A-D; p=0.35, r=0.2). This differential correlation may
be in part due to age-dependent upregulation of KCC2 in naive
brains during this developmental window (Example 2, Kang et al.,
2015). Naive P10 pups have been reported to have a higher
age-dependent expression of KCC2 compared to P7 (Example 2, Kang et
al., 2015). However, the percentage of KCC2 degradation following
ischemia at both P7 and P10 were similar (i.e.; .about.20%) and not
significantly different (One-way ANOVA; p=0.84). No significant
correlations between seizure suppression and NKCC1 expression were
detected at P7 or P10 (FIG. 6E-H), which was expected from the
inconsistent (FIGS. 6E & G) post-ischemic expression levels of
NKCC1 detected at both ages for all treatment groups (FIGS. 5B
& D and FIGS. 11B & D).
[0081] The efficacy of PB seizure suppression was significantly
dependent on the baseline seizure burden at P7 but not at P10
(Spearman's test: i.e.; P7; p=0.001, P10; p=0.30). P7 pups with
severe baseline seizure burden (i.e.; >1200/3600 sec) were less
responsive to ANA12+PB with a 54.9.+-.6.6% seizure suppression
(n=10, p<0.001) in contrast to their age-matched ligated-pups
with lower baseline seizure burden (<1200/3600 sec) that
responded with a 68.5.+-.8.3% seizure suppression to ANA12+PB
(n=11, p<0.001). Using this binning for moderate and severe
baseline seizure severity at P7 (i.e.; seizing for <or >1/3
of the total baseline hour), correlations between seizure
suppression and rescue of KCC2 degradation became evident. Ligated
pups at P7 that seized for less than 1/3rd of the hour before PB
administration, showed a significant positive correlation with
rescue of KCC2 degradation (p=0.02, r=0.8) similar to that detected
in P10 rescued pups. In contrast, in pups which seized for
>1/3rd of the hour, this correlation was not only lost
significance but became negative (p=0.31, r=-0.4). In summary, if
the baseline seizure burden was status-like at P7 (i.e.;
>1200/3600 sec), the ANA12+PB anti-seizure efficacy did not
correlate significantly with percent KCC2 expression (FIG. 6B). In
contrast, the percent change in the expression levels of NKCC1 did
not correlate significantly with the percent post-PB seizure
suppression (FIGS. 6F & H), and further binning by baseline
seizure severity did not reveal any significant correlations
either. Ligated pups at P10 responded significantly to PB
regardless of the severity of their baseline seizures burden. The
age-dependent seizure susceptibility and PB-resistance has been
previously reported and this study replicated that hallmark
characteristic for the model (Example 2, Kang et al., 2015).
Discussion
[0082] Using a small-molecule TrkB antagonist and a single systemic
(IP) dosing protocol, this study reports for the first time, the
proof-of-concept results for the pharmaco-modulation of KCC2
expression as a novel strategy for preventing the emergence of
PB-resistant seizures in neonatal ischemia. The salient findings
are: 1) a single post-insult dose of TrkB antagonist, ANA12,
significantly rescued the PB-resistance of ischemic seizures at P7;
2) the combination of a single dose ANA12+PB at 1 h, not only
prevented the acute post-ischemic degradation of KCC2 expression at
3 h but also maintained age-dependent KCC2 expression 24 h later;
3) the TrkB antagonist by itself could neither prevent the
occurrence of ischemic seizures nor rescue post-ischemic KCC2
degradation, suggesting that PB-resistant seizures can
independently degrade KCC2 by non-TrkB dependent pathways; 4) At
P10, ANA12 helped improve PB-efficacy; and 5) NKCC1 antagonist BTN
failed to further improve PB-efficacy as an adjunct therapy at both
ages. These findings highlight the pivotal role of KCC2 in seizure
susceptibility and the emergence of PB-resistant seizures in
neonatal ischemia. Therefore, transient pharmaco-modulation of KCC2
with the goal to restore the developmental profile of KCC2
upregulation may be a promising strategy for preventing the
emergence of PB-resistant seizures in HIE.
[0083] A single dose of ANA12 reversed PB-resistance at P7.
Age-dependent PB-resistance of ischemic seizures was the hallmark
of the neonatal ischemic seizure model used here (Example 2, Kang
et al., 2015). ANA12 successfully reversed PB-resistance at P7 at a
dose of 2.5 mg/kg. Therefore, the inability of old-generation TrkB
antagonists crossing the blood brain barrier was successfully
circumvented by the small molecule TrkB antagonist, ANA12. The
brain bioavailability of ANA12 after systemic injection, examined
in adult mouse brains indicates that ANA12 administered IP at 0.5
mg/kg dose was active as early as 0.5 h [400 nM] up to 6 h [10 nM]
(Cazorla et al., 121 J. CLIN. INVEST. 1846-57 (2011)).
Additionally, a single dose of 0.5 mg/kg ANA12, in the same study,
suppressed the ratio of phospho-/total TrkB activity in the whole
brain by 8% at 2 h and 25% at 4 h. The post-insult transient
opening of blood brain barrier in the immature brain has been shown
to result in the increased bioavailability of small and large
molecules, and may suggest a similar enhancement in the
pharmacokinetics of the single dose ANA12 given acutely in this
model of neonatal ischemia. In summary, acute TrkB inhibition by a
single dose of ANA12, achieved significant rescue of PB-resistance
even when PB was given 1 h after the occurrence of severe recurrent
seizures.
[0084] ANA12 required 5% DMSO to stay in solution. DMSO can
independently act as an anti-epileptic drug by modifying brain
bioelectric activity, specifically at above 50% concentration but
not at below 10%. To evaluate the anti-seizure effect of 5% DMSO
used as vehicle for ANA12 in this study, a separate
vehicle-treatment group of 5% DMSO was investigated. 5% DMSO
administered following ischemia did not have any anti-seizure
effect in this study, and the baseline seizure burdens remained
unaltered and similar to Example 2 (Kang et al., 2015).
[0085] A single dose of ANA12 with PB prevented both early and late
downregulation of KCC2 expression after ischemia. The BDNF-TrkB
pathway has been shown to be responsible for excitoxicity related
degradation of KCC2 in in vitro experiments. This degradation has
been shown to occur within minutes of an ischemic insult in vitro.
BDNF-TrkB pathway has also been implicated in epileptogenesis,
neuropathic pain, and psychiatric disorders. Ischemic insults
resulted in the downregulation of KCC2 expression levels in several
studies. Our previous studies have shown that neonatal ischemia
resulted in a transient downregulation of KCC2 expression that
recovered over the period of a few days and caught up with the
age-dependent KCC2 upregulation occurring during this developmental
window (Example 2, Kang et al., 2015). Similar KCC2 degradation has
also been reported to occur hours after the onset of status
epilepticus. However, those studies used chemoconvulsants to induce
seizures that also resulted in an early upregulation of KCC2
similar to a hypoxia-only model of neonatal seizures. The mechanism
underlying this early upregulation of KCC2 was not explored by
either group. However, recent research has reported that the
kainate-dependent upregulation of KCC2 was a homeostatic response
to increased neuronal firing during early seizures and occurs via
the ERK1/2 signaling cascade that may help improve inhibition.
[0086] In contrast, when status epilepticus continued to occur over
a period of >240min in the Puskarjov et al. study (Puskarjov et
al., 88 NEUROPHARMACOLOGY 103-09 (2015) (Epub 2014 Sep. 16, doi:
10.1016/j.neuropharm.2014.09.005), KCC2 degradation via the
calpain-mediated pathway became evident. These findings indicate
that early KCC2 homeostatic upregulation may fail in etiologies
associated with energy deprivation like ischemia. Additionally,
after severe and repeated seizures have occurred, seizures alone
may degrade KCC2 function through non-TrkB mediated pathways.
However, our results also indicate that if the PB-resistant
seizures are subdued early by blocking TrkB, the late onset KCC2
degradation can also be prevented.
[0087] Repeated seizures can independently lead to KCC2
degradation. Prevention of KCC2 degradation only occurred in the
presence of both TrkB inhibition and efficacious seizure
suppression. In contrast, the vehicle and ANA12-alone groups both
underwent significant KCC2 downregulation in the ipsilateral
hemisphere (FIG. 5) similar to untreated pups (Example 2, Kang et
al., 2015). ANA12-alone failed to prevent the post-ischemic KCC2
degradation in pups where ischemic seizures continued. This
indicates that prolonged recurrent seizures are capable of
degrading KCC2 expression likely through a non-TrkB mediated
pathway. Calpain-dependent downregulation of KCC2 had been shown to
occur following 3 h of status epilepticus induced seizures. Calpain
is a calcium-sensitive protease which is activated in conditions
associated with long duration status epilepticus. Similarly, the
failure of ANA12 to improve PB-efficacy when given 1 h after PB
showed that when many repeated ischemic seizures have already
occurred (i.e.; 2 hours of repetitive post-ischemic seizures),
ANA12 failed to reverse KCC2 downregulation even in the presence of
PB, indicating that TrkB pathway related KCC2 degradation starts
early after ischemia. Both of these observations support the
previous conclusions that severe seizures are independently
detrimental in ischemia and can lead to KCC2 downregulation
initiated by both TrkB and non-TrkB dependent pathways. Since the
calpain pathway has been shown to be triggered later during a
severe status-like seizure cascade in a slice culture model and in
acute slices derived from mice after kainate induced status, it may
be temporally different from the BDNF/TrkB pathway in the sequence
of initiation post-insult. Inability of calpain inhibitors to cross
the BBB is currently prohibitive to test the hypothesis in an in
vivo model.
[0088] At P10, with the age-dependent lower seizure burden as
compared to P7, and developmentally higher expression profile of
KCC2 in naive brains, the ANA12-alone group showed a trend towards
the recovery of KCC2 expression compared to the vehicle groups at
both 3 h and 24 h, however it was not statistically significant.
This indicates that TrkB inhibition, in the absence of PB, may be
able to marginally rescue KCC2 degradation by itself at P10 but not
at P7. This difference may be due to the age-dependent
susceptibility to ischemic seizure burden and the developmental
profile of KCC2 upregulation (i.e.; P10>P7).
[0089] Correlation between rescue of KCC2 downregulation and
efficacious seizure suppression. The results of this study
indicated that when seizures were efficaciously suppressed, there
was an associated significant rescue of KCC2 degradation at both P7
and P10 (FIG. 6). This was not true for NKCC1, highlighting the
critical role of Cl- extrusion played by KCC2 in determining the
efficacy of GABAA agonists like PB. However, it is important to
note that the currently available NKCC1 probes can only detect the
non-dominant isoform NKCC1a. The dominant isoform NKCC1b expressed
in the brain is a spice variant with missing exon 21 that remains
undetected with currently available probes. Unlike NKCC1a, NKCC1b
has been shown to have stable expression throughout development and
adulthood in human brains. These findings from human brain
research, indicate that the previous interpretations related to the
developmental downregulation of the immature form Cl- transporter,
NKCC1 with age, in animal research models may not be accurate. A
reliable pan-NKCC1 probe is not currently available, but is an area
of active research. Therefore, in the absence of a probe capable of
detecting simultaneous NKCC1a and NKCC1b expression levels,
interpretations related to the expression profile of NKCC1 and the
BTN-inefficacy replicated in this study, remain elusive. In
contrast, the KCC2 probe used in this study, detects both brain
isoforms with rigorous specificity (Williams et al., 1999). In
addition, KCC2 protein can stay internalized in cytosolic vesicles
or remain inactive in the plasmalemma, therefore, additional
strategies to increase the membrane insertion kinetics of KCC2
proteins may also be an appropriate alternative to test the
proof-of-concept approach investigated here.
[0090] The significant positive correlation of seizure suppression
to rescue of KCC2 degradation at P10 was absent at P7. However,
when data were binned by baseline seizure severity (<or
.gtoreq.1200 sec), there was a strong positive correlation of the
rescue of KCC2 degradation to the severity of the baseline seizure
burden before PB was administered at P7. This indicated that when a
P7 ligated pup seized for >1/3rd of the first hour (i.e.;
status-like severity) before PB administration, non-TrkB related
pathways like calpain-mediated KCC2 degradation, may get initiated
earlier, weakening the significant correlation detected in pups
with relatively lower seizure burdens. This observation highlights
the important role of seizure severity in the emergence of
refractoriness. Severe repeated seizures can degrade KCC2 by
multiple pathways. Therefore, both the etiology and severity of
seizures dictate the pharmacological consequences in neonates.
[0091] Role of KCC2 in brain development and long-term sequelae of
transient KCC2 down-regulation. KCC2 is known to play a role in
early development, spine formation, interneuron migration, and
synaptogenesis, which may occur independently to KCC2's function as
a Cl- extruder. KCC2 is robustly expressed in the dendritic spines
of cortical neurons. KCC2 also plays a crucial role in development
of interneurons and their response to injury. The stronger
tolerance of CA1 interneurons to ischemic injury compared to
pyramidal cells reported in the hippocampus, may suggest that the
higher density of KCC2 in CA1 interneurons may drive the
exceptional resistance of parvalbumin-positive interneurons to
excitotoxic injury associated with ischemia. Ischemic insults can
result in long-term damage to interneurons that become evident
after months. The transient ischemia-related KCC2 degradation
during early development detected in this model may have
neurological consequences that are not evident in the acute stages
but may manifest as late-onset co-morbidities.
[0092] A study conducted in human brains has reported that KCC2
expression was significantly lost in the brain samples of preterm
infants with white matter damage. Therefore, KCC2 degradation may
be a common pathology associated with perinatal brain insults that
are excitotoxic. Pharmaco-modulation of KCC2 with the goal of
restoring normal expression levels seems to be a viable path
forward not only for acutely preventing the emergence of
PB-resistant seizures in neonates as shown in the results but also
possibly preventing the long-term neurodevelopmental deficits.
These hypotheses require follow-on long-term studies. The known
action of ANA12 as an anti-depressant and anxiolytic in rodent
studies supports the further evaluation of the long-term effects of
ANA12 following neonatal use in this model. The prevention of KCC2
degradation documented in this study should be clearly
differentiated from KCC2 overexpression, because upregulation of
KCC2, above its normal developmental expression levels, that is not
physiological, may lead to a precocious maturation of neuronal
circuits in immature brains. Ischemic seizures are transient during
the neonatal period, and therefore a single acute intervention to
prevent KCC2 degradation similar to that reported here during the
critical stage may be sufficient.
[0093] Blocking NKCC1 fails to improve PB-efficacy. The efficacy of
BTN as an adjuvant anti-seizure agent has been controversial. The
recent termination of NEMO clinical trial [NCT01434225] reported
the inefficacy of BTN for HIE seizures. Our recent studies have
also reported a similar BTN inefficacy for acute ischemic seizures
and detected an age- and sex-specific aggravation of PB-subdued
seizures in P10 CD1 pups (Example 2, Kang et al., 2015). These
findings were replicated in the current study. In spite of the
improved PB-efficacy elicited by acute TrkB inhibition, BTN still
failed to act as an adjunct therapy. The expression level of NKCC1a
was not significantly altered regardless of the treatment paradigm
or anti-seizure efficacy, further supporting the importance of KCC2
rather than NKCC1 in regulating Cl- gradient and modulating
seizures. BTN has also been reported to have poor brain
bioavailability and short half-life. However, recent studies
evaluating the efficacy of a BTN pro-drug designed to have improved
brain bioavailability have reported no clear anti-seizure effect.
BTN also has been shown to have non-safety issues associated with
ototoxicity detected in the NEMO trial, adding to an unfavorable
risk-benefit ratio as an adjuvant in neonates. NKCC1 serves a
critical function in the circuit formation during early
development, and is also expressed systemically. The long-term
side-effects to blocking NKCC1 function during development,
although not fully investigated, indicate potential pitfalls. In
this study, transient inhibition of BDNF-TrkB pathway with a single
dose of ANA12 allowed for efficacious treatment of PB-resistant
seizures by preventing degradation of KCC2 in a model where BTN was
shown to be inefficacious (Example 2, Kang et al., 2015). The goal
of preventing KCC2 degradation was to help maintain the
developmental expression profile of KCC2 and prevent the emergence
of refractoriness. Helping maintain KCC2 function during ischemia
or preventing insult-related KCC2 degradation during development
may have the additional potential benefit of preventing long-term
co-morbidities associated with the transient loss of KCC2 function
during this critical period.
[0094] Conclusion This study reports the successful rescue of
PB-resistance associated with the rescue of KCC2 degradation in a
model of neonatal ischemic seizures. Pre-clinical studies of
neonatal seizures utilizing various translational models have
reported: a) differential alteration of KCC2 expression, and b)
differential severity of the seizure burdens in response to various
seizure induction methods. The main findings of this study suggest
a critical association between the anti-seizure efficacy of
GABA-agonists and post-ischemic KCC2 degradation. An ongoing debate
in clinical management of neonatal seizures is whether they can
independently harm the developing brain and how aggressively do
they need to be managed?When compared to the results from other
pre-clinical models, studies in this model suggest that the
etiology and severity of neonatal seizures dictate their
deleterious effects. Therefore, severe seizure burdens commonly
reported in HIE may independently cause KCC2 degradation that leads
to the emergence of refractory seizures. This study highlights the
potential of KCC2 pharmaco-modulation as a novel therapeutic
strategy in treating PB-resistant seizures in neonates. The
non-efficacy and safety issues that have arisen from the previous
strategy of antagonizing NKCC1 using BTN as an adjunct therapy in
HIE was based on results using non-ischemic pre-clinical models.
The novel approach proposed here uses a translationally relevant
pre-clinical model for HIE with comparable seizure burdens which
also demonstrates non-efficacy of BTN for ischemic seizures. The
model therefore, highlights the importance of methods of seizure
induction and associated seizure burdens in pre-clinical
modelling.
Example 2
Age- and Sex-Dependent Susceptibility to Phenobarbital-Resistant
Neonatal Seizures: Role of Chloride Transporters
[0095] Ischemia in the immature brain is an important cause of
neonatal seizures. Temporal evolution of acquired neonatal seizures
and their response to anticonvulsants are of great interest, given
the unreliability of the clinical correlates and poor efficacy of
first-line anti-seizure drugs. The expression and function of the
electroneutral chloride co-transporters KCC2 and NKCC1 influence
the anti-seizure efficacy of GABA.sub.A-agonists. To investigate
ischemia-induced seizure susceptibility and efficacy of the
GABA.sub.A-agonist phenobarbital (PB), with NKCC1 antagonist
bumetanide (BTN) as an adjunct treatment, we utilized permanent
unilateral carotid-ligation to produce acute ischemic-seizures in
postnatal day 7, 10 and 12 CD1 mice. Immediate post-ligation
video-electroencephalograms (EEGs) quantitatively evaluated
baseline and post-treatment seizure burdens. Brains were examined
for stroke-injury and western blot analyses to evaluate the
expression of KCC2 and NKCC1. Severity of acute ischemic seizures
post-ligation was highest at P7. PB was an efficacious anti-seizure
agent at P10 and P12, but not at P7. BTN failed as an adjunct, at
all ages tested and significantly blunted PB-efficacy at P10.
Significant acute post-ischemic downregulation of KCC2 was detected
at all ages. At P7, males displayed higher age-dependent seizure
susceptibility, associated with a significant developmental lag in
their KCC2 expression. This study established a novel neonatal
mouse model of PB-resistant seizures that demonstrates
age/sex-dependent susceptibility. The age-dependent profile of KCC2
expression and its post-insult downregulation may underlie the
PB-resistance reported in this model. Blocking NKCC1 with low-dose
BTN following PB treatment failed to improve PB-efficacy.
Introduction
[0096] Neonatal seizures are the most frequent clinical
manifestation of central nervous system dysfunction in newborns,
with an incidence of 1.5-3.5/1000 in term newborns, and an
incidence as high as 10-130/1000 in preterm newborns. Ischemia is a
major cause of neonatal seizures and first-line anticonvulsant
pharmacotherapy by commonly used anti-seizure drugs like PB often
proves insufficient. Both animal-model and human studies suggest
that neonatal seizures themselves may worsen brain injury, decrease
the threshold for subsequent seizures, and result in poor long-term
neurological co-morbidities. Electroclinical dissociation is now a
fairly well-accepted concept in human neonates, wherein GABA
agonists are able to block the clinical manifestations of seizures;
however, as displayed with electrographic monitoring, the brain
continues to seize.
[0097] Early in development, the depolarizing GABA.sub.Aergic
signaling that is instrumental in normal neuronal differentiation
and migration has been shown to be responsible for the inefficacy
of GABA.sub.A agonists like PB, as an anti-seizure agent.
Therefore, cation chloride co-transporters, specifically NKCC1 and
KCC2, could be used as potential targets for novel anti-seizure and
anti-epileptogenic treatments. NKCC1 is expressed in neurons and
astrocytes throughout the brain, systemically in the kidney and
inner hair cells of the ear, and is robustly involved in early
neural development. The co-transporter KCC2 is CNS-specific
predominantly in neurons and has a developmental expression profile
that increases exponentially during the third trimester and
continues to increase postnatally with advancing age.
[0098] BTN, a potent NKCC1 antagonist that has been used as a
diuretic in newborns, was also shown to be effective in reducing
kainic acid-induced and hypoxic seizures in neonatal animals by
blocking NKCC1 especially when used in combination with PB. BTN
later became the focus of clinical trials to test its efficacy in
seizing neonates with hypoxic ischemic encephalopathy. However in
pre-clinical studies, the anti-seizure effect of BTN has been shown
to depend on the experimental model. BTN enhanced, suppressed, or
had no effect on paroxysmal activity in vitro in different models,
none of which modeled ischemia. Additionally, a recent study showed
that kainic acid-induced seizures increased the surface expression
of KCC2, shifting E.sub.(GABA) close to the adult levels. However,
under ischemic conditions, a substantial neuron-specific
downregulation of KCC2 expression has been reported, which was also
detected in our mouse model of neonatal ischemia. Such discordant
model-specificity in the post-injury KCC2 expression may
significantly alter the efficacy of drugs that depend on the
Cl.sup.- gradient for their anti-seizure effects. In new
developments, the European clinical trial has recently been
terminated for non-efficacy of BTN with associated ototoxicity
following hypoxic ischemic encephalopathy (HIE) induced seizures in
neonates.
[0099] This study utilized cerebral ischemia alone to induce acute
ischemic seizures in the CD1 mouse strain, and investigated the
following: 1. Quantitative analyses of EEG recordings of early
post-stroke events at P7, P10, and P12 to determine the acute
age-dependent seizure susceptibilities and seizure burdens
following ischemia. 2. Response of the ischemic seizures to
standard anti-seizure agent PB, as well as novel agent BTN at doses
similar to the clinical trials, to evaluate for the first time, the
effect of the NKCC1 blocker BTN, as an adjunct in a model of acute
ischemic seizures. 3. Effect of neonatal ischemia on the
developmental expression profile of KCC2 and NKCC1.
Materials and Methods
[0100] Experimental design. This study was carried out in strict
accordance with the recommendations in the Guide for the Care and
Use of Laboratory Animals of the National Institutes of Health. The
protocol was approved by the Committee on the Ethics of Animal
Experiments of the Johns Hopkins University (Permit Number:
A3272-01). All surgery was performed under isoflurane general
anesthesia, and all efforts were made to minimize suffering. All
litters of CD1 mice were purchased from Charles River Laboratories
Inc. (Wilmington, Mass.). Newly born litters of pups arrived at
postnatal 3 or 4 days old, and were allowed to acclimate. Animals
were housed in polycarbonate cages on a 12 h light-dark cycle and
food provided ad libitum. Ninety two pups from 12 litters were
included in the video-EEG study (n=48 male and n=44 female). The
susceptibility to ischemic seizures was tested at 3 developmental
ages i.e., 7, 10 and 12 days old CD1 pups (n=80 ligated and 12
shams). The experimental paradigm is depicted in FIG. 12. The pups
underwent permanent ligation and sham surgeries followed by 3 h of
EEG recording each, as described in methods (total pups=92 of which
ligates P7=29, P10=24 and P12=27 and sham P7=5, P10=4 and P12=3;
see Table 2). From the ligated group of pups (total n=80), the
ligated-control pups also underwent 3 h EEG recordings to evaluate
natural progression of ischemic seizure burden over the duration of
treatment efficacy evaluated in this study (total pups n/n=26/80,
P7=8, P10=9 and P12=9). Following ligation, the ligated-treated
group of pups [total pups n/n=54/80; P7=20 (13 male & 7
female); P10=16 (8 male & 8 female) and P12=18 (9 male & 9
female)] were used to evaluate every baseline EEG (i.e., 1.sup.st h
of recording) that was then compared to post-PB (2.sup.nd h of
recording) and post-BTN (3.sup.rd h of recording) EEGs. Since the
severity of stroke-injury and seizure varies between pups, the
baseline EEG of each pup served as an important internal control.
Additionally, since the exact time-point of onset for ischemia in
human neonates is not known and may not be a single massive event,
the 1 h of non-treatment also served as an important delay expected
in the treatment of neonatal seizures that are detected hours after
stroke in humans. There was an age-dependent mortality associated
with survival to age P18 following the ischemic seizures in this
study [i.e.; 8 out of 29 pups at P7 (2 males and 6 females); 6 out
of 24 pups at P10 (3 males and 3 females); and none at P12].
[0101] Surgical procedure for ischemic insult and electrode
implantation. Animals were anesthetized with 2% isoflurane. The
right common carotid artery was permanently double-ligated with 6-0
surgisilk and the outer skin closed with 6-0 monofilament nylon to
induce ischemia (i.e.; ischemia was induced by unilateral common
carotid ligation alone. Unlike other rat models commonly used for
neonatal stroke, no global hypoxia is needed to induce acute
ischemic seizures). Since the ligated carotid is not transected in
our model, the intact pulsating carotid artery with silk ligatures
can lead to reperfusion over time in this model. The constrictive
efficacy (which drops cerebral perfusion to 40% or less) of the
silk ligatures is known to diminish, due to the loss of their
tensile strength in-vivo against the pulsating artery as a function
of time. Sham-control animals were treated identically except for
the carotid ligation, and did not seize (not shown). The animals
were then implanted with three sub-dermal EEG electrodes (1
recording, 1 reference, and 1 ground) on the skull overlying the
parietal cortex using bregma as a reference. Scalp wire electrodes
made for sub-dermal use in humans (IVES EEG; Model # SWE-L25-MA,
USA) were implanted, sub-dermally fixed with adhesive, in the pups.
The suggested spacing for human scalp electrodes is 5-8 mm, which
allows for optimal acquisition of EEG signal from mouse brains. In
this study, the recording and reference electrodes were implanted
less than 1 cm apart. Pups were then allowed to recover from
anesthesia in a 36.degree. C. isothermal chamber for 3 h recording
of video-EEG (FIG. 12). At the end of the recording session, the
pups were returned to the dams after removal of the sub-dermal
electrodes and application of local anesthetic medication.
[0102] In vivo synchronous video-EEG recording. After the
completion of the surgical procedures (i.e.; ligation+electrode
implantation (8min+8min.about.16min total), the EEG recordings were
initiated after the pups recovered from anesthesia. Bumetanide
(0.1-0.2 mg/kg dissolved in 100% alcohol, aliquoted and stored at
-20.degree. C.; protocol similar to previous study, phenobarbital
(25 mg/kg dissolved in 0.9% sodium chloride, made on the day of
experiment), or 0.9% sodium chloride was injected intraperitoneally
(IP) after baseline post-stroke EEGs were recorded (FIG. 12C).
Since these experiments were also designed to test BTN-efficacy as
an adjunct therapy to PB (based on the currently recruiting
clinical trials), ligated mice underwent one of two regimens: 1)
Treatment with saline only at 1 h and 2 h post-surgery; 2)
Treatment with PB at 1 h and BTN at 2 h post-surgery. The treatment
regimen of BTN before PB or BTN without PB was not relevant to this
translational design. Pharmacokinetics of PB and BTN additionally
supports our treatment regimen, because PB is a long acting drug
even in immature rodents [half-life .about.15 h whereas BTN has a
very short half-life of <30 min]. Data acquisition was done
using PAL-8400 software with synchronous video capture (Pinnacle
Technology Inc.). The data acquisition and conditioning system had
a 14-bit resolution, sampling rates of 500 Hz, high pass filters of
0.5 Hz and low pass filters of 1 kHz. The files were stored in .EDF
format and scored in real time using the review and scoring
software package. Manual scoring of all EEG files was done blinded
to treatment protocols by simultaneously scoring EEG traces and the
synchronous video in real time. Seizure burden scoring was done on
EEG with sampling rates of 400 Hz that had a pre-amplifier gain of
100. The filters of 1 Hz high-pass and 60 Hz low-pass were used to
remove ambient noise, and the binning was done in 10 sec intervals.
Seizures were defined as electrographic ictal events that consisted
of rhythmic spikes of high amplitude, diffuse peak frequency of
.gtoreq.7-8 Hz (i.e.; peak frequency detected by automated spectral
power analysis) lasting more than 6 seconds. Short duration burst
activity lasting <6 seconds (brief runs of epileptiform
discharge) was not included for seizure burden calculations in this
study.
[0103] Behavioral seizure scoring after EEG seizure detection.
After an EEG seizure was detected, its time and duration of
occurrence were noted, and the synchronous behavioral activity
recorded on video was scored according to a seizure rating scale
modified for mouse pups from a previously reported scale used for
adult mice. Behavioral seizures recorded on video for
EEG-identified seizures (FIGS. 13 A, B and C) were scored as
follows: 0=motionless/inactive; 1=flexor spasms; 2=jittery
movements; 3=repetitive grooming/scratching, circling towards side
of ischemia, with head bobbing; 4=limb clonus, unstable posture;
5=mice that exhibited level four behaviors for >30 seconds or
with loss of posture; and 6=severe tonic-clonic behavior with
inability to regain loss of posture. After video-EEG recording, the
mice were returned to the dam and littermates. Electrographic
seizures associated with grade 0-2 behaviors were grouped as
non-convulsive seizures and grade 3-6 behaviors were graded as
convulsive seizures.
[0104] Histology. All animals were anesthetized with chloral
hydrate (90 mg/ml; IP) before being transcardially perfused with
saline and 10% formalin in phosphate buffer (pH 7.4). The whole
brain was removed and submerged in the same fixative. The brains
were cryoprotected by first immersing in 15% sucrose for 24 h,
followed by 30% sucrose for 24 h. The brains were rapidly frozen
using dry ice and placed in -80.degree. C. storage. Coronal brain
sections (40 .mu.m thick) were cut on a cryostat in serial order to
create 5 series of sections and mounted on super frost plus glass
slides. One series of sections from the EEG recorded pups was
cresyl violet (CV) stained to quantitate ischemia injury using a
previously described method of computer-assisted comparison of
brain tissue area in ipsilateral versus contralateral hemispheres
of fixed CV stained mouse brain sections (i.e., Basic MCID). Stroke
injury seventies were quantitated at P18 for all brains processed
for CV (n=49/80; P7=15, P10=15 and P12=19) to compare differences
in infarct injury evolution between age groups. The remaining
brains from study were fresh frozen for western blot analysis.
[0105] Immunohistochemistry with triple labeling: Serial sections
from frontal and parietal cortex and dorsal hippocampi from a
separate cohort of naive CD1 pups aged P3 to P22 were labeled with
neuronal marker NeuN (1:100, Chemicon International; Catalog #
MAB377) and then processed for double labeling with NKCC1 [1:100,
Chemicon International: detecting 22 amino acid peptide sequence
near the C-terminus (exon 21); Catalog #AB3560P] and KCC2 (1:200,
Upstate: targeting N-terminal His-tag fusion protein corresponding
to residues 932-1043; Catalog #07-432). The NKCC1 antibody used in
this study is similar to those used in related published literature
and is incapable of detecting NKCC1b, due to the
post-transcriptional splicing of exon 21 that overlaps with the
targeted epitope site for the antibody. Currently no pan-NKCC1
(i.e.; splice isoforms a and b) antibodies are available. To block
nonspecific binding, sections were first incubated for 1 h at room
temperature (RT) in a solution containing 0.2% Triton X-100 and 10%
normal goat serum in PBS (Invitrogen). The sections were incubated
with primary antibodies overnight at 4.degree. C. After 3.times.10
min wash in PBS, slides were incubated at RT with secondary
antibodies (Alexa flour 488 and 594). After secondary antibody
incubation, sections were washed 3.times.10 min in PBS and
cover-slipped with an anti-fade medium for further image processing
(Axiovision, Zeiss). Confocal microscopy of FV 1000 system with
Olympus IX81 inverted microscope stand was used to acquire images
of immuno-stained sections. Z-stack images with 5 um step-size for
bilateral cortices was obtained from coronal sections cut at 40um
thickness. The Z-stacks were fused to obtain the final images.
[0106] Western blots. All animals for immunochemical
characterizations were anesthetized with chloral hydrate (90 mg/ml;
IP) before being transcardially perfused with ice-cold saline. The
whole fresh brains were removed, separated into left and right
cerebral hemispheres and frozen in liquid nitrogen and stored at
-80.degree. C. in preparation for further processing. Brain tissue
homogenates were made and suspended in RIPA buffer containing one
Complete Mini, Ethylenediaminetetraacetic acid (EDTA)-free protease
inhibitor cocktail tablet (Roche Indianapolis; Catalog #
04693159001) per 10 mL of buffer. Total protein amounts were
measured using the Bradford protein assay (Bio-Rad) and samples
diluted for equal amounts of protein in each sample. 50 ug of
protein samples were run on 4-12% gradient SDS gels (Bio-Rad) and
transferred onto polyvinylidene difluoride (PVDF) membranes.
Membranes were blocked for 1 h in odyssey buffer before overnight
incubation in primary antibodies, NKCC1 and KCC2. Blots were then
incubated for 1 h in secondary antibodies (Licor; IR Dye 800CW
Donkey anti-Rabbit IgG: Product # 926-32213 and IR Dye 680LT Goat
anti-Mouse IgG: Product # 926-68070). Protein bands were visualized
by chemiluminescence, using the Odyssey infrared imaging system 2.1
(LI-COR biosciences). The optical density of each sample was
normalized to the level of expression of the actin run on each
blot, for each antibody for statistical analysis. KCC2 and NKCC1
expression profiles in ipsilateral ischemic hemispheres were
normalized to contralateral non-ischemic hemispheres in the same
brain homogenized samples to compare differential effects of
ischemic injury in the model [n=3 for each age group represented in
FIG. 18B; n=2 for each age/sex in FIG. 18C; n=3 for each time-point
group shown in FIG. 19 A-C; n=13 in FIG. 19D (n pooled for
time-points 6-48 h post-ischemia].
[0107] Statistics. Group means for seizure severity between treated
and control mice were analyzed with independent sample student's
t-tests. Treatment efficacy following PB+BTN administration for
seizure burdens was analyzed using repeated measures one-way ANOVAs
followed by pairwise t-tests. Mauchly's test was used to confirm
the equal variances of the differences detected in repeated
measures ANOVA, which is known for testing a statistical assumption
of sphericity. No significance detected in Mauchly's test indicates
that the assumption of sphericity for the relevant F-test in
repeated measure ANOVA is not violated. Non-parametric correlations
among seventies of infarct lesion, seizure counts, and seizure
frequency (both electrographic and behavioral) were assessed by
Spearman's test. Differences with p<0.05 were considered
statistically significant.
Results
[0108] Age-dependent susceptibility to ischemic seizures and
response to anticonvulsants. EEG recordings in the post-ligation
period (FIG. 13) revealed ictal events along with interictal spikes
and non-convulsive epileptiform discharges between ictal events
(i.e., short duration bursts lasting 2-5 seconds). Ictal events on
EEG (i.e., lasting >6 sec) associated with video correlates
where pup was seen to be motionless or inactive (grade 0) or ictal
events with behavioral correlates of grade 1 or 2 [i.e.; scale 1-6]
were graded as non-convulsive (since these behaviors are also seen
in naive littermates), as described in methods. The baseline acute
ischemic seizures recorded in the 1'' post-ischemic hour were
significantly more severe at P7 (ligated control and
ligation-treated group pooled) compared to P10 and P12 (FIGS. 13D
& G black bars, p<0.0001 between P7 vs. P10 and P7 vs. P12).
The ligated-control group of P10 pups showed a significant decrease
in the number of ictal events in the 3rd h of EEG recording (FIG.
13E) not seen at P12.
[0109] Ligated-treated pups using the same 3 h recording paradigm
with PB (25 mg/kg) treatment at 1 h post-ligation and BTN (0.1-0.2
mg/kg) as an adjunct 2 h post-ligation followed similar trends for
baseline EEG severities as the ligated-control group (FIG. 13G, H
and I--black bars). However, interesting age-dependent drug
efficacies were detected within the treated group of pups. At P7,
PB and BTN both failed to have any significant anti-seizure effect
on total time spent seizing on EEG (FIG. 13G). PB, however, did
modulate the ischemic seizures by significantly reducing the number
of ictal events in the 2nd h recording (FIG. 13H, p=0.04). The lack
of overall PB-efficacy as an anti-seizure therapy resulted from a
sustained increase in the duration of post-treatment ictal events
(FIG. 13I) that was not seen in the ligated-control group (FIG.
13F). Adjunct BTN administration did not improve PB-efficacy, and
the longer ictal durations persisted at P7. In contrast, at P10, PB
administration 1 h after ligation, significantly reduced the
seizure burden by 61% (FIG. 13G, gray bar) by significantly
reducing the number of ictal events (FIG. 13H, gray bar). No
significant effect on the duration of the ictal events was detected
(FIG. 13I, gray bar). Follow-on BTN administration, failed to
improve PB-efficacy, and additionally PB-induced seizure
suppression was lost at P10 (FIG. 13G). This significant
aggravation of seizures was driven by an increase in the number of
ictal events (FIG. 13H) following BTN IP injection. At P12, post-PB
seizure suppressions were similarly effective compared to P10. PB
treatment after 1.sup.st h significantly reduced the seizure burden
by 64% due to a significant reduction in overall number of ictal
events. BTN again failed to act as an effective adjuvant as it did
not improve PB-efficacy (FIG. 13G).
[0110] When ligated-control and ligated-treated groups were
directly compared to each other by pairwise t-tests, no significant
difference was detected among the different groups for baseline
seizure burden at P7 (p=0.67, black bars FIG. 13D compared to 13G),
P10 (p=0.9, black bars FIG. 13D compared to 13G), or at P12 (p=0.1,
black bars FIG. 13D compared to 13G). Additionally, at P7,
post-treatment seizure burdens in ligated-treated group were not
significantly different from the ligated-control group in the
2.sup.nd and 3.sup.rd hour of recordings [P=0.6 (gray bars) and 0.6
(white bars) respectively]. In contrast, at P10 and P12, post-PB
(i.e.; 2.sup.nd hour) seizure burden was significantly lower in
ligated-treated group than in the ligated-control group during the
same period (p=0.007 and 0.03 respectively, gray bars).
[0111] Repeated measures ANOVAs using a within subjects design for
efficacious drug effects using the PB+BTN protocol in the
ligation-treated group were also evaluated. At P7, Mauchly's test
for sphericity was not significant (p=0.2) and within-subject drug
effect was not significant (df=2, F=0.62 and p=0.55). At P10,
Mauchly's test for sphericity was significant (p=0.02) and
within-subjects drug effect was also significant (df=2, F=6.54 and
p=0.01). The within-subjects contrast showed that the linear drug
effect was not significant, but the quadratic drug effect was
significant (p=0.01). At P12, Mauchly's test for sphericity was not
significant (p=0.9) and within subject drug effect was not
significant (df=2, F=3.690 and p=0.06). At P12, the within-subjects
contrast was not significant for either the linear or quadratic
drug effect. In summary, PB was a significantly efficacious
anti-seizure agent both at P10 and P12 when evaluated by
within-group pairwise t-tests and by independent sample t-tests
compared to the ligated-control group. However at P7, PB+BTN failed
to act as an efficacious anti-seizure therapy. At all three ages
evaluated, BTN failed to improve PB-efficacy, but significantly
blunted the PB-subdued ischemic seizures at P10. The overall lack
of BTN-efficacy in the model and PB inefficacy at P7 could not be
attributed to the clustering of seizures (not shown).
[0112] Non-convulsive vs. convulsive seizures at P7, P10 and P12.
The unique advantage of synchronous video-EEG in this study was its
ability to identify and quantitate the non-convulsive seizures for
the entire data set. Similar to humans, neonatal seizures in
rodents can be difficult to identify by behavioral parameters
alone. To evaluate if the treatment efficacy was different for
seizures graded as 0-2 (i.e., electrographic--ranging from inactive
to behavioral correlates associated with movements not overtly
convulsive--see methods) and 3-6 (i.e., convulsive), the data were
grouped by these seizure scores as the sum of [grade of the seizure
X count] in each epoch of the recording (FIG. 14). Electrographic
seizures were detected at every age investigated in this study, and
were not significantly different for the probability of occurrence
during the baseline EEG (i.e., early seizures in immediate
post-stroke period; FIG. 14A). Convulsive seizures showed an
age-dependent decrease of occurrences for baseline EEG (FIG. 14B);
however, this difference was not significant. Repeated measures
ANOVAs for PB+BTN treatment efficacy showed that the
within-subjects drug effects were significantly efficacious for
electrographic seizures at P10 and P12 but not at P7 (P7 df=2,
F=3.025, p=0.0'7, P10 df=2, F=5.224, p=0.03 and P12 df=2, F=9.144,
p=0.004). However, for grade 3-6 seizures, the within-subjects drug
effects for PB+BTN efficacy were not significant at P7 and P12 but
significant at P10 (i.e.; P7 df=2, F=1.224, p=0.3, P10 df=2,
F=6.604, p=0.02, and P12 df=2, F=3.262, p=0.07). For the pairwise
t-tests, PB significantly dropped the scores of both electrographic
and convulsive seizures at P10 and P12, but failed to curb either
at P7. In contrast, follow-on BTN treatment did not add any
significant therapeutic benefit on electrographic seizure scores at
any age and the post-BTN increase of seizure burden at P10 was
driven by significant increase in the occurrence of convulsive
seizures following effective PB-driven seizure suppression.
[0113] Correlation of baseline seizure severity to post-PB efficacy
as a function of postnatal age. To evaluate whether the severity of
seizure burden during baseline EEG with no drug on board
contributed to the PB-efficacy given 1 h later, correlations
between the numbers of ictal events during baseline recording and
during post-PB recording were evaluated in the ligated-treated
group (FIG. 15). At P7, there was a significant positive
correlation between the number of ictal events in the 1.sup.st and
2.sup.nd post-PB treatment hours (FIG. 15A; p=0.02). Since PB was
inefficacious at P7, this indicated that PB had no effect on
overall post-PB ictal counts in each pup. Interestingly,
correlations at P10 after PB-treatment compared to baseline showed
a significantly stronger positive correlation than at P7 (FIG. 15B;
p=0.001). Since PB was an efficacious anti-seizure agent at P10,
this may indicate that the anti-seizure efficacy of PB was
dependent on baseline seizure burdens (i.e.; if initial seizure
burden was high, follow-on post-treatment seizure burden remained
high. Additionally, FIG. 15B, group brackets representing low and
high baseline seizure burdens indicate that PB was very efficacious
in subduing all low seizure burdens (i.e.; .ltoreq.250 sec). This
is supported by additional correlations run for baseline vs.
post-PB seizure burdens which showed that low baseline seizure
burdens and PB efficacy at P10 were not significant (for baseline
seizure burden <=250 sec; r=0.35, p=0.39) but high baseline
seizure burdens at P10 showed significant positive correlations to
their post-PB seizure burdens (for baseline seizure burden>250
sec; r=0.61, p=0.03). In contrast, at P12, baselines vs. post-PB
correlations were not significant (FIG. 15C). Since PB was highly
efficacious at P12, this illustrates a strong anti-seizure efficacy
of PB, irrespective of baseline seizure burdens.
[0114] Age-dependent stroke injury. Stroke injury severities were
quantitated at P18 for all brains processed for histology (n=49;
P7=15, P10=15, and P12=19) and the remaining brains from study were
fresh frozen for western blot analysis. Ligations at P7 did not
result in a cystic infarct injury when evaluated at P18 (FIG. 16A).
Although the P7 ligated brains, when harvested for western blot
analysis at 6-8 h after ligation showed edema of the ipsilateral
hemisphere, no measurable atrophy was detected when P7 ligated
brains were harvested and processed for histology at P18 (FIG. 16).
Microscopic examination also did not reveal obvious cell death in
the watershed zones. Post-stroke diffuse cell death cannot be ruled
out in the P7 brains however; compared to the P10 and P12 ligated
pups, injury at P7 in CD1 ischemic pups may have white matter
injury, which was not evaluated in this study. Overall, P7 pups
showed a significant resistance to necrotic infarcts in the middle
cerebral artery perfusion territory detected at P10 and P12 (FIGS.
16A and B; p=0.005 P7 vs. P10 for both hemispheric and hippocampal
atrophy and p<0.0001 P7 vs. P12 for both hemispheric and
hippocampal atrophy). The mean hemispheric and hippocampal atrophy
in P10 were not significantly different from those in P12 (p=0.3
and 0.1 respectively).
[0115] To determine whether injury severity evaluated at P18
correlated with the seizure severity in the 1.sup.st three hours
after ischemic insult at the three ages examined, hippocampal and
hemispheric atrophies were compared to the time spent seizing on
EEG. No significant correlations were detected at any age.
Additionally, significant positive correlations between hemispheric
to hippocampal atrophy were detected for the P12 (p=0.008) ligated
group at P18, which has been reported previously for
ligated-control P12 mice at P40. Similar correlations were not
detected for the P10 ligated-treated group. No significant
correlations were detected between the seventies of ischemic
seizure burdens at baseline and the severity of the ischemic injury
at P18, in either the P10 or P12 pups. No significant sex
differences in injury severity were noted at P10 and P12 either.
Since stroke injuries evolve over time, injury assessments at
longer survival time-points may be more predictive of initial
seizure burdens.
[0116] Age- and Sex-Dependent Seizure Susceptibilities: is KCC2 a
Major Player?
[0117] To establish an age-dependent expression profile for the CD1
mouse strain used in this model, we examined KCC2 and NKCC1
expression in naive pup brains in ages advancing from P3 to P22
(FIG. 18A). As reported previously in both rodents and humans, we
saw an age-dependent increase in the expression of KCC2 examined
both by IHC and western blot analyses (FIGS. 18A and B). In
contrast, NKCC1 [i.e.; detecting NKCC1a, the non-dominant
spice-isoform (see methods section V.)] showed an age-dependent
decrease using the same samples (not shown). Additionally at P7, a
sex-dependent lag in the KCC2 expression levels was detected in
naive males compared to the age-matched females, which was not
detected at older ages when examined up to the age of P12 (FIG.
18C). Similar sex-dependent maturational lags have been previously
reported for males. No sex-dependent maturational lags were
detected with NKCC1 (not shown). These findings, in addition to the
higher susceptibility to ischemic seizures detected in P7 males
(see FIGS. 17A and B) may indicate KCC2 as the major player
underlying the age-dependent seizure susceptibility detected in
this model.
[0118] Sex-dependent susceptibility to ischemic seizures and
response to anticonvulsants. When the baseline EEG scores (i.e.;
seizure burden in the 1st hour post-ligation) for both
ligated-control and ligated-treated were pooled to further analyze
the effect of sex on the age-dependence of seizure susceptibility,
a one-way ANOVA showed a significance for males only. Post-hoc
(Bonferroni) comparisons showed P7 males to be significantly more
susceptible to seizures than either P10 (p=0.04) or P12 (p=0.01)
males (FIG. 17A). No significant differences were noted for females
(FIG. 17B). Based on this finding, future studies need to address
this sex and age-dependent ischemic-seizure susceptibility when
evaluating the efficacy of PB and BTN treatment.
[0119] PB-resistant ischemic seizures and an acute downregulation
of KCC2 expression. In this study, an acute post-ischemic
downregulation of KCC2 expression was detected in the ipsilateral
hemisphere compared to the contralateral hemisphere beginning from
a few hours to 48 h after ischemia at all ages tested (FIG. 19A-D).
The post-ischemic down-regulation of KCC2 expression in ipsilateral
hemisphere was .about.45% compared to uninjured contralateral
hemisphere (FIG. 19D; pairwise t-test, p=0.0002). A trend towards
recovery from the downregulation was also detected in the P7 age
group at 96 h after ischemia (FIG. 19A-D). Although in humans
NKCC1a represents a non-dominant isoform of NKCC1 splice variant,
the analogous expression profiles in rodents are not known due to
the current lack of a pan-NKKC1 antibody. Using the currently
available antibodies, our data matched the previously published
data where NKCC1 was shown to decrease with advancing age in naive
brains (not shown). There were paradoxical trends towards
post-ischemic increase in NKCC1 levels in the ipsilateral
hemisphere at <48 h in this study (not shown), as it was shown
in a lesion study. Similar findings have also been noted after
neonatal hypoxia-ischemia. These results indicate that neonatal
ischemia significantly alters the acute and sub-acute developmental
profiles of the adult-form chloride transporter KCC2; however,
NKCC1 developmental expression profiles remain relatively unaltered
or increased. While ischemia-related changes in cellular
populations were expected with the stroke lesions, both
co-transporters, KCC2 and NKCC1 would be similarly affected by
these changes at the three age groups evaluated (i.e.; P7 group
where no infarct lesions were seen at P18 vs. P10 and P12 where
they were commonly detected; see FIG. 16). Both of these
co-transporters were evaluated in the same homogenized brain
samples, and KCC2 showed consistent downregulation at 6 to 48 h and
recovery at 96 h. More importantly, the finding of PB-resistance at
P7 and the seizure-burden dependent efficacy at P10 validates the
P7-P10 CD1 mouse model of neonatal ischemia reported here as a
novel tool to test the efficacy of novel anti-seizure
pharmacotherapies in a clinically relevant model of seizure
induction.
Discussion
[0120] This study has several salient findings to report. 1. A new
mouse model of ischemic seizures that developed both primarily
PB-resistant seizures at P7 and PB-responsive seizures at P10. At
P10, PB-efficacy was dependent on the baseline seizure burden
(i.e.; lower the seizure burden better the anti-seizure efficacy).
The acute seizures recorded in the first three hours after
initiation of ischemia represented a status-like seizure burden
state well described for seventies typically seen clinically in
HIE. 2. The age-dependent seizure susceptibility after ischemia was
significantly higher at P7 than both at P10 and P12. This
susceptibility may represent the underlying age-dependent
upregulation of KCC2 expression in maturing brains. 3. An acute and
significant post-ischemic KCC2 downregulation was detected at all
ages tested. The post-ischemic KCC2 downregulation catches up with
the age-dependent developmental increase, representing recovery
from ischemic insult within a few days. Therefore, ischemic injury
significantly modulates the developmental profile of the adult-form
chloride co-transporter KCC2, and thus dictates the efficacy of
anti-seizure medications that follow. 4. The NKCC1 antagonist, BTN,
failed to act as an adjunct in the new model for the primary
PB-resistant seizures. Additionally, BTN blunted the anti-seizure
efficacy of PB treatment at P10 with the follow-on treatment
paradigm, by aggravating the PB-subdued seizures. Hence, NKCC1
blockage fails to rescue ischemic seizures regardless of the
anti-seizure efficacy of GABA.sub.A agonists which fail at P7 but
work at P10 and P12. 5. The sex-dependent seizure susceptibility
detected in P7 males may correlate with the developmental lag of
KCC2 expression in naive males compared to females at that age.
This lag goes away with advancing age.
[0121] Neonatal seizures, especially those associated with
ischemia, are known to be transient in the neonatal period. HIE
seizures also show hours of increasing seizure burdens alternating
with quiet or low-seizing periods with crests and troughs during
their natural temporal progression. How aggressively we treat these
transient seizures, which may be severe in some cases, with drugs
that may also alter the developmental profile of an immature brain,
is a subject of debate. The exact time of onset of ischemia in
neonates is rarely known, and clinical seizures are detected a few
hours to days later. This may be due to either, a failure to detect
the subtle early seizures since most of the EEG seizures are
non-clinical, or a slower paced evolution of the ischemic injury.
The dynamics of this evolution is however poorly understood and
difficult to quantitate clinically. Recent studies that have tried
to evaluate the issue are difficult to interpret with regards to
evolution because the baseline seizure burdens before onset of
treatment are rarely known or quantifiable without EEG. This
limitation in reported clinical studies persists due to the nature
of the disease and the lack of EEG data to accurately assess
pre-treatment seizure burdens of non-clinical seizures. Even so,
PB-inefficacy as first-line treatment is now widely reported.
[0122] Intrinsic features of immature networks make
GABA.sub.A-based pharmacotherapy more difficult. Following
recurrent seizures, it has been shown that intracellular chloride
ions accumulate, making GABA strongly excitatory. Recent research
has shown that KCC2 downregulation following excitotoxic injury may
underlie these findings. Developmentally, NKCC1 mediates influx of
chloride ions; however, this chloride co-transporter is neither
necessary nor sufficient, as these shifts of GABA polarity also
occur in NKCC1 KOs. Additionally, recent study has identified two
spice variants of NKCC1 in the human brain, NKCC1a [1-27] and
NKCC1b [1-27 (421)]. NKCC1b is the dominant splice variant in human
brains, which shows an age-dependent upregulation. No reliable
pan-NKCC1 antibodies, capable of detecting both variants, are
currently available for reevaluating the NKCC1 data reported here
(not shown) or the similar published data in animal models that may
have only quantitated NKCC1a, which was shown to downregulate with
advancing age. The activity-dependent downregulation of KCC2 after
NMDA-induced excitotoxicity may lead to appearance of
PB-resistance, and pre-clinical animal models of neonatal seizures
that do not result in KCC2 downregulation may not be relevant to
ischemic seizures. Reports of KCC2 downregulation in the
white-matter of premature babies with white matter lesions support
this hypothesis. Even in adult models of epilepsy, downregulation
of KCC2 has been detected in human cortices resected for refractory
seizures and in peritumoral neurons in mouse cortical slices,
further confirming that KCC2 is the key player in maintaining
chloride homeostasis in mature neurons. The expression profile of
the co-transporters in HIE brains remains unknown and has the
potential to add significant insights into pre-clinical animal
modeling. Therefore, preventing KCC2 downregulation following
injury may help delay the intracellular chloride accumulation
during repetitive ischemic seizures and thus increase the efficacy
of GABA.sub.A-agonists. The critical role of KCC2 for chloride
homeostasis and ultimately neuronal survival has been well
established in KCC2 knockout (KO) model of in vitro and in vivo.
Knockout of isoforms, KCC2a and b are lethal in mice due to
respiratory failure, while KCC2b KO mice can survive up to P17,
with frequent and severe spontaneous seizures. In addition, the now
known role of KCC2 in spine development and cortical interneuron
migration may indicate that post-excitotoxic downregulation of KCC2
may underlie the development of long-term sequelae like cognitive
and behavioral deficits. In contrast, the NKCC1 KO mouse does not
have spontaneous seizures, is non-lethal but deaf. Since our study
showed a strong correlation between the severity of the early
untreated ischemic seizures and PB-efficacy as a function of age,
this animal model of PB-resistant seizures would be a useful tool
to test this hypothesis further.
[0123] Using physiologic techniques, the intracellular chloride
shift has been shown to be primarily due to the downregulation and
internalization of the chloride exporter KCC2. It has been shown
that KCC2 downregulation occurs immediately within minutes after an
excitotoxic injury. This finding complements data from other
studies showing that this co-transporter is highly sensitive to
serine and tyrosine phosphorylation and seizures that control its
turnover. The diuretic NKCC1 antagonist, BTN, has been proposed as
a novel anti-seizure medication and is the basis of a current
clinical trial (NEMO, FP7-EU clinical trial: and
ClinicalTrials.gov; NCT00830531); however, BTN blocks seizures in
some but not all models of seizures. Even with a pre-treatment
protocol used in a chemoconvulsant model in neonatal rats, studies
have shown an age-dependent specificity of the lack of efficacy of
BTN where higher doses actually decreased the latency to
generalized seizures in the older pups (i.e.; P12). With the recent
reports of the termination of the European clinical trial reporting
BTN-inefficacy for HIE seizures associated with ototoxicity,
testing higher doses of BTN seems counterproductive. The results of
our experiments using a clinically relevant post-treatment protocol
support these reports. In the immature brain, it is possible that
the very early seizures, which occur before a significant KCC2
downregulation has begun, may be efficaciously blocked by BTN alone
or as an adjunct treatment after PB. In addition, BTN as an adjunct
treatment to PB may work efficaciously in neonatal seizures that
are not associated with KCC2 downregulation. However, after
recurrent seizures and KCC2 degradation, GABA strongly excites
neurons in the immature brain, and drugs like PB that act as
GABA.sub.A agonists fail to act. Similarly, our findings suggest
that PB efficacy is dependent on the baseline seizure burdens
specifically at P10 in our model. Our studies show that BTN fails
to improve PB-efficacy when given as a follow-on treatment at P7.
Our results also show that BTN can blunt PB-subdued seizures at
P10. The BTN aggravation in our study following PB-efficacy may not
be detected clinically, since BTN would not be administered to a
patient whose seizures have responded well to PB. However, this
finding is of scientific significance and deserves further
evaluation to understand the underlying mechanism.
[0124] In general, the use of BTN in critically ill patients
requires caution. Blocking NKCC1 function in the brain during
development may interfere with critical circuit formation. BTN
non-specificity as a NKCC1 antagonist and its ability to also block
KCC2 at higher doses should be an additional concern in a seizing
brain. The associated ototoxicity detected in the NEMO trial
associated with the expression of the same NKCC1 isoform in the
inner hair cells, should raise caution for all neonatologists who
use BTN for its approved use as a diuretic. In a model of hypoxic
seizures, the maximum concentration of BTN in the brain was
estimated to be 1.2 ng/ml following an acute dose of BTN injected
IP at 0.3 mg/kg. Recent review also discusses the various reasons
for the low bioavailability of BTN in brain, a significant caveat
to this line of pharmacotherapy. Although BTN prodrugs designed for
better brain bioavailability now exist, the anti-seizure efficacies
of those BTN prodrugs were recently investigated and show no clear
effect. Additionally, the issue of side-effects such as diuresis
and ototoxicity remain. The phenomenon of BTN aggravation of
seizures reported here occurred only after PB was efficacious in
subduing the ischemic seizures (not shown) in an immature brain
where KCC2 is already significantly downregulated. In addition to
the acute side-effects, BTN has also been shown to result in
deleterious long-term effects. A significant increase in the
percentage of rats having spontaneous seizures has been reported in
response to the acute administration of BTN following PB in a
pilocarpine model of temporal epilepsy. BTN may also attenuate the
seizure-induced activation of HPA by blocking NKCC1 in
periventricular neurons. However, this study was done in adult
rodents, and the effect of BTN on HPA axis in immature brains is
not known. Overall, further work is required to understand the
potential cause of post-BTN aggravation of seizures reported in
this model.
[0125] Early and effective treatment of neonatal seizures following
excitotoxic insults is of course the gold standard for management;
but early and efficacious treatment is not always attainable.
Inability to detect the exact onset of ischemia, failure to detect
subtle neonatal seizures, and the likelihood of preceding in-utero
ischemic insults may lead to the early KCC2 downregulation and a
lag in developmental profile that may underlie the increased
seizure susceptibility and PB-resistance of the first detected
seizures. Ischemia in the developing brain likely alters the
expression profile of a multitude of factors that modulate
transmembrane ionic gradients. This study shows that the pathology
underlying the occurrence of neonatal seizures may dictate drug
responses. Current clinical trials for BTN were initiated based on
earlier reports of BTN-efficacy from non-ischemic models that may
depend on the stable KCC2 expression following the initial insult.
However, our data show that KCC2 downregulation, beginning in-utero
following ischemia or possibly infection/inflammation, may make
early interventions with BTN futile. In conditions where KCC2 is
already downregulated before birth or lagging in the age-dependent
upregulation, blocking Cl- import through NKCC1 cannot compensate
for the role of KCC2 as a Cl- extruder. Additionally, we do not
clearly understand the BTN-induced aggravation of seizures detected
in our study that was also differentially modulated by sex. BTN
half-life in rodents is short and at .about.30 min, clearly less
than the 1 h of seizure burden quantitated in this study. BTN
aggravation, when noted, began within 5-10 min of the treatment and
lasted throughout the 1 h of recording (not shown). Recent evidence
of estradiol modulation of NKCC1 shows that many details of this
process and its sex-dependent modulation remain to be understood.
The overall age-dependent susceptibility of males to ischemic
seizures in this study and the additional window period of a lag in
KCC2 development in naive brains may add to the accumulating data
on male susceptibility to developmental injury and differential
effects of neonatal treatments by sex.
[0126] In conclusion, this study highlights the variability of drug
responses in animal models based on the mechanism by which the
seizures are induced. For BTN, these model-specific outcomes have
already been reported to be very variable. Dose-dependent efficacy
detected for higher doses of BTN has been shown to suppress
neonatal kindling when given as a pre-treatment protocol and has
been proposed as a reason behind the failure of lower dose regimens
reported in other models. However, pre-treatment protocols have
also resulted in an aggravation of seizure onset latencies with
higher doses in immature rats using chemoconvulsants. Additionally,
BTN failed to work at P7 in that study which highlights the
age-dependency for its efficacy. The findings of our study suggest
that when KCC2 levels remain unaltered or become enhanced after
kindling, such protocols may not be translationally relevant to the
HIE patients being recruited in the current clinical trials where
ischemia is the prominent underlying cause. This hypothesis is now
supported by outcomes reported in the recently published NEMO
study. The same caveat would apply to the translational value for
the BTN pre-treatment paradigms that alter the chloride gradients
in the naive brains prior to insult induction in a dose dependent
manner. The variable effects of BTN reported in recent literature
and in this current study, highlight the need for further
translational research using BTN. A recent study, Cleary et al has
reported beneficial dose-dependent effects of PB+BTN pre-treatment
in a rat model of hypoxic neonatal seizures. Since they also
reported a significant upregulation KCC2 in hippocampus on the day
of assessment of BTN treatment efficacy, these data strongly
suggest that KCC2 downregulation after ischemia may be a major
player in the development of PB-resistance. BTN efficacies were
shown to be age- and sex-specific even within the relatively narrow
age range investigated in this study. None of the pre-treatment
studies noted above examined sex differences. Our results also
indicate that neonatal stroke/seizures left undetected or untreated
for extended periods of time alter the acute and sub-acute
developmental profiles of the adult-form chloride co-transporter
KCC2 such that the later seizures may become resistant to treatment
with the conventional anticonvulsants that act as GABA.sub.A
agonists. Prolonged seizures are also known to alter GABA.sub.R
such that it reduces the efficacy of AEDs. In neonates, the
transient downregulation and the developmental lag in the KCC2
expression profile may additionally result in chronic alteration of
the way the immature brain is getting wired within that critical
developmental window. This study shows that a novel focus on
preventing KCC2 downregulation or enhancing KCC2 function following
neonatal insults may be critical in guiding future approaches for
treating PB-resistant seizures in neonates.
Example 3
Efficacy of KCC2 Agonist Treatment of Neonatal Seizures
[0127] FIG. 20 presents data using CLP290 (n=4) in an experimental
paradigm similar to ANA-12 indicating that a KCC2 agonist can act
independently as an anti-seizure agent and with PB to completely
block all ischemic seizures at a 20 mg/kg dose in the model when
compared to the vehicle injection (HPCD). Bottom trace in each
panel shows the seizure frequency as scored by electrographic
seizure activity on raw EEG in blue. Middle trace shows gamma
frequency seizure burst activity on 3 h EEG and top blue trace
shows low frequency activity due to movement artifacts during
ischemia induced status. This finding indicates that as a KCC2
agonist CLP290 also works efficiently to not only reverse
PB-resistant seizures but act as an anti-seizure agent by itself.
Arrow heads indicate time of 1 hourly drug injections from start of
the 3 h recording.
* * * * *