U.S. patent application number 17/232544 was filed with the patent office on 2022-03-03 for gsno reductase inhibitor as an adjunct therapy with remote ischemic conditioning and thrombolytic reperfusion therapies in stroke.
The applicant listed for this patent is KING ABDULAZIZ UNIVERSITY. Invention is credited to Muhammad Hussain AL-QAHTANI, Md. Nasrul HODA, Syed Kashif ZAIDI.
Application Number | 20220062262 17/232544 |
Document ID | / |
Family ID | 1000006150140 |
Filed Date | 2022-03-03 |
United States Patent
Application |
20220062262 |
Kind Code |
A1 |
ZAIDI; Syed Kashif ; et
al. |
March 3, 2022 |
GSNO REDUCTASE INHIBITOR AS AN ADJUNCT THERAPY WITH REMOTE ISCHEMIC
CONDITIONING AND THROMBOLYTIC REPERFUSION THERAPIES IN STROKE
Abstract
Stroke patients with comorbidities, such as diabetes,
hypertension, or other conditions associated with cardiovascular,
pulmonary or renal disease do not respond well to current
treatments for stroke. An inhibitor of S-nitrosoglutathione
reductase (GSNOR) is administered as an adjunct therapy with remote
ischemic conditioning (RIC) to treat a stroke in a subject having
at least one comorbidity, such as diabetes and hypertension. When
performed in conjunction with GRI therapy and RIC, intravenous
tissue plasminogen activator therapy (IVT) for thrombolysis and/or
endovascular thrombectomy (EVT) for clot retrieval are effective in
subjects with comorbidities, even after five hours post-onset of
stroke.
Inventors: |
ZAIDI; Syed Kashif; (Jeddah,
SA) ; HODA; Md. Nasrul; (Jeddah, SA) ;
AL-QAHTANI; Muhammad Hussain; (Jeddah, SA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KING ABDULAZIZ UNIVERSITY |
Jeddah |
|
SA |
|
|
Family ID: |
1000006150140 |
Appl. No.: |
17/232544 |
Filed: |
April 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17002216 |
Aug 25, 2020 |
11071733 |
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17232544 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4178 20130101;
A61P 9/10 20180101; A61K 31/47 20130101 |
International
Class: |
A61K 31/47 20060101
A61K031/47; A61P 9/10 20060101 A61P009/10; A61K 31/4178 20060101
A61K031/4178 |
Claims
1. A method of treating a diabetic subject suffering from a
blockage of brain microcirculatory flow caused by a thromboembolic
stroke, comprising the steps of administering to the diabetic
subject a therapeutically effective amount of S-nitrosoglutathione
reductase (GSNOR) inhibitor (GRI therapy) selected from the group
consisting of N6022, cavosonstat, N9115 and N6338, wherein the
therapeutically effective amount of the GSNOR inhibitor is
sufficient to inhibit brain microvascular tissue injury; and then
administering thereafter an intravenous tissue plasminogen
activator therapy (IVT) for thrombolysis, wherein the
therapeutically effective amount of the IVT is sufficient to
dissolve a thrombus or a blood clot from an artery, and wherein the
GRI therapy and the IVT reduce the risk of reperfusion injury in
the diabetic subject.
2. The method of claim 1, wherein the GSNOR inhibitor is N6022.
3. The method of claim 1, wherein at least 3 hours have passed
since a suspected time of onset of blockage of the brain
microcirculatory flow.
4-6. (canceled)
Description
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] The invention generally relates to a treatment for stroke
when remote ischemic conditioning is not effective. The invention
further relates to a method for repurposing an inhibitor of
S-nitrosoglutathione reductase as an adjunct therapy to delayed or
late IVT (beyond 3 hours) in stroke, particularly for ischemic
stroke with comorbidities.
Background
[0002] Acute ischemic stroke (AIS), often due to a thromboembolic
(TE) occlusion in cerebral arteries, results in reduced cerebral
blood flow (CBF), brain tissue hypoxia/ischemia and subsequent
progression of cerebral infarction and brain cell death. To date,
the FDA has approved only two reperfusion therapies in AIS:
intravenous tissue plasminogen activator therapy (IVT) for
thrombolysis within 3 hours of stroke and endovascular thrombectomy
(EVT) for mechanical clot retrieval. These two therapies re-open
large occluded blood vessels and reinstate CBF in them. However,
neither IVT nor EVT guarantee the restoration of microcirculatory
reflow, which is critically needed to eventually reoxygenate the
ischemic region. Thus, despite a successful reperfusion therapy in
stroke, brain tissue hypoxia may persist, which remains a critical
barrier in improving post-stroke outcomes. Therefore, ischemic
stroke remains a significant cause of adult mortality and a leading
cause of adult disability worldwide. The FDA and NIH/NINDS stroke
research progress related committees strongly recommend the
development and repurposing of existing therapies to treat stroke
(Fisher et al. Stroke 2009, 40(6):2244-2250). It is also
recommended that the new promising adjunct therapy should be
effective alone to reduce ischemia-reperfusion (IR) injury, should
be safe to utilize as a field therapy without requiring any
state-of-art facilities such as in remote community hospitals for
primary care, during transport in ambulance, Emergency Department
and any acute care situation. Most importantly, a new and/or
repurposed therapy also should not contradict and prevent the use
of IVT/EVT.
[0003] Nitric oxide (NO) is a freely diffusible endogenous small
molecule which is produced by the NO-synthase (NOS) enzymes.
Endothelial NOS (eNOS) remains the major producer of
vascular/endothelial NO which relaxes vascular smooth muscles cells
and improves tissue oxygenation. Endogenous NO can be present as
nitrate (NO3.sup.-)/nitrite (NO2.sup.-) or preserved as
s-nitrosylated proteins (--SH to --SNO) such as glutathione
GSH>GSNO and hemoglobin Hb>HbSNO. Thus, GSNO and HbSNO were
recognized as the major chariots and sources of bioactive
endogenous NO in the form of -SNO adducts. Exogenous NO-inhalation,
which increases levels of GSNO/HbSNO, was one of the earliest
therapies incidentally identified to treat angina and later tested
in ischemic stroke (Lundberg et al. Nat Rev Drug Discov.
2008;7(2):156-167). However, NO-inhalation therapy increases risk
of blood pressure lowering which can cause hemorrhagic
transformation (HT) to worsen stroke outcomes. On the other hand,
NO in the form of conventional NO3.sup.-/ NO2.sup.- therapy
recently showed no benefits in stroke clinical trial (Bath et al.
Cochrane Database Syst Rev. 2017;4:CD000398).
[0004] Others and we recently reported that remote ischemic
conditioning (RIC) modulates endogenous NO-metabolome, enhances CBF
and improves outcomes after stroke/ ischemic brain injuries (Hess
et al. Acta Neurochir Suppl. 2016;121:45-48; Hoda et al. Stroke.
2012;43(10):2794-2799; Hoda et al. Transl Stroke Res.2014,
5(4):484-90). However, it remains a concern that in a larger
population of stroke patients often accompanied with most common
comorbidities (such as hypertension and diabetes), RIC may fail to
protect. Indeed, it is evident from a latest preliminary small
clinical trial report that RIC therapy remained neutral in effect
in stroke patients, likely indicating that the "no-effects" outcome
in comorbid stroke patients might have neutralized or skewed the
overall outcome of the trial (Pico et al. Int J Stroke.
2016;11(8):938-943).
[0005] Thus, there is a need for an adjuvant treatment that can
enhance benefits of the current FDA-approved reperfusion therapies
to further improve functional outcomes after stroke. Compounding
this lack of treatment options, most exogenous therapies have
failed in stroke trials in the past four decades aside from
IVT/EVT, so the problem of microcirculatory no-reflow persists. In
particular, there is a need for an effective treatment for patients
with comorbidities to improve recovery from stroke and reduce
ischemic brain injury by promoting reperfusion and restoring
microcirculatory flow because comorbidities such as diabetes alters
the vascular patency and increases the risk of HT during ischemic
stroke, particularly in response to late IVT.
SUMMARY OF THE INVENTION
[0006] The invention is a method of treatment for a stroke and is
particularly suited for treating a subject who also has at least
one comorbid condition, such as diabetes and/or hypertension. The
treatment comprises administering a therapeutically effective
amount of an S-nitrosoglutathione reductase (GSNOR) inhibitor,
wherein the therapeutically effective amount is sufficient to
inhibit brain tissue injury. The GSNOR inhibitor is an adjunct
therapy to be administered in conjunction with the RIC therapy,
particularly in stroke with comorbidities such as stroke patients
accompanied with diabetes and/or hypertension. GSNOR inhibitors
that may be used include N6022, cavosonstat, N91115, N6338, as well
as any other GSNOR inhibitor. The GSNOR inhibitor may be
administered prior to performing RIC therapy or concurrently with
the RIC therapy. The RIC therapy is performed according to an
FDA-approved protocol and/or by using an FDA-approved device.
[0007] In one embodiment, the method further comprises the step of
administering a therapeutically effective amount of IVT, wherein
the therapeutically effective amount is sufficient to achieve
thrombolysis, and/or administering EVT for clot retrieval. The
combination of GRI therapy and RIC therapy with EVT and/or IVT is
effective even when the "window of opportunity" of 3 hours has
passed since the time of onset of stroke, which is when the risk of
HT would otherwise will increase due to late IVT.
[0008] In another embodiment, the subject having comorbid
conditions is suspected to be suffering from a stroke or is at the
risk of suffering an imminent stroke with comorbidities and lacking
FDA-approved reperfusion interventions (IVT/EVT) for at least 3 h.
Stroke in subjects having comorbidities is particularly dangerous
and more likely to result in exacerbated brain injury and HT than
subjects not having comorbidities. In particular, the patient is
suffering from at least one comorbid condition, such as diabetes,
type II diabetes, hypertension, cardiac disease, coronary artery
disease, arteriosclerosis, atherosclerosis, myocardial infarct,
congestive heart failure, peripheral vascular disease, dementia,
cerebrovascular disease, chronic pulmonary disease, congestive
obstructive pulmonary disease, kidney disease, kidney failure,
chronic liver disease and metabolic syndrome. In one embodiment,
the subject is suffering from the comorbid conditions of diabetes
and hypertension, and the GSNOR inhibitor is administered as an
adjunct therapy to RIC therapy to enhance the efficacy and benefits
of RIC. Optionally, IVT and/or EVT are also administered to
dissolve and/or remove a thrombus or blood clot from an artery,
thereby restoring perfusion of the brain region in which blood flow
was being blocked by the thrombus/clot.
[0009] As a result of the treatment, ischemic injury and brain
tissue infarct can be reduced, minimized or avoided, and functional
activity that would otherwise be impaired is restored, at least in
part. Functional activities in humans include cognitive, speech and
motor skills that are needed for normal everyday living and
contribute to quality of life.
[0010] In one embodiment, the invention is a method of restoring
brain microcirculatory flow in a subject suffering from at least
one comorbid condition, comprising the step of administering in
combination of an RIC therapy and a therapeutically effective
amount of an GSNOR inhibitor, wherein the therapeutically effective
amount is sufficient to allow reperfusion of a brain region wherein
the brain microcirculatory flow was previously blocked. The GSNOR
inhibitor may be administered prior to initiation of the RIC
therapy, or the GSNOR inhibitor may be administered concurrently
with delivery of the RIC therapy.
[0011] In another embodiment, the invention is a method of
restoring brain microcirculation in a subject suffering from at
least one comorbid condition, comprising the step of administering
in combination RIC therapy and a therapeutically effective amount
of an GSNOR inhibitor, wherein the therapeutically effective amount
is sufficient to allow reperfusion of a brain region wherein the
brain microcirculatory flow was previously blocked. Optionally, the
RIC and GRI therapy may be followed by administration of IVT for
thrombolysis and/or EVT for clot retrieval.
[0012] Other features and advantages of the present invention will
be set forth in the description of invention that follows, and in
part, will be apparent from the description or may be learned by
practice of the invention. The invention will be realized and
attained by the compositions and methods particularly pointed out
in the written description and claims hereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0014] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate embodiments of
the invention and, together with a general description of the
invention given above, and the detailed description given below,
serve to explain the invention.
[0015] FIG. 1A-1D shows the effect of embolic middle cerebral
artery (MCA) occlusion (eMCAo) on
[0016] GSNOR protein expression and activity. 1A shows
semi-quantitative densitometry of an immunoblot for GSNOR protein
normalized to .beta.-actin protein in whole brain tissue for
sham-operated group. 1B shows a representative immunoblot for GSNOR
expression in whole brain tissue. 1C shows an activity assay based
on the rate of GSNO-dependent consumption of NADH in brain. 1D
shows an activity assay of rate of GSNO-dependent consumption of
NADH in plasma.
[0017] FIGS. 2A-2D show the effect of GRI therapy in aged male mice
following photothrombotic (PT) stroke, a thrombotic model of
moderate injury. 2A shows a representative image of whole brain
collected immediately after stroke to confirm the site of occlusion
in MCA region. 2B shows a Z-projection of T2-weighted magnetic
resonance image 48 h after stroke. Site of thrombotic occlusion and
stroke injury in 2A and 2B, respectively, are indicated with arrow.
2C shows densitometry of coronal sections from brains of treated
mice, and 2D shows representative TTC-stained set of coronal
sections (2-.mu.m thick) obtained from the whole brain and stained
freshly.
[0018] FIGS. 3A-3D show that a sex difference was found in the
basal level of plasma GSNOR activity. 3A shows GSNOR activity in
males and females. 3B shows plasma GSNOR activity in aged females
after PT-stroke. 3C shows densitometry of infarct volume measured
in coronal sections, and 3D shows a representative TTC-stained set
of coronal sections (2 mm thick) obtained from the whole brain and
stained freshly.
[0019] FIGS. 4A-4D show analyses of mice in a stroke model of
permanent MCA occlusion (pMCAo). 4A shows a representative grey
scale laser speckle contrast image (LSCI) immediately after stroke.
A narrow beam walk test was used to determine any deterioration of
motor function for walking time to traverse through the beam
length, shown in 4B, and number of foot slips, shown in 4C. 4D
shows % CBF for males and ovariectomized females (OVX-Fem) treated
with vehicle (Veh) or GRI therapy.
[0020] FIGS. 5A-5B show representative LSCI images from mice
treated with vehicle or GRI therapy. Males are represented in 5A
and OVX-female mice are represented in 5B.
[0021] FIGS. 6A-6D show representative real-time perfusion plots.
6A is vehicle-treated male; 6B is a GRI therapy treated male; 6C is
a vehicle-treated OVX-female; and 6D is a GRI therapy treated
OVX-female.
[0022] FIGS. 7A-7B show analyses of males and OVX-females treated
with vehicle or GRI therapy. 7A shows TTC-staining of fresh brain
tissue and 7B shows infarct volume analysis.
[0023] FIGS. 8A-8B show representative LSCI images and real-time
perfusion curve during ischemia (8A) and reperfusion (8B), in the
transient MCA occlusion (tMCAo) model of stroke and restoration of
CBF.
[0024] FIGS. 9A-9D show representative real-time electrogram traces
for mice treated with combinations of vehicle or GRI and RIC-mock
or RIC therapy (RIC-Thr).
[0025] FIG. 10A-10B analyses of mice treated with combination
vehicle or GRI and RIC-mock or RIC therapy. 10A shows a plot of the
linear regression and 10B shows reoxygenation of brain tissues
(PbtO2) for each treatment combination.
[0026] FIGS. 11A-11D show inflammatory gene expression in brains of
mice treated with combinations of vehicle or GRI and RIC-mock or
RIC therapy (N=6/gp). 11A shows measurement of iNOS expression; 11B
shows measurement of iCAM; 11C shows measurement of IL-1.beta.; and
11D shows measurement of IL-6.
[0027] FIGS. 12A-12D show that GRI therapy enhanced the
neuroprotective efficacy of RIC therapy in reperfused tMCAo stroke
model in streptozotocin (STZ)-induced diabetic mice. 12A shows %
infarct volume following treatment, and 12B shows % swelling, and
12C shows infarct volume in GRI+RIC combination therapy group,
vehicle-mock operated control, GRI therapy singly and RIC therapy
alone. 12D shows representative TTC-stained fresh brain tissue
images wherein GRI therapy in combination with RIC therapy enhanced
neuroprotection in the tMCAo model with the comorbidity of
diabetes.
[0028] FIGS. 13A-13B show analyses of mice treated with vehicle,
GRI, IVT or GRI+IVT. 13A shows stroke injury/edema volume
quantification. 13B shows representative coronal slices from the
MCA region.
[0029] FIGS. 14A-14B show neurological deficit scoring (NDS) and
survival. 14A shows NDS based on a modified Bederson Scale
performed for all surviving mice at 96 h post-eMCAo in treatment
groups of vehicle, GRI, IVT or GRI+IVT. 14B shows a Kalan-Meier
survival curve for all 4-groups followed for 2-weeks.
[0030] FIGS. 15A-15B show tests for long-term functional outcomes
for mice treated with vehicle, GRI, IVT or GRI+IVT. 15A shows
recovery of muscular-strength (hanging wire test) and 15B shows
protection of learning-memory function (novel object recognition
test).
[0031] FIG. 16 shows representative series of fixed brain samples
from mice treated with vehicle, GRI, IVT or GRI+IVT at day-15
post-eMCAo.
DETAILED DESCRIPTION
[0032] The following descriptions and examples illustrate some
exemplary embodiments of the disclosed invention in detail. Those
of the skill in the art will recognize that there are numerous
variations and modifications of this invention that are encompassed
by its scope. Accordingly, the description of a certain exemplary
embodiment should not be deemed to limit the scope of the present
invention. The invention is a method of treatment for stroke and,
in particular, it is a treatment for stroke in a subject or patient
who has at least one comorbid condition. This is a patient
population that is particularly predisposed to stroke, with
comorbid conditions that complicate medical management of
conventional treatments, and will be likely treated with RIC, EVT,
IVT and/or any possible combinations of these 3 therapies The
method of the invention enhances and preserves endogenous --SNO, a
bioactive form of NO that improves microvascular flow during
hypoxia/ischemia without altering blood pressure. A key benefit of
the invention is that it extends the effective safe window of
thrombolysis with intravenous tissue plasminogen activator (IVT)
therapy beyond the current 3 hours post-onset of stroke when the
risk of hemorrhage/HT is elevated and IVT is not approved beyond
this window of 3 hours, particularly in stroke patients with
comorbidities.
[0033] In one embodiment, the invention is a method of treatment
for a stroke in a subject in need thereof, comprising administering
a therapeutically effective amount of an GSNOR inhibitor, wherein
the therapeutically effective amount is sufficient to inhibit brain
tissue injury, as an adjunct therapy with RIC therapy. Known GSNOR
inhibitors include N6022, cavosonstat, N91115 and N6338. Any of
these inhibitors are suitable for the method of the invention. The
GSNOR inhibitor may be administered prior to performing RIC therapy
or it may be administered concurrently with the RIC therapy. The
RIC is performed according to an FDA-approved protocol and/or by
using an FDA-approved device.
[0034] In another embodiment, the method further comprises the step
of administering a therapeutically effective amount of IVT, wherein
the therapeutically effective amount is sufficient to achieve
thrombolysis, and/or administering EVT for clot retrieval. The
method is surprisingly effective even when the "window of
opportunity" (e.g., 3 hrs) has passed since a suspected time of
onset of the stroke and the chance of hemorrhage would otherwise by
increased.
[0035] In another embodiment, the subject is suspected to be
suffering from a stroke or is expected to have higher brain injury
and worsened outcomes (such as stroke with diabetes, hypertension
etc.) and/or hemorrhagic transformation. Stroke in subjects having
comorbidities is particularly dangerous and likely to result in
exacerbated brain injury worsening outcomes due to the comorbid
condition(s). In particular, the treatment may be administered
prophylactically if a stroke or higher brain injury or hemorrhagic
transformation appears imminent when a patient also suffers from at
least one comorbid condition.
[0036] The method of the invention comprises a combination therapy
that was surprisingly effective in patients whose symptoms are
resistant to conventional treatments. This resistance arises from
the complex underlying conditions of comorbidity, such as, type II
diabetes, hypertension, cardiac disease, coronary artery disease,
arteriosclerosis, atherosclerosis, myocardial infarct, congestive
heart failure, peripheral vascular disease, dementia,
cerebrovascular disease, chronic pulmonary disease, congestive
obstructive pulmonary disease, kidney disease, kidney failure,
chronic liver disease and metabolic syndrome. A patient with even
one of these conditions is frequently refractory to all currently
available treatments procedures, and presents a higher risk of
complications and poor outcomes, both from the stroke itself and/or
procedures to treat stroke. In one embodiment, the subject has one
of these comorbid conditions, and in other embodiments, the subject
has many of these comorbid conditions.
[0037] In one embodiment, the subject is suffering from the
comorbid conditions of diabetes and hypertension, and the GSNOR
inhibitor is administered as an adjunct therapy to RIC therapy.
Optionally, IVT and/or EVT are also administered to dissolve and/or
remove a thrombus or blood clot from an artery, thereby restoring
perfusion of the brain region in which blood flow was being blocked
by the thrombus/clot. As a result of the treatment, ischemic injury
and brain tissue infarct can be reduced, minimized or avoided, and
functional activity that would otherwise be impaired is preserved
or restored, at least in part.
[0038] In one embodiment, the invention is a method of restoring
brain microcirculatory flow in a subject suffering from at least
one comorbid condition, comprising the step of administering in
combination with RIC therapy and a therapeutically effective amount
of an GSNOR inhibitor, wherein the therapeutically effective amount
is sufficient to allow reperfusion of a brain region wherein the
brain microcirculatory flow was previously blocked. The GSNOR
inhibitor may be administered prior to initiation of the RIC
therapy, or the GSNOR inhibitor may be administered concurrently
with RIC therapy.
[0039] In another embodiment, the invention is a method of
restoring brain microcirculatory flow in a subject suffering from
at least one comorbid condition, comprising the step of
administering in combination a therapeutically effective amount of
an GSNOR inhibitor, wherein the therapeutically effective amount is
sufficient to allow reperfusion of a brain region wherein the brain
microcirculatory flow was previously blocked, and RIC therapy, and
these are followed by administration of IVT for thrombolysis and/or
EVT for clot retrieval.
[0040] As used herein, the term "GRI therapy" refers to
administration or delivery of a GSNOR inhibitor.
[0041] As used herein, the term "RIC therapy" refers to
administration or delivery of remote ischemic conditioning (in
regimens of pre, per and/or post conditionings), a medical
procedure that aims to reduce the severity of ischemic injury to an
organ such as the heart or the brain, most commonly in the
situation of a heart attack or a stroke, or during procedures such
as heart surgery when the heart may temporary suffer ischemia
during the operation, by triggering the body's natural protection
against tissue injury. The procedure involves repeated, temporary
cessation of blood flow to a limb to create ischemia in the tissue.
This "conditioning" activates the body's natural protective
physiology against reperfusion injury and the tissue damage caused
by low oxygen levels. It typically can be administered or performed
using a simple blood pressure cuff, or it can be performed using a
device designed for this purpose, such as one currently in review
at the FDA.
[0042] As used herein, the term "intravenous tissue plasminogen
activator therapy" or "IVT" refers to a medical procedure involving
administration of an FDA-approved intravenous thrombolytic (IVT).
Tissue plasminogen activator (tPA), is an intravenous medicine
given during ischemic stroke, i.e., in a stroke caused by a blood
clot, wherein IVT can dissolve the stroke-causing clot. Studies
have shown that certain stroke patients who receive tPA within 3
hours, have better outcomes. IVT is currently FDA-approved for use
only within the initial 3 hours window of post-ictus.
[0043] As used herein, the term "endovascular thrombectomy" or
"EVT" refers to a medical procedure for mechanical removal of a
thrombus (blood clot) under image guidance using a catheter type
device. A thrombectomy is most commonly performed for an arterial
embolism, which is an arterial blockage often caused by atrial
fibrillation, a heart rhythm disorder, blood clot etc.
[0044] As used herein, the term "biopharmaceutical compound" may be
considered different from a "synthetic pharmaceutical compound",
and may include a non-immunogenic adeno-associated virus carrying a
transgene to genetically intervene and inhibit GSNO-reductase for
long-term benefits via genetic intervention.
[0045] As used herein, the term "pharmaceutically acceptable" means
any molecular entity or composition that does not produce an
adverse, allergic, or other untoward or unwanted reaction when
administered to an individual. As used herein, the term
"pharmaceutically acceptable composition" is synonymous with
"pharmaceutical composition". A pharmaceutical composition of the
invention may be used for human and veterinary applications. In a
preferred embodiment of the invention, a composition of the
invention is administered to humans, most preferably to children.
The pharmaceutical compositions of the invention may be
administered to an individual alone, or in combination with other
active ingredients.
[0046] The invention is a method of treatment to enhance and
preserve endogenous --SNO, a bioactive form of NO that improves
microvascular flow during hypoxia/ischemia without altering blood
pressure (Stamler et al. Science 1997, 276(5321):2034-2037; Shu et
al. Cell Mol Life Sci 2015, 72(23):4561-4575). Thus, the invention
is a method of treatment or therapy to protect against stroke
injury and improve benefits of RIC therapy in stroke with
comorbidity. Of interesting note here, --SNO, the endogenously
preserved vasoactive NO, s-nitrosylates tPA to enhance the effects
of IVT in stroke treatment (Stamler et al. Proc Natl Acad Sci USA.
1992, 89(17):8087-8091). GSNOR is an endogenous enzyme and the
master regulator which balances the endogenous level of --SNO by
degrading/reducing GSNO (Jahnova et al. Plants (Basel). 2019;8(2)).
Thus, an increased activity of GSNOR may deplete endogenous NO
level during stroke and therapies such as RIC therapy. Without
being bound by theory, it is believed that the method of the
invention counteracts this depletion of endogenous NO that is
caused by increased GSNOR activity.
[0047] Acute ischemic stroke, often due to TE occlusion, is the
most common (--87%) type of strokes. Microcirculatory reflow after
stroke is essential for the reoxygenation of ischemic penumbra and
to prevent infarct progression. Despite reperfusing "large" vessels
with the FDA-approved stroke therapies, reoxygenation of brain
frequently remains incomplete, and hence, a critical barrier to
improve post-stroke outcomes. Microcirculation is notably enhanced
with freely diffusible vasculo-humoral NO, which improves tissue
perfusion during hypoxia; however, a recent clinical trial
concluded no benefits from a conventional NO3.sup.-/NO2.sup.-
therapy in stroke. Moreover, a risk of blood pressure BP lowering
remains warranted with the long-term use of exogenous NO-therapy.
Therefore, an objective of the invention is the enhancement and
preservation of "endogenous" NO-metabolome for microvascular
protection in stroke.
[0048] Endothelial NO synthase (eNOS) activity prominently
constitutes the vascular NO-pool that enhances microcirculation.
Genetic impairment or deletion of eNOS in mice augmented
microvascular dysfunction and brain tissue hypoxia during ageing
and also exacerbated injury after acute stroke; thus, the critical
role of endothelial NO was established in microvascular perfusion
and resultant neuroprotection. Following injury, eNOS expression
decreases in micro-vessels and activity is concomitantly impaired;
however, NO generated or delivered remotely was found transportable
to a distant ischemic organ resulting in improved microcirculation
via hypoxic vasodilation. Thus, myocardial overexpression of eNOS
protected against ischemia-reperfusion (IR) injury in liver in an
experimental model. This mechanism of NO preservation and carriage
also involves s-nitrosylation/trans-nitrosylation of thiol group of
proteins (--SH >--SNO) such as glutathione (GSH) and hemoglobin
(Hb) within red blood cells; thereby, s-nitrosylated GSH (GSNO) and
Hb (HbSNO) were recognized as the major endogenous chariots of
bioactive NO which protect against hypoxia and IR- injuries.
Intravenous GSNO-therapy and increased HbSNO with NO-inhalation
improved outcomes in stroke models. Furthermore, --SNO-therapy
reduced embolization in human patients, attenuated secondary
ischemia after sub-arachnoid hemorrhage, and of particular
interest, induced hypoxic vasodilation to enhance cerebral blood
flow in rodents; however, a risk of increased injury remains
associated at certain doses of exogenous GSNO in stroke.
[0049] The enzyme GSNO reductase (GSNOR or ADH5) is a class III
alcohol dehydrogenase which degrades --SNO/GSNO. Genetic deletion
and pharmacological inhibition of GSNOR protected against IR-injury
due to increased endogenous level of --SNO. A GSNOR inhibitor
preserved vasculo-protective benefits of low-level endogenous NO in
hypertension, one of the most common comorbidities associated with
ageing and stroke. GRI therapy, in hypertensive rats with impaired
NO production, preserved flow-mediated dilation and protected
against microvascular and conduit artery dysfunction. GRI therapy
also protected against mechanical reperfusion injury in brain;
however, the effect of stroke on GSNOR expression/activity
particularly in a clinically relevant TE-stroke model has been
previously unknown, as have any potential benefits of its
modulation in different stroke models, ages, sexes and reperfusion
dynamics. The examples of the invention will demonstrate the
effectiveness of the methods disclosed herein.
[0050] Vasculo-humoral NO is decreased during acute stroke in both,
rodents and human patients. Others and we have reported that
therapies capable of modulating endogenous NO-metabolome, such as
RIC, enhances cerebral blood flow, protects against ischemic brain
injuries, and improved efficacies of late IVT in stroke. Of note,
NO following s-nitrosylation of tissue plasminogen activator (tPA)
promotes thrombolysis. Herein, the examples of the invention will
further demonstrate that TE-stroke upregulates GSNOR
expression/activity, which in turn, will attenuate endogenous --SNO
level and augment stroke injury. Furthermore, examples of the
invention demonstrate that GRI therapy in stroke will preserve
endogenous --SNO to augment microcirculation, improve benefits of
reperfusion to reoxygenate brain and attenuate injury to improve
functional outcomes.
[0051] GSNOR inhibitors are known and have been used in
investigational studies of ischemia and reperfusion. For example,
Khan et al. (Brain Res. 2020 Aug. 15;1741:146879.) teaches that
mice were treated with N6022 (5 mg/kg) at 2 hr post
ischemia/reperfusion, daily for 3 days or 2 weeks. Neurological
score, survival rate and motor/cognitive skills were improved.
However, these results were not performed in clinically relevant
thromboembolic stroke model nor were they validated in different
stroke models. While Khan mimics conditions of a specific minor
population of stroke patients reperfused following mechanical EVT,
EVT alone does not guarantee microvascular perfusion and brain
tissue reoxygenation. Furthermore, Khan fails to teach or suggest
IVT, RIC, or any combination of any adjuvants with RIC/IVT. Landman
et al. (Stroke Vol 50; 7, 2019 p19534-1939) teaches the hypothesis
that RIC induces the release of humoral factors including NO, which
activate afferent neural and humoral pathways and shows that RIC
reduces oxidative damage and suppresses inflammatory responses in
the brain. However, Landman fails to make any connection of RIC to
other therapies or consider subjects having comorbidities.
[0052] The GSNOR inhibitor may be administered via any suitable
route, including but not limited to intravenous, intranasal,
nebulization, oral, intramuscular, caudal, intrathecal, and
subcutaneous. Compositions comprising GSNOR inhibitors include any
pharmaceutically-acceptable carrier or buffer suitable for
administration to a subject. In a preferred embodiment, the pH of
the composition is in the range of 6.5 to 8.2 or 6.5 to 8.0 or 6.5
to 7.0. In another preferred embodiment, the pH of the composition
is in the range of 6.6 to 8.2 or 6.6 to 8.0 or 6.6 to 7.0. In a
preferred embodiment, the pH is 6.6, 6.7, 6.8 or 6.9. In a more
preferred embodiment, the pH is 6.7 or 6.8. In a further preferred
embodiment, the pH is 6.8. In another more preferred embodiment,
the pH is 7. In a particularly preferred embodiment, the pH is 6.8
and the buffer strength is 0.05 M. In another particularly
preferred embodiment, the pH is 7.0 and the buffer strength is 0.02
M. Various buffers may be used to prepare a pharmaceutical
composition of the invention, provided that the resulting
preparation is pharmaceutically acceptable. Such buffers include,
without limitation, acetate buffers, citrate buffers, phosphate
buffers, neutral buffered saline, phosphate buffered saline and
borate buffers. It is understood that acids or bases can be used to
adjust the pH of a composition as needed. In a preferred
embodiment, the buffer is sodium citrate buffer. In a more
preferred embodiment, the sodium citrate buffer comprises
tri-sodium citrate, citric acid and purified water. In another
preferred embodiment, the buffer is phosphate buffer. Preferably,
the buffer strength is in the range of 0.01 to 0.1 M or 0.01 to 1
M. More preferably the buffer strength is 0.01 M to 0.06 M. In a
further preferred embodiment, the buffer strength is 0.02 M to 0.06
M, more preferably 0.02 M to 0.05 M and most preferably 0.02 M or
0.05 M. The active ingredients and/or excipients can be soluble or
can be delivered as a suspension in the desired carrier or diluent.
In a preferred embodiment of the invention, the liquid
pharmaceutical composition is a solution.
[0053] Before exemplary embodiments of the present invention are
described in greater detail, it is to be understood that this
invention is not limited to any particular embodiments described
herein and may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting, since the
scope of the present invention will be limited only by the appended
claims.
[0054] Where a range of values is provided, it is understood that
each intervening value between the upper and lower limit of that
range (to a tenth of the unit of the lower limit) is included in
the range and encompassed within the invention, unless the context
or description clearly dictates otherwise. In addition, smaller
ranges between any two values in the range are encompassed, unless
the context or description clearly indicates otherwise.
[0055] 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.
Representative illustrative methods and materials are herein
described; methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present invention.
[0056] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference, and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual dates of public availability and may need
to be independently confirmed.
[0057] It is noted that, as used herein and in the appended claims,
the singular forms "a", "an", and "the" include plural referents
unless the context clearly dictates otherwise. It is further noted
that the claims may be drafted to exclude any optional element. As
such, this statement is intended to serve as support for the
recitation in the claims of such exclusive terminology as "solely,"
"only" and the like in connection with the recitation of claim
elements, or use of a "negative" limitations, such as "wherein [a
particular feature or element] is absent", or "except for [a
particular feature or element]", or "wherein [a particular feature
or element] is not present (included, etc.) . . . ".
[0058] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
EXAMPLES
[0059] The following Examples provide exemplary designs and methods
for fabricating and using microgrippers of the invention. These
Examples describe materials and methods for using embodiments
illustrated in FIGS. 1-16. Additional details can be found in the
section entitled "Brief Description of the Drawings".
[0060] The following Examples of the invention are provided to
demonstrate that TE-stroke enhances GSNOR expression/activity,
which in turn, will attenuate endogenous --SNO level and augment
stroke injury. The Examples also demonstrate that GRI therapy for
stroke will preserve endogenous --SNO to augment microcirculation,
improve benefits of reperfusion to reoxygenate brain and attenuate
injury to improve functional outcomes.
Models of Stroke
[0061] The following models of stroke were used in the Examples of
the invention to demonstrate methods of treatment.
The eMCAo Model of TE-Stroke
[0062] Human fibrinogen-supplemented clot was prepared to induce
TE-stroke as reported by Hoda et. al. Briefly, under isoflurane
anesthesia, the right common carotid artery (CCA), external carotid
artery (ECA) and internal carotid artery (ICA) were exposed with
blunt dissection. A modified microcatheter containing a 9.0
.+-.0.5-mm long clot was inserted into the right ECA, advanced into
the ICA and the clot was gently delivered into the MCA region along
with 100-.mu.L of 1.times. sterile phosphate buffered saline (PBS).
The catheter was retracted, the ECA-arteriotomy was ligated to
prevent bleeding, and wounds were sutured. Sham-operated mice
underwent a similar procedure including infusion of 100 .mu.L of
1.times. sterile PBS without a clot. Mice were returned to clean
warm recovery cages until conscious and were given free access to
NAPA-gel and chow diets along with water ad-libitum until
sacrifice.
The Photothrombotic (PT)-Model of Stroke
[0063] Aged mice (16 .+-.1-mo) were subjected to PT-stroke via a
novel lateral side surgical approach without craniotomy resulting
in the thrombotic occlusion of distal main trunk of MCA. For the
purpose, mice were anesthetized with isofluorane, prepared for
surgery, and rose Bengal dye (50 mg/kgbwt in 200 uL of 1.times.
sterile PBS) was intravenously (IV) infused. Mice were immediately
put on a robotic stereotaxic frame, the head was rotated to face
right side up, and the skull was exposed. The distal trunk of MCA
was visualized underneath the skull bone (without craniotomy) using
a 532 nm laser source (8-mm beam diameter; 4.5 mW) integrated to an
ID8-iris (ThorLabs, NJ, USA). The MCA region above the zygomatic
arc was exposed with the laser for 20-min from a distance of
.about.2-3-mm. Following 20-min laser exposure, thrombotic
occlusion of the distal trunk of MCA was confirmed using laser
doppler flowmeter showing >60% drop in CBF and finally the
lateral side skin wound was closed.
The pMCAL Model of Stroke
[0064] The procedure for pMCAo was performed in mice as reported by
Hoda et al. (Stroke 2012, 43(10):2794-2799). Briefly, under
anesthesia, the junction of left zygomatic arch and squamous bone
was drilled to make a 2-3 mm burr hole to expose the main trunk of
MCA. The left MCA distal to the lenticulostriate branches was
electrocauterized carefully without bleeding. The wound was closed,
and mice were put under post-op recovery as described above.
Sham-operated group underwent similar surgical procedures but
without electrocauterization.
The Reperfused tMCAo Model of Stroke
[0065] The tMCAo surgical procedure is similar to TE-stroke
surgery, however, the transient occlusion is achieved using a
suture instead of a clot as reported by Hoda et al (supra).
Briefly, a 6-0 monofilament suture with .about.3-mm silicone-coated
tip designed to induce tMCAo in mice (Doccol Corp, USA) was
introduced into the ICA via an ECA stump, directed and gently
advanced into the MCA region until the suture was wedged to occlude
the proximal stem of MCA. The occlusion was maintained prior to
withdrawing the filament after 45 minutes, the ECA- arteriotomy was
ligated and the reperfusion was allowed following suture
withdrawal. The sham-operated group underwent similar procedures
without occlusion, and mice recovered as above.
Materials and Methods
Animal Models and Experimental Approach
[0066] All mice under C57/B6 background were bred, housed, used and
related experimental procedures were performed in accordance with
the approved protocol of Institutional Animal Care and Use
Committees (IACUCs) of St. Joseph's Hospital and Medical Center
(SJHMC), and Augusta University, Augusta, Ga. Effect of stroke on
GSNOR expression/activity was first determined in a clinically
relevant partially-humanized TE-stroke, embolic middle cerebral
artery occlusion (eMCAo) model in aged (16.+-.1-mo-old) male mice.
Next, an effective dose-finding study of GRI therapy (stroke injury
size) was performed in aged (16.+-.1-mo-old) male mice in a
photothrombotic (PT) model of stroke with the reduction in infarct
volume as the primary outcome. The safety and benefits of the best
dose of GRI therapy in reducing infarct volume as the primary
outcome was also confirmed in reproductively senescent aged
(16.+-.1-mo-old) female mice subjected to PT-stroke. To test the
benefits of GRI therapy during permanent ischemia, GRI was also
tested in adult (14.+-.1-weeks) mice of both sexes subjected to
permanent model of middle cerebral artery ligation (pMCAL). All
females were ovariectomized at the age of 12-weeks, i.e., 2-weeks
prior to stroke surgery. A 2.times.2 factorial design was adopted
[2 Sexes (MALE vs. FEMALE).times.2 GRI (NO vs. YES)] such that all
4 groups of both sexes were subjected to pMCAL followed by
randomization into two different treatment cohorts: GRI therapy or
equal volume of Vehicle treatment. All mice were tested for
behavioral outcome on a narrow beam test, relative CBF measurement
using laser speckle contrast imaging (LSCI) and infarct volume
size. Next, benefit of GRI therapy was tested with and without RIC
therapy in preserving benefits of endogenous NO in a reperfused
stroke model in diabetic male mice (14.+-.1-weeks). RIC therapy has
been reported to enhance CBF in stroke models likely via enhancing
endogenous NO but remains untested in comorbid (such as diabetic)
stroke, a patient population which are least benefited from the
FDA-approved reperfusion therapies such as endovascular
thrombectomy (ET). To mimic a clinical ET-like scenario, GRI
therapy was tested in a reperfused transient MCA occlusion (tMCAo)
model of stroke. A 2.times.2 factorial design was adopted [2 GRI
(NO vs. YES).times.2 RIC (NO vs. YES)] such that all 4 groups were
subjected to tMCAo followed by randomization into 4 different
treatment cohorts: Vehicle, GRI, RIC and GRI+RIC. Outcome measures
such as brain tissue oxygenation (PbtO2), acute inflammatory genes
response, neurological deficit, edema as percent (%) swelling and
infarct volumes were assessed. Lastly, GRI therapy was also tested
with and without late-IVT, an FDA-approved thrombolytic reperfusion
therapy, in eMCAo for which again a 2.times.2 factorial design was
adopted [2 GRI (NO vs. YES).times.2 IVT (NO vs. YES)] as above:
Vehicle, GRI, IVT and GRI+IVT groups. Adult male mice (16
.+-.2-weeks) were used in this experiment.
Treatments After Stroke
[0067] Briefly, different doses of GRI therapy (N6022; Axon
Medchem, USA) at various time points, as indicated for each of the
following Examples, were tested in different sets of experiments
and stroke models. In general, GRI therapy was IV-infused via the
tail vein following stroke, and IP-repeated after stroke as
indicated. RIC and IVT therapies were performed as indicated. All
control groups were either infused with equal volume of vehicle
(for GRI- and IVT-therapies) or underwent RIC-mock for
sham-operation of RIC therapy.
Laser Doppler Flowmetry (LDF) and Laser Speckle Contrast Analysis
(LASCA) Imaging
[0068] Cortical LDF (Perimed Inc, Sweden) was performed to confirm
the induction of stroke in anesthetized mice. Briefly, mice were
anesthetized as above and a shallow indent was made in the parietal
skull using a low-speed drill to place the LDF probe holder
(PH07-6, Perimed Inc) aligning the holder hole at the stereotaxic
coordinates (AP: 2-mm and lateral 3-mm with respect to bregma). The
needle of LDF-probe (PH407, Perimed Inc) was inserted into the
probe holder and the signal was recorded to confirm a significant
drop in CBF following stroke. Mice were anesthetized as above, body
temperature was maintained at 37.+-.0.5 .degree. C., the skull was
shaved, and a midline skin incision was made to primarily expose
the MCA region. Perfusion images were acquired using PeriCam high
resolution LASCA-Imager (PSI-Z system, Perimed Inc.) with a 70-mW
built-in laser diode for illumination and 1388.times.1038 pixels
CCD camera installed 10 cm above the skull (speed 19 Hz, and
exposure time 6 mSec, 1.3 x 1.3 cm). Acquired video and images were
analyzed for the dynamic changes in CBF. Overall perfusion of the
ipsilateral ischemic hemisphere was determined, normalized with the
equal size of region of interest (ROI) from the uninjured
contralateral hemisphere and the relative CBF was calculated.
MRI Acquisition
[0069] All MRI experiments were conducted using a 7-Tesla
horizontal magnet with a clear bore of 20-cm in diameter interfaced
to a Bruker Avance console. Anesthetized mice underwent the
following MRI-fast spin echo (RARE factor=8) with effective echo
time of 47 ms to create corresponding T2-weighted images to
determine absolute edema/stroke injury volume. Ex vivo diffusion
tensor imagining (DTI) was performed with overnight MRI of fixed
brains through a pulse-gradient spin echo sequence to determine the
diffusion parameters including apparent diffusion coefficient
(ADC), tensor trace and fractional anisotropy (FA).
MRI Data Analysis
[0070] Image post-processing for edema was performed using ImageJ
coupled with in-house designed ImageJ macro scripts. T2-weighted
images were used to determine the volume of cerebral edema by
drawing an irregular ROI to encircle the regions exhibiting edema
in each impacted image slice. The impact volume was determined by
summation of the ROIs in all the impacted image slices and then
multiplying with the thickness of the image slice. DTI analysis was
done using the vendor-supplied software Paraysion 5.1 (Bruker
Inc.), from which the associated diffusion weighted, FA and ADC
images were generated.
Analysis of GSNOR Expression and Activity after Stroke
[0071] Plasma was isolated from freshly drawn blood after cardiac
puncture under deep isoflurane anesthesia, and mice were sacrificed
to collect brain tissue. Plasma and brain tissue samples were
snap-frozen until assay.
Immunoblot Assay for GSNOR Expression
[0072] Gel electrophoresis followed by immunoblot for the GSNOR
expression in brain tissue samples (perfused with 50-mL of chilled
1X sterile PBS) was performed using conventional methods. Briefly,
brain homogenates in modified RIPA buffer (Upstate, Lake Placid,
NY), supplemented with 40-mM NaF, 2-mM Na.sub.3VO.sub.4, 0.5-mM
phenylmethylsulfonyl fluoride and 1:100 (v/v) of proteinase
inhibitor cocktail (Sigma), were electrophoresed and immunoblotted
separately against antibodies for anti-GSNOR (Sigma), and anti-beta
actin (.beta.-Actin; Santa Cruz Biotech, USA) as loading control.
Immuno-densitometric signal for GSNOR was quantified in arbitrary
units (AU) using ImageJ NIH free software and was normalized with
the corresponding expression of .beta.-Actin.
GSNOR Activity Assay
[0073] A method of GSNO dependent NADH consumption was used to
determine GSNOR activity as reported. Briefly, brain tissue was
homogenized in a buffer containing 50mM Tris-HCl (pH 8.0), 150 mM
NaCl, 1-mM EDTA, 0.1% Triton X-100 and 1:100 protease inhibitor
cocktail and centrifuged (10, 000 g.times.10 minutes at 4.degree.
C.) to obtain supernatant. Next, plasma and brain tissue
supernatant were diluted to a protein concentration of 1 mg/mL and
0.1 mg/mL, respectively, in a reaction buffer containing 20 mM
Tris-HCl (pH 8.0), 0.5 mM EDTA and 0.7504 NADH following which
samples were incubated in triplicate with or without 100 .mu.M GSNO
in 96-well plate. The consumption of NADH was monitored with
fluorescence spectrophotometry at an excitation wavelength of 340
nm and emission at 460 nm. GSNOR activity was calculated by
subtracting the rate of NADH consumption incubated in the presence
of GSNO minus the rate of NADH consumption without GSNO in
samples.
Euthanasia and 2,3,5-Triphenyltetrazolium Chloride (TTC)
Staining
[0074] TTC-stain differentiates between metabolically active (live
or penumbra) and inactive (dead or core) tissues after stroke. TTC
is a white powder and the solution remains colorless, which is
reduced to red 1,3,5-triphenylformazan (TPF) by the enzymatic
action of various dehydrogenases primarily mitochondrial
dehydrogenase from the living tissues, while the core remains as
white. Therefore, larger white area indicates higher injury and
infarction volume. After performing ATT, mice were overdosed and
deeply anesthetized with high isofluorane (5%). Brains were very
quickly perfused with cold 25 ml of 0.01 M phosphate-buffered
saline (PBS), harvested fresh and immediately placed in a metallic
mouse brain matrix. Looking at the infarcted area, 5 blades were
placed in alternate gaps to cut and obtain 2-.mu.m.times.4 coronal
slices. Sections were individually placed in a 35-mm dish
containing pre-warmed (37.degree. C.) 3-ml of 5% TTC in PBS (Sigma)
for 20-25 minutes at 37.degree. C., followed by 2.times. washing
with cold PBS and fixation with 10% formalin. In order to image,
fixed sections were taken out of the dish and placed in order on a
high-resolution Cannon Scanner. After scanning, images were cropped
and saved for analysis. Corrected infarct volume was estimated
using gray scale image and Scion Image software and presented as
the Corrected % infarct volume normalized to the uninjured
side.
Behavioral Tests
Beam Walk Test for Motor Balance and Coordination
[0075] Motor balance and coordination test was performed using the
narrow balance beam walk. The beam apparatus consisted of a
graduated beam, was fixed 20-cm above the tabletop on two poles
(6-mm flat width.times.125-cm long). A black box was fixed at the
end of narrow beam as the finish point containing nesting (bedding)
material from the home cage to attract the mouse. A lamp (with
60-watt light bulb) is used to shine light above the start point,
serving as an aversive stimulus. Mice were trained to walk on the
barrow beam prior to injury and before performing the post-stroke
test. During training, mice were encouraged to keep moving across
the beam by gently poking or pushing on their back. Training trials
were repeated for 3 trials.times.3 consecutive days or until each
animal crosses the beam 3.times. without stopping or turning
around, and without any assistance. On a final day of testing, each
mouse was tested to walk 100-cm on the beam with 3-trials.times.5
min interval between trials. Performance of each mouse on the
narrow beam was quantified by measuring the time it took for the
mouse to traverse the 100-cm distance on the narrow beam and the
number of foot slips that occurred in each trial. The mean value of
3 trials was calculated for the time taken in traversing the 100-cm
distance on the beam, and the number of foot faults.
Neurologic Deficit Score (NDS)
[0076] Neurologic deficits after stroke in mice were assessed by
blinded investigators blinded on a 5-point modified Bederson
NDS-scale. The highest number indicates the worst outcomes while
lower number indicates better neurological outcomes: 0, no deficit
(normal mice); 1, forelimb flexion deficit on contralateral side;
2, flexion deficit along with decreased resistance to lateral push
and torso-turning to the ipsilateral side when held by tail; 3, all
deficits as in Score 2 including very significant circling to the
affected side during the move inside the cage and reduced
capability to bear weight on the affected side; 4, all deficits as
above but rarely willing to move spontaneously and preferring to
stay at rest.
Hanging Wire Test for Forelimb Coordination and Muscular
Strength
[0077] This test was used as a measure of grasping ability,
forelimb strength and muscular coordination movements. The
apparatus is consisted of a 55-cm long 1-mm diameter single metal
cord stretched firmly between two metal stands, 50-cm above a
standard mouse cage filled with soft paper bedding material. In the
training process, mice are gently held and taken closer to the wire
to grab it with their two forelimbs. Mice were trained for 3
trials.times.3 consecutive days before the actual test, or until
they are trained to grab it with two forelimbs. For the actual
test, the latency to lose the grip on the wire falling into the
cage was recorded for 3 trials.times.5-min interval between trials
for each mouse.
Novel Object Recognition (NOR) Test for Cognitive Function
[0078] Object recognition memory is one of the domains of cognition
that is often impaired in aged (non-demented) individuals as well
as in patients with other forms of dementia. Importantly, the
rodent NOR-task has been described as a model of (non-spatial)
recognition memory. This form of memory is believed to consist of a
recollected (episodic) and a familiarity component, i.e., behaviors
that are demonstrated in the NOR-task when subjects explore a novel
object more than a familiar one. The NOR-task has become a popular
method for rodent studies with particular relevance to dementia and
neuropsychiatric disorders.
[0079] Behavioral assessment by NOR test that reliably evaluates
the nonspatial working memory of the subcortical region was
performed 4-wks after stroke. Briefly, mice were habituated in an
activity box and familiarized with two identical objects placed at
a set distance apart. On the day of trial, each mouse was
individually given similar trial and the time spent (T.sub.f) with
the familiar object was recorded. After the familiar object trial,
the mouse was then removed from the environment for a set amount of
time and one of the two previously used (familiar) objects was
replaced with a novel object that was different from the familiar
object in shape, texture, and appearance. The time spent (T.sub.n)
by the mouse with the novel object upon exposure during the probe
trial was then recorded in one 5-min trial. This test is based on
the natural tendency of mouse to investigate a novel object rather
than a familiar one, which reflects the use of learning and
recognition memory processes. The capability of the mouse to
discriminate between a familiar vs. novel object was determined as
the discrimination index, which is calculated by
DI=(T.sub.n-T.sub.f)/(T.sub.n+T.sub.f). A lower DI-value reflects
poor cognitive function, while a higher DI-values demonstrates a
better learning and memory function.
Data Analysis
Data and Statistical Analysis
[0080] Quantitative data were presented as mean .+-.standard
deviation (SD). Wherever needed, unpaired t-test was performed to
determine statistical significance. A log transformation was used
prior to statistical analysis as needed to stabilize variance
across groups. Non-normal data between groups were compared using
nonparametric asymptotic two-sided Wilcoxon rank sum test. In
experiments with 2.times. factorial design, a factorial analysis of
variance (ANOVA) with interaction between two therapies followed by
post-hoc comparisons using independent t-tests was used to analyze
the outcome measures. Bonferroni multiple corrections were applied
in the post-hoc analysis. In the absence of a significant
interaction, the main effects were considered to be additive when
combined. A p-value <0.05 was considered statistically
significant. All statistical analyses were conducted using STATA
15.
Data
[0081] FIG. 3E, FIG. 4B, FIG. 4D, FIGS. 6A-D, FIG. 8B, FIG. 9A-D,
and others in this description are real time curves/plots from the
instrument. They are presented to show the real time output as an
example from one animal. For each animal subject, the values are
averaged and calculated as either a percentage (%)(e.g., for
CBF/perfusion) or in mmHg for oxygen (after converting the
millivolt output) and then they are plotted as the data for each
group.
Example 1
[0082] Effect of eMCAo on GSNOR protein expression and
activity.
[0083] The eMCAo model was chosen because it is the most clinically
relevant rodent model of TE stroke. Aged (16.+-.1-mo) mice were
subjected to eMCAo/stroke using a partially-humanized TE-clot. At 6
h post-eMCAo, blood was collected via cardiac puncture to isolate
plasma and mice were quickly flushed-perfused with 25-mL of chilled
0.1M PBS prior to collecting brain samples. Both plasma and brain
samples were snap-frozen in liquid nitrogen until assays. FIGS. 1A
and 1B show whole brain assay and the resultant relative normalized
densitometry data confirmed that the protein expression of GSNOR
was significantly (p<0.01) increased as early as 6h post-stroke
as compared to sham-operated group. An activity assay based on the
rate of GSNO-dependent consumption of NADH further confirmed that
the increase in protein expression of GSNOR translated into
significant (p<0.001) acute increase in the enzymatic activity
of GSNOR as early as 6 h after stroke. FIG. 1C shows GSNOR activity
in brain and
[0084] FIG. 1D shows GSNOR activity in plasma.
Example 2
[0085] Effect of GRI Therapy in Aged Mice of both Sexes following
PT Stroke.
[0086] The PT stroke model was chosen because it is a thrombotic
model of moderate injury. FIG. 2A shows a representative image of
whole brain collected immediately after stroke to confirm the site
(black arrow) of occlusion in MCA region. FIG. 2B shows a
Z-projection of a T2-weighted MR-image 48 h after stroke. The site
of stroke injury is indicated with a white arrow. Different doses
of GRI therapy or equal volume of vehicle to aged male mice
(N=8/gp) given intravenously 1 h after PT-stroke (1, 2.5, 5.0 and
10 mg/kg) showed a dose-dependent neuroprotective effect. However,
a plateau in the neuroprotective effect was seen beginning from the
GRI dose >2.5 mg/kg. Densitometry showing absolute infarct
volume in FIG. 2C was performed on tissues sections, including
those shown in FIG. 2D. FIG. 2D shows representative TTC-stained
set of coronal sections (2-.mu.m thick) obtained from the whole
brain from the aged male mice and stained freshly. GRI therapy
significantly protected against the stroke and prevented the
infarct/injury progression as compared to vehicle treated group
(P<0.0001). Data are presented as Mean .+-.SD. Statistical
significance was determined at P<0.05 and were indicated with
asterisks or different letters indicating statistically different
Means.
[0087] A sex difference was found in the basal level of plasma
GSNOR activity, showing higher GSNOR activity in female mice
compared to age-matched males (P<0.05; N=6/gp) as shown in FIG.
3A. Therefore, GRI therapy was also tested in aged female mice
(N=10/gp). (F-H) The effective dose of GRI therapy (2.5 mg/kg) for
aged male mice was tested in aged female mice as described above
for males. As evident in FIG. 3B, there was a significant increase
(P<0.0001) in plasma GSNOR activity in aged females, too, after
PT-stroke. GRI therapy after PT-stroke in females significantly
inhibited the post-stroke plasma GSNOR activity (P<0.01). GRI
therapy was also effective in reducing the stroke injury
significantly compared to vehicle treated group (P<0.05). FIG.
3D shows representative TTC-stained set of coronal sections
(2-.mu.m thick) obtained from the whole brain of aged female mice
and stained freshly. Data are presented as Mean.+-.SD. Statistical
significance was determined at P<0.05 and were indicated with
asterisks or different letters indicating statistically different
Means.
Example 3
[0088] GRI Therapy Protects against Permanent Ischemia Induced by
pMCAo Independent of Sex
[0089] Adult mice of both sexes (14.+-.1-weeks; N=10/gp; females
were ovariectomized, OVX) were subjected to pMCAL-stroke model with
electrocauterization leading to permanent occlusion of distal MCA
trunk. Mice were anesthetized with isofluorane, prepared for
surgery, and the right parietal bone of the skull was exposed. A
craniotomy was performed to expose the distal part of MCA trunk and
the artery was permanently electrocauterized using a bipolar
electrocautery. FIG. 4A shows a representative LSCI image.
Immediately after stroke, a significant drop in CBF occurred. Mice
were IV-treated with either GRI therapy (2.5 mg/kg) or equal volume
of vehicle at 1 h after stroke and the treatment was repeated daily
for 2 days. Mice were evaluated for behavioral outcomes at 72 h
post-stroke. A 2 sexes (Male vs. Female) by 2 treatments (GRI vs.
Veh) ANOVA was used to analyze results. All the data are expressed
as mean.+-.SD, pairs of means indicated with different letters are
significantly different (P<0.05). Narrow beam walk test
determined that there was no significant difference in the
deterioration of motor function between two sexes in the context of
walking time to traverse through the beam length and number of foot
slips. FIG. 4B shows the time to traverse the beam, and FIG. 4C
shows the mean number of foot slips. GRI therapy was equally
effective in improving motor function in both sexes.
[0090] Mice were also evaluated for CBF changes and infarct volume
analysis at 72 h post-stroke. A 2 sexes (Male vs. Female) by 2
treatments (GRI vs. Veh) ANOVA was used to analyze results. Changes
in CBF showed a trend toward slight differences in vehicle-treated
OVX-females as evident from the plot shown in FIG. 4D.
[0091] At 72 h post-pMCAo, % CBF loss in the injured side of brain
in both sexes was evident, as shown in FIG. 5A for males, and FIG.
5B for OVX-females, but was also significantly higher in
OVX-females compared to males. GRI therapy improved restoration of
CBF in both sexes significantly compared to their corresponding
vehicle treated groups. The GRI therapy treated groups in both
sexes showed no significant difference in % CBF when compared
together, demonstrating that the effect of GRI therapy in enhancing
CBF remains sex independent.
[0092] Real-time perfusion was measured in mice at 72 h
post-stroke. FIG. 6A shows vehicle-treated males and FIG. B shows
GRI-treated males. FIG. 6C shows vehicle-treated OVX-females
(OVX-F) and FIG. 6D shows GRI-treated OVX-females. As with the %
CBF and LSCI, vehicle-treated OVX-females showed a slight trend
towards difference in real-time perfusion plots. At 72 h
post-pMCAo, GRI therapy treated groups in both sexes showed no
significant difference in real-time perfusion plots when compared
together, demonstrating that the effect of GRI therapy in enhancing
CBF remains sex independent.
[0093] TTC-staining of fresh brain tissue, shown in FIG. 7A, and
infarct volume analysis, shown in FIG. 7B, further demonstrates
that vehicle treated OVX-females were significantly more prone to
stroke injury compared to their vehicle treated male counterparts.
GRI therapy significantly prevented the infarct progression
sex-independently in both groups.
Example 4
[0094] GRI Therapy Improves Acute Brain Tissue Oxygenation
following Reperfusion and Enhances Benefits of RIC therapy in
STZ-induced Diabetic Mice.
[0095] This example is a demonstration of reperfusion and
restoration of oxygenation (PbtO2) in reperfused tMCAo stroke
model. Diabetes was induced in adult male mice (14.+-.1 weeks of
age) as confirmed after 7 days of STZ-injection, followed by tMCAo
stroke surgery at 3 weeks post-STZ injection. Stroke was induced
with 60-min suture occlusion followed by reperfusion with suture
withdrawal. GRI therapy or vehicle was IV-infused 10-min prior to
reperfusion and RIC therapy/ RIC-mock was performed 1 h after
reperfusion. A 2 GRI (GRI vs. Veh) by 2 RIC (RIC therapy vs.
RIC-mock) ANOVA was used to analyze results. All the data are
expressed as Mean.+-.SD, and pairs of Means indicated with
different letters are significantly different (P<0.05).
Representative LSCI, shown in FIG. 8A, and real-time perfusion
curve during ischemia and reperfusion, shown in FIG. 8B,
demonstrated successful induction of stroke and restoration of CBF.
These results were comparable to those shown in Example 3 using the
tMCAo model. PbtO2 was measured at 6h post-reperfusion (N=6/gp)
using a 50-um thick oxygen sensor integrated to an UniAmp
multi-channel system (Unisense, Denmark). Measurement signal was
averaged for 3 min during a period of stable output as shown in the
representative real-time electrogram traces in FIGS. 9A-9D. FIG. 9A
is representative of a mouse treated with vehicle and receiving
mock RIC. FIG. 9B is representative of a mouse receiving vehicle
and RIC therapy. FIG. 9C is representative of a mouse receiving GRI
therapy and mock RIC. FIG. 9D is representative of a mouse
receiving GRI therapy and RIC therapy. PbtO2 values were calculated
from the calibration curve and associated regression equation,
shown in FIG. 10A. As evident from the data plot in FIG. 10B, GRI
therapy alone improved PbtO2 significantly as compared to
vehicle-treated RIC-mock operated group. RIC therapy alone did not
improve PbtO2 in diabetic stroke as compared to vehicle-treated
stroke group; however, in combination therapy group, a prior
treatment with GRI followed by RIC therapy showed enhanced efficacy
of both, GRI and RIC-therapies, in improving benefits of
reperfusion to increase PbtO2.
[0096] Mice were immediately sacrificed after PbtO2 measurement and
brain tissue samples were analyzed for inflammatory gene expression
(N=6/gp). RT-PCR primers shown in Table 1 were used to measure
changes in expression of iNOS (FIG. 11A), iCAM-1 (FIG. 11B), IL-1(3
(FIG. 11C) and IL-6 (FIG. 11D). The result for each inflammatory
gene was normalized to expression of .beta.-actin and expressed as
a fold change.
TABLE-US-00001 TABLE 1 Nucleotide Sequences of Mouse Primers used
for RT-PCR. Genbank Gene Accession No. Primer sequence SEQ ID NO
iNOS NM_010927.3 GAT GTG CTG CCT CTG GTC TT SEQ ID NO: 1 TT GGG ATG
CTC CAT GGT CAC SEQ ID NO: 2 iCAM-1 NM_010493 ACG CAG AGG ACC TTA
ACA GTC TAC SEQ ID NO: 3 GCT TCA CAC TTC ACA GTT ACT TGG SEQ ID NO:
4 IL-1.beta. NM_010554.4 GCA CCT TAC ACC TAC CAG AGT SEQ ID NO: 5
AAA CTT CTG CCT GAC GAG CTT SEQ ID NO: 6 IL-6 NM_031168.1 TAG TCC
TTC CTA CCC CAA TTT CC SEQ ID NO: 7 TTG GTC CTT AGC CAC TCC TTC SEQ
ID NO: 8 .beta.-actin NM_007393 TGA CAG ACT ACC TCA TGA AGA TCC SEQ
ID NO: 9 ACA TAG CAC AGC TTC TCT TTG ATG SEQ ID NO: 10
[0097] GRI therapy alone showed a trend only to downregulate most
inflammatory genes and were not significantly different from the
vehicle-treated stroke group. Surprisingly, RIC therapy alone
enhanced inflammatory responses in stroke-induced diabetic mice;
however, in the combination GRI+RIC therapy group, prior treatment
with GRI significantly downregulated and prevented RIC-induced
inflammatory responses in diabetic stroke mice as compared to the
RIC therapy alone group.
Example 5
[0098] GRI Enhances Neuroprotective Efficacy of RIC therapy in
tMCAo -STZ-induced Diabetic Mice.
[0099] Diabetic adult male mice (as described in Example 4) were
subjected to tMCAo (N=10/gp) with or without GRI and/or
RIC-therapies and outcome measures were performed at 24h
post-stroke. A 2.times.2 factorial ANOVA was used to analyze
results, data are expressed as Mean.+-.SD, indicating Means with
different letters are significantly different (P<0.05).
[0100] Looking at the results for mice receiving a vehicle
treatment instead of GRI and/or RIC, treatments including any sham
component (vehicle-mock operated control, GRI-treatment singly and
RIC therapy alone) did not improve any of the neurological outcomes
of mean NDS, % infarct volume, or % swelling, shown in FIG. 12A,
12B, and 12C, respectively. However, when low-dose GRI was combined
with RIC therapy, all outcome measures were significantly improved
in combination therapy group as compared to all other 3 groups.
Representative TTC-stained fresh brain tissue images, shown in FIG.
12D, further supports the finding that GRI in combination with the
NO-enhancing RIC therapy increases neuroprotection in diabetic
stroke.
Example 6
[0101] GRI Therapy Protects against TE Stroke Injury and Enhances
Benefits of Late-IVT Therapy.
[0102] In this example, the TE-clot eMCAo model is used to
demonstrate a method of the invention. Adult male WT-mice
(16.+-.2-weeks old; N=20/gp) were subjected to eMCAo using a
partially humanized TE-clot (9.+-.1-mm). Mice were randomized to
4-groups: 2 GRI (GRI vs. Veh) by 2 IVT (IVT vs. Veh). GRI therapy
(2.5 mg/kgbwt) or equal volume of vehicle was IV-infused at 1 h
post-eMCAo. The same dose of GRI therapy or equal volume of vehicle
was also repeated and IP-injected after IVT or IVT-veh at 6 h and
24 h post-eMCAo. Late IVT-therapy (tPA as Altepase formulation; 10
mg/kg in 250 uL) or equal volume of vehicle was infused at 5 h
post-eMCAo as 10% of volume IV bolus and remaining over a period of
20 min. A 2 GRI (GRI vs. Veh) by 2 IVT (IVT vs. Veh) ANOVA was used
to analyze results, as appropriate. All the data are expressed as
mean .+-.SD, and pairs of means indicated with different letters
are significantly different (P<0.05).
[0103] For stroke injury/edema volume measurement, 6 surviving mice
from each group were randomly selected for T2-Weighted MRI at 24 h
post-eMCAo. As evident from the volume quantification shown in FIG.
13A, early GRI therapy alone after stroke prevented the edema
progression significantly as compared to Veh-treated group and also
in comparison to late-IVT therapy. The representative coronal
slices from the MCA region shown in FIG. 13B further support the
quantitative finding shown in FIG. 13A. Moreover, as anticipated,
late-IVT therapy did not prevent the edema progression/stroke
injury significantly as compared to Veh-treated group. However,
treatment with GRI therapy after eMCAo and prior to tPA-injection
enhanced the benefits of late IVT and prevented edema progression
significantly as compared to both, Veh- and IVT-treated groups.
[0104] Neurological deficit scoring (NDS) based on a modified
Bederson Scale was performed for all surviving mice at 96 h
post-eMCAo. As evident from the mean NDS values shown in FIG. 14A,
early GRI therapy alone after stroke reduced the later neurological
deficit significantly as compared to both Veh- and late-IVT-treated
groups. Late-IVT therapy failed to improve the neurological score;
however, GRI therapy in combination with late-IVT therapy
significantly improved the neurological outcomes at 96 h
post-stroke. Kalan-Meier survival curve for all 4 groups followed
for 2 weeks. As evident from the curve shown in FIG. 14B, both
groups without a GRI treatment showed higher mortality within the
initial 5 days (acute/sub-acute phase) after stroke with a
prominent trend in mortality in late-IVT treated group. A higher
trend in mortality in late-IVT treated group is likely attributed
to frequent hemorrhagic transformation, as indicated (asterisk) in
the representative coronal section for IVT-group in FIG. 14B.
[0105] On day 13 & 14, all surviving mice were tested for
long-term functional outcomes such as the recovery of muscular
strength (hanging wire test), shown in FIG. 15A, and protection of
learning/memory function (NOR test), shown in FIG. 15B. In
agreement with the benefits of acute protection, GRI therapy alone
and in combination with late-IVT therapy significantly improved and
protected the long-term motor and learning/memory functions,
respectively, compared to both Veh- and late-IVT-treated groups. As
such, late-IVT beyond 4.5 h post-stroke remained ineffective in
improving long-term functional outcomes as compared to Veh-treated
mice.
[0106] At day-15 post-eMCAo, all mice were sacrificed after
behavioral outcome tests and brains were harvested. Q-ball MRI
(DTI, T1-Intensity and DWI sequences) of randomly assigned
representatives of each group was performed ex vivo using fixed
brain samples. Results are shown in representative series of
sections for each treatment group in FIG. 16. As evident from the
appearance of the injured hemisphere (stroked ipsilateral side),
both Veh- and late-IVT-treated groups demonstrated higher cerebral
atrophy, larger ventricle size and greater loss in white matter
(WM; corpus callosum) integrity. In contrast, GRI-treated groups
with and without late-IVT showed preservation of WM-integrity,
reduced cerebral atrophy and decrease in ventricle size
enlargement.
[0107] The foregoing examples of the invention demonstrate the
following novel findings: [0108] 1. The changes in expression and
activity GSNOR have never been investigated and reported in stroke
models, particularly in the most clinically relevant partially
humanized TE stroke model. For the first time, these examples
demonstrate that GSNOR expression/activity is increased within 3-6
hours after stroke. [0109] 2. A dose-escalation study for GRI
therapy with validation in different stroke models, different ages
and sexes, and various dynamics of reperfusion, has never been
performed. For the first time, the Examples of the invention
demonstrate differential effects of GRI therapy in various
clinically relevant settings, such as different dynamics of
reperfusion (permanent occlusion, partial reperfusion, complete
reperfusion simulating IVT and EVT), aged vs young rodents, and
male vs female. [0110] 3. The GRI therapy has not previously been
tested in any kind of comorbid (diabetic, hypertensive) stroke
models with or without reperfusion. For the first time, the
Examples of the invention demonstrate therapeutic benefits of GRI
therapy in comorbid strokes models in both, reperfused and
non-reperfused stroke models. [0111] 4. RIC therapy is a modulator
of endogenous NO level and a recent outcome report informs that the
overall benefit of RIC therapy was neutral, i.e., without any
benefits. Of note, a large population of stroke patients have
comorbidities (Pico et al. Int J Stroke. 2016;11(8):938-943 (see
also, pubmed.ncbi.nlm.nih.gov/31500849,
pubmed.ncbi.nlm.nih.gov/31621833, pubmed.ncbi.nlm.nih.gov/27412192,
and pubmed.ncbi.nlm.nih.gov/29748420)). The Examples of the
invention provide the first demonstration that RIC therapy alone is
not protective in comorbid stroke models. These Examples also
demonstrate for the first time that in comorbid
(diabetic/hypertensive) strokes, performing GRI therapy prior to
RIC enhances the benefits of RIC, turning it into an effective
therapy in ischemic stroke with comorbidity. [0112] 5. Since it is
important to demonstrate the safety of GRI therapy with the
FDA-approved IVT, The Examples also demonstrate for the first time
that the GRI therapy in combination with IVT therapy in the most
clinically relevant partially humanized TE stroke model enhances
the benefits of IVT and extends its safe window of thrombolysis.
[0113] 6. Using state-of-art live animal imaging (MRI and LASCA)
and in vivo brain tissue oxygenation recording techniques, the
Examples of the invention demonstrate for the first time that GRI
therapy improves microcirculation and brain tissue oxygenation.
[0114] In summary, the methods of the invention demonstrate the
efficacy of GRI therapy as an adjunct therapy administered with RIC
therapy in different models of stroke, particularly comorbid
stroke, and under various clinically relevant conditions. GRI
therapy showed a dose dependent differential effect, enhanced
neurovascular protection and behavioral outcomes, and remain safe
and beneficial with FDA-approved therapies and therapies under
trial such as RIC.
[0115] While the invention has been described in terms of its
several exemplary embodiments, those skilled in the art will
recognize that the invention can be practiced with modification
within the spirit and scope of the appended claims. Accordingly,
the present invention should not be limited to the embodiments as
described above but should further include all modifications and
equivalents thereof within the spirit and scope of the description
provided herein.
ACKNOWLEDGMENT
[0116] "This project was funded by the National Plan for Science,
Technology and Innovation (MAARIFAH)--King Abdulaziz City for
Science and Technology--the Kingdom of Saudi Arabia--award number
(14-MED2597). The authors also, acknowledge with thanks Science and
Technology Unit, King Abdulaziz University for technical support".
Sequence CWU 1
1
10120DNAArtificial SequenceSynthetic oligonucleotide 1gatgtgctgc
ctctggtctt 20220DNAArtificial SequenceSynthetic oligonucleotide
2ttgggatgct ccatggtcac 20324DNAArtificial SequenceSynthetic
oligonucleotide 3acgcagagga ccttaacagt ctac 24424DNAArtificial
SequenceSynthetic oligonucleotide 4gcttcacact tcacagttac ttgg
24521DNAArtificial SequenceSynthetic oligonucleotide 5gcaccttaca
cctaccagag t 21621DNAArtificial SequenceSynthetic oligonucleotide
6aaacttctgc ctgacgagct t 21723DNAArtificial SequenceSynthetic
oligonucleotide 7tagtccttcc taccccaatt tcc 23821DNAArtificial
SequenceSynthetic oligonucleotide 8ttggtcctta gccactcctt c
21924DNAArtificial SequenceSynthetic oligonucleotide 9tgacagacta
cctcatgaag atcc 241024DNAArtificial SequenceSynthetic
oligonucleotide 10acatagcaca gcttctcttt gatg 24
* * * * *