U.S. patent application number 14/531296 was filed with the patent office on 2015-02-19 for n,n'-di-1 naphthylguanidine hcl (nagh) and n,n'-di-p-nitrophenylguanidine hcl (nad) treatment for stroke at delayed timepoints.
This patent application is currently assigned to UNIVERSITY OF SOUTH FLORIDA. The applicant listed for this patent is Jon C. Antilla, Michelle Cortes-Salva, Javier Cuevas, Keith R. Pennypacker, Alison Willing. Invention is credited to Jon C. Antilla, Michelle Cortes-Salva, Javier Cuevas, Keith R. Pennypacker, Alison Willing.
Application Number | 20150051291 14/531296 |
Document ID | / |
Family ID | 49515039 |
Filed Date | 2015-02-19 |
United States Patent
Application |
20150051291 |
Kind Code |
A1 |
Pennypacker; Keith R. ; et
al. |
February 19, 2015 |
N,N'-DI-1 NAPHTHYLGUANIDINE HCL (NAGH) AND
N,N'-DI-P-NITROPHENYLGUANIDINE HCL (NAD) TREATMENT FOR STROKE AT
DELAYED TIMEPOINTS
Abstract
A composition and method of treating stroke by administration of
a novel sigma agonist is presented. Twenty-four hours after MCAO,
systemic administration of several novel sigma agonists including:
Bromo-DTG; Chloro-DTG; N,N'-di-1-Naphthylguanidine hydrochloride
(NAGH); N,N'-di-p-Nitrophenylguanidine HCL (NAD) or vehicle were
injected subcutaneously daily for 3 days. Rats treated with
Bromo-DTG and Chloro-DTG had no significant improvements in any of
the motor or cognitive tests while NAGH treated rats showed
improved vertical movement and had significantly less motor
asymmetry and bias than vehicle treated rats. Sigma receptor
agonist NAGH also was found to exert its long-term neuroprotective
effects by preserving both gray matter and white matter tracts.
Both NAD and NAGH, when administered 24 hours after experimental
stroke, reduced neural damage and enhanced behavioral recovery
thirty days later which suggests that NAGH and NAD potentially
extend the therapeutic window of stroke several fold over the
current treatments.
Inventors: |
Pennypacker; Keith R.;
(Wesley Chapel, FL) ; Willing; Alison; (Tampa,
FL) ; Cuevas; Javier; (Lutz, FL) ; Antilla;
Jon C.; (Tampa, FL) ; Cortes-Salva; Michelle;
(Fairfax, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pennypacker; Keith R.
Willing; Alison
Cuevas; Javier
Antilla; Jon C.
Cortes-Salva; Michelle |
Wesley Chapel
Tampa
Lutz
Tampa
Fairfax |
FL
FL
FL
FL
VA |
US
US
US
US
US |
|
|
Assignee: |
UNIVERSITY OF SOUTH FLORIDA
Tampa
FL
|
Family ID: |
49515039 |
Appl. No.: |
14/531296 |
Filed: |
November 3, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US2013/039301 |
May 2, 2013 |
|
|
|
14531296 |
|
|
|
|
61641514 |
May 2, 2012 |
|
|
|
Current U.S.
Class: |
514/634 ;
564/238 |
Current CPC
Class: |
A61K 31/155 20130101;
A61P 9/10 20180101; C07C 279/18 20130101; A61P 9/00 20180101 |
Class at
Publication: |
514/634 ;
564/238 |
International
Class: |
C07C 279/18 20060101
C07C279/18 |
Claims
1. A method of treating stroke at a delayed timepoint comprising:
administering a therapeutically effective amount of a sigma agonist
selected from the group consisting of N,N'-di-1-Naphthylguanidine
hydrochloride (NAGH) or N,N'-di-p-Nitrophenylguanidine
hydrochloride (NAD) to a patient in need thereof.
2. The method of claim 1, wherein the sigma agonist is administered
at least 12 hours after stroke.
3. The method of claim 1, wherein the sigma agonist is NAGH.
4. The method of claim 3, wherein the therapeutically effective
amount is between about 0.86 mg/kg and 8.6 mg/kg.
5. The method of claim 4, wherein the NAGH is administered at least
once per day.
6. The method of claim 5, wherein the NAGH is administered for at
least 3 days
7. The method of claim 1, wherein the sigma agonist is NAD.
8. The method of claim 7, wherein the therapeutically effective
amount is between about 2.8 mg/kg and 8.6 mg/kg.
9. The method of claim 8, wherein the NAD is administered at least
once per day.
10. The method of claim 9, wherein the NAD is administered for at
least 3 days.
11. A method of reducing infarct volume long term in a patient
suffering from a neurological disorder comprising: administering a
therapeutically effective amount of a sigma agonist selected from
the group consisting of N,N'-di-1-Naphthylguanidine hydrochloride
(NAGH) or N,N'-di-p-Nitrophenylguanidine hydrochloride (NAD) to a
patient in need thereof
12. The method of claim 11, wherein the neurological disorder is
stroke.
13. The method of claim 11, wherein the sigma agonist is
administered at least 12 hours after stroke.
14. The method of claim 11, wherein the sigma agonist is NAGH.
15. The method of claim 14, wherein the therapeutically effective
amount is between about 0.86 mg/kg and 8.6 mg/kg.
16. The method of claim 15, wherein the NAGH is administered at
least once per day.
17. The method of claim 16, wherein the NAGH is administered for at
least 3 days.
18. The method of claim 11, wherein the sigma agonist is NAD.
19. The method of claim 18, wherein the therapeutically effective
amount is between about 2.8 mg/kg and 8.6 mg/kg.
20. The method of claim 19, wherein the NAD is administered at
least once per day.
21. A compound comprising formula (I): ##STR00001## wherein R.sup.1
and R.sup.3 are cyclic compounds selected from the group consisting
of phenyl and naphthyl; wherein R.sup.2 and R.sup.4 are selected
from the group consisting of Cl, Br, hydrogen, and NO.sub.2; or a
salt thereof.
22. The compound of claim 21, wherein the composition is selected
from the group consisting of N,N'-di-1-Naphthylguanidine (NAGH),
N,N'-di-p-Nitrophenylguanidine (NAD), N,N'
-di-p-Bromophenylguanidine (Bromo-DTG),
N,N'-di-m-Chlorophenylguanidine (Chloro-DTG), and salts thereof
23. The compound of claim 21, wherein R.sup.1 and R.sup.3 are both
phenyl.
24. The compound of claim 23, wherein R.sup.2 and R.sup.4 are both
Cl.
25. The compound of claim 24, wherein each Cl group is meta to the
guanidine group.
26. The compound of claim 23, wherein the R.sup.2 and R.sup.4 are
both Br.
27. The compound of claim 26, wherein each Br group is para to the
guanidine group.
28. The compound of claim 23, wherein the R.sup.2 and R.sup.4 are
both NO.sub.2.
29. The compound of claim 28, wherein each NO.sub.2 group is para
to the guanidine group.
30. The compound of claim 21, wherein the compound comprises
formula (II): ##STR00002## or a salt thereof.
31. The compound of claim 21, wherein R.sup.1 and R.sup.3 are both
napthyl.
32. The compound of claim 32, wherein R.sup.2 and R.sup.4 are
hydrogen.
33. The compound of claim 21, wherein the compound comprises
formula (III): ##STR00003## or a salt thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority to
International Patent Application No. PCT/US2013/039301, filed on
May 2, 2013 which claims priority to U.S. Provisional Patent
Application No. 61/641,514, entitled
"N,N'-Di-P-Nitrophenylguanidine HCL Reduces Infarct Volume and
Increases Functional Recovery After Stroke", filed on May 2, 2012,
the contents of which are herein incorporated by reference.
FIELD OF INVENTION
[0002] This invention relates to stroke treatment. Specifically,
the invention discloses a novel treatment of stroke using sigma
receptors.
[0003] BACKGROUND OF THE INVENTION
[0004] Stroke poses a significant healthcare burden in this country
as it is the fourth leading cause of death and results in a
significant amount of chronic disability (Towfighi and Saver 2011).
Despite decades of research, there is presently no approved
drug-based therapy that stimulates recovery of neurological
functions in stroke patients (Endres, Engelhardt et al. 2008).
[0005] Several pharmacological drugs, such as carbamylate
erythropoietin (Villa, van Beek et al. 2007), granulocyte-colony
stimulating factor (Schabitz and Schneider 2006) and amphetamine
(Barbay and Nudo 2009) enhance functional recovery and are
currently being evaluated in clinical trials for stroke recovery.
However, results from pharmacological studies suggest that multiple
mechanisms contribute to different aspects of functional recovery
and therefore several mechanisms must be targeted in developing
stroke drug therapies to provide optimal functional restoration.
Recently one research group found a selective sigma receptor
agonist, SA4503 enhanced brain plasticity and improved sensorimotor
function without decreasing infarct size (Ruscher, Shamloo et al.
2011). The researchers concluded that SA4503 may exert
neuroprotective effects by enhancing cellular trafficking of
biomolecules essential for brain repair.
[0006] Sigma receptors (.sigma.Rs) are predominantly expressed at
the endoplasmic reticulum (ER) and represent a structurally unique
class of intracellular proteins that function as chaperones (Katz,
Su et al. 2011). Ajmo and collaborators (Ajmo, Vernon et al. 2006)
showed that the .sigma.-1/.sigma.-2 ligand N,N'-di-o-tolyl
guanidine (DTG) decreases infarct size in a rat model of ischemic
stroke. The authors suggested that this neuroprotective effect of
DTG was probably due in part to the preservation of intracellular
calcium homeostasis. The authors also showed that DTG dampened
pro-inflammatory signals at delayed time points by decreasing the
number of activated microglia and astrocytes, thereby preventing
expansion of the ischemic core. When administered beginning 24
hours after permanent middle cerebral artery occlusion (MCAO),
1,3-di-o-tolylguanidine (DTG) decreased infarct size 96 hrs
post-MCAO, but failed to improve long-term functional recovery
(Leonardo, Hall et al. 2010).
[0007] Most rodent stroke studies have focused on mechanisms of
neuronal injury and neuroprotection, and have largely neglected
white matter injury following stroke. The inventors believe that
drugs targeted only to gray matter will not be efficient in
minimizing damage or sustaining functional recovery following
stroke. White matter comprises 50% of human brain, is more
sensitive to oxidative stress than neurons and is as equally
affected as neurons in most cases of stroke (Ho, Reutens et al.
2005). It does not matter how many neurons are spared by a
treatment, if there remains extensive damage to oligodendrocytes
and white matter tracts, functional recovery will not be
possible.
[0008] Sigma receptor (.sigma.1R) agonist 1,3 di-o-tolylguanidine
[DTG] decreased infarct volume in rats 96 hours after stroke, but
the effect was not maintained long-term, and therefore, did not
improve behavioral outcomes (Leonardo, Hall et al. 2010). Of the
thousands of promising treatments tested in animals, the clot
busters, tissue plasminogen activator (tPA) which restore blood
flow remain as the only FDA approved drug. However, most patients
do not receive tPA as they seek treatment beyond the narrow
therapeutic window. Moreover, these drugs do not treat any of the
pathophysiologic processes occurring after stroke. So it is an
unmet need to target pathological process occurring at delayed time
points after stroke.
[0009] What is needed is a composition and method of treating
neurological disorders such as stroke which improves optimal
long-term functional recovery.
SUMMARY OF INVENTION
[0010] The inventors have developed a novel method and compositions
for treating stroke at a delayed timepoint comprising administering
a therapeutically effective amount of a novel sigma agonist
selected from the group consisting of N,N'-di- 1-Naphthylguanidine
hydrochloride (NAGH) or N,N'-di-p-Nitrophenylguanidine
hydrochloride (NAD) to a patient in need thereof. The sigma agonist
can be administered at least 12 hours after stroke. The sigma
agonist may be NAGH and be administered in a therapeutically
effective amount of between about 0.86 mg/kg and 8.6 mg/kg at least
once per day for at least 3 days.
[0011] In an alternative embodiment, the sigma agonist may be NAD
and be administered in a therapeutically effective amount of
between about 2.8 mg/kg and 8.6 mg/kg at least once per day for at
least 3 days.
[0012] In another embodiment, a method of reducing infarct volume
long term in a patient suffering from a neurological disorder is
presented comprising administering a therapeutically effective
amount of a novel sigma agonist selected from the group consisting
of N,N'-di- 1-Naphthylguanidine hydrochloride (NAGH) or
N,N'-di-p-Nitrophenylguanidine hydrochloride (NAD) to a patient in
need thereof. The neurological disorder may be stroke and the sigma
agonist may be administered at least 12 hours after stroke. In an
embodiment, the sigma agonist is NAGH administered in a
therapeutically effective amount of between about 0.86 mg/kg and
8.6 mg/kg at least once per day for at least 3 days.
[0013] In an alternative embodiment, the sigma agonist may be NAD
and be administered in a therapeutically effective amount of
between about 2.8 mg/kg and 8.6 mg/kg at least once per day for at
least 3 days.
[0014] In an embodiment, the inventors administered a newly
synthesized mixed sigma 1/2 receptor agonist,
N,N'-di-1-Naphthylguanidine hydrochloride (NAGH) beginning about 24
hour post-stroke and performed a battery of behavioral tests to
evaluate its effects on long-term behavioral outcomes and the
pathological processes occurring at delayed time points after
stroke onset. The data show that NAGH improved motor function,
decreased the number of activated microglia, and protect
demyelination following stroke in particular in the cortical area
without decreasing the infarct volume. Taken together, the results
showed that NAGH improved behavioral recovery 30 days after MCAO
via preservation of both gray matter and white matter tracts.
[0015] Although many preclinical studies have focused on a small
time window after the experimental stroke to evaluate functional
outcome, the inventors evaluated long-term behavioral outcomes at
30 days after MCAO to assess functional recovery. Male
Sprague-Dawley rats were subcutaneously injected daily with either
a mixed sigma 1/2 receptor agonist; N,N'-di-1-Naphtylguanidine
hydrochloride (NAGH) (n=42/group) or with vehicle (40% DMSO) for
three days starting at 24 hours post-stroke.
[0016] The inventors examined the long-term therapeutic effects of
NAGH on infarct volume and white matter tracts as well as motor
recovery. Infarct volume in the stroke group receiving vehicle only
(n=25) was 17.57.+-.4.43%, whereas animals receiving NAGH (n=27)
showed infarcts involving 13.58.+-.2.3% of the hemisphere
(p>0.05). The NAGH treated rats show improved vertical movement
and had significantly less motor asymmetry and bias than vehicle
treated rats (*p<0.05). Further, the results demonstrated a
larger loss of Luxol-fast blue staining (*p<0.05) within the
subcortical and striatal white matter in animals treated with DMSO
compared to NAGH treated animals.
[0017] The inventors discovered that sigma receptor agonist NAGH
exerts its long-term neuroprotective effects by preserving both
gray matter and white matter tracts. Pharmacological targeting of
the sigma receptor provides new opportunities to open new avenues
in developing stroke treatment.
[0018] Another newly synthesized mix of .sigma.1/.sigma.2Rs
agonist, N,N'-di-p-nitrophenylguanidine HCL (NAD) was developed
based on the structure of DTG that also induced sustained
functional recovery, enhanced behavioral recovery, and decreased
infarct size when compared to DTG.
[0019] The systemic administration of
N,N'-di-p-nitrophenylguanidine HCL (NAD) when administered 24 hours
after experimental stroke was found to reduce neural damage and
more importantly, enhance behavioral recovery thirty days later.
This agent has the potential to extend the therapeutic window
several-fold over the current one to open treatment to many stroke
patients. Additionally, this agent provides neural protection
leading to recovery of functions, such as motor skills. The
approved treatment, tissue plasminogen activator acts only as a
clot buster and does not possess any neural protective
properties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] For a fuller understanding of the invention, reference
should be made to the following detailed description, taken in
connection with the accompanying drawings, in which:
[0021] FIG. 1 is a diagram of the experimental design used to test
the compounds used in the present invention.
[0022] FIG. 2 is a series of images depicting comparison of daytime
vertical movement with or without NAGH treatment at 1 month.
Percent baseline of three parameters of vertical movement i.e. VA
(A), VMN (C), and VT (E) during the daytime testing period at 1
month post-treatment. Time course of these parameters over the
entire 1-hr test at 1 month post-treatment (B, D, and F
respectively). Note that activity decreases steadily over the first
four observation periods and then remains relatively stable for the
remainder of the test.
[0023] FIG. 3 is a series of images depicting comparison of
Nighttime vertical movement with or without NAGH treatment at 1
month. Percent baseline of three parameters of vertical movement
i.e. VA (A), VMN (C), and VT (E) during the Nighttime testing
period at 1 month post-treatment. Time course of these parameters
over the entire 12-hr test at 1 month post-treatment (B, D, and F
respectively). The same pattern of decreasing activity was also
observed at this time.
[0024] FIG. 4 is a series of images depicting the results of the
behavioral tests including Elevated body swing test (A), Corner
test (B), Grip strength test (C), Rotarod test (D), Step test (E),
and fore limb use asymmetry (cylinder test) (F) Step test at
baseline and at 30 days after MCAO. Values are mean.+-.SEM, n=27 to
28. *p<0.05 Vs control.
[0025] FIG. 5 is a series of images depicting Nissl-Thionin stained
coronal brain sections. Nissl-Thionin staining was used to
determine infarct volumes of rats following MCAO treated with DMSO
(A) or NAGH (B). Infarct size was determined at 6 pre-determined
levels beginning 1.7 mm anterior to bregma to 3.3 mm posterior to
bregma at 1 mm intervals. The area (mm2) of intact tissue in the
ischemic and normal hemispheres was measured using Image Pro Plus.
The relative infarct volume was determined as the difference
between the sum of the normal hemisphere and the sum of the
infarcted hemisphere divided by the sum of the normal hemisphere
multiplied by 100. Lesion size in the infarcted hemisphere of the
NAPG treated animals was significantly smaller than lesion sized in
the DMSO treated group (*p<0.05) NAGH failed to decrease infarct
volume compared to the MCAO only group. (C) Percent contralateral
infarct area of animals treated with NAGH and DMSO. Values are
mean.+-.SEM, n=27-28 p>0.05
[0026] FIG. 6 is a series of images depicting the effects of NAGH
on myelinating oligodendrocytes. Luxol Fast blue stained brain from
DMSO treated animal revealed reduced staining in the cortex and
striatum (A and B) compared with those of NAGH treated animals (C
and D). (E), the quantification of the percentage of white matter
area stained by LFB of the each hemisphere. Values are mean .+-.SE.
*p<0.05 vs. DMSO. Scale bar=100 .mu.m.
[0027] FIG. 7 is a series of images depicting Ibal Immunostaining
of rat brains 30 days after MCAO. (A) Representative Ibal staining
brain from DMSO and NAGH treated rats after MCAO. NAGH treated
brains showed decreased number of activated microglia in the
ipsilateral hemisphere (B). Values are mean .+-.SE. p>0.05
[0028] FIG. 8 is a series of images confirming NAGH acts as a sigma
receptor agonist. A) Concentration-response relationship for NAGH
(closed circles) and DTG (open circles) inhibition of microglial
migration evoked by 100 .mu.M ATP. Solid line represents best fit
to the NAGH data using a two-component Langmuir-Hill equation with
the Emax, IC50 and Hill coefficient being 0.43, 0.75 .mu.M and 2.2
for the high affinity component and 0.57, 84 .mu.M and 0.80 for the
low affinity component, respectively. Dashed line represents best
fit to the DTG data using a two-component Langmuir-Hill equation
with the Emax, IC50 and Hill coefficient being 0.38, 0.76 .mu.M,
and 0.59 for the high affinity component and 0.62, 80 .mu.M, and
1.7 for the low affinity component, respectively. Points represent
mean (.+-.SEM) and for all points, n>9. B) Bar graph of the mean
(.+-.SEM) number of microglial cells migrating in response to 100
.mu.M ATP when ATP was added alone (DMEM) or in the presence of 100
.mu.M NAGH. Microglia were further exposed to no .sigma. antagonist
(Control), or the .sigma.-1 antagonist, BD-1047 (10 .mu.M,
BD-1047), or the .sigma.-2 antagonist, rimcazole (10 .mu.M,
Rimcazole). Asterisks denote significant difference between DMEM
and NAGH within Control, BD-1047 and Rimcazole groups,
respectively. Pound symbols indicate significant difference between
Control, BD-1047 and Rimcazole within DMEM and NAGH groups,
respectively. P <0.05 and n =24 for all groups.
[0029] FIG. 9 is a diagram of the different compounds tested. DTG
is 1,3-di-o-tolylguanidine; Bromo-DTG is
N,N'-di-p-Bromophenylguanidine hydrochloride, Chloro-DTG is
N,N'-di-m-Chlorophenylguanidine hydrochloride, NAGH is N,N'-di-
1-Naphthylguanidine hydrochloride; and NAD is
N,N'-di-p-Nitrophenylguanidine hydrochloride.
[0030] FIG. 10 is an image showing the effect of treatment with
Bromo-DTG on Thionin staining throughout the brain hemispheres
compared to animals without Bromo-DTG treatment.
[0031] FIG. 11 is a graph showing the infarct volume in the stroke
group receiving vehicle only versus Bromo-DTG treatment.
[0032] FIG. 12 is an image showing the effect of treatment with
Chloro-DTG on Thionin staining throughout the brain hemispheres
compared to animals without Chloro-DTG treatment.
[0033] FIG. 13 is a graph showing the infarct volume in the stroke
group receiving vehicle only versus Chloro-DTG treatment.
[0034] FIG. 14 is an image showing results of MCAO on Thionin
staining throughout the brain hemispheres in animals without NAD
treatment thionin staining
[0035] FIG. 15 is an image showing treatment with NAD resulted in
prominent Thionin staining throughout the brain hemispheres whereas
animals without NAD treatment thionin staining was preserved mostly
in the contralateral hemisphere.
[0036] FIG. 16 is a graph showing the infarct volume in the stroke
group receiving vehicle only (n=8) was 30.49 .+-.8.71%, whereas
animals receiving NAD (n=8) showed infarcts involving only 13.93
.+-.3.79% of the hemisphere.
[0037] FIG. 17 is a graph showing overall animals treated with NAD
were less active then the non-treated animals at 30 days after
MCAO. Day time activity at 1 month post MCAO.
[0038] FIG. 18 is a graph showing overall animals treated with NAD
were less active then the non-treated animals at 30 days after
MCAO. Nighttime time activity at 1 month post MCAO.
[0039] FIG. 19 is a graph showing overall animals treated with NAD
were less active then the non-treated animals at 30 days after
MCAO. Horizontal activity during the day time testing period.
[0040] FIG. 20 is a graph showing overall animals treated with NAD
were less active then the non-treated animals at 30 days after
MCAO. Horizontal activity during the nighttime testing period.
[0041] FIG. 21 is a graph showing the elevated body swing test at
baseline and at 30 days after MCAO comparing control animals and
those receiving NAD. Values are mean.+-.SEM, n=9 to 12. *p<0.05
Vs control.
[0042] FIG. 22 is a graph showing the elevated body swing test at
baseline and at 30 days after MCAO comparing controls against
Bromo-DTG or NAGH.
[0043] FIG. 23 is a graph showing the grip strength test comparing
control versus NAD. Values are mean .+-.SEM, n=9 to 12. *p<0.05
Vs control.
[0044] FIG. 24 is a graph showing the step test at baseline and at
30 days after MCAO for control versus NAD. Values are mean.+-.SEM,
n=9 to 12. *p<0.05 Vs control.
[0045] FIG. 25 is a graph showing the corner test at baseline and
at 30 days after MCAO for control versus NAD. Values are
mean.+-.SEM, n=9 to 12. *p<0.05 Vs control
[0046] FIG. 26 is a graph showing the rotorod test at baseline and
at 30 days after MCAO for control versus NAD. Values are
mean.+-.SEM, n=9 to 12. *p<0.05 Vs control
[0047] FIG. 27 is a graph showing the rotorod test at baseline and
at 30 days after MCAO comparing control versus Bromo-DTG,
Chloro-DTG or NAGH.
[0048] FIG. 28 is a graph showing the cylinder test at baseline and
at 30 days after MCAO comparing control versus Bromo-DTG,
Chloro-DTG or NAGH.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0049] In the following detailed description of the preferred
embodiments, reference is made to the accompanying drawings, which
form a part hereof, and within which are shown by way of
illustration specific embodiments by which the invention may be
practiced. It is to be understood that other embodiments may be
utilized and structural changes may be made without departing from
the scope of the invention.
[0050] Definitions
[0051] NAGH is used interchangeably herein with NAPH, NAPH-DTG and
N-N-NAPH and is an acronym for N,N'-di- 1-Naphthylguanidine
hydrochloride.
[0052] NAD is used interchangeably herein with NDH, NDH-DTG and
Nitro-DTG and is an acronym for N,N' -di-p-Nitrophenylguanidine
hydrochloride.
[0053] All numerical designations, such as pH, temperature, time,
concentration, and molecular weight, including ranges, are
approximations which are varied up or down by increments of 1.0 or
0.1, as appropriate. It is to be understood, even if it is not
always explicitly stated that all numerical designations are
preceded by the term "about". It is also to be understood, even if
it is not always explicitly stated, that the reagents described
herein are merely exemplary and that equivalents of such are known
in the art and can be substituted for the reagents explicitly
stated herein.
[0054] The term "about" or "approximately" as used herein refers to
being within an acceptable error range for the particular value as
determined by one of ordinary skill in the art, which will depend
in part on how the value is measured or determined, i.e. the
limitations of the measurement system, i.e. the degree of precision
required for a particular purpose, such as a pharmaceutical
formulation. For example, "about" can mean within 1 or more than 1
standard deviation, per the practice in the art. Alternatively,
"about" can mean a range of up to 20%, preferably up to 10%, more
preferably up to 5% and more preferably still up to 1% of a given
value. Alternatively, particularly with respect to biological
systems or processes, the term can mean within an order of
magnitude, preferably within 5-fold, and more preferably within
2-fold, of a value. Where particular values are described in the
application and claims, unless otherwise stated, the term "about"
meaning within an acceptable error range for the particular value
should be assumed.
[0055] Concentrations, amounts, solubilities, and other numerical
data may be expressed or presented herein in a range format. It is
to be understood that such a range format is used merely for
convenience and brevity and thus should be interpreted flexibly to
include not only the numerical values explicitly recited as the
limits of the range, but also to include all the individual
numerical values or sub-ranges encompassed within that range as if
each numerical value and sub-range is explicitly recited. As an
illustration, a numerical range of "about 1 to about 5" should be
interpreted to include not only the explicitly recited values of
about 1 to about 5, but also include the individual values and
sub-ranges within the indicated range. Thus, included in this
numerical range are individual values such as 2, 3, and 4 and
sub-ranges such as from 1-3, from 2-4 and from 3-5, etc. This same
principle applies to ranges reciting only one numerical value.
Furthermore, such an interpretation should apply regardless of the
range or the characteristics being described.
[0056] "Composition" is used herein interchangeably with
"compound", "drug", "formulation" and "molecule".
[0057] "Patient" is used to describe an animal, preferably a human,
to whom treatment is administered, including prophylactic treatment
with the compositions of the present invention. "Patient" includes
both mammals and non-mammals including, but not limited to, humans;
non-human primates; bovines; domestic animals such as rabbits,
dogs, and cats; laboratory animals such as rats, mice and guinea
pigs; and non-mammals such as birds and fish.
[0058] The "therapeutically effective amount" for purposes herein
is thus determined by such considerations as are known in the art.
A therapeutically effective amount of a composition, or mixture of
compositions thereof, is that amount necessary to provide a
therapeutically effective result in vivo. The amount of sigma
agonist or any combination of sigma agonists thereof must be
effective to achieve a response, including but not limited to total
prevention of (e.g., protection against) and to improved survival
rate or more rapid recovery, or improvement or elimination of
symptoms associated with neurological disorders or other indicators
as are selected as appropriate measures by those skilled in the
art. In accordance with the present invention, a suitable single
dose size is a dose that is capable of preventing or alleviating
(reducing or eliminating) a symptom in a patient when administered
one or more times over a suitable time period. An "effective
amount" for therapeutic purposes is the amount of the composition
of sigma receptor ligand required to provide a clinically
significant decrease in neurodegenerative disease, such as those
resulting from ischemic stroke. One of skill in the art can readily
determine appropriate single dose sizes for systemic administration
based on the size of a mammal and the route of administration.
[0059] "Administration" or "administering" is used to describe the
process in which the compositions or any combination of
compositions thereof of the present invention are delivered to a
patient. Compositions of this invention can be administered as
pharmaceutical compositions, including those suitable for oral,
including buccal and sub-lingual; rectal; nasal; topical;
pulmonary; vaginal; or parenteral, including intramuscular,
intra-arterial, intrathecal, subcutaneous, and intravenous.
[0060] The pharmaceutical compositions of the subject invention can
be formulated according to known methods for preparing
pharmaceutically useful compositions. Furthermore, as used herein,
the phrase "pharmaceutically acceptable carrier" means any of the
standard pharmaceutically acceptable carriers. The pharmaceutically
acceptable carrier can include diluents, adjuvants, and vehicles,
as well as implant carriers, and inert, non-toxic solid or liquid
fillers, diluents, or encapsulating material that does not react
with the active ingredients of the invention. Examples include, but
are not limited to, phosphate buffered saline, physiological
saline, water, and emulsions, such as oil/water emulsions. The
carrier can be a solvent or dispersing medium containing, for
example, ethanol, polyol (for example, glycerol, propylene glycol,
liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetable oils. Formulations are described in a number
of sources that are well known and readily available to those
skilled in the art. For example, Remington's Pharmaceutical
Sciences (Martin E W [1995] Easton Pa., Mack Publishing Company,
19th ed.) describes formulations which can be used in connection
with the subject invention.
[0061] For ease of administration, the subject compounds may be
formulated into various pharmaceutical forms. As appropriate
compositions there may be cited all compositions usually employed
for systemically administering drugs. To prepare the pharmaceutical
compositions of this invention, the sigma agonist, as the active
ingredient is combined with a pharmaceutically acceptable carrier,
which may take a wide variety of forms depending on the form of
preparation desired for administration. Compounds of this invention
can be administered as pharmaceutical compositions, including those
suitable for oral, including buccal and sub-lingual; rectal; nasal;
topical; pulmonary; vaginal; or parenteral, including
intramuscular, intra-arterial, intrathecal, subcutaneous, and
intravenous. In some embodiments, intravenous or intra-arterial
administration is a preferred manner of providing a daily dosing
regimen that can be adjusted according to the degree of affliction.
For example, in preparing the compositions in oral dosage form, any
of the usual pharmaceutical media may be employed, such as, for
example, water, glycols, oils, alcohols and the like in the case of
oral liquid preparations such as suspensions, syrups, elixirs and
solutions; or solid carriers such as starches, sugars, kaolin,
lubricants, binders, disintegrating agents and the like in the case
of powders, pills, capsules and tablets. Because of their ease in
administration, tablets and capsules often represent the most
advantageous oral dosage unit form, in which case solid
pharmaceutical carriers are obviously employed. For parenteral
compositions, the carrier will usually comprise sterile water, at
least in large part, though other ingredients, for example, to aid
solubility, may be included. Injectable solutions, for example, may
be prepared in which the carrier comprises saline solution, glucose
solution or a mixture of saline and glucose solution. In the
compositions suitable for percutaneous administration, the carrier
optionally comprises a penetration enhancing agent and/or a
suitable wettable agent, optionally combined with suitable
additives of any nature in minor proportions, which additives do
not cause any significant deleterious effects on the skin. Said
additives may facilitate the administration to the skin and/or may
be helpful for preparing the desired compositions.
[0062] The amount of the compound in the drug composition will
depend on absorption, distribution, metabolism, and excretion rates
of the drug as well as other factors known to those of skill in the
art. Dosage values may also vary with the severity of the condition
to be alleviated. The compounds may be administered once, or may be
divided and administered over intervals of time. It is to be
understood that administration may be adjusted according to
individual need and professional judgment of a person
administrating or supervising the administration of the compounds
used in the present invention.
[0063] The dose of the compounds administered to a subject may vary
with the particular composition, the method of administration, and
the particular disorder being treated. The dose should be
sufficient to affect a desirable response, such as a therapeutic or
prophylactic response against a particular disorder or condition
such as stroke. For example, the dosage of a compound of the
invention administered can be between about 0.01 mg/kg to about
10.0 mg/kg. Typically, the dosage of NAGH can be in the range of
about 0.86 mg/kg to about 8.6 mg/kg when given on a daily schedule.
It is contemplated that one of ordinary skill in the art can
determine and administer the appropriate dosage of compounds
disclosed in the current invention according to the foregoing
considerations.
[0064] Dosing frequency for the composition includes, but is not
limited to, at least about 1 day, at least about 2 days, at least
about 3 days, at least about 4 days, at least about 5 days, at
least about 6 days and at least about 7 days. In some embodiments,
the interval between each administration is constant. For example,
the administration can be carried out daily, every two days, every
three days, every four days, every five days, or weekly. For
example, in an embodiment using NAGH, the dosing frequency for the
composition is once per day for between about 3 to about 7 days
after stroke starting at least about 12 hours post-stroke. In some
embodiments, the administration can be carried out twice daily,
three times daily, or more frequent. Administration can also be
continuous and adjusted to maintaining a level of the compound
within any desired and specified range.
[0065] When used to treat symptoms of a neurological disease such
as ischemia or stroke, the compounds may be administered at any
time after occurrence of the neurological disease. A defined time
period may pass before administration of the compound. In the case
of stroke, this defined time period may be at least 3 hours
post-stroke, at least 6 hours post-stroke, at least 9 hours
post-stroke, at least 12 hours post-stroke, at least 24 hours
post-stroke, at least 48 hours post-stroke, at least 72 hours
post-stroke, at least 96 hours post-stroke, at least 7 days
post-stroke, at least 14 days post-stroke, at least 21 days
post-stroke, and at least 30 days post-stroke. Administration of
the compound includes all time periods between those listed above
to the hour.
[0066] The compounds used in the present invention may be
administered individually, or in combination with or concurrently
with one or more other compounds used in other embodiments of the
present invention. Additionally, compounds used in the present
invention may be administered in combination with or concurrently
with other therapeutics for neurological disorders such as
stroke.
[0067] The term "agonist" as used herein is defined as a molecule,
such as a compound, drug, enzyme activator, or hormone, which
enhances the activity of another molecule or the activity of the
sigma receptor site.
[0068] The term "antagonist" as used herein is defined as a
molecule, such as a compound, drug, enzyme activator, or hormone,
which diminishes or prevents the action of another molecule or the
activity of the sigma receptor site.
[0069] "Neurological disorder" and "neurological disease" are used
interchangeably throughout. Neurological disorder/neurological
disease as used herein refers to a disorder which is caused by
damage to the central nervous system and results in any abnormal
physical or mental behavior or experience where the death or
dysfunction of neuronal cells is involved in the etiology of the
disorder. Both stroke and ischemia are considered neurological
disorders as used herein.
[0070] The term "stroke" as used herein is defined broadly as the
development of neurological deficits associated with impaired blood
flow to the brain regardless of cause. Potential causes include,
but are not limited to, thrombosis, hemorrhage and embolism. Other
injuries that can result in stroke include aneurysm, angioma, blood
dyscrasias, cardiac failure, cardiac arrest, septic shock, head
trauma, spinal cord trauma, seizure, bleeding from a tumor or other
blood loss.
[0071] The term "ischemic" or "ischemic episode" means any
circumstance that results in a deficit of blood supply to a tissue,
especially the central nervous system (CNS) or brain tissue. When
the ischemia is associated with a stroke, it can be either global
or focal ischemia. The term "ischemic stroke" means a type of
stroke that is of limited extent and caused by a blockage of blood
flow. Non-limiting examples include cerebral ischemia, ischemia
after cardiac arrest, stroke, multi-faceted dementia, and
complications from surgery. Cerebral ischemic episodes result from
a deficiency in blood supple to the brain. The spinal cord is also
considered part of the CNS, and is equally susceptible to ischemia
resulting from diminished blood flow. The term "focal ischemia" is
used to refer to a condition resulting from a blockage of a single
artery that supplies blood to the brain or spinal cord, resulting
in damage to the cells in the territory supplied by that artery.
Conversely, "global ischemia" refers to a condition that results
from a general diminution of blood flow to the entire tissue, such
as the entire brain, forebrain, or spinal cord, thereby causing the
death of neurons in selectively vulnerable regions throughout these
tissues.
[0072] "Treatment" or "treating" as used herein refers to any of:
the alleviation, amelioration, elimination and/or stabilization of
a symptom, as well as delay in progression of a symptom of a
particular disorder, namely a neurodegenerative disease or disorder
such as stroke. For example, "treatment" of stroke may include any
one or more of the following: amelioration and/or elimination of
one or more symptoms associated with stroke, reduction of one or
more symptoms of stroke, stabilization of symptoms of stroke, and
delay in progression of one or more symptoms of stroke.
[0073] The term "long term" as used herein refers to a time period
that is greater than 25 hours. In some embodiments, the term
applies to a time period of at least 3 days, at least 5 days, at
least 7 days, at least 10 days, at least 14 days, at least 18 days,
at least 21 days, at least 25 days, at least 30 days, at least 2
months, at least 3 months, at least 4 months, at least 5 months, at
least 6 months, at least 7 months, at least 8 months, at least 9
months, at least 10 months, at least 11 months, at least 12 months,
and greater than one year. The time periods listed are exemplary
and include all intervening days not specifically listed in 1 day
increments.
[0074] The term "delayed timepoint" as used herein refers to a
timepoint that is at least 3 hours after occurrence of a
neurological disorder such as stroke. In the case of stroke, the
delayed timepoint may be at least 3 hours post-stroke, at least 6
hours post-stroke, at least 9 hours post-stroke, at least 12 hours
post-stroke, at least 24 hours post-stroke, at least 48 hours
post-stroke, at least 72 hours post-stroke, at least 96 hours
post-stroke, at least 7 days post-stroke, at least 14 days
post-stroke, at least 21 days post-stroke, and at least 30 days
post-stroke. Administration of the compound includes all time
periods between those listed above to the hour. In some
embodiments, the delayed timepoint is 24 hours post-stroke.
[0075] The inventors have previously shown that human umbilical
cord blood cells provided long-term neuroprotection and improved
functional recovery at 30 days in animals following stroke via its
neuroprotective and anti-inflammatory properties (Chen, Sanberg et
al. 2001; Vendrame, Cassady et al. 2004; Vendrame, Gemma et al.
2005). Sigma receptor agonists may mimic these effects and play a
potential role in expanding the therapeutic window for stroke
treatment. It was shown previously that sigma receptor agonists
reduced neurodegeneration and enhanced neurosurvival following
ischemic insult by decreasing ischemia-induced intracellular
calcium elevations (Zhang and Cuevas 2002; Zhang and Cuevas 2005;
Katnik, Guerrero et al. 2006), depressing inflammatory responses
(Leonardo, Hall et al. 2010), and modulating immune responses
specially by regulating microglial activation (Hall, Herrera et al.
2009). A recent study found that sigma receptor genes are
upregulated in rat brains following injury when the rats were
housed in an enriched environment (Ruscher, Shamloo et al. 2011).
Moreover, this enriched environment increases neurogenesis in the
hippocampus during stroke recovery (Ohlsson and Johansson 1995;
Nygren, Wieloch et al. 2006). It was reported further that this
enriched environment upregulates the genes associated with lipid
synthesis and transport (Ruscher, Shamloo et al. 2011). It is well
known that the sigma receptor is a membrane spanning cholesterol
binding protein detected in the lipid rafts which act as a vehicle
for cellular trafficking (Palmer, Mahen et al. 2007). They have
also been found responsible for delivering myelin into the plasma
(Jahn, Tenzer et al. 2009). Sigma receptors may play a greater role
in regulating myelin trafficking following ischemic injury.
[0076] It was shown previously that MCAO produces locomotor and
stereotypic hyperactivity with decreased resting time leading the
animals to be more active than before the stroke (Janac, Radenovic
et al. 2006). A battery of behavioral tests was performed to
determine whether the efficacy of NAGH in reducing stroke-induced
neural injury leads to functional recovery. There were significant
improvements in function on a number of behavioral measures in rats
treated with NAGH. The most significant finding was that NAGH
treatment resulted in improvements in motor asymmetry in the EBST
while non-treated animals exhibited functional impairment at 30
days post MCAO; this test has been used many times to evaluate
sensorimotor asymmetry after focal brain ischemia (Borlongan, Hida
et al. 1998). Treated animals were significantly less active than
non-treated animals during both the dark and the light phases of
the light cycle.
[0077] Previous studies provided much evidence that vertical
movement and body asymmetry are strongly correlate to the size of
infarct volume and neuronal recovery following stroke (Shen and
Wang 2010; Encarnacion, Horie et al. 2011). Significant improvement
was shown in all parameters of vertical movement except daytime
VMN, and a significant improvement in body asymmetry in animals
treated with NAGH.
[0078] It has been found that microglial cells are highly
susceptible to ischemic injury (Jin, Yang et al. 2010). It has been
further reported that microglia transform from a resting state to
an activated state in response to neuronal injury (Kettenmann,
Hanisch et al. 2011). The exact role of microglial cells after
injury and repair is still unclear despite important progress in
the understanding of microglial activation, proliferation,
phagocytosis function, cytokines and growth factors production. It
was found that stimulation of sigma receptors can reduce the
production of cytokines and microglial activation in response to
various stimuli including adenosine triphosphate (ATP), monocyte
chemoattractant protein 1 (MCP-1), and lipopolysaccharide (LPS)
(Hall, Leonardo et al. 2009). DTG inhibited ATP and UTP-evoked
microglial migration when cells are pre-incubated in media
containing metaphit, a pan-selective irreversible sigma receptor
antagonist (Cuevas, Rodriguez et al. 2011). When the effects of DTG
on microglial cell survival in response to in vitro ischemia was
examined, it was found that DTG significantly reduced microglial
cell death 24 hours after ischemia suggesting that DTG can be glial
protective at delayed time points.
[0079] Almost all cases of ischemic stroke in humans involve white
matter (Dewar, Underhill et al. 2003). Recent evidence suggests
development of new drugs that only target excitotoxicity
(Ikonomidou and Turski 2002), oxidative stress (Wang and Shuaib
2007), or inflammation (Kennedy and Vinten-Johansen 2006) are
likely to fail, at least in part due to the lack of emphasis on
white matter injury. Moreover, lower blood flow and less collateral
blood supply in the deep white matter tracts make it more
vulnerable to ischemia than gray matter (Lo, Dalkara et al. 2003).
It was previously shown that human umbilical cord blood cells
promote the expression of myelin-associated and antioxidant genes
in cultured primary oligodendrocytes exposed to hypoxia-ischemia
(Rowe, Leonardo et al. 2010). Therefore, a gene chip microarray
analysis is needed to examine changes in gene expression in
oligodendrocytes induced by NAGH. Previous results found that brain
ischemia enhances proliferation of immature oligodendrocytes in the
regions surrounding the lateral ventricle but decreases the number
of mature oligodendrocytes in the per-infarct area (Mandai,
Matsumoto et al. 1997).
[0080] The inventors sought to develop new sigma agonists, using
DTG as the base molecule for manipulation, that induce more
sustained functional recovery than DTG. The inventors investigated
the following novel sigma ligands: N,N'-di-p-Bromophenylguanidine
hydrochloride (Bromo-DTG; 8.2 mg/kg/day),
N,N'-di-m-Chlorophenylguanidine hydrochloride (Chloro-DTG; 8.5
mg/kg/day), N,N'-di-1-Napthylguanidine hydrochloride (NAGH 0.86
mg/kg/day) and N,N'-di-p-Nitrophenylguanidine hydrochloride (NAD
2.8 mg/kg/day).
[0081] While all of the above drugs decreased infarct volume, the
NAGH-DTG agonist is the only sigma receptor agonist in this series
that induces long term motor recovery. The inventors found animals
receiving NAGH spent significantly less time moving vertically
during their day time activity. Nighttime vertical movement was
also significantly enhanced in stroke animals receiving NAGH
(p<0.05 vs. vehicle group). Moreover, Luxol Fast blue
demonstrated that white matter tracts were more intact in the NAGH
treated group, particularly in cortex. The data here showed that
NAGH tended to decrease the number of Iba1-expressing cells in the
ipsilateral hemisphere of the treated rats compared to the vehicle
treated rats. The effect may be more robust with a different dosing
regimen of the NAGH. Taken together, the data suggest that NAGH
protected both neurons and oligodendrocytes from MCAO-induced cell
death and is a good molecule for development as a stroke
therapy.
[0082] Animals:
[0083] All animal procedures were performed according to the
University of South Florida guidelines for the care and use of
animals and protocols were reviewed and approved by the
Institutional Animal Care and Use Committee and adhered to the NIH
guidelines for the care and use of animals. Adult male Sprague
Dawley rats (Harlan) weighing about 250-300 g at the time of
surgery were used for the study. All animals were housed two per
cage in a temperature controlled room, under a 12 hour light-dark
cycle, and given access to water and food ad libitum. Animals were
monitored daily for wellness and biweekly for weight loss. For the
NAGH studies, a total of 84 animals were randomly assigned to 2
groups: MCAO only (n=42) and rat treated with NAGH (n=42) beginning
24 hour after MCAO.
[0084] Prior to MCAO induction and then again at 1 month post HUCB
transplantation, animals were tested with a battery of standard
functional tests as previously described (Vendrame M, Cassady C J,
Newcomb J, Butler T, Pennypacker K R, Zigova T, et al. Stroke.
2004; 35:2390-5; Willing A E, Lixian J, Milliken M, Poulos S,
Zigova T, Song S, et al. J Neurosci Res. 2003; 73(3):296-307), and
as seen below. This was used as a baseline for treatment.
[0085] MCAO
[0086] MCAO surgeries were performed as described elsewhere
(Vendrame M, Cassady C J, Newcomb J, Butler T, Pennypacker K R,
Zigova T, et al. Stroke. 2004;35:2390-2395). Briefly, rats were
anesthetized with isoflurane, and the right common carotid and
external carotid arteries were exposed using blunt dissection
techniques. The vagus nerve was separated from common carotid
artery and the external carotid artery was ligated proximally, and
transected near the bifurcation. An embolus 40 mm in length (4.0
siliconized monofilament) was inserted into the external carotid
artery and fed distally into the origin of the middle cerebral
artery (.about.25 mm from insertion) through the internal carotid
artery. Blood samples were taken and analyzed using an i-STAT
handheld analyzer for hemoglobin, hematocrit, ionized calcium,
glucose, sodium, potassium, pH, pCO2, HCO3, TCO2, base excess, pO2,
and O2. Cerebral blood flow was recorded using a Laser Doppler
Monitor (Moor Instruments) to verify occlusion. Afterwards, the
embolus was ligated permanently into the vessel and the neck
incision was closed. Animals were treated with Ketoprofen IM (10
mg/kg) for three days post-surgery.
[0087] Drug Dosing Schedule
[0088] For the NAGH studies, vehicle or NAGH were prepared fresh
each day. Daily subcutaneous injections (s.c) were administered
beginning about 24 hours after MCAO and continued daily to about 72
hours post-surgery (FIG. 1). Animals received a total of 3
injections. A total of 84 animals were randomly assigned into 2
groups: Rats treated with vehicle (40% DMSO in sterile saline,
n=42), or rats treated with 0.86 mg/kg NAGH (n=42).
[0089] For the studies with other compounds based on the structure
of DTG, twenty-four hours after MCAO, the rats began a sigma
agonist treatment regimen. Vehicle or drug was prepared fresh each
day. Testing procedures are summarized in FIG. 1. Daily
subcutaneous injections (s.c) were administered daily for 3 days
(ending 72 hours post-surgery) with either
N,N'-di-p-Bromophenylguanidine hydrochloride (Bromo-DTG; 8.2
mg/kg/day), N,N' -di-m-Chlorophenylguanidine hydrochloride
(Chloro-DTG; 8.5 mg/kg/day), N,N'-di- 1-Naphthylguanidine
hydrochloride (NAGH; 0.86 mg/kg/day),
N,N'-di-p-nitrophenylguanidine HCL (NAD; 2.8 mg/kg), or vehicle.
Animals received a total of 3 injections. The structures of the
compounds are seen in FIG. 9.
[0090] For NAD testing, a total of 32 animals were randomly
assigned into 2 groups: Vehicle (n=16), and rat treated with 2.8
mg/kg NAD (n=16). Vehicle consisted of 40% DMSO in sterile
saline.
[0091] Behavioral Testing
[0092] After surgery, animals were tested was performed once before
surgery and 30 days post-surgery for motor coordination and
behavior. All behavioral data are reported as mean +sem.
[0093] Spontaneous Activity
[0094] Spontaneous Activity was measured during both the day (1 hr)
and night (12 hrs) using an automated activity monitor (VersaMax,
Accuscan Instruments, Inc). Spontaneous Activity was measured prior
to stroke to establish the normal baseline for each individual
animal as well as 1 month after the MCAO. The animals were placed
in a Plexiglas box and allowed to roam freely. There is a three
dimensional infrared array in this box and when a beam is broken,
activity is recorded. A total of sixteen parameters were measured
including: Vertical movement, Horizontal movement, and Stereotypic
activity were selected for analysis: Horizontal activity (HA),
Total distance travelled (TD), Number of movements (MVN), Movement
time (MVT), Vertical activity (VA), Number of vertical movements
(VMN), vertical time (VT), Stereotype counts (SC), Number of
Stereotypy (SN), Stereotypy time (ST), Clockwise rotation (CR),
counterclockwise rotation (CCR), Margin distance (MD), Marginal
time (MT), Center distance (CD), and Center time (CT). The data are
presented as mean+SEM of percent of baseline behavior.
[0095] Rotorod Test
[0096] Rotorod test, which measures motor coordination, was
conducted using Omni-rotor Rotorod (Omnitech, Inc). Animals were
trained on the accelerating rotorod apparatus for 3 consecutive
days before times were collected. Animals were placed on the
revolving rod and instrument was set to accelerate to a maximum
speed of 40 rpm over 180 seconds. Latency to fall (in seconds) was
recorded. This test was performed in triplicate once per day for 3
days.
[0097] Step Test
[0098] Rats were held tightly with one forelimb placed on a hard
flat table top and then pulled for a length of 1 meter in the
direction of the placed forelimb at constant velocity. The test was
repeated with the opposite forelimb. This was repeated three times
for each forelimb and the percent of contralateral forelimb paw was
obtained. The bias was standardized as the absolute number of steps
with the contralateral paw minus 100.
[0099] Elevated Body Swing Test
[0100] The animal was placed in a Plexiglas box
(40.times.40.times.35.5 cm), allowed to habituate for 2 minutes and
attain a neutral position, defined as having all four paws on the
ground. The animal was held approximately 1 inch from the base of
its tail and elevated to an inch above the Plexiglas cage surface.
A swing was recorded whenever the animal moved its head out of the
vertical axis to the right or left over 20 times. Swings were
recorded using a hand counter. There is no bias when there are an
equal number of swings to the left and right (i.e., 10 in each
direction). The bias was standardized as the absolute value of the
number of swings on the impaired side minus 10.
[0101] Passive Avoidance Test
[0102] On the first day of testing, animals were placed in the
lighted chamber of a shuttlebox with a circular doorway opened to
the darkened chamber. When the animals passed into the darkened
chamber, they received a 0.9 mA foot shock for 2 second duration
after which they were returned to their home cage. On day 2, foot
shock was turned off and animals were placed into the same light
chamber. The latency to enter the darkened chamber was recorded.
The trial ended when the animal passed into the darkened side or 60
seconds had elapsed.
[0103] Cylinder Test
[0104] The animals were placed in a transparent cylinder about 20
cm in diameter and 30 cm in height with an open top. The number
times each paw was placed on the sides of the cylinder was counted
a 5 minute period. The test was videotaped and a mirror was placed
behind the cylinder allowing the experimenter to see the placement
of paws when the animal was facing away from the camera.
[0105] Corner Test
[0106] A 30 degree angle was formed using the edges of two
plexiglass boards. The animals were placed halfway into the corner
facing the corner and the total number of left and right turns was
recorded. A total of 10 trials were conducted and videotaped. A
non-lesioned animal turned randomly toward the left or right, while
a lesioned animal preferentially turned toward the non-impaired
(left) side. Results were reported as the percentage of ipsilateral
(injury site, right side) versus contralateral turns.
[0107] Grip Test
[0108] Motor deficits were assessed using a grip strength meter
(Bioseb, # BIO-GS3). The animals were held so the front left paw
could firmly grasp the bar of the strength meter, and then pulled
straight backward until its front paw released its grasp. Grip
strength was recorded in Newtons (N). This test was repeated 3
times.
[0109] Histology and Immunochemistry
[0110] Tissue collection: Upon completion of the behavioral test at
30 days post MCAO, rats were euthanized with isoflurane and
perfused transcardially with ice-cold phosphate buffer (PB)
followed by 4% paraformaldehyde (PFA) in PB. Brains were carefully
removed, post-fixed in 4% PFA for 24 hour, and equilibrated in 30%
sucrose, and sectioned frozen in the coronal plane at 30 .mu.m
using a cryostat and cryopreserved at -20.degree. C. for
immunohistochemistry and histology.
[0111] Brain Infarction Quantification
[0112] Infarct size was determined by Nissl thionin staining Six
coronal sections were chosen from each animal representing 1 mm
intervals from 1.7 mm anterior to bregma, to -3.3 mm posterior to
bregma. Slides were washed in PBS and hydrated in a series of
alcohols, then stained with Thionin to label Nissl bodies for 3
minutes. Slides were then dehydrated through a series of alcohols,
cleared in Xylene, and coverslipped with DPX mountant (Electron
Microscopy Sciences, Ft. Washington, Pa.). Photographs of the
sections were taken on an Olympus IX71 microscope with DP manager
software (Olympus America Inc., Melville, N.Y.) at 1.25.times..
Infarct size was determined with the indirect method. We measured
the ipsilateral healthy (intact) tissue and the area of the
contralateral hemisphere a using ImageJ software. The infarct area
was obtained by deducting the ipsilateral volume from the total
contralateral volume and was expressed as percent of contralateral
hemisphere.
[0113] Evaluation of White Matter Injury
[0114] To evaluate white matter injury, coronal brain sections from
each the animals were stained with Luxol Fast Blue (LFB;
Sigma-Aldrich). For Luxol Fast Blue (LFB) staining, slides were
dehydrated in 95% ethanol and incubated in LFB solution at
56.degree. C. overnight. The following day slides were rinsed in
95% ethanol and distilled water and differentiated in 0.1% lithium
carbonate solution for 20 s followed by 70% ethyl alcohol for 30 s,
and finally rinsed in distilled water. After complete gray and
white matter differentiation, slides were dehydrated and placed
under coverslips with DPX. The quantitative analysis for
demyelination in LFB staining section was performed as described
previously with some modification (Virgili, Espinosa-Parrilla et
al. 2011; Yu, Gong et al. 2012). LFB stained area was measured on
the randomly selected sections (n=21-23 animals/group) to assess
demyelination. The extent of the LFB positive area was quantified
using a pixel grayscale approach in ImageJ software (National
Institute of Health, USA). Briefly, images were converted into a
gray scale format and a contour was drawn around each individual
hemisphere. The threshold was set for LFB-positive area by
determining the maximum threshold of corpus callosum as "highest
gray value". Once calibrated, the amount of LFB staining was
measured as the percentage of hemispheric area using ImageJ
software.
[0115] Immunohistochemistry
[0116] Anti-Ibal (1:500; Wako 019-19741) immunocytochemistry was
used for the specific identification of microglia. The slide was
rinsed in PBS, quenched for peroxidase using 1% H202 in methanol
for 15 minutes, and incubated overnight at 40 C with primary
antibody. The slides were then incubated with biotinylated
secondary antibody (1:200 goat anti-rabbit; Vector Labs BA-1000)
for 1 hour at room temperature, followed by avidin bound peroxidase
complex (ABC-kit, Vector) for 1 hour. Following several washes in
PBS, slides were treated with 3-3'diaminobenzidine
tetrahydrochloride (DAB) for about 3 to 5 minutes (Pierce, PI
34065). The development of the color reaction was stopped by
washing with PBS. Slides were then dehydrated and coverslipped.
Iba-1 positive cells were quantified (n=12) as previously detailed
by Smirkin et al. (Smirkin, Matsumoto et al. 2010) with little
modification. Four images (Upper lateral/medial and lower
lateral/medial) in the region around the ischemic core in the
ipsilateral hemisphere and the corresponding fields from the
contralateral hemisphere were captured using an Olympus IX71
microscope at 20.times. objective with DP manager software. Total
number of Iba-1 positive cells were counted manually by two
observer using ImagePro plus software.
[0117] Statistical Analysis
[0118] Data are expressed as group mean.+-.SEM and were analyzed
using GraphPad Prism 5.01 software (GraphPad Software, San Diego,
Calif.). All analyses were performed using a Student's t-test. A
value of p<0.05 was considered significant for all analyses.
[0119] Results for NAGH:
[0120] NAGH Improves Vertical Activity
[0121] Daytime Spontaneous Activity: Spontaneous activity was
measured prior to MCAO and NAGH treatment and then 1 month post
MCAO and NAGH treatment. Daytime spontaneous activity was measured
at 5-min intervals over a 1-hr test period. When daytime
spontaneous activity was examined for differences between the DMSO
group and the NAGH-treated groups, the overall analyses showed that
the NAGH-treated animals were less active on all measured
parameters except for VMN, CCR, CD, and CT (Data not shown). All
vertical movement parameters (VA, VMN, VT) significantly correlate
with the infarct volume, while most of horizontal movement
parameters except the distance travelled in the margin, did not
correlate with the infarct volume (Shen and Wang 2010). These
researchers also did not find any correlation between the size of
infarction and all stereotypy parameters (SC, SN, and ST).
[0122] When the inventors analyzed the three parameters of vertical
movement, i.e. VA, VMN, and VT, significant differences were found
between the groups on VA and VT but not VMN (p>0.05); the
animals in the NAGH treatment group were significantly less active
on VA (p=0.03) and VT (p=0.01) (FIG. 2). However, the NAGH
treatment group was more active than DMSO group on VMN (p=0.15).
The overall horizontal activities during daytime were similar and
there were no significant differences between the animals that
received NAGH and DMSO group. Although there was a decrease in
activity in animals treated with NAGH across each parameter of
stereotypy activity (i.e. SC, SN, and ST) these failed to reach a
statistically significant level (p>0.05).
[0123] Nighttime Spontaneous Activity: Nighttime spontaneous
activity was measured at 1-hr intervals over a 12-hr test period.
Overall pattern of behavior during nighttime was similar to that
observed during the day, i.e. there was less activity observed in
animals treated with NAGH on all measured parameters than the DMSO
group except for VMN (Data not shown). Further, there was a
significant difference on all measured vertical movement parameters
between the groups (FIG. 3). The animals in the NAGH treatment
group were significantly less active on VA (p=0.01), and VT
(p=0.03) and significantly more active on VMN (p=0.015). Although
there was a decrease in activity in animals treated with NAGH
across each horizontal parameters monitored, these changes did not
reach statistical significance. All parameters of stereotypic
behavior (SC, SN, and ST) did not show any significant difference
between groups.
[0124] NAGH Improves Body Asymmetry
[0125] A battery of behavioral tests was performed to determine the
effects of NAGH on behavioral outcome following MCAO. The overall
behavioral data showed there were no significant differences
between the NAGH group and the vehicle group on all tests except on
EBST which is a measure of motor asymmetry (FIG. 4). On EBST, there
was no significant difference in the bias between the animals
treated with NAGH at baseline and those treated with DMSO
(p=0.073). However, at 30 days the animals treated with NAGH showed
significantly less bias (p=0.009) compared to DMSO group.
[0126] NAGH Failed to Significantly Reduce Infarct Volume
[0127] Ischemic damage was determined by total infarct size using
Nissl-Thionin staining Although there was reduced thionin staining
in the ipsilateral corpus striatum and cortical area animals
treated with DMSO (FIG. 5A) than that of NAGH treated animals (FIG.
5B), the difference did not reach statistical significance
(p=0.86). Infarct volume in the stroke group receiving vehicle only
(n=25) was 17.57 .+-.4.4.3%, whereas animals receiving NAGH (n=27)
showed infarcts involving only 13.58 .+-.2.3% of the hemisphere
(FIG. 5C).
[0128] NAGH Exerts its Neuroprotective Effects via Preservation of
White Matter Tracts
[0129] To determine treatment effects of NAGH on myelinating
oligodendrocytes in the rat brain following MCAO, the inventors
histologically stained white matter tracts with Luxol Fast Blue.
After MCAO, the demyelination in the DMSO treated group (n=23)
showed a statistically significant reduction in LFB staining in the
ischemic hemisphere (9.13.+-.0.5%) compared to non-infarcted
hemisphere (11.4 .+-.0.49%, p<0.05) that persisted at 30 days
post-stroke (FIG. 6A, B, E). In animals receiving NAGH (n=22),
myelinated fibers were intensely stained by LFB in the striatum and
cortex. The levels of LFB did not change between the ischemic and
non-ischemic hemisphere 10.32.+-.0.48% and 9.81.+-.0.38%
respectively (p>0.05), suggesting white matter tracts were more
intact in treated animals (FIG. 6C, D, E). Indeed, the percent
change in LFB staining from contralateral to ipsilateral hemisphere
is 18+5.6% in the DMSO treated group compared to -0.73+7.7% in the
NAGH treated group (p<0.05). These results demonstrated severe
and long-term white matter demyelination following MCAO.
[0130] NAGH Reduces Ibal Positive Cells in the Infarcted
Regions
[0131] To test whether NAGH alters activation of immune cells in
the brain following MCAO, Ibal expression was measured in the
ischemic brain since Ibal labels microglia regardless of their
activation state. In general, there are more intensely stained
amoeboid or round shaped cells in the peri-infarct and ischemic
core following MCAO, suggesting that these immunopositive cells
were activated microglia (FIG. 7). Ibal positive cells were mostly
located in the cortex, striatum, and adjacent to the lateral
ventricle. The observations demonstrate that the peri-infarct and
ischemic core of DMSO treated rats contain more activated microglia
than the NAGH treated animals even 30 days after stroke, although
the difference was not statistically significant (p>0.05).
[0132] NAGH blocks microglial migration via actions at both
.sigma.-1 and .sigma.-2 receptors.
[0133] FIG. 8A illustrates the concentration-response relationship
for NAGH (closed circles) and DTG (open circles) inhibition of
microglial migration evoked by 100 .mu.M ATP. Solid line represents
best fit to the NAGH data using a two-component Langmuir-Hill
equation with the
[0134] Emax, 1050 and Hill coefficient being 0.43, 0.75 .mu.M and
2.2 for the high affinity component and 0.57, 84 .mu.M and 0.80 for
the low affinity component, respectively. Dashed line represents
best fit to the DTG data using a two-component Langmuir-Hill
equation with the Emax, 1050 and Hill coefficient being 0.38, 0.76
.mu.M, and 0.59 for the high affinity component and 0.62, 80 .mu.M,
and 1.7 for the low affinity component, respectively. Points
represent mean (.+-.SEM) and for all points, n>9. FIG. 8B
depicts a bar graph of the mean (.+-.SEM) number of microglial
cells migrating in response to 100 .mu.M ATP when ATP was added
alone (DMEM) or in the presence of 100 .mu.M NAGH. Microglia were
further exposed to no .sigma. antagonist (Control), or the
.sigma.-1 antagonist, BD-1047 (10 .mu.M, BD-1047), or the .sigma.-2
antagonist, rimcazole (10 .mu.M, Rimcazole). Asterisks denote
significant difference between DMEM and NAGH within Control,
BD-1047 and Rimcazole groups, respectively. Pound symbols indicate
significant difference between Control, BD-1047 and Rimcazole
within DMEM and NAGH groups, respectively. P<0.05 and n=24 for
all groups.
[0135] Results for Other Sigma Agonists
[0136] With regard to the other sigma agonists, they were also
tested to determine if they would induce long-term recovery of
function and minimize lesion size in the brain when administered
24-72 hours after MCAO. While DTG induces short-term improvements
in neurohistological outcome (Hall A A, Leonardo C C, Collier L A,
Rowe D D, Willing A E, Pennypacker K R. Neurosci.
2009;164:470-477), the therapeutic efficacy of this sigma receptor
agonist is minimal because the early benefits are not maintained
long term (Leonardo, C. C., A. A. Hall, et al. (2010). Transl
Stroke Res 1(2): 135-145).
[0137] Results from Bromo-DTG and Chloro-DTG did not show
significant effects over vehicle, as seen in FIGS. 10 through 13.
With NAD, there was a tendency for infarct volume of treated
animals to decrease, as seen in FIGS. 14 and 15, but this was not
significant (23.78.+-.7.78% compared to 28.84.+-.8.78% in MCAO only
group, as seen in FIG. 16. Behaviorally, animals treated with NAD
showed less activity than MCAO-only animals, as seen in FIGS. 17
through 20.
[0138] The overall behavioral data showed there was no significant
difference between the NAD and vehicle treated groups except on the
Grip Strength test and Step test, as seen in FIGS. 21 through 28.
The average grip strength of NAD treated animals was significantly
higher than that of non-treated rats (p=0.0007). Similarly, on the
step test, the untreated animals took fewer steps with their
impaired paw compared to their unimpaired paw (86.7+6.2%) at 30
days post-stroke, while NAD treated animals showed no decrement
(100.3+5.6%, p<0.05).
[0139] Further analysis is needed to determine the extent of the
behavioral recovery and determine if it is related to increased
neuronal survival (infarct size), preservation of oligodendrocytes
and white matter tracts or modulation of post-stroke inflammation
(microglia and astrocytes).
[0140] In conclusion, examining the available data gathering from
testing each compound, the NAGH agonist was found to be the only
sigma receptor agonist in the test group that reduced infarct
volume and increased functional recovery, as seen in FIGS. 22, 27,
28. The NAGH treated rats had significantly less motor asymmetry
than vehicle treated rats (*p<0.05). On the cylinder test, the
number of paw hits on the side of the cage decreased dramatically
for both paws (data not shown), but the extent of the decrease was
larger for the left (contralateral) paw. However, changes in
asymmetry on the cylinder test failed to gain significance.
[0141] With regard to infarct volume, NAGH protected both neurons
and oligodendrocytes from MCAO-induced cell death. Infarct size was
significantly less in the treated group (*p<0.05). (FIG. 5)
Similarly, Luxol Fast blue demonstrated that white matter tracts
were more intact in the treated group, particularly in cortex.
(FIG. 6)
[0142] As such, NAGH is a promising drug that appears to have
long-term clinical efficacy as a treatment for stroke. NAGH may
protect mature oligodendrocyte damage following MCAO which is a
promising therapeutic approach to restore myelin.
[0143] In the preceding specification, all documents, acts, or
information disclosed do not constitute an admission that the
document, act, or information of any combination thereof was
publicly available, known to the public, part of the general
knowledge in the art, or was known to be relevant to solve any
problem at the time of priority.
[0144] The disclosures of all publications cited above are
expressly incorporated herein by reference, each in its entirety,
to the same extent as if each were incorporated by reference
individually.
[0145] While there has been described and illustrated specific
embodiments of methods for treatment of stroke, it will be apparent
to those skilled in the art that variations and modifications are
possible without deviating from the broad spirit and principle of
the present invention. It is also to be understood that the
following claims are intended to cover all of the generic and
specific features of the invention herein described, and all
statements of the scope of the invention which, as a matter of
language, might be said to fall there between.
* * * * *