U.S. patent application number 14/175861 was filed with the patent office on 2014-10-30 for method and compositions for treating stroke with fever.
This patent application is currently assigned to NONO INC.. The applicant listed for this patent is NONO INC.. Invention is credited to Michael Tymianksi.
Application Number | 20140323406 14/175861 |
Document ID | / |
Family ID | 38923841 |
Filed Date | 2014-10-30 |
United States Patent
Application |
20140323406 |
Kind Code |
A1 |
Tymianksi; Michael |
October 30, 2014 |
METHOD AND COMPOSITIONS FOR TREATING STROKE WITH FEVER
Abstract
The invention provides methods of treating stroke and related
conditions exacerbated by fever and/or hyperglycemi by
administering peptides or peptidomimetics that inhibit binding of
NMDAR 2B to PSD-95 to a patient.
Inventors: |
Tymianksi; Michael;
(Toronto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NONO INC. |
Etobicoke |
|
CA |
|
|
Assignee: |
NONO INC.
Etobicoke
CA
|
Family ID: |
38923841 |
Appl. No.: |
14/175861 |
Filed: |
February 7, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12307581 |
Sep 1, 2009 |
8685925 |
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PCT/US07/15747 |
Jul 10, 2007 |
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14175861 |
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60833572 |
Jul 26, 2006 |
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60830189 |
Jul 11, 2006 |
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Current U.S.
Class: |
514/15.1 ;
435/40.52; 506/10 |
Current CPC
Class: |
G01N 2800/042 20130101;
A61K 38/08 20130101; A61K 38/177 20130101; G01N 33/5088 20130101;
G01N 2800/2871 20130101; A61K 38/07 20130101; A61P 9/10 20180101;
A61P 25/00 20180101; G01N 2500/02 20130101 |
Class at
Publication: |
514/15.1 ;
435/40.52; 506/10 |
International
Class: |
A61K 38/08 20060101
A61K038/08; G01N 33/50 20060101 G01N033/50 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under NIH
Grant Number NS048956. The Government may have certain rights in
this invention.
Claims
1-25. (canceled)
26. A method of screening a compound that inhibits binding of
PSD-95 to NDMAR, comprising administering the compound to a animal
having ischemia exacerbated by hyperthermia and/or hyperglycemia;
and determining whether the compound reduces infarction volume
resulting from the ischemia relative to a control animal not
treated with the compound.
27. The method of claim 26, wherein the ischemia is a permanent
ischemia.
28. The method of claim 27, wherein the animal has hyperglycemia
before administering the compound.
29. The method of claim 27, wherein the animal has an
infection.
30. The method of claim 27, wherein the animal has diabetes.
31. The method of claim 20, wherein the animal has eaten less than
twelve hours before administering the compound
32. A method for reducing the damaging effect of stroke in a
patient having stroke or other injury to the CNS exacerbated by
fever comprising administering to the an effective amount of a
peptide having an amino acid sequence comprising ESDV (SEQ ID NO:3)
or ETDV (SEQ ID NO:5), wherein the peptide is linked to an
internalization peptide or derivatized to improve the ability of
the peptide to cross a membrane, and thereby reducing the damaging
effect of the stroke or traumatic injury to the CNS, wherein the
patient has a fever of at least 38.degree. C. on initiating
treatment.
33-35. (canceled)
36. The method of claim 32, wherein a peptide having an amino acid
sequence comprising KLSSIETDV (SEQ ID NO:10) is administered.
37. The method of claim 36, wherein a peptide having an amino acid
sequence comprising YGRKKRRQRRRKLSSIETDV (SEQ ID NO:11) is
administered.
38. The method of claim 36, wherein a peptide having an amino acid
sequence consisting of YGRKKRRQRRRKLSSIETDV (SEQ ID NO:11) is
administered.
39. The method of claim 32, wherein a peptide having an amino acid
sequence comprising KLSSIESDV (SEQ ID NO:9) is administered.
40. The method of claim 32, wherein a peptide having an amino acid
sequence comprising YGRKKRRQRRRKLSSIESDV (SEQ ID NO:12) is
administered.
41. The method of claim 40, wherein a peptide having an amino acid
sequence comprising YGRKKRRQRRRKLSSIESDV (SEQ ID NO:12) is
administered.
42. (canceled)
43. The method of claim 32, wherein the patient has stroke
exacerbated by a fever of at least 39.degree. C. degrees for a
period within 6-24 hours after onset of stroke.
44-45. (canceled)
46. The method of claim 32, wherein the patient has stroke
exacerbated by a fever of at least 40.degree. C. at least for a
period between 6-24 hours after onset of stroke.
47. The method of claim 32, wherein the fever is due to concurrent
infection.
48. The method of claim 32, wherein the fever is due to location of
the stroke in a region of the brain affecting body temperature
regulation or set-point.
49. The method of claim 32, wherein the subject is also suffering
from hyperglycemia substantially concurrent with onset of
stroke.
50. The method of claim 49, wherein the hyperglycemia is due to
diabetes.
51. The method of claim 32, wherein the stroke is an ischemic
stroke.
52. The method of claim 32, wherein the method reduces the
infarction volume resulting from the stroke by at least 15%.
53-92. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a nonprovisional of U.S. Ser. No.
60/830,189, filed Jul. 11, 2006 and U.S. Ser. No. 60/833,572, filed
Jul. 26, 2007, both incorporated by reference in their entirety for
all purposes.
BACKGROUND OF THE INVENTION
[0003] Stroke is predicted to affect more than 600,000 people in
the United States a year. In a 1999 report, over 167,000 people
died from strokes, with a total mortality of 278,000. In 1998, 3.6
billion was paid to just those Medicare beneficiaries that were
discharged from short-stay hospitals, not including the long term
care for >1,000,000 people that reportedly have functional
limitations or difficulty with activities of daily living resulting
from stroke (Heart and Stroke Statistical update, American Heart
Association, 2002). No therapeutics has yet been approved to reduce
brain damage resulting from stroke.
[0004] Stroke is characterized by neuronal cell death in areas of
ischemia, brain hemorrhage and/or trauma. Cell death is triggered
by glutamate over-excitation of neurons, leading to increased
intracellular Ca.sup.2+ and increased nitric oxide due to an
increase in nNOS (neuronal nitric oxide synthase) activity.
[0005] Glutamate is the main excitatory neurotransmitter in the
central nervous system (CNS) and mediates neurotransmission across
most excitatory synapses. Three classes of glutamate-gated ion
channel receptors (N-methyl-D-aspartate (NMDA),
alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and
Kainate) transduce the postsynaptic signal. Of these, NMDA
receptors (NMDAR) are responsible for a significant portion of the
excitotoxicity of glutamate. NMDA receptors are complex having an
NR1 subunit and one or more NR2 subunits (2A, 2B, 2C or 2D) (see,
e.g., McDain, C. and Caner, M. (1994) Physiol. Rev. 74:723-760),
and less commonly, an NR3 subunit (Chatterton et al. (2002) Nature
415:793-798). The NR1 subunits have been shown to bind glycine,
whereas NR2 subunits bind glutamate. Both glycine and glutamate
binding are required to open the ion channel and allow calcium
entry into the cell. The four NR2 receptor subunits appear to
determine the pharmacology and properties of NMDA receptors, with
further contributions from alternative splicing of the NR1 subunit
(Kornau et al. (1995) Science 269:1737-40). Whereas NR1 and NR2A
subunits are ubiquitously expressed in the brain, NR2B expression
is restricted to the forebrain, NR2c to the cerebellum, and NR2D is
rare compared to the other types.
[0006] Because of the key role of NMDA receptors in the
excitotoxicity response, they have been considered as targets for
therapeutics. Compounds have been developed that target the ion
channel (ketamine, phencyclidine, PCP, MK801, amantadine), the
outer channel (magnesium), the glycine binding site on NR1
subunits, the glutamate binding site on NR2 subunits, and specific
sites on NR2 subunits (Zinc--NR2A; Ifenprodil, Traxoprodil--NR2B).
Among these, the non-selective antagonists of NMDA receptor have
been the most neuroprotective agents in animal models of stroke.
However, clinical trials with these drugs in stroke and traumatic
brain injury have so far failed, generally as a result of severe
side effects such as hallucination and even coma. Other criticisms
of past animal stroke studies include that the efficacy of many
neuroprotectants was determined in mild ischemia models
(ischemia-reperfusion instead of permanent ischemia), and under
conditions of food deprivation, which can not adequately mimic the
more severe human situation. Also, most drugs were administered
pre-ischemia whereas human trials necessitate a post-treatment
paradigm (Gladstone et al., 2002; STAIR Committee, 1999).
[0007] Another key difference between human stroke and experimental
ischemia is that some stroke victims also suffer from aggravating
premorbid or comormid conditions or stroke-related complications.
Prominent among these is hyperglycemia (Alvarez-Sabin., 2003),
especially in diabetic patients (Paolino, 2005), but also in
non-diabetics (Alvarez-Sabin., 2003). However, hyperglycemia is
actively avoided in laboratory stroke studies as it is known to
exacerbate cerebral infarction (Li, 1997, 1998, 2000, 2001;
Farrokhnia, 2005), and experimental animals are routinely fasted to
minimize intra-ischemic blood glucose elevations (Elsersy, 2004;
Horiguchi, 2003; Belayev, 2005a). Fever is another complication
that afflicts some stroke victims, and is an independent predictor
of poor outcome (Azzimondi, 1995; Reith, 1996; Boysen, 2001;
Ginsberg, 1998). Hyperthermia has long been known to exacerbate
both global and focal experimental ischemic injury (Busto, 1987b,
1989a, 1989b; Ginsberg, 1992; Morikawa, 1992; Chen, 1993;
Minamisawa, 1990a, 1990b, 1990c; Chen, 1991) and, precisely for
this reason, has been strongly avoided in studies of
neuroprotective drugs.
[0008] The present inventor has reported that postsynaptic
density-95 protein (PSD-95) couples NMDARs to pathways mediating
excitotoxicity and ischemic brain damage (Aarts et al., Science
298, 846-850 (2002)). This coupling was disrupted by transducing
neurons with peptides that bind to modular domains on either side
of the PSD-95/NMDAR interaction complex. This treatment attenuated
downstream NMDAR signaling without blocking NMDAR activity,
protected cultured cortical neurons from excitotoxic insults and
reduced cerebral infarction volume in rats subjected to transient
focal cerebral ischemia. The analysis was performed under
conditions of transient ischemia and prior fasting to avoid
exacerbating fever and hyperglycemia.
SUMMARY OF THE CLAIMED INVENTION
[0009] The invention provides the use of a peptide having an amino
acid sequence comprising T/SXV/L or a peptidomimetic thereof for
manufacture of a medicament for treatment of the damaging effect of
stroke or other injury to the CNS exacerbated by fever or
hyperglycemia.
[0010] The invention further provides a method of screening a
compound that inhibits binding of PSD-95 to NDMAR, comprising
administering the compound to a animal having ischemia exacerbated
by hyperthermia and/or hyperglycemia; and determining whether the
compound reduces infarction volume resulting from the ischemia
relative to a control animal not treated with the compound.
[0011] The invention further provides a method for reducing the
damaging effect of stroke in a patient having stroke or other
injury to the CNS exacerbated by fever or hyperglycemia comprising
administering to the an effective amount of a peptide having an
amino acid sequence comprising T/S-[X]-V/L, or peptidomimetic
thereof, and thereby reducing the damaging effect of the stroke or
other injury.
[0012] The invention further provides for the use of a peptide
having an amino acid sequence comprising T/SXV/L or a
peptidomimetic thereof in the manufacture of a medicament for
treating the damaging effect of stroke or other CNS injury in a
patient having fever or hyperglycemia.
[0013] The invention further provides for the use of a peptide
having an amino acid sequence comprising T/SXV/L or a
peptidomimetic thereof in the manufacture of a medicament for the
prophylactic treatment of the damaging effect of stroke or other
CNS injury in a patient with fever or hyperglycemia.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1. Effects of ischemia models on core temperature.
Animals in A-E were implanted with intra-peritoneal telemetric
temperature probes and exposed to the indicated conditions. Gray
bars indicate duration of animal surgery. A. Sham procedure (n=4).
B. Sham procedure with administration of Tat-NR2B9c.sub.(SDV)
(n=6). C. Pial vessel occlusion (n=5). D. Permanent MCAO without
cage cooling. (n=8). E. Permanent MCAO with cage cooling using a
feedback system (n=8). CT: Core temperature. Cage: Cage
temperature. Symbols in A-E indicate the means.+-.SE of the
indicated number of animals. F. Effects of the indicated conditions
on temporalis muscle, core, and brain temperature at the indicated
times post pMCAO. Core-Saline: animals treated with saline 1b post
pMCAO (n=6). Temporalis-Saline: Concurrent temporalis muscle
temperatures from saline treated animals. Core-SDV: animals treated
with Tat-NR2B9c.sub.(SDV) 1 h post pMCAO (n=6). Brain-SDV:
Concurrent direct brain temperature measurements from
NR2B9c.sub.(SDV)-treated animals.
[0015] FIG. 2. Effect of Tat-NR2B9c.sub.(SDV) post-treatment in the
permanent pial vessel occlusion model. A. The three sites of pial
vessel occlusion. B. Resulting typical infarct in TTC stained
brain. C. Effect of the indicated drug and drug concentration on
infarct size. Number of animals per group is provided in Table 1.
Asterisk: significantly different from both saline and ADA controls
(ANOVA, p<0.05). ADA: Tat-NR2B9c.sub.(ADA). SDV:
Tat-NR2B9c.sub.(SDV). D. Representative infarcts in TTC-stained
coronal sections from each group.
[0016] FIG. 3. PSD-95 inhibitors do not affect the hyperthermic
response following pMCAO. A-E: Core temperatures before, during
(gray bars) and following pMCAO surgery in animals treated with the
indicated PSD-95 inhibitor at the indicated dose. N=8 for each
group.
[0017] FIG. 4. Representative TTC stained coronal brain sections
taken from animals 24 h after subjecting them to sham surgery (A)
or pMCAO (B-F). Animals in B-F were treated with the indicated
PSD-95 inhibitor at the indicated dose at 1 h after pMCAO.
[0018] FIG. 5. Reduction of pMCAO infarct volumes by post-treatment
with PSD-95 inhibitors. Ai and Aii: Effects of PSD-95 inhibitors on
hemispheric (Ai) and cortical (Aii) infarct volumes in the first
blinded study. Bi and Bii: Effects of PSD-95 inhibitors on
hemispheric (Bi) and cortical (Bii) infarct volumes in the second
blinded study. Animals were treated with the PSD-95 inhibitors at
the indicated doses at 1 h after pMCAO. Asterisks: significantly
different from saline controls (ANOVA followed by multiple
comparisons using the Bonferroni correction). Inset: study
paradigm. Number of animals per group is provided in Table 2.
[0019] FIG. 6. Infarct areas from 8 coronal brain sections from
which the volumes in FIG. 5 were derived. Each symbol indicates the
mean.+-.SE area in a given stereotactic plane for the conditions
indicated.
[0020] FIG. 7. A. Composite neurobehavioral scores at 2 and 24 hrs
after pMCAO for the indicated conditions. B-G: Plots of animal cage
activity before and after the permanent MCAO procedure in the
different PSD-95 inhibitor groups. Zero indicates time of MCAO.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0021] A "fusion polypeptide" refers to a composite polypeptide,
i.e., a single contiguous amino acid sequence, made up of two (or
more) distinct, heterologous polypeptides which are not normally
fused together in a single amino acid sequence.
[0022] The term "PDZ domain" refers to a modular protein domain of
about 90 amino acids, characterized by significant sequence
identity (e.g., at least 60%) to the brain synaptic protein PSD-95,
the Drosophila septate junction protein Discs-Large (DLG), and the
epithelial tight junction protein 201 (Z01). PDZ domains are also
known as Discs-Large homology repeats ("DHRs") and GLGF repeats.
PDZ domains generally appear to maintain a core consensus sequence
(Doyle, D. A., 1996, Cell 85: 1067-76). Exemplary PDZ
domain-containing proteins and PDZ domain sequences disclosed in
U.S. application Ser. No. 10/714,537, which is herein incorporated
by reference in its entirety.
[0023] The term "PL protein" or "PDZ Ligand protein" refers to a
naturally occurring protein that forms a molecular complex with a
PDZ-domain, or to a protein whose carboxy-terminus, when expressed
separately from the full length protein (e.g., as a peptide
fragment of 3-25 residues, e.g. 3, 4, 5, 8, 10, 12, 14 or 16
residues), forms such a molecular complex. The molecular complex
can be observed in vitro using the "A assay" or "G assay"
described, e.g., in U.S. application Ser. No. 10/714,537, or in
vivo.
[0024] The term "NMDA receptor," or "NMDAR," refers to a membrane
associated protein that is known to interact with NMDA. The term
thus includes the various subunit forms described in the Background
Section. Such receptors can be human or non- (e.g., mouse, rat,
rabbit, monkey).
[0025] A "PL motif" refers to the amino acid sequence of the
C-terminus of a PL protein (e.g., the C-terminal 3, 4, 5, 6, 7, 8,
9, 10, 12, 14, 16, 20 or 25 contiguous residues) ("C-terminal PL
sequence") or to an internal sequence known to bind a PDZ domain
("internal PL sequence").
[0026] A "PL peptide" is a peptide of comprising or consisting of,
or otherwise based on, a PL motif that specifically binds to a PDZ
domain.
[0027] The terms "isolated" or "purified" means that the object
species (e.g., a peptide) has been purified from contaminants that
are present in a sample, such as a sample obtained from natural
sources that contain the object species. If an object species is
isolated or purified it is the predominant macromolecular (e.g.,
polypeptide) species present in a sample (i.e., on a molar basis it
is more abundant than any other individual species in the
composition), and preferably the object species comprises at least
about 50 percent (on a molar basis) of all macromolecular species
present. Generally, an isolated, purified or substantially pure
composition comprises more than 80 to 90 percent of all
macromolecular species present in a composition. Most preferably,
the object species is purified to essential homogeneity (i.e.,
contaminant species cannot be detected in the composition by
conventional detection methods), wherein the composition consists
essentially of a single macromolecular species.
[0028] A "peptidomimetic" and refers to a synthetic chemical
compound which has substantially the same structural and/or
functional characteristics of a peptide of the invention. The
peptidomimetic can contain entirely synthetic, non-natural
analogues of amino acids, or, is a chimeric molecule of partly
natural peptide amino acids and partly non-natural analogs of amino
acids. The peptidomimetic can also incorporate any amount of
natural amino acid conservative substitutions as long as such
substitutions also do not substantially alter the mimetic's
structure and/or inhibitory or binding activity. Polypeptide
mimetic compositions can contain any combination of nonnatural
structural components, which are typically from three structural
groups: a) residue linkage groups other than the natural amide bond
("peptide bond") linkages; b) non-natural residues in place of
naturally occurring amino acid residues; or c) residues which
induce secondary structural mimicry, i.e., to induce or stabilize a
secondary structure, e.g., a beta turn, gamma turn, beta sheet,
alpha helix conformation, and the like.
[0029] The term "specific binding" refers to binding between two
molecules, for example, a ligand and a receptor, characterized by
the ability of a molecule (ligand) to associate with another
specific molecule (receptor) even in the presence of many other
diverse molecules, i.e., to show preferential binding of one
molecule for another in a heterogeneous mixture of molecules.
Specific binding of a ligand to a receptor is also evidenced by
reduced binding of a detectably labeled ligand to the receptor in
the presence of excess unlabeled ligand (i.e., a binding
competition assay).
[0030] Statistically significant refers to a p-value that is
<0.05, preferably <0.01 and most preferably <0.001.
II. General
[0031] The invention provides peptides and peptidomimetics useful
for reducing damaging effects of stroke and other neurological
conditions exacerbated by fever and/or hyperglycemia. The subsets
of patients afflicted with one or both of these exacerbating
factors have a significantly poorer outcome compared with patients
in which these factors are present. The invention is based in part
on results described in the examples in which certain peptides were
found to reduce infarction volume in a rat model of permanent
ischemia notwithstanding severe hyperthermia (.gtoreq.39.degree.
C.) and lack of prior fasting. Surprisingly, subjects with stroke
and fever or hyperglycemia can be treated as effectively as
subjects not suffering from such comorbid complications. Peptides
used in such methods have an amino acid sequence including or based
on the PL motif of NMDAR 2B receptor (i.e., PL peptides). Although
an understanding of mechanism is not required for practice of the
invention, it is believed that such peptides act at least in part
by inhibiting interaction between NMDARs with postsynaptic density
95 protein (i.e., PSD-95 inhibitors). The peptides may also inhibit
interactions between PSD-95 and nNOS. Unlike glutamate antagonists
that have previously failed clinical trials, such peptides can
disrupt neurotoxic signaling during ischemia without incurring the
negative consequences of loss of NDMAR function.
III. Peptides and Peptidomimetics
[0032] Peptides and peptidomimetics useful in the invention inhibit
interaction between domain 2 of postsynaptic density-95 protein
(PSD-95 d2) containing a PDZ domain (Stathakism, Genomics
44(1):71-82 (1997)) and the C-terminus of NR2B subunit of the
neuronal N-methyl-D-aspartate receptor (NMDAR) containing a PL
motif (Mandich et al., Genomics 22, 216-8 (1994)). Such peptides
include or are based on a PL motif from the C-terminus of this
subunit and have an amino acid sequence comprising [S/T]-X-[V/L]
(SEQ ID NO.: 1). This sequence preferably occurs at the C-terminus
of the peptides of the invention. Preferred peptides have an amino
acid sequence comprising [E/D/N/Q]-[S/T]-[D/E/Q/N]-[V/L] (SEQ ID
NO.: 2) at their C-terminus. Exemplary peptides comprise: ESDV (SEQ
ID NO.: 3), ESEV (SEQ ID NO.: 4), ETDV (SEQ ID NO.: 5), ETEV (SEQ
ID NO.: 6), DTDV (SEQ ID NO.:7), and DTEV (SEQ ID NO.: 8). Two
particularly preferred peptides are KLSSIESDV (SEQ ID NO.: 9), and
KLSSIETDV (SEQ ID NO.:10).
[0033] Any of the peptides of the invention can be linked,
preferably at their N-terminus, to an internalization peptide that
facilitates translocation through the plasma membrane of a cell.
For example, the HIV TAT internalization peptide YGRKKRRQRRR can be
used. An internalization peptide derived from Antennapedia can also
be used (see Bonfanti, Cancer Res. 57, 1442-6 (1997)). Two
preferred peptides including the HIV Tat internalization peptide
are
TABLE-US-00001 (SEQ ID NO.: 11, Tat-NR2B9c.sub.(TDV))
YGRKKRRQRRRKLSSIETDV, and (SEQ ID NO.: 12, Tat-NR2B9c.sub.(SDV))
YGRKKRRQRRRKLSSIESDV.
[0034] Peptides of the invention without an internalization peptide
usually have 3-25 amino acids, Peptide lengths (also without an
internalization peptide) of 5-10 amino acids, and particularly 9
amino acids are preferred.
[0035] Appropriate pharmacological activity of peptides or
peptidomimetics can be confirmed, if desired, using the animal
model described in the Examples. Optionally, peptides or
peptidomimetics can also be screened for capacity to inhibit
interactions between PSD-95 and NDMAR 2B using assays described in
e.g., US 20050059597, which is herein incorporated by reference.
Useful peptides typically have IC50 values of less than 50 uM, 25
.mu.M, 10 uM, 0.1 .mu.M or 0.01 .mu.M in such an assay. Preferred
peptides typically have an IC50 value of between 0.001-1 .mu.M, and
more preferably 0.05-0.5 or 0.05 to 0.1 .mu.M
[0036] Peptides such as those just described can optionally be
derivatized (e.g., acetylated, phosphorylated and/or glycoslylated)
to improve the binding affinity of the inhibitor, to improve the
ability of the inhibitor to be transported across a cell membrane
or to improve stability. As a specific example, for inhibitors in
which the third residue from the C-terminus is S or T, this residue
can be phosphorylated before use of the peptide.
[0037] Peptides of the invention, optionally fused to
internalization domains, can be synthesized by solid phase
synthesis or recombinant methods. Peptidomimetics can be
synthesized using a variety of procedures and methodologies
described in the scientific and patent literature, e.g., Organic
Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley &
Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby
(1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol.
Divers. 3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234.
IV. Stroke and Related Conditions
[0038] A stroke is a condition resulting from impaired blood flow
in the CNS regardless of cause. Potential causes include embolism,
hemorrhage and thrombosis. Some neuronal cells die immediately as a
result of impaired blood flow. These cells release their component
molecules including glutamate, which in turn activates NMDA
receptors, which raise intracellular calcium levels, and
intracellular enzyme levels leading to further neuronal cell death
(the excitotoxicity cascade). The death of CNS tissue is referred
to as infarction. Infarction Volume (i.e., the volume of dead
neuronal cells resulting from stroke in the brain) can be used as
an indicator of the extent of pathological damage resulting from
stroke. The symptomatic effect depends both on the volume of an
infarction and where in the brain it is located. Disability index
can be used as a measure of symptomatic damage, such as the Rankin
Stroke Outcome Scale (Rankin, Scott Med J; 2:200-15 (1957)) and the
Barthel Index. The Rankin Scale is based on assessing directly the
global conditions of a patient as follows.
TABLE-US-00002 0 No symptoms at all 1 No significant disability
despite symptoms; able to carry out all usual duties and
activities. 2 Slight disability; unable to carry out all previous
activities but able to look after own affairs without assistance. 3
Moderate disability requiring some help, but able to walk without
assistance 4 Moderate to severe disability; unable to walk without
assistance and unable to attend to own bodily needs without
assistance. 5 Severe disability; bedridden, incontinent, and
requiring constant nursing care and attention.
[0039] The Barthel Index is based on a series of questions about
the patient's ability to carry out 10 basic activities of daily
living resulting in a score between 0 and 100, a lower score
indicating more disability (Mahoney et al., Maryland State Medical
Journal 14:56-61 (1965)).
[0040] An ischemic stroke refers more specifically to a type of
stroke that caused by blockage of blood flow to the brain. The
underlying condition for this type of blockage is most commonly the
development of fatty deposits lining the vessel walls. This
condition is called atherosclerosis. These fatty deposits can cause
two types of obstruction. Cerebral thrombosis refers to a thrombus
(blood clot) that develops at the clogged part of the vessel
"Cerebral embolism" refers generally to a blood clot that forms at
another location in the circulatory system, usually the heart and
large arteries of the upper chest and neck. A portion of the blood
clot then breaks loose, enters the bloodstream and travels through
the brain's blood vessels until it reaches vessels too small to let
it pass. A second important cause of embolism is an irregular
heartbeat, known as arterial fibrillation. It creates conditions in
which clots can form in the heart, dislodge and travel to the
brain. Additional potential causes of ischemic stroke are
hemorrhage, thrombosis, dissection of an artery or vein, a cardiac
arrest, shock of any cause including hemorrhage, and iatrogenic
causes such as direct surgical injury to brain blood vessels or
vessels leading to the brain or cardiac surgery. Ischemic stroke
accounts for about 83 percent of all cases of stroke.
[0041] Transient ischemic attacks (TIAs) are minor or warning
strokes. In a TIA, conditions indicative of an ischemic stroke are
present and the typical stroke warning signs develop. However, the
obstruction (blood clot) occurs for a short time and tends to
resolve itself through normal mechanisms.
[0042] Hemorrhagic stroke accounts for about 17 percent of stroke
cases. It results from a weakened vessel that ruptures and bleeds
into the surrounding brain. The blood accumulates and compresses
the surrounding brain tissue. The two general types of hemorrhagic
strokes are intracerebral hemorrhage and subarachnoid hemorrhage.
Hemorrhagic stroke result from rupture of a weakened blood vessel
ruptures. Potential causes of rupture from a weakened blood vessel
include a hypertensive hemorrhage, in which high blood pressure
causes a rupture of a blood vessel, or another underlying cause of
weakened blood vessels such as a ruptured brain vascular
malformation including a brain aneurysm, arteriovenous malformation
(AVM) or cavernous malformation. Hemorrhagic strokes can also arise
from a hemorrhagic transformation of an ischemic stroke which
weakens the blood vessels in the infarct, or a hemorrhage from
primary or metastatic tumors in the CNS which contain abnormally
weak blood vessels. Hemorrhagic stroke can also arise from
iatrogenic causes such as direct surgical injury to a brain blood
vessel. An aneurysm is a ballooning of a weakened region of a blood
vessel. If left untreated, the aneurysm continues to weaken until
it ruptures and bleeds into the brain. An arteriovenous
malformation (AVM) is a cluster of abnormally formed blood vessels.
A cavernous malformation is a venous abnormality that can cause a
hemorrhage from weakened venous structures. Any one of these
vessels can rupture, also causing bleeding into the brain.
Hemorrhagic stroke can also result from physical trauma.
Hemorrhagic stroke in one part of the brain can lead to ischemic
stroke in another through shortage of blood lost in the hemorrhagic
stroke.
[0043] Several other neurological conditions can also result in
neurological death through NDMAR-mediated excitotoxicity. These
conditions include epilepsy, hypoxia, traumatic injury to the CNS
not associated with stroke such as traumatic brain injury and
spinal cord injury, Alzheimer's disease and Parkinson's diseaseV.
Conditions Exacerbating Stroke
[0044] A subset of stroke patients have exacerbating fever and/or
hyperglycemia, which are comorbid conditions, that in the absence
of treatment by the present methods predispose patients to a poorer
outcome than is the case for all stroke patients, particularly
stroke patients lacking such an exacerbating comorbidity.
[0045] Fever (also known as pyrexia) means an increase in internal
body temperature to a level at least 0.5.degree. C. above normal
(37.degree. C., 98.6.degree. F.). In some patients the fever is at
least 38, 39 or 40.degree. C. Fever is related to hyperthermia,
which is an acute condition resulting from an increase in body
temperature over the body's normal thermoregulatory set-point (due
to excessive heat production or insufficient thermoregulation, or
an altered thermoregulatory set point or any combination thereof).
Fever exacerbates stroke by promoting infarct formation.
[0046] Fever, as an exacerbating comorbid condition with stroke,
can result from several circumstances. Some patients have an
infection before the stroke occurs resulting in fever at the time
of the stroke, at the time of treatment, (usually 1-6 hr after the
stroke) and usually persisting at least 24 hours after treatment.
Other patients do not have fever at the onset of stroke, but
develop fever because the stroke affects an area of the brain that
controls temperature of the patient. Such a fever can develop
between onset of the stroke and initiation of treatment, and can
persist for at least 24 hours after treatment. Such a fever can
also develop after treatment has begun and persist for a period of
at least 24 hours after initiation of treatment. This type of
spontaneous fever has been associated with large strokes in humans
(Azzimondi et al., 1995; Reith et al., 1996; Ginsberg and Busto,
1998; Boysen and Christensen, 2001). Other patients have fever as a
result of being exposed to high temperatures at the time of onset
of stroke. Such fever typically persists through initiation of
treatment of the patient, but may diminish thereafter.
[0047] Hyperglycemia or high blood sugar is a condition in which an
excessive amount of glucose circulates in the blood plasma. Blood
glucose levels can be measured in either milligrams per deciliter
(mg/dL) or in millimoles per liter (mmol/L). In general, normal
fasting blood glucose levels range from about 80 to 120 mg/dL or 4
to 7 mmol/L. Fasting blood glucose levels above about 126 mg/dL or
7 mmol/L are hyperglycemicAlthough the mechanism by which
hyperglycemia exacerbates stroke is controversial, one mechanism is
the promotion of tissue acidosis (lowered pH), and/or the
activation many intracellular responses such as the activities of
protein kinases and protein phosphorylation, intracellular calcium
metabolism, and hormone metabolism including glucocorticoids.]
[0048] Hyperglycemia as an exacerbating comorbidity with stroke can
also result from several circumstances. One circumstance is
concurrent presence of diabetes, both type I and II. Although
glycemic levels can be controlled to some extent by administration
of insulin, diabetes patients are particularly vulnerable to swings
in glycemic levels. Hyperglycemia can also be the result of eating
a large meal, particularly one rich in simple carbohydrates, before
onset of a stroke.
VI. Methods of Treatment
[0049] The peptides or petidomimetics described above are used to
treat patients with stroke exacerbated by fever and/or
hyperglycemia, as described above. Treatment is usually initiated
as soon as possible after initiation of the stroke. Occasionally,
treatment can be initiated at or before onset of stroke in patients
known to be at high risk. Risk factors include hypertension,
diabetes, family history, smoking, previous stroke, and undergoing
surgery. Usually, treatment is first administered within one to six
hours after initiation of stroke. Optionally, the temperature of
the patient and/or blood glycemic level of the patient is measured
before commencing treatment to determine presence or absence of
fever and/or hyperglycemia. Presence of diabetes or other metabolic
condition disposing the subject to hyperglycemia can also be
determined. Optionally, the temperature and blood glycemic level of
the patient are monitored at several intervals or at least daily
after receiving treatment. Often a single dose of peptide or
peptidomimetic of the invention is sufficient. However, multiple
doses can also be administered at intervals of 6-24 hr.
[0050] The response of the patient to the treatment can be
monitored by determining infarction volume before and at various
times after treatment Early ischemia is detectable using MRI
diffusion imaging. Combinations of MRI protocols, including
perfusion imaging, can be used to determine tissue at risk and
predict infarction volume. The methods preferably achieve a
reduction in infarction volume of at least 10, 15, 20, 25, 30, 35,
40, or 50% relative to the mean infarction volume in a population
of comparable patients not receiving treatment by the methods of
the invention. The response of the patient can also be measured
from a disability index determined one day to one week after
initiating treatment. The patient preferably shows an improvement
(i.e., less disability) in disability index of at least 4, 10, 15,
20, 25, 30, 35, 40, or 50% relative to the mean disability index in
a population of comparable patients not receiving treatment by the
methods of the invention The patient preferably scores a zero or
one on the Rankin stroke index or over 75 on the Barthel index.
VII. Pharmaceutical Compositions, Dosages and Routes of
Administration
[0051] The peptides and peptidomimetics of the invention can be
administered in the form of a pharmaceutical composition.
Pharmaceutical compositions are manufactured under GMP conditions.
Pharmaceutical compositions can be provided in unit dosage form
(i.e., the dosage for a single administration) containing any of
the dosages indicated above. Pharmaceutical compositions can be
manufactured by means of conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping or lyophilizing processes. In particularly, lypholyized
peptides or peptidomimetics of the invention can be used in the
formulations and compositions described below.
[0052] Pharmaceutical compositions can be formulated in
conventional manner using one or more physiologically acceptable
carriers, diluents, excipients or auxiliaries that facilitate
processing of peptides or peptidomimetics into preparations which
can be used pharmaceutically. Proper formulation is dependent on
the route of administration chosen.
[0053] Administration can be parenteral, intravenous, oral,
subcutaneous, intraarterial, intracranial, intrathecal,
intraperitoneal, topical, intranasal or intramuscular. Intravenous
administration is preferred.
[0054] Pharmaceutical compositions for parenteral administration
are preferably sterile and substantially isotonic. For injection,
peptides or peptidomimetics can be formulated in aqueous solutions,
preferably in physiologically compatible buffers such as Hanks's
solution, Ringer's solution, or physiological saline or acetate
buffer (to reduce discomfort at the site of injection). The
solution can contain formulatory agents such as suspending,
stabilizing and/or dispersing agents.
[0055] Alternatively the peptides or peptidomimetics can be in
powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0056] For transmucosal administration, penetrants appropriate to
the barrier to be permeated are used in the formulation. This route
of administration can be used to deliver the compounds to the nasal
cavity or for sublingual administration.
[0057] For oral administration, the compounds can be formulated by
combining the peptides or peptidomimetics with pharmaceutically
acceptable carriers as tablets, pills, dragees, capsules, liquids,
gels, syrups, slurries, suspensions and the like, for oral
ingestion by a patient to be treated. For oral solid formulations
such as, for example, powders, capsules and tablets, suitable
excipients include fillers such as sugars, such as lactose,
sucrose, mannitol and sorbitol; cellulose preparations such as
maize starch, wheat starch, rice starch, potato starch, gelatin,
gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP);
granulating agents; and binding agents. If desired, disintegrating
agents can be added, such as the cross-linked polyvinylpyrrolidone,
agar, or alginic acid or a salt thereof such as sodium alginate. If
desired, solid dosage forms can be sugar-coated or enteric-coated
using standard techniques. For oral liquid preparations such as,
for example, suspensions, elixirs and solutions, suitable carriers,
excipients or diluents include water, glycols, oils, alcohols.
Additionally, flavoring agents, preservatives, coloring agents and
the like can be added.
[0058] In addition to the formulations described previously, the
compounds can also be formulated as a depot preparation. Such long
acting formulations can be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds can be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0059] Alternatively, other pharmaceutical delivery systems can be
employed. Liposomes and emulsions can be used to deliver peptides
and petidomimetics. Certain organic solvents such as
dimethylsulfoxide also can be employed, although usually at the
cost of greater toxicity. Additionally, the compounds can be
delivered using a sustained-release system, such as semipermeable
matrices of solid polymers containing the therapeutic agent.
[0060] Sustained-release capsules can, depending on their chemical
nature, release the peptides or peptidomimetics for a few weeks up
to over 100 days. Depending on the chemical nature and the
biological stability of the therapeutic reagent, additional
strategies for protein stabilization can be employed.
[0061] As the peptides or peptidomimetics of the invention can
contain charged side chains or termini, they can be included in any
of the above-described formulations as the free acids or bases or
as pharmaceutically acceptable salts. Pharmaceutically acceptable
salts are those salts which substantially retain the biologic
activity of the free bases and which are prepared by reaction with
inorganic acids. Pharmaceutical salts tend to be more soluble in
aqueous and other protic solvents than are the corresponding free
base forms.
[0062] The peptides or peptidomimetics of the invention are used in
an amount effective to achieve the intended purpose (e.g.,
reduction of damage effect of the damaging stroke and related
conditions). A therapeutically effective amount means an amount of
peptide or peptidomimetic sufficient to significantly reduce the
damage resulting from stroke in a population of patients (or animal
models) treated with the peptides or peptidomimetics of the
invention relative to the damage in a control population of stroke
patients (or animal models) not treated with the peptides or
peptidomimetics of the invention. The amount is also considered
therapeutically effective if an individual treated patient achieves
an outcome more favorable than the mean outcome (determined by
infarction volume or disability index) in a control population of
comparable patients not treated by methods of the invention. The
amount is also considered therapeutically effective if an
individual treated patient shows a disability of two or less on the
Rankin scale and 75 or more on the Barthel scale. A dosage is also
considered therapeutically effective if a population of treated
patients shows a significantly improved (i.e., less disability)
distribution of scores on a disability scale than a comparable
untreated population, see Lees et at 1., N Engl J Med 2006;
354:588-600A therapeutically effective regime means a combination
of a therapeutically effective dose and a frequency of
administration needed to achieve the intended purpose as described
above. Usually a single administration is sufficient.
[0063] Preferred dosage ranges include 0.001 to 20 .mu.mol peptide
or peptidomimetic per kg patient body weight, optionally 0.03 to 3
.mu.mol peptide or peptidomimetic per kg patient body weight to
.mu.mol peptide or peptidomimetic per kg patient body weight within
6 hours of stroke. In some methods, 0.1-20 .mu.mol peptide or
peptidomimetic per kg patient body weight within 6 hours are
administered. In some methods, 0.1-10 .mu.mol peptide or
peptidomimetic per kg patient body weight is administered within 6
hours, more preferably about 0.3 .mu.mol peptide or peptidomimetic
per kg patient body weight within 6 hours. In other instances, the
dosages range is from 0.005 to 0.5 .mu.mol peptide or
peptidomimetic per kg patient body weight. Dosage per kg body
weight can be converted from rats to humans by dividing by 6.2 to
compensate for different surface area to mass ratios. Dosages can
be converted from units of moles to grams by multiplying by the
molar weight of a peptide. Suitable dosages of peptides or
peptidomimetics for use in humans can include 0.001 to 5 mg/kg
patient body weight, or more preferably 0.005 to 1 mg/kg patient
body weight or 0.05 to 1 mg/kg, or 0.09 to 0.9 mg/kg. In absolute
weight for a 75 kg patient, these dosages translate to 0.075-375
mg, 0.375 to 75 mg or 3.75 mg to 75 mg or 6.7 to 67 mg. Rounded to
encompass variations in e.g., patient weight, the dosage is usually
within 0.05 to 500 mg, preferably 0.1 to 100 mg, 0.5 to 50 mg, or
1-20 mg.
[0064] The amount of peptide or peptidomimetic administered depends
on the subject being treated, on the subject's weight, the severity
of the affliction, the manner of administration and the judgment of
the prescribing physician. The therapy can be repeated
intermittently while symptoms detectable or even when they are not
detectable. The therapy can be provided alone or in combination
with other drugs.
[0065] Therapeutically effective dose of the present peptides or
peptidomimetics can provide therapeutic benefit without causing
substantial toxicity. Toxicity of the peptides or peptidomimetics
can be determined by standard pharmaceutical procedures in cell
cultures or experimental animals, e.g., by determining the
LD.sub.50 (the dose lethal to 50% of the population) or the
LD.sub.100 (the dose lethal to 100% of the population). The dose
ratio between toxic and therapeutic effect is the therapeutic
index. Peptides or peptidomimetics exhibiting high therapeutic
indices are preferred (see, e.g., Fingl et al., 1975, In: The
Pharmacological Basis of Therapeutics, Ch. 1, p. 1).
VIII. Screening Methods
[0066] The invention further provides methods of screening
peptides, peptidomimetics and other compounds for activity useful
in reducing damaging effects of stroke. The methods are
particularly useful for screening compounds known to inhibit
interactions between PSD-95 and NMDRA 2B. Compounds are
administered to an animal model of stroke, in which the animal has
fever and/or hyperglycemia at the time of administering the
compound. Fever can be induced by the ischemic event. For example,
the rats subject to permanent focal ischemia described in the
Examples spontaneously develop fever probably due to the ischemia
affecting an area of the brain affecting brain controlling
temperature regulation [Experimental animals can also be caused to
have a fever by the introduction of pyrogenic substances such as
bacterial products (endotoxins) that cause them to have a fever, or
by increasing the ambient temperature using heating lamps, heating
blankets, or other heating devices, to a degree that exceeds the
animal's ability to thermoregulate through usual physiological
mechanisms such as sweating or, vasodilation The animals can be
subject to hyperglycemia simply by feeding them within 6 or 12
hours of initiating isehemia. After administering compounds to the
animals, infarction volume and/or disability index are determined.
Infarction volumes are usually determined 24 hr post treatment but
can be determined at a later time such as 3, 7, 14 or 60 days.
Disability index can be monitored over time, e.g., at 2 hr post
treatment, 24 hr post treatment, one week and one month post
treatment. Compounds showing a statistically significant reduction
in infarction volume and/or disability index relative to control
animals not treated with the compounds are identified as having
activity useful for practicing the methods of the invention.
[0067] Compounds suitable for screening in the methods include
peptides, peptidomimetics and small molecules (i.e., less than 500
Da) known to inhibit interactions of PSD-95 and NDMAR 2B. Other
peptides, peptidomimetics and small molecules known to inhibit
interactions between other pairs of NDMAR and PDZ domain proteins
shown in Table A can also be screened.
TABLE-US-00003 TABLE A NMDA RECEPTORS WITH PL SEQUENCES C-terminal
C-terminal internal Name GI# 20 mer sequence 4 mer sequence PL? PL
ID NMDAR1 307302 HPTDITGPLNLSDPSVST STVV X AA216 VV (SEQ ID NO: 13)
(SEQ ID NO: 14) NMDAR1-1 292282 HPTDITGPLNLSDPSVST STVV X AA216 VV
(SEQ ID NO: 13) (SEQ ID NO: 14) NMDAR1-4 472845 HPTDITGPLNLSDPSVST
STVV X AA216 VV (SEQ ID NO: 13) (SEQ ID NO: 14) NMDAR1-3b 2343286
HPTDITGPLNLSDPSVST STVV X AA216 VV (SEQ ID NO: 13) (SEQ ID NO: 14)
NMDAR1-4b 2343288 HPTDITGPLNLSDPSVST STVV X AA216 VV (SEQ ID NO:
13) (SEQ ID NO: 14) NMDAR1-2 11038634 RRAIEREEGQLQLCSRH HRES RES
(SEQ ID NO: 15) (SEQ ID NO: 16) NMDAR1-3 11038636 RRAIEREEGQLQLCSRH
HRES RES (SEQ ID NO: 15) (SEQ ID NO: 16) NMDAR2C 6006004
TQGFPGPCTWRRISSLES ESEV X AA180 EV (SEQ ID NO: 16) (SEQ ID NO: 4)
NMDAR3 560546 FNGSSNGHVYEKLSSIES ESDV X AA34.1 DV (SEQ ID NO: 17)
(SEQ ID NO: 3) NMDAR3A 17530176 AVSRKTELEEYQRTSRT TCES CES (SEQ ID
NO: 18) (SEQ ID NO: 20) NMDAR2B 4099612 FNGSSNGHVYEKLSSIES ESDV X
DV (SEQ ID NO: 17) (SEQ ID NO: 3) NMDAR2A 558748 LNSCSNRRVYKKMPSIE
ESDV X AA34.2 SDV (SEQ ID NO: 19) (SEQ ID NO: 3) NMDAR2D 4504130
GGDLGTRRGSAHFSSLE ESEV X SEV (SEQ ID NO: 20) (SEQ ID NO: 4)
[0068] Compounds to be screened can be both naturally occurring and
synthetic, organic and inorganic, and including polymers (e.g.,
oligopeptides, polypeptides, oligonucleotides, and
polynucleotides), small molecules, antibodies, sugars, fatty acids,
nucleotides and nucleotide analogs, analogs of naturally occurring
structures (e.g., peptide mimetics, nucleic acid analogs, and the
like), and numerous other compounds. Compounds can be prepared from
diversity libraries, such as random or combinatorial peptide or
non-peptide libraries. Libraries include chemically synthesized
libraries, recombinant (e.g., phage display libraries), and in
vitro translation-based libraries. Examples of chemically
synthesized libraries are described in Fodor et al., 1991, Science
251:767-773; Houghten et al., 1991, Nature 354:84-86; Lam et al.,
1991, Nature 354:82-84; Medynski, 1994, Bio/Technology 12:709-710;
Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251;
Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926;
Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426;
Houghten et al., 1992, Biotechniques 13:412; Jayawickreme et al.,
1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., 1993,
Proc. Natl. Acad. Sci. USA 90:11708-11712; WO 93/20242; and Brenner
and Lerner, 1992, Proc. Natl. Acad. Sei. USA 89:5381-5383. Examples
of phage display libraries are described in Scott and Smith, 1990,
Science 249:386-390; Devlin at al., 1990, Science, 249:404-406;
Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718);
Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993,
Gene 128:59-65; WO 94/18318 dated Aug. 18, 1994. In vitro
translation-based libraries include those described in WO 91/05058;
and Mattheakis et al, 1994, Proc. Natl. Acad. Sci. USA
91:9022-9026. By way of examples of nonpeptide libraries, a
benzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl.
Acad. Sci. USA 91:4708-4712) can be adapted for use. Peptoid
libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA
89:9367-9371) can also be used. Another example of a library that
can be used, in which the amide functionalities in peptides have
been permethylated to generate a chemically transformed
combinatorial library, is described by Ostresh et al. (1994, Proc.
Natl. Acad. Sci. USA 91:11138-11142).
EXAMPLES
Materials and Methods
PSD-95 Inhibitors
[0069] Synthetic peptides (Advanced Protein Technology Centre,
Hospital for Sick Kids, Toronto, Ontario or BACHEM California),
were designed to inhibit the interactions of NMDARs with the
submembrane scaffolding protein PSD-95PSD-95 binds NMDAR NR2
subunits as well as nNOS through its second PDZ domain (PDZ2;
reviewed in Hung, 2002), thus keeping nNOS in a close functional
association with NMDARs (Brenman, 1996; Brenman, 1997). The
interaction between NMDAR NR2B subunits and the PDZ2 domain of
PSD-95 depends on a conserved C-terminus T/SXV motif of NR2B
(Kornau, 1995). This interaction can be disrupted by the
intracellular introduction of exogenous proteins that contain a 9
residue peptide sequence containing the SXV PDZ-domain binding
motif of NR2B (KLSSIESDV (SEQ ID NO.: 9); termed NR2B9c.sub.(SDV))
(Aarts, 2002). Here, an additional sequence was synthesized
containing the TXV motif (KLSSIETDV (SEQ ID NO.: 10); termed
NR2B9c.sub.(TDV)), which also interacts with the PDZ2 domain of
PSD-95 (Kornau, 1995). These peptides associate with PSD-95 through
Type-I PDZ domain interactions (Reviewed in Aarts, 2004). A control
peptide was also synthesized, in which the residues in positions 0
and -2 of the C-terminal T/SXV motif were mutated to alanines
(KLSSIEADA (SEQ ID NO.: X); termed NR2B.sub.(ADA)), rendering this
peptide incapable of binding PSD-95 (Kornau, 1995; Aarts,
2002).
[0070] NR2B9c.sub.(SDV), NR2B9c.sub.(TDV) or NR2B.sub.(ADA) on
their own are not anticipated to enter cells and therefore, each
peptide was fused to a corresponding cell-membrane protein
transduction domain (PTD) of the HIV-1-Tat protein (YGRKKRRQRRR
(SEQ ID NO.: 21); Tat) to obtain the 20 amino acid peptides
Tat-NR2B9c.sub.(SDV), Tat-NR2B9c.sub.(TDV) and Tat-NR2B.sub.(ADA).
The Tat PTD transduces cell membranes in a rapid, dose-dependent
manner (Schwarze, 1999). This approach was previously used to
successfully introduce small peptides and fusion proteins into CNS
neurons in-vitro and in-vivo (Aarts., 2002; Arundine, 2004), and
many others by now have shown that protein transduction can be used
to deliver systemically administered proteins into the brain during
and after stroke (Asoh, 2002; Borsello, 2003; Cao, 2002; Denicourt,
2003; Dietz, 2002; Eum, 2004; Kilic, 2002, 2003; Kim, 2005).
[0071] The peptides were prepared daily from lyophilized powder by
dissolving them in normal saline to the final desired
concentration. They were administered intravenously via a slow (4-5
min) injection by individuals blinded to the treatment group.
[0072] Experiments were performed in male adult Sprague-Dawley rats
weighing 250-300 g (Charles River Laboratory, Canada). All
procedures conformed to guidelines established by the Canadian
Council on Animal Care and with the approval of the University
Health Network animal care committee. All animals were housed in
groups of 1-2 animals in cages with free access to food and water
and in rooms having an ambient temperature of 20.+-.1.degree. C.
and 12:12 hr light/dark cycle.
Surgical Preparation
[0073] Each animal was weighed and then anesthesia was induced. For
the permanent pial vessel occlusion model, rats were anesthetized
with ketamine (100 mg/kg), acepromazine (2 mg/kg), and xylazine (50
mg/kg), supplemented with one third the initial dose as required.
For the two permanent middle cerebral artery occlusion (pMCAO)
studies, anesthesia was induced with 3.5% halothane in a mixture of
nitrous oxide and oxygen (Vol. 2:1) and maintained with 0.8%
halothane in the mixture. Rats were endotracheally intubated and
mechanically ventilated (60 strokes/min, tidal volume of 30-35 ml).
A rectal temperature probe was inserted. Polyethylene catheters
(PE-50) were introduced into the left femoral artery and vein for
blood pressure recording, blood sampling, and drug injection. Mean
arterial blood pressure was measured with the use of an indwelling
femoral arterial catheter connected to a pressure transducer and
was recorded continuously. Serial measurements were made of
arterial blood gases, pH, and blood glucose.
Core, Brain and Temporalis Temperature Measurements and Temperature
Control
[0074] A day prior to undergoing ischemia, the animals were
implanted with intraperitoneal transmitters (E-Mitter; Mini
Mitter/Respironics, Oregon, USA) permitting core temperature (CT)
and activity monitoring. Monitoring was performed continuously from
20 hours before, to 24 hours after, the pMCAO (Mini-Mitter
VitalView telemetric monitoring software; Mini Mitter/Respironics,
Oregon, USA). During the animal surgery the animals were away from
the telemetric receivers, so CT was measured with a rectal probe
and maintained at 36.5.degree. C. to 37.5.degree. C. using a
heating lamp or homeothermic blanket. To measure brain temperatures
following pMCAO, the right skull was exposed and a 1 mm diameter
hole drilled at the following stereotactic coordinates: from
bregma, AP 3.3 mm, ML 4.0 mm, DV 3.0 mm. A blunt-tip 19 gauge metal
cannula (10 mm length) was inserted to a depth of 5 mm and fixed in
place with dental glue. Brain temperature was recoded at 0, 1, 2, 4
and 24 h using a small thermocouple probe inserted into the metal
cannula. Temporalis muscle temperatures were measured with the same
thermocouple probe inserted into the muscle through an 18 gauge
needle. Cage temperatures were continuously recorded using an
external temperature probe and software (Tektronix WaveStar
software; Tektronix, Texas, USA) running on a digital oscilloscope
and a separate computer. In some experiments, cage temperature was
continuously adjusted based on the animal's CT using a custom-built
feedback control setup that drove a Peltier-based cooling device
(Igloo KoolMate 18; Texas, USA). The cooling device was
automatically activated whenever the animal's CT exceeded a
threshold of 37.1.degree. C.
Permanent Distal Middle Cerebral Artery Pial Vessel Occlusion
[0075] This was carried out as described elsewhere (Forder, 2005).
In Brief, the right ECA was cannulated with PE 10 polyethylene
tubing. The skull was exposed via a midline incision, and a 6- to
8-mm cranial window was made over the right somatosensory cortex (2
mm caudal and 5 mm lateral to bregma). The pial arteries were
visualized by injecting small boluses (10-20 .mu.L) of the vital
dye patent blue violet (10 mMol/L; Sigma) in normal saline, into
the ECA (FIG. 2A). The same three pial arteriolar MCA branches were
electrically cauterized and dye injections were repeated to ensure
the interruption of flow through the cauterized arterioles. The
incision was then closed and the animal returned to its cage and
allowed free access to food and water. This permanent ischemia
model produces a highly reproducible (Forder, 2005) small
infarction that is limited to the cortex underlying the coagulated
terminal pial arteries (FIG. 2A,B).
Permanent Middle Cerebral Artery Occlusion
[0076] The left middle cerebral artery was occluded by the
intraluminal suture method described by Longa (1989). In brief, the
left common carotid artery (CCA) was exposed through a midline neck
incision and was dissected free from surrounding nerves and fascia,
from its bifurcation to the base of the skull. The occipital artery
branches of the external carotid artery (ECA) were then isolated,
and these branches were dissected and coagulated. The ECA was
dissected further distally and coagulated along with the terminal
lingual and maxillary artery branches, which were then divided. The
internal carotid artery (ICA) was isolated and carefully separated
from the adjacent vagus nerve, and the pterygopalatine artery was
ligated close to its origin. The tip of a 4-cm length of 3-0
monofilament nylon suture (Harvard Apparatus) was rounded by
burning to achieve a tip diameter of 0.33-0.36 mm and tip length of
0.5-0.6 mm and coated with poly-L-lysine (Belayev at al., 1996).
The suture was introduced through the CCA and advanced into the ICA
and thence into the circle of Willis (about 18-20 mm from the
carotid bifurcation), effectively occluding the middle cerebral
artery. The silk suture around the CCA was tightened around the
intraluminal nylon suture to secure it and permanently occlude the
middle cerebral artery. Sham operated animals underwent the
identical surgical procedure, including permanent ligation of the
CCA, but without suture insertion to occlude the MCA. The pMCAO
procedure was considered to be adequate if the animal's
neurobehavioral score (below) exceeded 10 at 2 h after pMCAO.
Animals were allowed to recover from anesthesia at room
temperature.
Neurobehavioral Evaluation
[0077] Behavioral scoring was performed at 2 h and again at 24 h
after pMCAO. The battery consisted of two tests that have been used
previously (Aarts, 2002) to evaluate various aspects of
neurological function: the postural reflex test (Bederson, 1986b),
and the forelimb placing test (De Ryck, 1989). Neurological
function was graded on a scale of 0 to 12 (normal score, 0; maximal
score, 12).
Infarct Volume Evaluation.
[0078] At 24 h after pMCAO the animals were deeply anaesthetized
employing halothane inhalation and the brains were quickly removed,
sliced into 8 standard coronal sections, and incubated for 30 min
in 2% triphenyltetrazolium chloride (TTC; Sigma, St. Louis, USA) in
saline at 37.degree. C. This standard technique (Hatfield., 1991;
Bederson, 1986a; Joshi, 2004) reveals the infracted area as a pale,
unstained portion of the brain section. Each section was digitally
photographed, and the infarcts were then traced onto templates
representing the 8 standardized coronal slices. The use of the
templates corrects for any brain edema produced by the infarct,
allowing for a more accurate determination of infarct volume. Each
infarct area was then digitally traced from the templates (MOD
Version 6.0, Imaging Research Inc., St. Catharines, Ontario,
Canada) and the 8 infarct areas per brain were integrated in order
to obtain the volume.
Data Analysis
[0079] All animal surgery including drug infusions, behavioural
assessments and infarct volume determinations were performed by
individuals blinded to the treatment group. Exclusions of animals
from analysis of the pMCAO data (Results section) were based on
pre-established criteria, applied prospectively by individuals
blinded to the treatment group. The pre-established exclusion
criteria were: all deaths prior to animal sacrifice, failure to
maintain CT pre pMCAO and for 10 min post-MCAO at
37.0.+-.1.0.degree. C., failure to maintain pCO.sub.2 between 35
and 45 mmHg or mean arterial blood pressure (MABP) above 100 mm Hg
during surgery, failure of the neurobehavioral score to exceed 10
at 2 h after pMCAO, technical surgical complications, and the lack
of any basal ganglia infarct on morphological evaluation. Data are
expressed in mean.+-.S.E.M. Differences between groups were
analyzed using ANOVA followed by multiple comparisons using the
Bonferroni correction.
Results
Effects of Overnight Fasting on Blood Glucose
[0080] The fasting of animals is common practice in experimental
stroke studies (e.g., Belayev, 2005b; Aronowski, 2003; Nakashima,
1995; Kuge, 1995), and can be practiced, in large part, due to the
adverse impact of hyperglycemia on the efficacy of neuroprotective
compounds. To measure the effect of overnight fasting on blood
glucose, SD rats were either permitted access to water only
(fasting period of 16 hours; n=8; body weight 278.38.+-.14.86 g) or
toboth food and water overnight (non-fasting; n=12; body weight
276.50 g.+-.13.69 g). Blood glucose was determined in the morning.
Animals that were permitted free overnight access to both food and
water exhibited significantly higher blood glucose levels than
animals that had free overnight access to water, but not food
(5.50.+-.0.08 mMol/1 vs. 3.71.+-.0.21 mMol/l, respectively;
t.sub.18=9.134; p<0.001).
Effects of PSD-95 Inhibitors on Core Temperature
[0081] Previous experience has shown that the neuroprotective
effects of some anti-ischemic drugs might have been, at least in
part, related to drug-induced hypothermia (e.g., MK-801; Corbett,
1990). Conversely, if a drug induces hyperthermia, its protective
effects might be diminished (Noor, 2005; Memezawa, 1995). To
determine whether PSD-95 inhibition affected core temperature (CT),
it was measured in rats (n=6) implanted with telemetric
intra-peritoneal temperature monitors. CTs were measured from 20 h
before until 24 h after a single intravenous injection of 3 nMole/g
of Tat-NR2B9c.sub.(SDV), the highest PSD-95 inhibitor dose used in
the present study. Changes in CT were compared to those of animals
undergoing sham surgery (Methods) with a saline infusion (vehicle,
n=4). Animals in both groups exhibited a mild increase in CT after
termination of anesthesia (.about.0.5.degree. C. increase) which
returned to baseline within 10-15 hours. However, there were no
differences in CT at any time point between the sham (vehicle) and
peptide-infused animals (FIG. 1A,B). Transient elevations in CT
have been noted in rodents after general anesthesia (Hansen et al.,
2002; Weinandy et al., 2005), and have been attributed to
anaesthetic stress.
Effect of pMCAO on Core Temperature.
[0082] Spontaneous sustained hyperthermia is a recognized
consequence of severe ischemia following MCAO (Roberts-Lewis.,
1993; Zhao., 1994; Reglodi., 2000; Legos, 2002; Abraham, 2002,
2003), possibly due to hypothalamic injury (Zhao, 1994), or to
early microglial activation in the temperature-regulatory centers
of the hypothalamus (Abraham., 2003). To achieve consistent and
sustained hyperthermia after pMCAO, the filament used for MCA
occlusion was modified according to Abraham, who demonstrated that
the degree of post-ischemic hyperthermia and the magnitude of
cerebral infarction are related to the size of the occluding
filament (Abraham, 2002). pMCAO was induced using a filament with a
tip diameter of 0.33-0.36 mm (Methods). This caused the animals'
temperature to rise rapidly after pMCAO, peaking at
.about.39.5.degree. C. approximately 2 h after pMCAO and remaining
elevated at or above 39.degree. C. for the duration of the 24 h
observation period (FIG. 1D).
[0083] It was next determined whether the spontaneous hyperthermia
was the result of the animals' sustaining a defect in their ability
to thermoregulate. Using a feedback-controlled cage temperature
regulator (Methods) induction of normothermia was attempted in the
ischemic animals (n=8) by cooling the cage whenever their CT
exceeded a threshold of 37.1.degree. C. (FIG. 1E). The animals were
compared with a cohort which underwent pMCAO, but whose cage was
maintained at room temperature (n=8; FIG. 1D). The hyperthermic
response to pMCAO was resistant to cooling by ambient temperature
reduction, and the animals maintained a CT.gtoreq.39.degree. C.
even when the cage temperature dropped to .about.8.degree. C. (FIG.
1E). Prior studies suggest that blunting this hyperthermic response
requires extreme measures, consisting of shaving of large areas of
fur, placing the animals at 4.degree. C., and topically applying
70% alcohol (Reglodi, 2000). Thus it was concluded that the robust
and sustained hyperthermia after pMCAO was not due to the inability
of the animals to thermoregulate but rather, was due to an
alteration in the animal's temperature set-point (fever).
Effect of Pial Vessel Occlusion on Core Temperature
[0084] As the pMCAO model produced hyperthermia, a permanent
ischemia model was sought that would not alter CT so that
neuroprotection using PSD-95 inhibitors could be evaluated
independently of the hyperthermia produced by pMCAO. To this end a
pial vessel occlusion model (Methods) was used that produces a
small infarction (FIG. 2A,B) in order to not impact CT. Animals
subjected to this ischemic insult (n=5) exhibited no significant
changes in CT as compared with sham surgery animals (FIG.
1A,C).
Relationship Between Core and Brain Temperature in pMCAO.
[0085] Although brain temperature can be most directly correlated
with the extent of ischemic damage (Busto, 1987a, 1987c, 1989b;
Dietrich, 1992; Minamisawa, 1990a; Morikawa., 1992), it is unlikely
that any systemically-administered drug can affect brain
temperature independently of Core temperature. To determine whether
core temperature measurements were reflective of brain temperature
in this study, some animals that had undergone pMCAO with core
temperature monitoring also underwent temperature measurements
directly from brain and, in some experiments, from temporalis
muscle as a surrogate measure of brain temperature. Core
temperature measurements correlated to within 0.5.degree. C. with
brain and temporalis muscle temperatures in animals receiving
either saline (n=6; FIG. 1F) or 3 nMole/g of Tat-NR2B9c.sub.(SDV)
(n=6; FIG. 1F). Thus CT measurements were used for the remainder of
the study.
Exclusions from Data Analysis.
[0086] Exclusion criteria (Methods) were applied by individuals
blinded to the treatment groups. All exclusions from the pial
vessel occlusion study are detailed in Table 1. No animals were
excluded after dosing with the PSD-95 inhibitor. Exclusions from
analysis of the two pMCAO studies are listed in Table 2. In brief,
perioperative mortalities were the main reasons for excluding
animals from analysis. Overall mortality rates were 8.7% and 16.4%
for animals in the first and second independent pMCAO studies,
respectively, with no apparent relationship to the drug infusions
or identities. Necropsies revealed that mortalities were primarily
associated with subarachnoid and/or brain haemorrhages induced by
arterial perforations by the pMCAO filament.
Effect of PSD-95 Inhibitors on Infarction Volume.
[0087] In all experiments, the PSD-95 inhibitors were administered
1 h after the onset ischemia, as treatment after stroke onset
likely has the most clinical relevance.
[0088] The effects of Tat-NR2B9c.sub.(SDV) in the pial vessel
occlusion model (FIG. 2A) were first evaluated. The animals were
treated with vehicle (saline), low (0.3 nMole/g) or high (3
nMole/g) doses of the PSD-95 inhibitor at 1 h after the vessel
occlusion. As a farther control in this screening study,
Tat-NR2B9c.sub.(ADA) was used, a peptide incapable of binding
PSD-95 (Kornau, 1995; Methods), and which does not affect
excitotoxic vulnerability (Aarts, 2002; Arundine et al., 2004) or
infarct size (Aarts, 2002). Treatment of the animals with either
vehicle or Tat-NR2B9c.sub.(ADA) resulted in infarcts localized to
the cortex underlying the pial vessel occlusion, occupying about
9-10% of the hemisphere volume (FIG. 2B). Treatment of the animals
with Tat-NR2B9c.sub.(SDV) (3 nMole/g) reduced the infarcts by about
60% (FIG. 2C,D).
[0089] Next, the PSD-95 inhibitors in pMCAO was used. All of the
animals that underwent pMCAO exhibited hyperthermia to the same
degree, with CT exceeding 39.5.degree. C. in the first 8 h, and
remaining at about 39.degree. C. thereafter (FIG. 3A-E). Injection
of the PSD-95 inhibitors had no impact on the hyperthermic response
post pMCAO as there were no significant differences between the
treatment groups in either the peak or the mean temperature
elevation (ANOVA, p>0.15 for each).
[0090] Animals that had undergone pMCAO without treatment sustained
large hemispheric infarcts that, at 24 h, occupied the majority of
the cortical surface and deep structures (FIG. 4B). However,
treatment with the PSD-95 inhibitor Tat-NR2B9c.sub.(SDV) 1 h after
pMCAO (0.3-3.0 nM/g) attenuated the total infarct volume (e.g.,
FIG. 4C,D) by as much as 40% (FIG. 5A.sub.i), with the effects
being most pronounced in the cortical component of the infarct
(.about.45% cortical infarct reduction; FIG. 5A.sub.ii). The
reduction in tissue infarction was observable in all sterotactic
planes used to quantify the infarct volumes (FIGS. 4C,D.
6A.sub.i,A.sub.ii).
[0091] Neuroprotection by the Tat-NR2B9c.sub.(SDV) peptide has been
reported by us previously in-vitro (Aarts, 2002; Arundine., 2004)
and in-vivo using a model of transient, reversible MCAO (Aarts,
2002). The terminal amino acids in the -0 and -2 positions are
critical, and mutation of even one residue prevents or reduces the
association of the NR2B C-terminus with PSD-95 (Bassand, 1999).
However, it is predicted that peptides ending with the C-terminal
TDV consensus sequence should also bind similar protein targets,
including PSD-95 (Kornau, 1995, 1997; Niethammer, 1996; Bassand,
1999). If so, then they can exhibit similar neuroprotective
effects, though this has never been determined in any disease
model. To test this hypothesis, Tat-NR2B9c.sub.(TDV) was used at
0.3 mM/g and at 3.0 nM/g in the same study and under the same
conditions as Tat-NR2B9c.sub.(SDV). As shown in FIGS. 4E,F,
5A.sub.i,A.sub.ii, and 6A.sub.i,A.sub.ii, this peptide also reduced
hemispheric infarction volume by .about.35%, and cortical
infarction by 40-45%.
[0092] Next, the reproducibility of the novel findings arising from
the first study was evaluated. A second, confirmatory, study was
carried out similarly to the first. The confirmatory study focused
on replicating the effects of the lower concentration of the
Tat-NR2B9c.sub.(SDV) peptide, and the effects of the
Tat-NR2B9c.sub.(TDV) peptides. As surgical technique is a key
variable in animal stroke models, the surgery in the two
independent studies was performed by different, blinded, surgeons.
As in the first study, all peptides were administered at 1 h after
pMCAO. The team conducting the confirmatory study was blinded to
the results of the first study.
[0093] The confirmatory study yielded similar results to the first,
with both the Tat-NR2B9c.sub.(SDV) and Tat-NR2B9c.sub.(TDV)
peptides having had a similar effect on reducing both hemispheric
and cortical infarction volumes (FIGS. 5B.sub.i,B.sub.ii,
6B.sub.i,B.sub.ii).
Effect of PSD-95 Inhibitors on Cage Activity and on Neurobehavioral
Scores.
[0094] A disadvantage of the pMCAO intraluminal thread occlusion
model combined with hyperthermia and in unfasted animals is that
the experimental animals suffer from an extensive brain insult
causing severe neurological deficits (composite neurological score
>11 in untreated animals; FIG. 7A). Unlike in transient MCAO, in
which this score improves spontaneously by 24 h (Belayev., 2001;
Aarts, 2002), the untreated animals in this series of experiments
remained profoundly impaired (24 h composite neurological scores of
.about.11). The animals treated with the PSD-95 inhibitors showed a
trend towards improved neurological scores 24 h after pMCAO, but
these results did not reach statistical significance (paired
Student's t-test, P>0.05; FIG. 7A). However, telemetric
monitoring of cage activity (Colbourne, 1999; Barber, 2004)
revealed that by 24 h animals treated with the PSD-95 inhibitors
had similar levels of cage activity as compared with shams (FIG.
7B) and pre-MCAO levels (FIG. 7D-G), whereas the activity in the
untreated animals dropped off (FIG. 7C). Longer post-pMCAO recovery
times can be necessary to fully evaluate neurological recovery
after this profound type of ischemic injury. However, this was not
pursued in the present study due to concerns about the longer-term
survivability of untreated animals.
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[0194] All publications and patents cited in this specification are
incorporated by reference as if each individual publication or
patent were specifically and individually indicated to be
incorporated by reference. Further, any polypeptide sequence,
polynucleotide sequences or annotation thereof, are incorporated by
reference herein. 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.
[0195] Although the present invention has been described with
reference to the specific embodiments thereof various changes can
be made and equivalents can be substituted without departing from
the true spirit and scope of the invention. In addition, many
modifications can be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
TABLE-US-00004 TABLE 1 Mortalities and Exclusions from Pial Vessel
Occlusion Study: Animals Surgical Post-op Surgical Number Group
entered Deaths* Deaths Technique** analyzed SALINE 9 2 0 1 6
Tat-NR2B9c 9 1 0 1 7 (SDV) (0.3 nMole/g) Tat-NR2B9c 9 0 0 2 7 (SDV)
(3.0 nMole/g) Animals excluded due to surgical deaths* or due to
difficulties with surgical technique** (difficulty obtaining clean
pial cauterization) were all excluded before the drug infusion.
There were no exclusions after dosing.
TABLE-US-00005 TABLE 2 Mortalities and Exclusions from pMCAO Study
Surgeon # (1/2) excluded CT <36 because MABP <100 or
>38.degree. C. 2 h mm pre and Neuro- Hg 10 min No basal Animals
Surgical Post-op score during pCO.sub.2 <35 after ganglia Number
Group entered Deaths Deaths** <=10 surgery or >45 pMCAO
infarcts analyzed SALINE 19/11 1*/0 2/3*** 0/0 0/0 0/0 0/0 2/0 14/8
Tat-NR2B9c.sub.(SDV) 10/9 0/0 1/1 0/0 1/0 0/0 0/0 1/0 7/8 (0.3
nMole/g) Tat-NR2B9c.sub.(SDV) 9 0 1 0 0 0 0 0 8 (3.0 nMole/g)
Tat-NR2B9c.sub.(TDV) 9/10 0/0 0/2 0/0 0/0 0/0 0/0 0/0 9/8 (0.3
nMole/g) Tat-NR2B9c.sub.(TDV) 10/11 0/0 0/3 0/0 2/0 0/0 0/0 0/0 8/8
(3.0 nMole/g) Sham 4 0 0 0 0 0 0 0 4 Surgery pMCAO with 8 0 0 0 0 0
0 0 8 feedback Cooling The two numbers in each cell (X/Y) represent
animal numbers for surgeons from pMCAO study #1 and #2
respectively. *Expired before MCAO, during arterial catheterisation
**Necropsy revealed brain/subarachnoid hemorrhage likely incurred
from MCAO filament insertion. ***One animal euthanized due to
respiratory problems 5 h post pMCAO surgery. Necropsy revealed
tracheal injury related to intubation.
TABLE-US-00006 TABLE 3-1 Physiological Parameters of All Groups
from 1.sup.st Study. SDV SDV TDV TDV Groups Control 0.3 nmol/g 3
nmol/g 0.3 nmol/g 3 nmol/g Sham N 8 8 8 9 8 4 BW (g) 275.25 .+-.
7.83 291.13 .+-. 4.30 302.87 .+-. 8.22 296.44 .+-. 5.15 300.75 .+-.
4.67 291.25 .+-. 4.05 24 hrs 248.75 .+-. 8.04 250.00 .+-. 3.55
242.80 .+-. 4.12 254.63 .+-. 2.73 259.38 .+-. 4.86 277.25 .+-. 4.71
NS @ 2 hrs 11.25 .+-. 0.25 10.50 .+-. 0.27 10.88 .+-. 0.13 11.00
.+-. 0.00 11.25 .+-. 0.16 0 24 hrs 11.00 .+-. 0.33 10.37 .+-. 0.26
10.00 .+-. 0.42 10.55 .+-. 0.41 10.37 .+-. 0.42 0 MABP 126.13 .+-.
5.80 119.63 .+-. 5.14 126.75 .+-. 4.39 126.00 .+-. 5.54 123.63 .+-.
6.27 108.75 .+-. 3.88 (mmHg) 10 min 139.38 .+-. 4.88 136.75 .+-.
4.85 134.38 .+-. 7.42 121.44 .+-. 5.23 140.63 .+-. 7.64 132.25 .+-.
8.44 70 min 134.88 .+-. 3.60 139.38 .+-. 5.14 103.38 .+-. 5.06
129.44 .+-. 6.54 102.13 .+-. 2.39 120.75 .+-. 5.59 pH 7.43 .+-.
0.01 7.42 .+-. 0.01 7.44 .+-. 0.01 7.42 .+-. 0.02 7.44 .+-. 0.01
7.43 .+-. 0.01 10 min 7.42 .+-. 0.01 7.42 .+-. 0.01 7.43 .+-. 0.01
7.44 .+-. 0.01 7.43 .+-. 0.01 7.43 .+-. 0.01 70 min 7.41 .+-. 0.01
7.43 .+-. 0.01 7.41 .+-. 0.01 7.41 .+-. 0.01 7.40 .+-. 0.01 7.42
.+-. 0.01 pCO.sub.2 38.5 .+-. 1.27 37.5 .+-. 0.42 37.38 .+-. 0.78
38.11 .+-. 0.81 39.75 .+-. 0.70 39.25 .+-. 0.63 (mmHg) 10 min 40.00
.+-. 0.91 41.38 .+-. 0.98 40.75 .+-. 1.03 39.33 .+-. 0.73 40.25
.+-. 0.99 38.25 .+-. 0.25 70 min 39.88 .+-. 1.13 39.00 .+-. 1.35
37.00 .+-. 0.53 38.67 .+-. 0.93 40.38 .+-. 0.84 38.50 .+-. 1.50
pO.sub.2 142.13 .+-. 4.80 137.75 .+-. 7.18 130.63 .+-. 5.63 131.44
.+-. 9.11 130.63 .+-. 6.24 130.25 .+-. 15.39 (mmHg) 10 min 140.00
.+-. 5.83 132.38 .+-. 6.77 129.75 .+-. 4.67 124.22 .+-. 6.34 136.00
.+-. 2.57 122.00 .+-. 5.67 70 min 136.38 .+-. 4.93 139.38 .+-. 7.15
136.13 .+-. 5.28 137.67 .+-. 4.75 132.25 .+-. 4.65 125.5 .+-. 8.58
BW--body weight; NS--neurobehavior score. Mean .+-. SEM
TABLE-US-00007 TABLE 3-2 Physiological Parameters of All Groups
from 2.sup.nd Study. SDV TDV TDV Groups Control 0.3 nmol/g 0.3
nmol/g 3 nmol/g N 8 8 8 8 BW (g) 293.75 .+-. 4.07 287.75 .+-. 6.84
293.50 .+-. 2.28 296.25 .+-. 3.25 NS@2 hrs 10.75 .+-. 0.16 10.86
.+-. 0.13 10.63 .+-. 0.18 10.60 .+-. 0.24 24 hrs 10.88 .+-. 0.13
10.43 .+-. 0.28 10.63 .+-. 0.18 11.00 .+-. 0.00 MABP 116.00 .+-.
5.29 114.50 .+-. 3.76 119.00 .+-. 4.11 124.13 .+-. 6.29 (mmHg) 10
min 134.00 .+-. 8.76 134.38 .+-. 4.16 140.50 .+-. 4.69 146.63 .+-.
4012 70 min 137.25 .+-. 5.52 125.75 .+-. 4.94 129.25 .+-. 8.09
130.25 .+-. 12.26 pH 7.39 .+-. 0.01 7.39 .+-. 0.01 7.38 .+-. 0.02
7.41 .+-. 0.01 10 min 7.40 .+-. 0.01 7.41 .+-. 0.01 7.38 .+-. 0.01
7.38 .+-. 0.02 70 min 7.39 .+-. 0.01 7.41 .+-. 0.02 7.41 .+-. 0.01
7.39 .+-. 0.02 pCO.sub.2 40.13 .+-. 1.19 39.13 .+-. 1.25 39.25 .+-.
1.06 38.63 .+-. 1.05 (mmHg) 10 min 40.88 .+-. 0.93 38.63 .+-. 0.89
42.75 .+-. 0.59 37.50 .+-. 0.80 70 min 39.13 .+-. 1.25 39.50 .+-.
1.22 41.75 .+-. 1.13 40.88 .+-. 1.42 pO.sub.2 119.13 .+-. 7.83
142.75 .+-. 4.34 133.13 .+-. 5.15 141.13 .+-. 10.44 (mmHg) 10 min
121.25 .+-. 6.09 137.75 .+-. 5.55 132.00 .+-. 4.92 120.13 .+-. 6.53
70 min 120.50 .+-. 3.01 126.38 .+-. 4.79 121.00 .+-. 4.39 124.63
.+-. 4.44
Sequence CWU 1
1
2413PRTArtificialPL protein motif 1Xaa Xaa Xaa 1 24PRTArtificialPL
protein motif 2Xaa Xaa Xaa Xaa 1 34PRTArtificialPL protein motif
3Glu Ser Asp Val 1 44PRTArtificialPL protein motif 4Glu Ser Glu Val
1 54PRTArtificialPL protein motif 5Glu Thr Asp Val 1
64PRTArtificialPL protein motif 6Glu Thr Glu Val 1
74PRTArtificialPL protein motif 7Asp Thr Asp Val 1
84PRTArtificialPL protein motif 8Asp Thr Glu Val 1
99PRTArtificialPL protein motif 9Lys Leu Ser Ser Ile Glu Ser Asp
Val 1 5 109PRTArtificialPL protein motif 10Lys Leu Ser Ser Ile Glu
Thr Asp Val 1 5 1120PRTHuman immunodeficiency virusTat
Internalization Peptide 11Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg
Arg Lys Leu Ser Ser Ile 1 5 10 15 Glu Thr Asp Val 20 1220PRTHuman
immunodeficiency virusTat Internalization Peptide 12Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Lys Leu Ser Ser Ile 1 5 10 15 Glu Ser
Asp Val 20 1320PRTArtificialNMDA C-terminal 20mer sequence 13His
Pro Thr Asp Ile Thr Gly Pro Leu Asn Leu Ser Asp Pro Ser Val 1 5 10
15 Ser Thr Val Val 20 144PRTArtificialNMDA C-terminal 4mer sequence
14Ser Thr Val Val 1 1520PRTArtificialNMDA C-terminal 20mer sequence
15Arg Arg Ala Ile Glu Arg Glu Glu Gly Gln Leu Gln Leu Cys Ser Arg 1
5 10 15 His Arg Glu Ser 20 1620PRTArtificialNMDA C-terminal 20mer
sequence 16Thr Gln Gly Phe Pro Gly Pro Cys Thr Trp Arg Arg Ile Ser
Ser Leu 1 5 10 15 Glu Ser Glu Val 20 1720PRTArtificialNMDA
C-terminal 20mer sequence 17Phe Asn Gly Ser Ser Asn Gly His Val Tyr
Glu Lys Leu Ser Ser Ile 1 5 10 15 Glu Ser Asp Val 20
1820PRTArtificialNMDA C-terminal 20mer sequence 18Ala Val Ser Arg
Lys Thr Glu Leu Glu Glu Tyr Gln Arg Thr Ser Arg 1 5 10 15 Thr Cys
Glu Ser 20 1920PRTArtificialNMDA C-terminal 20mer sequence 19Leu
Asn Ser Cys Ser Asn Arg Arg Val Tyr Lys Lys Met Pro Ser Ile 1 5 10
15 Glu Ser Asp Val 20 2020PRTArtificialNMDA C-terminal 20mer
sequence 20Gly Gly Asp Leu Gly Thr Arg Arg Gly Ser Ala His Phe Ser
Ser Leu 1 5 10 15 Glu Ser Glu Val 20 2111PRTHuman immunodeficiency
virus 21Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10
229PRTArtificialPL protein motif 22Lys Leu Ser Ser Ile Glu Ala Asp
Ala 1 5 234PRTArtificialNMDA C-terminal 4mer sequence 23His Arg Glu
Ser 1 244PRTArtificialNMDA C-terminal 4mer sequence 24Thr Cys Glu
Ser 1
* * * * *