Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
COMPOSITIONS COMPRISING NEUREGULIN FOR THE PREVENTION AND
TREATMENT OF NEUROINJURY AND METHODS OF USE THEREOF
Field
[00021 The present invention relates generally to medical treatments and, in
particular, to compositions and methods for preventing and treating
neuroinjuries, such as
acute CNS injuries, with neuregulin (NRG).
-
BACKGROUND
(0003] Traumatic brain injury (TBI) is a leading cause of morbidity and death
in both
industrialized and developing countries. TBI is a major and increasing cause
of long-term
disability in individuals surviving head injuries sustained in military
combat. TBI can result
from a closed head injury or a penetrating head injury. The incidence of all
closed head
injuries admitted to hospitals is conservatively estimated to be 200 per
100,000 populations in
the United States. The incidence of penetrating head injury in the United
States is estimated
to be 12 per 100,000, the highest of any developed country in the world. Acute
neuronal
injury following TBI results in the rapid necrosis of neuronal tissue at the
site of injury
[Werner, C. et aL, Br J Anaesth, 2007. 99(1): p. 4-9]. This primary injury is
exacerbated in
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the ensuing hours and days via the progression of secondary injury
mechanism(s) leading to
significant neurological dysfunction [Xiong, Y., et al., Biochem Biophys Res
Commun, 2001.
286(2): p. 401-5; Xiong, Y., et al., dr Neurotrautna, 1998. 15(7): p. 531-44;
Sullivan, PG., et
al., Exp Neurol, 2000. 161(2): p. 631-7; Sullivan, P.G., et al., Neuroscience,
2000. 101(2): p.
289-95.] The delayed progression of deterioration of neuronal tissues gives
hope that a
clinical intervention can be applied in a realistic timeframe following the
initial trauma. TBI
is characterized by neuroinfiammatory pathological sequelae which contribute
to brain edema
and delayed neuronal cell death [Schumacher, M., et al., Pharmacol Ther, 2007.
116(1): p.
77-106; Stein, S.C., et al., Neurocrit Care, 2004. 1(4): p. 479-881 To date,
there is no
targeted pharmacological treatment that effectively limits the progression of
secondary injury.
SUMMARY
[0004] One aspect of the present invention relates to a method for preventing
or
ameliorating secondary neuronal injury and inflammation following traumatic
brain injury
(TB!). The method comprises the step of administering into a subject in need
of such
treatment an effective amount of a pharmaceutical composition containing a
NRG, a variant
of NRG, or an expression vector encoding a NRG or a variant of NRG.
[0005] In one embodiment, the method comprises administering into the subject
an
effective amount of a pharmaceutical composition containing (1) a NRG or a
variant of NRG,
and (2) an expression vector encoding a NRG or a variant of NRG.
[0006] Another aspect of the present invention relates to a method for
preventing and
treating acute CNS injuries. The method comprises the step of administering
into a subject in
need of such treatment an effective amount of a pharmaceutical composition
containing a
NRG, a variant of NRG, or an expression vector encoding a NRG or a variant of
NRG.
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[00071 Yet another aspect of the present invention relates to a kit for
preventing or
ameliorating secondary neuronal injury and inflammation following traumatic
brain injury
(TB1). The kit contains (1) a NRG, a variant of NRG, or an expression vector
encoding a
NRG or a variant of NRG, and (2) an instruction on how to use the NRG, the
variant of NRG
or the expression vector.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Figure 1 is a composite of pictures showing ErbB4 receptor expression
in
apoptotic and degenerating neurons. After ErbB4 immunohistochemistry, brain
sections from
rats MCAO were stained with Fluoro-Jade, a marker for degenerating neurons.
Many
neurons in the cortex were Fluoro-Jade-positive (A) ErbB4 positive cells
(B)
were co-localized in Fluoro-Jade-positive neurons (C) Similarly, TUNEL
staining
(D) and erbB4 (0 , were double-labeled (F) in a
subpopulation of cells in
the ipsilateral brain. Arrows indicate examples of double-labeled cells. Scale
bar is 40 fiM in
panels A-C and 20 p.M in panels D-F.
[0009] Figure 2 is a composite of pictures showing erbB4 expression in
maorophageshnicroglia but not astrocytes following MCAO. Sections from the
ipsilateral
hemisphere were double labeled with antibodies against erbB4 (panel A) and
GFAP (panel
B). Cells in the pen-infarct regions did not show co-localization of erbB4 and
GFAP (panel
C). Co-localization of erbB4 and Mac-1/CD1
lb indicated that erbB4 is found in
a subset of macrophages/microglia (panel D). Double arrows indicate examples
of double
labeled cells). Scale bar is 40 p.M in panel A-C and 20 p.M in panel D.
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[00101 Figure 3 is a composite of pictures and graphs showing that NRG1B
treatment
reduce MCAO/reperfusion-induced brain infarction. Representative 2,3,5-
triphenyltetrazolium chloride (TTC) stained brain sections are shown from rats
injected with
vehicle (panel a; n--11), NRG-1B (panel b; n=7) or NRG-1ct (panel c; n=3)
before MCAO.
Infarct volumes in brains from vehicle and NRG-1 treated animals are shown in
the graph
(panel d). Values are presented as mean SEM; * denotes significantly
different from
respective vehicle treated animals (P<0.01).
[00111 Figure 4 is a composite of pictures showing that NRG1B suppresses
MCAO/reperfusion-induced apoptotic damage in rat brain. Rats were subjected to
MCAO
for 1.5 hours followed by reperfusion for 24 hours (representative views are
shown for
TUNEL labeling of rat brain sections; n=5 for each condition). TUNEL staining
is found in
the cortex (panel a) and striatum (panel b) following MCAO while no TUNEL
staining is
seen in the cortex (panel c) and reduced levels are seen in the striatum
(panel d) in NRG113-
treated rats. The coronal brain image (--bregrna + 1.2 ram) indicates the
areas observed in the
sections (panel e). Scale bar is 100 uM.
[00121 Figure 5 is a composite of pictures and graphs showing that NRG1
treatment
reduces MCAO/reperfusion-induced brain infarction. Representative TTC stained
coronal
brain sections are shown where rats were injected with vehicle (panel a) or
NRG1
immediately after MCAO (panel b) and 4 hours after reperfusion (panel e).
Infarct volumes
in brains from rats treated with vehicle (n=10) or NRG1 immediately after MCAO
(RU; n=8),
4 hours after reperfusion (R4; n=6) or 12 hours after reperfusion (R12; n=8)
are show in the
graph (panel d). Values are presented as mean SD of all infarct volumes for
each
experimental condition; * denotes significantly different from respective
vehicle treated
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animals (P<0.01). The time line (panel e) illustrates the MCAO protocol and
NRG1
injections.
100131 Figure 6 is a graph showing that NRG1 administration resulted in a
significant
improvement in neurological outcome (* denotes P<0.01). NRG1 was administered
after
MCAO and 4 hours of reperfusion. Neurological function was graded on a scale
of 0-4
(normal score 0, maximal deficit score 4). All animals were tested prior to
surgery (controls;
n=14) and after treatment with NRG1 or vehicle. The NRGI treated group (n=9)
displayed a
33% improvement in neurological score compared with vehicle treated rats
(n=5).
100141 Figure 7 is a composite showing that NRG113 prevents microglial and
astrocytic activation following MCAO. Rats were subjected to MCAO followed by
reperfusion for 24 hours (n=5 for each condition). NRGH3 or vehicle was
injected into the
ECA. Sections were labeled for immunohistochemistry with an antibody against
ED-1.
While no staining was seen in the contralateral side (panel a), ED-1 labeled
cells are present
in the ipsilateral hemisphere (panel b) following MCAO in vehicle-treated
animals. Few ED-
1 positive cells are found in animals treated with NRG-1B (panel c). Examples
of ED-1
positive cells are indicated by the arrows. Scale bar is 50 p.M. To assess
astrocytic
activation, sections were labeled for immunohistochemistry with an antibody
against GFAP.
Compared to the contralateral control (panel a), heavy GFAP staining is found
at the border
or infarct (panel e) following MCAO in vehicle-treated animals. However, when
rats were
treated with NRG113, GFAP expression was dramatically reduced in the pen-
infarct regions
(panel 1). * denotes infarct core or the corresponding region in the
contralateral control; #
denotes non-ischernic tissues or the corresponding region in the contralateral
control. Scale
bar is 100 RM.
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[0015] Figure 8 is a composite of pictures and graphs showing that NRG1B
reduces
MCAO/reperfusion-induced IL-1B mRNA levels. Rats were treated with NRG1I3 or
vehicle
then subjected to MCAO. RNA was isolated and IL-113 mRNA expression was
measured by
RT-PCR. The expression of IL-1 (panel a) and GAPDH (panel b) mRNA is shown
(n=4 for
each condition). Panel c shows the average percentage of change SEM in IL-I
mRNA
levels from NRG-13-treated rat compared to vehicle-treated controls after
normalization to
GAPDH (* denotes P<0.05). 1= ipsilateral hemisphere; C contralateral
hemisphere.
DETAILED DESCRIPTION
[00161
[00171 The practice of the present invention will employ, unless otherwise
indicated,
conventional methods of molecular biology, cell biology, Neurology,
biochemistry and
microbiology within the skill of the art. Such techniques are explained fully
in the literature.
[00181 One aspect of the present invention relates to a method for preventing
or
ameliorating secondary neuronal injury and inflammation following traumatic
brain injury
(TBI). The method comprises the step of administering into a subject in need
of such
treatment an effective amount of a pharmaceutical composition containing a
NRG, a variant
of NRG, or an expression vector encoding a NRG or a variant of NRG.
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NRG and NRG variant
[00191 The term "neuregulin (NRG)," as used herein, refers to a family of
proteins,
including NRG1 (Entrez GeneID 3804), NRG2 (Entrez GeneID 9542), NRG3 (Entrez
GeneID 10718) and NRG4 (Entrez GeneID 145957), that are involved in the
development of
the nervous system. The term "NRG1," as used herein, also includes all NRG-1
isoforms,
including acetylcholine receptor inducing activity (ARIA), glial growth factor
(GGF),
heregulin and neu differentiation factor (NDF) [ Falls, DL., et al., Cell,
1993. 72(5): p.
801-15; Wen, D., et al., Cell, 1992. 69(3): p. 559-72.] NRG-1 isoforms are
synthesized as
transmembrane precursors consisting of either an iminunoglobulin-like or
cysteine-rich
domain, an EGF-like domain, a transmembrane domain and a cytoplasmic tail
[Fischbach, et
al., Annu Rev Neurosci, 1997. 20: p. 429-58,18, 22; Falls, DL., Exp Cell Res,
2003. 284(1):
p. 14-30; Talmage, D.A., etal., J Comp Neurol, 2004. 472(2): p. 134-9.]. NRG-1
isoforms
are generated from one gene by alternative mRNA splicing, and most of them are
synthesized
as part of a larger transmembrane precursor. The two major classes of NRG-1
include a and
isoforms. The NRG-1 3 isofonns predominate in the nervous system, while a
isoforms are
prevalent in mesenchymal cells. The r3 isoforms are 100 to 1,000 fold more
potent in
stimulating AChR synthesis in skeletal muscle and Schwann cell proliferation
[Buonanno, A.,
et al., Curr Opin Neurobiol, 2001. 11(3): p. 287-96.] The effects of NRG-1
appear to be
mediated by interaction with a class of tyrosine kinase receptors related to
the epidermal
growth factor (EGF) receptor which includes erbB2, erbB3 and erbB4 [ Burden,
S., et al.,
Neuron, 1997. 18(6): p. 847-551. The EGF-like domain of NRG-1 appears to be
sufficient
for activation of erbB receptors and downstream signal transduction pathways
[Holmes,
W.E., et al., Science, 1992. 256(5060): p. 1205-10.]. NRG-1 stimulates the
tyrosine
phosphorylation of these receptors and the subsequent activation of various
signal
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transduction mechanisms including Map kinase, P13 kinase and CDK5 Fu, A.K., et
al., Nat
Neurosci, 2001.4(4): p. 374-81; Alroy, 1., et al., FEBS Lett, 1997.410(1): p.
83-6.
[00201 Neuregulin 2 (NRG2) is a novel member of the neuregulin family of
growth
and differentiation factors. Through interaction with the ErbB family of
receptors, NRG2
induces the growth and differentiation of epithelial, neuronal, glial, and
other types of cells.
The gene consists of 12 exons and the genomic structure is similar to that of
neuregulin 1
(NRG1). NRG1 and NRG2 mediate distinct biological processes by acting at
different sites in
tissues and eliciting different biological responses in cells. The NRG2 gene
is located close to
the region for demyelinating Charcot-Marie-Tooth disease locus, but is not
responsible for
this disease. Alternative transcripts encoding distinct isoforms have been
described. (Chang H
et al. Nature (1997) 387: 509-12; Can-away KL et al.(1997) Nature 387: 512-6).
[00211 Neuregulin 3 (NRG3) binds to the extracellular domain of the ERBB4
receptor
tyrosine kinase but not to the related family members ERBB2 or ERBB3. NRG3
binding
stimulates tyrosine phosphorylation of ERBB4. Variants of the NRG3 gene have
been linked
to a susceptibility to schizophrenia(Zhang D, et al. Proc. Natl. Acad. Sci.
U.S.A. (1997) 94:
9562-7; Chen PL et al. Am. J. Hum. Genet. (2009) 84: 21-34).
[00221 Neuregulin 4 (NRG4) activates type-1 growth factor receptors (EGFR) to
initiating cell-to-cell signaling through tyrosine phosphorylation. Loss of
expression of
NRG4 is frequently seen in advanced bladder cancer while increased NRG4
expression
correlates to better survival(Harari D et al. Oncogene (1999) 18: 2681-9;
Memon AA et al.,
Br. J. Cancer (2004) 91: 2034-41).
NO231 As used herein, a "variant of a NRG" is a polypeptide that differs from
a native
NRG polypeptide in one or more substitutions, deletions, additions and/or
insertions, such
that the bioactivity or immunogenicity of the native NRG polypeptide is not
substantially
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diminished. In other words, the bioactivity of a variant NRG polypeptide may
be enhanced or
diminished by less than 50%, and preferably less than 20%, relative to the
native NRG
polypeptide. Variant NRG polypeptides include those in which one or more
portions, such as
an N-terminal leader sequence or transmembrane domain, have been removed.
Other
preferred variants include variants in which a small portion (e.g., 1-30 amino
acids, preferably
5-15 amino acids) has been removed from the N- and/or C-terminal of the mature
protein.
[00241 Modifications and changes can be made in the structure of a NRG
polypeptide
and still obtain a molecule having biological activity and/or immunogenic
properties.
Because it is the interactive capacity and nature of a NRG polypeptide that
defines that
polypeptide's biological activity, certain amino acid sequence substitutions
can be made in a
NRG polypeptide sequence (or, of course, its underlying DNA coding sequence)
and
nevertheless obtain a polypeptide with like properties.
[00251 In making such changes, the hydropathic index of amino acids can be
considered. The importance of the hydropathic amino acid index in conferring
interactive
biologic function on a polypeptide is generally understood in the art. It is
believed that the
relative hydropathic character of the amino acid residue determines the
secondary and tertiary
structure of the resultant polypeptide, which in turn defines the interaction
of the polypeptide
with other molecules, such as enzymes, substrates, receptors, antibodies,
antigens, and the
like. It is known in the art that an amino acid can be substituted by another
amino acid
having a similar hydropathic index and still obtain a functionally equivalent
polypeptide. In
such changes, the substitution of amino acids whose hydropathic indices are
within +/-2 is
preferred, those that are within +1-1 are particularly preferred, and those
within +/-0.5 are
even more particularly preferred.
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[0026] Substitution of like amino acids can also be made on the basis of
hydrophilicity, particularly where the biological functional equivalent
polypeptide or
polypeptide fragment, is intended for use in immunological embodiments. U.S.
Patent
4,554,101, incorporated hereinafter by reference, states that the greatest
local average
hydrophilicity of a polypeptide, as governed by the hydrophilicity of its
adjacent amino acids,
correlates with its immunogenicity and antigenicity, i.e. with a biological
property of the
polypeptide.
[0027] As detailed in U.S. Patent 4,554,101, the following hydrophilicity
values have
been assigned to amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0 1);
glutamate (+3.0 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); proline
(-0.5 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-
1.0); methionine (-1.3);
valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan
(-3.4). It is understood that an amino acid can be substituted for another
having a similar
hydrophilicity value and still obtain a biologically equivalent, and in
particular, an
immunologically equivalent polypeptide. In such changes, the substitution of
amino acids
whose hydrophilicity values are within 2 is preferred, those that are within
1 are
particularly preferred, and those within - 0.5 are even more particularly
preferred.
100281 As outlined above, amino acid substitutions are generally therefore
based on
the relative similarity of the amino acid side-chain substituents, for
example, their
hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary
substitutions which take
various of the foregoing characteristics into consideration are well known to
those of skill in
the art and include: arginine and lysine; glutamate and aspartate; serine and
threonine;
glutamine and asparagine; and valine, leucine and isoleucine (See Table 1,
below). The
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present invention thus contemplates functional or biological equivalents of a
NRG as set forth
above.
TABLE 1 Amino Acid Substitutions
Original Exemplary Residue
Residue Substitution
Ala Gly; Ser
Arg Lys
Asn Gin; His
Asp Giu
Cys Ser
Gln Asn
Glu Asp
Gly Ala
His Asn; Gin
Ile Leu; Val
Leu Ile; Val
Lys Arg
Met . Lett; Tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr Tip; Phe
Val lie; Lett
[00291 A NRG variant may also, or alternatively, contain nonconservative
changes.
In a preferred embodiment, variant NRG polypeptides differ from a native NRG
sequence by
substitution, deletion or addition of five amino acids or fewer. NRG variants
may also (or
alternatively) be modified by, for example, the deletion or addition of amino
acids that have
minimal influence on the immunogenicity, secondary structure, tertiary
structure, and
hydropathic nature of the native NRG polypeptide.
[00301 NRG variants preferably exhibit at least about 70%, more preferably at
least
about 90% and most preferably at least about 95% sequence homology to the
original NRG
polypeptide.
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[0031.] NRG variant also includes a polypeptide that is modified from the
original
NRG polypeptide by either natural process, such as post-translational
processing, or by
chemical modification techniques which are well known in the art.
Modifications can occur
anywhere in a polypeptide, including the peptide backbone, the amino acid side-
chains and
the amino or carboxyl termini. It will be appreciated that the same type of
modification may
be present in the same or varying degrees at several sites in a given
polypeptide. Also, a
given polypeptide may contain many types of modifications. Polypeptides may be
branched,
for example, as a result of ubiquitination, and they may be cyclic, with or
without branching.
Cyclic, branched, and branched cyclic polypeptides may result from post-
translation natural
processes or may be made by synthetic methods. Modifications include
acetylation,
acylation, ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of
a fluorophore or a chromophore, covalent attachment of a heme moiety, covalent
attachment
of a nucleotide or nucleotide derivative, covalent attachment of a lipid or
lipid derivative,
covalent attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond
formation, demethylation, formation of covalent cross-links, formation of
cysteine, formation
of pyroglutamate, fonnylation, gamma-carboxylation, glycosylation, GPI anchor
formation,
hydroxylation, iodination, rnethylation, myristoylation, oxidation,
pegylation, proteolytic
processing, phosphorylation, prenylation, racemization, selenoylation,
sulfation, transfer-
RNA mediated addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0032] The term "NRG variant" also includes fusion proteins containing a NRG-
related polypeptide and a non-NRG-related polypeptide. Within a fusion
protein, the NRG-
related polypeptide can correspond to all or a portion of a NRG. In a
preferred embodiment,
a fusion NRG comprises at least one biologically active portion of a NRG.
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[00331 As used herein, a "biologically active portion" of a NRG includes a
fragment
of a NRG comprising amino acid sequences sufficiently homologous to or derived
from the
amino acid sequence of the NRG, which includes fewer amino acids than the full
length
NRG, and exhibits at least one biological activity of the NRG. Typically, a
biologically
active portion of a NRG comprises a domain or motif with at least one activity
of the NRG.
A biologically active portion of a NRG can be a polypeptide, which is, for
example, 10, 25,
50, 100, 200 or more amino acids in length.
[0034] Within the fusion protein, the NRG-related polypeptide and the non-NRG-
related polypeptide are fused in-frame to each other. The non-NRG-related
polypeptide can
be fused to the N-terminus or C-terminus of the NRG-related polypeptide. A
peptide linker
sequence may be employed to separate the NRG-related polypeptide from non-NRG-
related
polypeptide components by a distance sufficient to ensure that each
polypeptide folds into its
secondary and tertiary structures. Such a peptide linker sequence is
incorporated into the
fusion protein using standard techniques well known in the art, Suitable
peptide linker
sequences may be chosen based on the following factors: (1) their ability to
adopt a flexible
extended conformation; (2) their inability to adopt a secondary structure that
could interact
with functional epitopes on the NRG-related polypeptide and non-NRG-related
polypeptide;
and (3) the lack of hydrophobic or charged residues that might react with the
polypeptide
functional epitopes. Preferred peptide linker sequences contain gly, asn and
ser residues.
Other near neutral amino acids, such as thr and ala may also be used in the
linker sequence.
Amino acid sequences which may be used as linkers are well known in the art.
The linker
sequence may generally be from 1 to about 50 amino acids in length. Linker
sequences are
not required when the NRG-related polypeptide and non-NRG-related polypeptide
have non-
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essential N-terminal amino acid regions that can be used to separate the
functional domains
and prevent steric interference.
Expression Vectors
[0035] The expression vectors of the present invention include plasmid vectors
and
viral vectors. The plasmid vectors typically include a circular double-
stranded DNA loop into
which additional DNA segments can be ligated. The plasmid vectors of the
invention
comprise one or more regulatory sequences operably linked to a polynucleotide
encoding a
NRG or NRG variant in a form suitable for expression of the polynucleotide in
a target cell.
[0036] As used herein, the term "regulatory sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence in a
particular host
organism. The term "regulatory sequence" is intended to include promoters,
enhancers and
other expression control elements (e.g., polyadenylation signals). Regulatory
sequences
include those which direct constitutive expression of a nucleotide sequence in
many types of
host cells and those which direct expression of the nucleotide sequence only
in certain host
cells (e.g., tissue-specific regulatory sequences).
[0037] A nucleic acid sequence is "operably linked" to another nucleic acid
sequence
when the former is placed into a functional relationship with the latter. For
example, a DNA
for a presequence or secretory leader peptide is operably linked to DNA for a
polypeptide if it
is expressed as a preprotein that participates in the secretion of the
polypeptide; a promoter or
enhancer is operably linked to a coding sequence if it affects the
transcription of the
sequence; or a ribosome binding site is operably linked to a coding sequence
if it is positioned
so as to facilitate translation. Generally, "operably linked" means that the
DNA sequences
being linked are contiguous and, in the case of a secretory leader, contiguous
and in reading
phase. However, enhancers do not have to be contiguous. Linking is
accomplished by
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ligation at convenient restriction sites. If such sites do not exist,
synthetic oligonucleotide
adaptors or linkers are used in accordance with conventional practice.
100381 It will be appreciated by those skilled in the art that the design of
the
expression vector can depend on such factors as the type of target cell, the
level of expression
of protein desired, and the like. The expression vectors of the invention can
be introduced
into the target cells to thereby produce proteins or peptides, such as NRGs
and NRG variants.
[0039] In one embodiment, the expression vector of the invention is a
mammalian
expression vector. Examples of mammalian expression vectors include pCDM8 and
pMT2PC, When used in mammalian cells, the expression vector's control
functions are often
provided by viral regulatory elements. For example, commonly used promoters
are derived
from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40.
[0040] In another embodiment, the mammalian expression vector is capable of
directing expression of the polynucleotide preferentially in a particular cell
type (e.g.,
neurons) using tissue-specific regulatory elements. Examples of neuron-
specific promoters
(e.g., the neurofila.ment promoter)
[0041] A number of methods have been developed to deliver the plasmid vector
to the
target cells. Examples of the delivery methods include, but are not limited
to, liposomes-
mediated gene transfer, polycationic condensed DNA linked or unlinked to
killed
adenovirus, ligand linked DNA, eukaryotic cell delivery vehicles cells,
deposition of
photopolymerized hydrogel materials, handheld gene transfer particle gun,
ionizing radiation,
nucleic charge neutralization or fusion with cell membranes.
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100421 The viral vectors include, but are not limited to, retroviral vector,
lentiviral
vectors, adenoviral vectors, adeno-associated viral (AAV)vectors, herpes viral
vectors, and
alphavirus vectors. The viral vector can also be an astrovirus, coronavirus,
orthomyxovirus,
papovavinis, paramrKovirus, parvovirus, picornavirus, poxvirus, or togavirus
viral vector.
100431 In certain embodiment, a regulatable expression system is employed to
control
the level and duration of NRG expression from an expression vector. These
systems are
briefly described below:
[0044] Tet-on/off system. The Tet-system is based on two regulatory elements
derived
from the tetracycline-resistance operon of the E. coli Tn10 transposon: the
tet repressor
protein (TetR) and the Tet operator DNA sequence (tet0) to which TetR binds.
The system
consists of two components, a "regulator" and a "reporter" plasmid. The
"regulator" plasmid
encodes a hybrid protein containing a mutated let repressor (rtetR) fused to
the VP16
activation domain of herpes simplex virus. The "reporter" plasmid contains a
tet-responsive
element (TRE), which controls the "reporter gene of choice. The rtetR-VP16
fusion protein
can only bind to the TRE, therefore activates the transcription of the
"reporter" gene, in the
presence of tetracycline. The system has been incorporated into a number of
viral vectors
including retrovirus, adenovirus and AAV.
[00451 Ecdysone system. The ecdysone system is based on the molting induction
system found in Drosophila, but modified for inducible expression in mammalian
cells. The
system uses an analog of the drosophila steroid hormone ecdysone, muristerone
A, to activate
expression of the gene of interest via a heterodimeric nuclear receptor.
Expression levels
have been reported to exceed 200-fold over basal levels with no effect on
mammalian cell
physiology.
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[0046] Progesterone system. The progesterone receptor is normally stimulated
to bind
to a specific DNA sequence and to activate transcription through an
interaction with its
hormone ligand. Conversely, the progesterone antagonist mifepristone (RU486)
is able to
block hormone-induced nuclear transport and subsequent DNA binding. A mutant
form of
the progesterone receptor that can be stimulated to bind through an
interaction with RU486
has been generated. To generate a specific, regulatable transcription factor,
the RU486..
binding domain of the progesterone receptor has been fused to the DNA-binding
domain of
the yeast transcription factor GALA and the transactivation domain of the HSV
protein VP16.
The chimeric factor is inactive in the absence of RU486. The addition of
hormone, however,
induces a conformational change in the chimeric protein, and this change
allows binding to a
GAL4-binding site and the activation of transcription from promoters
containing the GAL4-
binding site.
[0047] Rapamycin system. Irnmunosuppressive agents, such as FK506 and
rapamycin, act by binding to specific cellular proteins and facilitating their
dimerization. For
example, the binding of rapamycin to FK506-binding protein (FKBP) results in
its
heterodimerization with another rapamycin binding protein FRAP, which can be
reversed by
removal of the drug. The ability to bring two proteins together by addition of
a drug
potentiates the regulation of a number of biological processes, including
transcription. A
chimeric DNA-binding domain has been fused to the FKBP, which enables binding
of the
fusion protein to a specific DNA-binding sequence. A transcriptional
activation domain also
has been fused to FRAP. When these two fusion proteins are co-expressed in the
same cell, a
fully functional transcription factor can be formed by heterodimerization
mediated by
addition of rapamycin. The dimerized chimeric transcription factor can then
bind to a
synthetic promoter sequence containing copies of the synthetic DNA-binding
sequence. This
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system has been successfully integrated into adenoviral and AAV vectors. Long-
term
regulatable gene expression has been achieved in both mice and baboons.
[0048] The plasrnid and viral expression vectors may be used to provide long-
term in
vivo expression (weeks and even months) of NRG or NRG variant.
Pharmaceutical Compositions
[0049] The pharmaceutical composition of the present invention contains one or
more
NRGs, NRG variants, expression vectors encoding NRGs or NRG variants, or
combinations
thereof. In certain embodiments, the pharmaceutical composition further
contains a
pharmaceutically acceptable carrier.
[00501 As used herein the language "pharmaceutically acceptable carrier" is
intended
to include any and all solvents, solubilizers, fillers, stabilizers, binders,
absorbents, bases,
buffering agents, lubricants, controlled release vehicles, diluents,
emulsifying agents,
humectants, lubricants, dispersion media, coatings, antibacterial or
antifungal agents, isotonic
and absorption delaying agents, and the like, cOmpatible with pharmaceutical
administration.
The use of such media and agents for pharmaceutically active substances is
well-known in the
art. Except insofar as any conventional media or agent is incompatible with
the active
compound, use thereof in the compositions is contemplated. Supplementary
agents can also
be incorporated into the compositions.
100511 The another aspect of the invention includes methods for preparing
pharmaceutical compositions for modulating the expression or activity of a
polypeptide or
polynucleotide corresponding to an NRG or NRG variant of the invention. Such
methods
comprise formulating a pharmaceutically acceptable carrier with an agent which
modulates
expression or activity of a NRG or NRG variant. Such compositions can further
include
additional active agents. Thus, the invention further includes methods for
preparing a
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pharmaceutical composition by formulating a pharmaceutically acceptable
carrier with an
agent, which modulates expression or activity of a NRG or NRG variant and one
or more
additional bioactive agents.
[00521 A pharmaceutical composition of the invention is formulated to be
compatible
with its intended route of administration. Examples of routes of
administration include
parenteral, e.g., intravenous, intraarterial, intramuscular, intradermal,
subcutaneous, oral (e.g.,
inhalation), transdermal (topical), and transmucosal administration. Solutions
or suspensions
used for parenteral, intraderrnal, or subcutaneous application can include the
following
components: a sterile diluent such as water for injection, saline solution,
fixed oils,
polyethylene glycols, glycerine; propylene glycol or other synthetic solvents;
antibacterial
agents such as benzyl alcohol or methyl parabens; antioxidants such as
ascorbic acid or
sodium bisulfate; ehelating agents such as ethylenediaminetetraacetic acid;
buffers such as
acetates, citrates or phosphates and agents for the adjustment of tonicity
such as sodium
chloride or dextrose. pH can be adjusted with acids or bases, such as
hydrochloric acid or
sodium hydroxide. The parenteral preparation can be enclosed in ampoules,
disposable
syringes or multiple dose vials made of glass or plastic.
[00531 Pharmaceutical compositions suitable for injectable use include sterile
aqueous solutions or dispersions and sterile powders for the extemporaneous
preparation of
sterile injectable solutions or dispersion. For intravenous administration,
suitable carriers
include physiological saline, bacteriostatic water, Cremophor ELTM (BASF,
Parsippany, NJ)
or phosphate buffered saline (PBS). In all cases, the injectable composition
should be sterile
and should be fluid to the extent that easy syringability exists. It must be
stable under the
conditions of manufacture and storage and must be preserved against the
contaminating
action of microorganisms such as bacteria and fungi. The proper fluidity can
be maintained,
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for example, by the use of a coating such as lecithin, by the maintenance of
the requited
particle size in the case of dispersion and by the use of surfactants.
Prevention of the action
of microorganisms can be achieved by various antibacterial and antifungal
agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the
like. In many
cases, it will be preferable to include isotonic agents, for example, sugars,
polyalcohols such
as manitol, sorbitol and sodium chloride in the composition. Prolonged
absorption of the
injectable compositions can be brought about by including in the composition
an agent which
delays absorption, for example, aluminum mono stearate and gelatin.
[0054] Sterile injectable solutions can be prepared by incorporating the
active
compound (e.g., a NRG or NRG variant) in the required amount in an appropriate
solvent
with one or a combination of ingredients enumerated above, as required,
followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the active
compound into a
sterile vehicle which contains a basic dispersion medium and the required
other ingredients
from those enumerated above. In the case of sterile powders for the
preparation of sterile
injectable solutions, the preferred methods of preparation are vacuum drying
and freeze-
drying which yields a powder of the active ingredient plus any additional
desired ingredient
from a previously sterile-filtered solution thereof.
[0055] Oral compositions generally include an inert diluent or an edible
carrier. They
can be enclosed in gelatin capsules or compressed into tablets. For the
purpose of oral
therapeutic administration, the active compound can be incorporated with
excipients and used
in the form of tablets, troches, or capsules. Oral compositions can also be
prepared using a
fluid carrier for use as a mouthwash, wherein the compound in the fluid
carrier is applied
orally and swished and expectorated or swallowed. Pharmaceutically compatible
binding
agents, and/or adjuvant materials can be included as part of the composition.
The tablets,
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pills, capsules, troches and the like can contain any of the following
ingredients, or
compounds of a similar nature: a binder such as microcrystalline cellulose,
gum tragacanth or
gelatin; an excipient such as starch or lactose; a disintegrating agent such
as alginic acid,
Primogel, or corn starch; a lubricant such as magnesium stearate or Stertes; a
glidant such as
colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or
a flavoring
agent such as peppermint, methyl salicylate, or orange flavoring.
[0056] For administration by inhalation, the compounds are delivered in the
form of
an aerosol spray from a pressured container or dispenser, which contains a
suitable propellant,
e.g., a gas such as carbon dioxide, or a nebulizer.
[00571 Systemic administration can also be by transnmcosal or transdennal
means.
For transmucosal or transdennal administration, penetrants appropriate to the
barrier to be
peimeated are used in the formulation. Such penetrants are generally known in
the art, and
include, for example, for transmucosal administration, detergents, bile salts,
and fusidic acid
derivatives. Transmucosal administration can be accomplished through the use
of nasal
sprays or suppositories. For transdennal administration, the bioactive
compounds are
formulated into ointments, salves, gels, or creams as generally known in the
art.
[0058] In one embodiment, the therapeutic moieties, which may contain a
bioactive
compound, are prepared with carriers that will protect the compound against
rapid
elimination from the body, such as a controlled release formulation, including
implants and
microencapsulated delivery systems. Biodegradable, biocompatible polymers can
be used,
such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters,
and polylactic acid. Methods for preparation of such formulations will be
apparent to those
skilled in the art. The materials can also be obtained commercially. Liposomal
suspensions
(including Liposomes targeted to infected cells with monoclonal antibodies to
viral antigens)
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can also be used as pharmaceutically acceptable carriers. These can be
prepared according to
methods known to those skilled in the art.
100591 It is especially advantageous to formulate oral or parenteral
compositions in
dosage unit form for ease of administration and uniformity of dosage. Dosage
unit form, as
used herein, includes physically discrete units suited as unitary dosages for
the subject to be
treated; each unit contains a predetermined quantity of active compound
calculated to
produce the desired therapeutic effect in association with the required
pharmaceutical carrier.
The specification for the dosage unit forms of the invention are dictated by
and directly
dependent on the unique characteristics of the active compound and the
particular therapeutic
effect to be achieved, and the limitations inherent in the art of compounding
such an active
compound for the treatment of individuals.
[0060] Toxicity and therapeutic efficacy of the active ingredient (e.g., a NRG
or NRG
variant) in the pharmaceutical composition can be determined by standard
pharmaceutical
procedures in cell cultures or experimental animals, e.g., for determining the
LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose therapeutically
effective in 50% of
the population). The dose ratio between toxic and therapeutic effects is the
therapeutic index
and it can be expressed as the ratio LD50/ED50. Active ingredients which
exhibit large
therapeutic indices are preferred. While active ingredients that exhibit toxic
side effects may
be used, care should be taken to design a delivery system that targets such
active ingredients
to the site of affected tissue in order to minimize potential damage to
uninfected cells and,
thereby, reduce side effects.
100611 The data obtained from the cell culture assays and animal studies can
be used
in formulating a range of dosage for use in humans. The dosage of such
compounds lies
preferably within a range of circulating concentrations that includes the ED50
with little or no
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toxicity. The dosage may vary within this range depending upon the dosage form
employed
and the route of administration utilized. For any compound used in the method
of the
invention, the therapeutically effective dose can be estimated initially from
cell culture
assays. A dose may be formulated in animal models to achieve a circulating
plasma
concentration range that includes the IC50 (i.e., the concentration of the
test compound which
achieves a half-maximal inhibition of symptoms) as determined in cell culture.
Such
information can be used to more accurately determine useful doses in humans.
Levels in
plasma may be measured, for example, by high performance liquid
chromatography.
[0062] The plasmid and viral vectors of the present invention can be delivered
to a
subject by, for example, intravenous administration, intraportal
administration, intrabiliary
administration, intra-arterial administration, direct injection into the liver
parenchyma, by
intramusclular injection, by inhalation, by perfusion, or by stereotactic
injection. The
pharmaceutical preparation of the plasmid and viral vectors can include an
acceptable diluent,
or can comprise a slow release matrix in which the plasmid and viral vectors
are imbedded.
Alternatively, where the viral vector can be produced intact from recombinant
cells, e.g.,
retroviral vectors, the pharmaceutical preparation can include one or more
cells which
produce the gene delivery system.
[0063] The pharmaceutical compositions can be included in a container, pack,
or
dispenser together with instructions for administration.
[0064] In a preferred embodiment, the method for preventing or ameliorating
secondary neuronal injury and inflammation following TBI comprises
administering into a
subject in need of such treatment an effective amount of a pharmaceutical
composition
containing NRG I. In one embodiment, NRG1 is administered within 24 hours of
the TBI. In
another embodiment, NRG1 is administered intravascularly or intramuscularly at
doses
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between 0.05-5000 ug/kg body weight; preferably, 0.5-1000 ug/kg, more
preferably, 1-500
ug/kg, most preferably, 5-100 ug/kg. In another embodiment, NRG1 is
administered daily for
a period of 3-14 days.
[0065] hi another embodiment, the method comprises administering into the
subject
an effective amount of a phaunaceutical composition containing (1) a NRG or a
variant of
NRG, and (2) an expression vector encoding a NRG or a variant of NRG. The NRG
or
variant of NRG provides short-term effect in preventing or ameliorating
secondary neuronal
injury and inflammation following TBI. The expression vector expresses the NRG
or variant
of NRG in vivo and provides long-term effect in preventing or ameliorating
secondary
neuronal injury and inflammation following TBI. The NRG and NRG expressing
vector may
be injected concurrently or separately.
[0066) Another aspect of the present invention relates to a method for
preventing or
treating acute CNS injuries. The method comprises the step of administering
into a subject in
need of such treatment an effective amount of a pharmaceutical composition
containing a
NRG, a variant of NRG, or an expression vector encoding a NRG or a variant of
NRG.
[0067] In a preferred embodiment, the method for preventing or treating acute
CNS
injuries comprises the step of administering into a subject in need of such
treatment an
effective amount of a phainiaceutical composition containing NRG1. In one
embodiment,
NRG1 is administered within 24 hours of the acute CNS injury. In another
embodiment,
NRG1 is administered intravascularly or intramuscularly at doses between 0.05-
5000 ug/kg
body weight; preferably, 0.5-1000 ug/kg, more preferably, 1-500 ug/kg, most
preferably, 5-
100 ug/kg. In another embodiment, NRGI is administered daily for a period of 3-
14 days.
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Kits
[0068] The invention also encompasses kits for preventing and treating acute
CNS
injuries, and kits for preventing or amelioratring secondary neuronal injury
and inflammation
following traumatic brain injury (TBD. The kits comprise one or more effective
doses of a
NRG, an NRG variant, an expression vector encoding a NRG or NRG variant, or
combinations thereof along with a label or labeling with instructions on using
the NRG, NRG
variant, or expression vector to prevent or ameliorate secondary neuronal
injury and
inflammation following 'FBI according to the methods of the invention. In
certain
embodiments, the kits can comprise components useful for carrying out the
methods such as
devices for delivering the NRG, NRG variant, or expression vector. In certain
embodiments,
the kit can comprise components useful for the safe disposal of devices for
delivering the
NRG, NRG variant, or expression vector, e.g., a sharps container for used
syringes.
[0069] In one embodiment, the NRG, NRG variant, or expression vector in the
kit is
formulated for intravascular administration. In another embodiment, the NRG,
NRG variant,
or expression vector in the kit is formulated for intramusclular
administration. In another
embodiment, the NRG, NRG variant, or expression vector in the kit is
formulated for
subcutaneous administration. The NRG, NRG variant, or expression vector may be
formulated for slow release. In one embodiment, the NRG, NRG variant, or
expression
vector is embedded in an implantable inert matrix.
[00701 The present invention is further illustrated by the following examples
which
should not be construed as limiting.
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EXAMPLE 1: ErbB4 Receptors Are Expressed Anoptotic and Degenerating Neurons
_
[00711 After erB4 immunohistochemistry, brain sections harvested from rats
subjected to middle cerebral artery occlusion (MCAO) were stained with Fluoro-
Jade, a
marker for degenerating neurons, and with antibodies against erbB4. As shown
in :Figure 1,
many neurons in the cortex were Fluoro-Jade-positive (panel A) ErbB4
positive cells
(panel B) were co-localized in Fluoro-Jade-positive neurons,(panel c4,
Similarly, TUNEL staining (panel D) 'and erbB4 (panel
E). were double-labeled
(panel F) in a subpopulation of cells in the ipsilateral brain.
EXAMPLE 2: ErbB4 Expression Is Upregulated In Macrophages/Microglia But Not In
Astrocytes Following MCAO
100721 Sections from the ipsilateral hemisphere of rats subjected to MCAO were
double labeled with antibodies against erbB4 (Figure 2, panel A) and glial
fibrillary acidic
protein (GFAP) (Figure 2, panel B). Cells in the pen-infarct regions did not
show co-
localization of erbB4 and GFAP (Figure 2, panel C). Co-localization of erbB4
and
Mac-1/CD11 b indicated that erbB4 is found in a subset of
macrophages/microglia
(Figure 2, panel D).
EXAMPLE 3: NRG-113 Treatment Reduces IvICAO/Reperfusion-Induced Brain
Infarction
100731 Figure 3 shows representative 'TTC stained brain sections from rats
injected
with vehicle (panel a; n=11), NRG113 (panel b; n=7) or NRGla (panel c; n=3)
before MCAO.
NRG1B (2.5 ug/kg) or NRGla (2.5 jig/kg) was given by a single intra-arterial
injection
immediately before MCAO. Adult male Sprague-Dawley rats weighing 250-300 g
were used
for this study. A total of 164 rats were used in this study. Rats were
anesthetized with a
ketamine/xylazine solution (10mg/kg, IP) and subjected to left MCAO. MCAO was
induced
by the intraluminal suture method where the left common carotid artery (CCA)
was exposed
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through a midline incision and was carefully dissected free from surrounding
nerves and
fascia. The occipital artery branches of the external carotid artery (ECA)
were then isolated,
and the occipital artery and superior thyroid artery branches of the ECA were
coagulated. The
ECA was dissected further distally. 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 with a 6-0 silk suture. Then, a 40 mm 3-0 surgical
monofilament nylon
suture (Harvard Apparatus, Holliston, Massachusetts) was coated with poly-L-
lysine with its
tip rounded by heating near a flame. The filament was inserted from the ECA
into the ICA
and then into the circle of Willis to occlude the origin of the left MCA. The
suture was
inserted 18 to 20 mm from the bifurcation of the CCA to occlude the MCA. After
1.5 hour of
ischemia, the nylon suture was withdrawn and the ischemic brain tissue was
reperfused for 24
hours before sacrificing. Core body temperature was monitored with a rectal
probe and
maintained at 37 C with a Homeothermic Blanket Control Unit (Harvard
Apparatus) during
anesthesia. To determine the effects of NRG-1 on ischemic stroke, rat were
injected
intravascularly with a single bolus 10 ul dose of vehicle (1% BSA in PBS) or
NRG-13 (1-50
umol/L NRG-1 (EGF-like domain, R&D Systems, Minneapolis, Minnesota) dissolved
in 1%
BSA/PBS) through a Hamilton syringe at a rate of 5 ul/min. This resulted in
the
administration of 0.5 ¨ 2.5 jig of NRG-1/kg body weight. NRG-1 or vehicle was
administered
by bolus injection into the ICA through ECA. Solutions were administered
either before
MCAO or immediately following 1.5 hours of MCAO and either 0, 4 or 12 hours of
reperfiision. Animals were sacrificed 24 hours after reperfusion or after 14
days for the long-
term studies. Animals were killed 24 hours later and the brains were sliced
into 2 mm
sections and stained with 2,3,5-triphenyltetrazolium chloride (TTC). Infarct
volumes in
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brains from vehicle and NRG1 treated animals are shown in the graph (panel d).
The data
demonstrate that NRG1B treatment reduces MCAO/reperfusion-induced brain
infarction.
EXAMPLE 4: NRG-1B Suppresses MCAO/Reperfusion-Induced Apoptotic Damage In Rat
Brain
[0074] Rats were subjected to MCAO for 1.5 hours followed by reperfusion for
24
hours. Figure 4 shows representative views of TUNEL labeling of rat brain
sections (n=5 for
each condition). TUNEL assay was performed with a DeadEND Fluoronietric TUNEL
System (Promega, Madison, Wisconsin) according to the manufacturer's
instructions. Slides
were then washed with PBS and mounted with Vectashield Mounting Medium
containing
DAPI. All sections were examined by fluorescence microscopy in three random
middle
cerebral artery served areas in the inner border of the infarct in the
ischemic front-parietal
cortex of each rat. In animals given vehicle or neuregulin-1, cortex and
striatum were
examined in three or more 20 p.m sections per animal. TUNEL staining was found
in the
cortex (panel a) and striatum (panel b) following MCAO while no TUNEL staining
was seen
in the cortex (panel c) and reduced levels were seen in the striatum (panel d)
in NRG1B-
treated rats.. The coronal brain image (¨bregma + 1.2 mm) indicates the areas
observed in
the sections (panel e).
EXAMPLE 5: NRG-1 Treatment Reduces MCAO/Reperfusion-Induced Brain Infarction
[09751 Figure 5 shows representative TTC stained coronal brain sections from
rats
injected with vehicle (panel a) or NRG1 immediately after MCAO (panel b) and 4
hours after
reperfusion (panel c). Infarct volumes in brains from rats treated with
vehicle (n=10) or
NRG1 immediately after MCAO (RO; n=8), 4 hours after reperfusion (R4; n=6) or
12 hours
after reperfusion (R12; n=8) are show in the graph (panel d). The time line
(panel e)
illustrates the MCAO protocol and NRG1 injections.
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EXAMPLE 6: NRG-1 Administration Resulted In A Significant Improvement In
Neurological Outcome
100761 NRG1 was administered after MCAO and 4 hours of reperfusion. As shown
in Figure 6, neurological function was graded on a scale of 0-4 (normal score
0, maximal
deficit score 4). All animals were tested prior to surgery (controls; n=14)
and after treatment
with NRG-1 or vehicle. The NRG1 treated group (n=9) displayed a 33%
improvement in
neurological score compared with vehicle treated rats (n=5).
EXAMPLE 7: NRG1B Prevents Microglial And Astrocytic Activation Following MCAO
[00771 Rats were subjected to MCAO followed by reperfiision for 24 hours (n=5
for
each condition). NRG1J3 or vehicle was injected intraarterially as described
above. Sections
were labeled for immunohistochemistry with an antibody against ED-1. As shown
in Figure
7, while no staining was seen in the contralateral side (panel a), ED-1
labeled cells are present
in the ipsilateral hemisphere (panel b) following MCAO in vehicle-treated
animals. Few ED-
1 positive cells are found in animals treated with NRG1B (panel c). To assess
astrocytic
activation, sections were labeled for immunohistochemistry with an antibody
against GFAP.
Compared to the contralateral control (panel a), heavy GFAP staining is found
at the border
or infarct (panel e) following MCAO in vehicle-treated animals. However, when
rats were
treated with NRG-113, GFAP expression was dramatically reduced in the pen-
infarct regions
(panel I).
EXAMPLE 8: NRG1B Reduces MCAO/Reperfusion-Induced IL-113 mRNA Levels
[0078] Rats were treated with NRG113 or vehicle then subjected to MCAO. RNA
was
isolated and IL1.13 mRNA expression was measured by RT-PCR. Figure 8 shows the
mRNA
expression levels of IL-1 (panel a) and GAPDH (panel b) (n=4 for each
condition). Panel c
shows the average percentage of change SEM in IL-1 mRNA levels from NRG113-
treated
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rat compared to vehicle-treated controls after normalization to GAPDH.
100791 The above description is for the purpose of teaching the person of
ordinary
skill in the art how to practice the present invention, and it is not intended
to detail all those
obvious modifications and variations of it which will become apparent to the
skilled worker
upon reading the description. It is intended, however, that all such obvious
modifications and
variations be included within the scope of the present invention, which is
defined by the
following claims. The claims are intended to cover the claimed components and
steps in any
sequence which is effective to meet the objectives there intended, unless the
context
specifically indicates the contrary.
