Note: Descriptions are shown in the official language in which they were submitted.
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THERAPEUTIC METHODS COMPRISING USE OF A NEUREGULIN
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods for treatment of certain neurological-
related injuries and disorders comprising use of a neuregulin, or a fragment
or
S derivative of a neuregulin, or a nucleic acid encoding a neuregulin or
neuregulin
fragment or derivative.
2. Background
Nerve cell death (degeneration) can cause potentially devastating and
irreversible effects for an individual and may occur e.g. as a result of
stroke, heart
10 attack or other brain or spinal chord ischemia or trauma. Additionally,
neurodegenerative disorders involve nerve cell death (degeneration) such as
Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic
Lateral
Sclerosis, Down's Syndrome and Korsakoffs disease.
Therapies have been investigated to treat nerve cell degeneration and related
15 disorders, e.g., by limiting the extent of nerve cell death that may
otherwise occur to
an individual as well as promoting repair, remodeling and reprogramming after
stroke
or other neuronal injury. See, e.g., F. Seil, Curr Opin Neuro, 10:49-51
(1997); N. L.
Reddy et al., JMed Chem, 37:260-267 (1994); and WO 95/20950.
Certain growth factors have been reported to exhibit neuroprotective
20 properties. In particular, nerve growth factor (NGF) has been evaluated in
certain
neuroprotective models. See, for example, G. Sinson et al., JNeurosurg,
86(3):511-
518 (1997); and G. Sinson et al., JNeurochem, 65(5):2209-2216 (1995).
Osteogenic
protein-1 (OP-1) has been evaluated in a rat model of cerebral
hypoxia/ischemia for
neuroprotective activity. G. Perides, Neurosci Lett, 1871):2I-24 (1995). Glial
cell
25 Line-derived neurotrophic factor (GDNF) was reported to exhibit trophic
activity on
certain populations of central neurons. Y. Wang et al., JNeurosci, 17(11):4341-
4348
(1997). Small molecules also have been investigated as neuroprotective agents,
such
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as MK-801. See B. Meldrum , Cereb Brain Metab Rev, 2:27-57 (1990); D. Choi,
Cereb Brain Metab Rev, 2:27-57 ( 1990).
However, no effective pharmacotherapies are in regular clinical use for
ischemia-induced brain injury or other such injuries and disorders. See, for
example,
5 Y. Wang et al., supra; G. Sinson et al., JNeurochem, JNeurochem, 65(5):2209
( 1995).
It thus would be highly desirable to have new neuroprotective agents,
particularly agents to limit the extent or otherwise treat nerve cell death
(degeneration)
that occur with stroke, heart attack or brain or spinal cord trauma, or to
treat
10 Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotraphic
Lateral
Sclerosis, Down's Syndrome and Korsakoffs disease. It also would be desirable
to
have agents that promote repair, remodeling or reprogramming after stroke or
other
neuronal injury.
SUMMARY OF THE INVENTION
15 The present invention provides methods for treatment and/or prophylaxis of
certain neurological-related disorders, particularly treatment or prophylaxis
of the
effects of stroke, brain or spinal cord injury or ischemia, heart attack,
optic nerve and
retinal injury and ischemia and other acute-type conditions disclosed herein
as well as
chronic-type conditions, specifically epilepsy, Alzheimer's disease,
Parkinson's
20 disease, Huntington's disease, Amyotrophic Lateral Sclerosis, Down's
Syndrome,
KorsakofFs disease, cerebral palsy and/or age-dependent dementia. Methods of
the
invention also include therapies for promoting repair, remodeling or
reprogramming
after stroke or other neuronal injury.
The methods of the invention comprise administration of an effective amount
25 of neuregulin, or fragment or derivative of a neuregulin, or a nucleic acid
encoding a
neuregulin or a neuregulin fragment or derivative (i.e. gene therapy), to a
patient
suffering from or susceptible to such conditions.
Neuregulins are members of the epidermal growth factor (EGF) superfamily
and include glial growth factor (GGF), acetylcholine receptor-inducing
activity
30 (ARIA), neu differentiation factor (NDF) and heregulins (HRF). See D. E.
Wen et al.,
Cell, 69:559-572 (1992); W.E. Holmes et al., Science, 256:1205-1210 (1992);
M.A.
Marchionni et al., Nature, 362:312-318 (1993); and D.L. Falls, Cell, 72:801-
815
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(1993). A variety of neuregulins and fragments and derivatives thereof can be
employed in the methods of the invention. For example, suitable agents have
been
disclosed in U.S. Patent 5,530,109 and PCT/US93/07491. Neuregulins also have
been reported in U.S. Patent 5,367,060. Preferred neuregulins include regions
shown
5 in FIGS. 1-2 (SEQ ID NOS. 2 and 4), also known as the E sequence. Preferred
neuregulins or fragments or derivatives also include those that contain the C,
C/D or
C/D' sequences as shown in Figures 7, 8 and 9 respectively of the drawings, or
those
neuregulins or fragments or derivatives that have substantial homology to the
peptide
sequences shown in Figures 7, 8 or 9, e.g. at least about 70 percent homology,
or at
10 least about 80 percent homology, or more preferably at least about 90 or 95
percent
homology to the peptide sequences shown in Figures 7, 8 or 9. Preferred
nucleic
acids and fragments and derivatives for use in the methods of the invention
include
those nucleic acids that include one or more nucleic acids sequences shown in
Figures
7, 8 and 9 of the drawings, or those nucleic acids that that have substantial
homology
15 to the nucleic acid sequences shown in Figures 7, 8 or 9, e.g. at least
about 70, 80, 90
or 95 percent homology to the nucleic acid sequences shown in Figures 7, 8 or
9. A
particularly preferred neuregulin is encoded by DNA obtainable from the clone
pGGF2HBS 11 (ATCC Deposit No. 75347). Also preferred are neuregulins encoded
by DNA obtainable from GGF2BPP5, GGF2BPP2 and GGF2BPP4.
20 Typical patients that may be treated in accordance with the methods of the
invention are persons suffering from brain or spinal cord trauma or ischemia,
stroke,
heart attack, hypoxia, hypoglycemia, post-surgical neurological deficits,
decreased
blood flow or nutrient supply to retinal tissue or optic nerve, retinal trauma
or
ischemia or optic nerve injury. Patients suffering from chronic-type
conditions also
25 may be treated in accordance with the invention, specifically subjects
suffering from
or susceptible to epilepsy, Parkinson's disease, Huntington's disease,
Amyotrophic
Lateral Sclerosis, Alzheimer's disease, Down's Syndrome, Korsakoffs disease,
cerebral palsy and/or age-dependent dementia.
Also, as discussed above, a neuregulin or fragment or derivative thereof or
30 nucleic acid encoding same, may be administered to promote repair,
remodeling or
reprogramming to a subject that has suffered stroke or other neuronal injury
such as
traumatic brain or spinal cord injury. In such cases, the therapeutic agent
may be
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suitably administered to the subject over an extended period following the
injury, e.g.
at least about 1, 2, 3, 4, 6, 8, 12 or 16 weeks following the injury.
Other aspects of the invention are disclosed infra.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a nucleotide sequence (SEQ ID NO:1 ) encoding a preferred
neuregulin region (E segment of human GGF) and the amino acid sequence (SEQ ID
N0:2) of that preferred region.
FIG. 2 shows a nucleotide sequence (SEQ ID N0:3) encoding a preferred
neuregulin region (E segment of bovine GGF) and the amino acid sequence (SEQ
ID
10 N0:4) of that preferred region.
FIG. 3 shows nucleotide sequences (SEQ ID NOS:6-7) encoding further
neuregulin regions (B segment of human and bovine GGF) and amino acid
sequences
(SEQ ID NOS:S and 8) of those regions. Line 1 is the predicted amino acid
sequence
of bovine B segment, line 2 is a nucleotide sequence of bovine B segment, line
3 is a
15 nucleotide sequence of human B segment (nucleotide base matches are
indicated with
a vertical line), and line 4 is the predicted amino acid sequence of human B
segment
shown where it differs from the bovine sequence set forth in line 1 of the
figure.
FIG. 4 shows nucleotide sequences (SEQ ID NOS:10-11) encoding fiu-ther
neuregulin regions (A segment of human and bovine GGF) and amino acid
sequences
20 (SEQ ID NOS:9 and 12) of those regions. Line 1 is the predicted amino acid
sequence of bovine A segment, line 2 is a nucleotide sequence of bovine A
segment,
line 3 is a nucleotide sequence of human A segment (nucleotide base matches
are
indicated with a vertical line), and line 4 is the predicted amino acid
sequence of
human A segment shown where it differs from the bovine sequence set forth in
line 1
25 of the figure.
FIG. 5 shows a nucleotide sequence (SEQ ID N0:13) encoding a further
neuregulin region (A' segment of bovine GGF) and the predicted amino acid
sequence
(SEQ ID N0:14) of that region.
FIG. 6 shows nucleotide sequences (SEQ ID NOS:16-17) encoding further
30 neuregulin regions (G segment of bovine and human GGF) and amino acid
sequences
(SEQ ID NOS:15 and 18) of that region. Line 1 is the predicted amino acid
sequence
of bovine G segment, line 2 is a nucleotide sequence of bovine G segment, line
3 is a
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nucleotide sequence of human G segment (nucleotide base matches are indicated
with
a vertical line), and line 4 is the predicted amino acid sequence of human G
segment
shown where it differs from the bovine sequence set forth in line 1 of the
figure.
FIG. 7 shows nucleotide sequences (SEQ ID NOS:20-21 ) encoding further
5 neuregulin regions (C segment of bovine and human GGF) and amino acid
sequences
(SEQ ID NOS:19 and 22) of those regions. Line 1 is the predicted amino acid
sequence of bovine C segment, line 2 is a nucleotide sequence of bovine C
segment,
line 3 is a nucleotide sequence of human C segment (nucleotide base matches
are
indicated with a vertical line), and line 4 is the predicted amino acid
sequence of
10 human C segment shown where it differs from the bovine sequence set forth
in line 1
of the figure.
FIG. 8 shows nucleotide sequences (SEQ ID NOS:24-25) encoding further
neuregulin regions (C/D segment of human and bovine GGF) and amino acid
sequences (SEQ ID NOS:23 and 26) of those regions. Line 1 is the predicted
amino
15 acid sequence of bovine C!D segment, line 2 is a nucleotide sequence of
bovine C/D
segment, line 3 is a nucleotide sequence of human C/D segment (nucleotide base
matches are indicated with a vertical line), and line 4 is the predicted amino
acid
sequence of human C/D segment shown where it differs from the bovine sequence
set
forth in line 1 of the figure.
20 FIG. 9 shows nucleotide sequences (SEQ ID NOS:28-29) encoding a further
neuregulin region (C/D' segment of the human and bovine GGF) and the amino
acid
sequence (SEQ ID N0:27) of that region. Line 1 is the predicted amino acid
sequence
of the CID' segment, line 2 is a nucleotide sequence of bovine C/D' segment
and line
3 is a nucleotide sequence of human C/D' segment (nucleotide base matches are
25 indicated with a vertical line).
FIG. 10 shows nucleotide sequences (SEQ ID NOS:31-32) encoding a further
neuregulin region (D segment of the human and bovine GGF) and the amino acid
sequence (SEQ ID N0:30) of that region. Line 1 is the predicted amino acid
sequence
of the D segment, line 2 is a nucleotide sequence of bovine D segment and line
3 is a
30 nucleotide sequence of human D segment (nucleotide base matches are
indicated with
a vertical line).
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FIG. 11 shows nucleotide sequence (SEQ ID N0:34) encoding a further
neuregulin region (D' segment of bovine GGF) and the amino acid sequence (SEQ
ID
N0:33) of that region.
FIGS. 12A-12B show nucleotide sequences (SEQ ID NOS:36-37) encoding
5 further neuregulin regions (H segment of human and bovine GGF) and amino
acid
sequences (SEQ ID N0:35 and 38) of that region. Line I is the predicted amino
acid
sequence of bovine H segment, line 2 is a nucleotide sequence of bovine H
segment,
line 3 is a nucleotide sequence of human H segment (nucleotide base matches
are
indicated with a vertical line), and line 4 is the predicted amino acid
sequence of
10 human H segment shown where it differs from the bovine sequence set forth
in line 1
of the figure.
FIG. 13 shows a nucleotide sequence (SEQ ID N0:40) encoding a further
neuregulin region (K segment of bovine GGF) and the amino acid sequence (SEQ
ID
N0:39) of that region.
15 FIGS. 14A-14C show nucleotide sequences (SEQ ID NOS:42-43) encoding a
further neuregulin region (L segment of bovine and human GGF) and amino acid
sequences (SEQ ID N0:41 and 44) of that region. Line 1 is the predicted amino
acid
sequence of bovine L segment, line 2 is a nucleotide sequence of bovine L
segment,
line 3 is a nucleotide sequence of human L segment (nucleotide base matches
are
20 indicated with a vertical line), and line 4 is the predicted amino acid
sequence of
human L segment shown where it differs from the bovine sequence set forth in
line 1
of the figure.
FIG. 15 shows nucleotide sequences (SEQ TD NOS:46-47) encoding further
neuregulin regions (F segment of bovine and human GGF) and amino acid
sequences
25 (SEQ ID NOS:45 and 48) of that region. Line 1 is the predicted amino acid
sequence
of bovine F segment, line Z is a nucleotide sequence of bovine F segment, line
3 is a
nucleotide sequence of human F segment (nucleotide base matches are indicated
with
a vertical line), and line 4 is the predicted amino acid sequence of human F
segment
shown where it differs from the bovine sequence set forth in line 1 of the
figure.
30 FIGS. 16A-16C show the nucleotide sequence (SEQ ID N0:49) and deduced
amino acid sequence (SEQ ID NO:50) of GGF2BPP4.
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FIGS. 17A-17B show the nucleotide sequence (SEQ ID NO:S 1 ) and deduced
amino acid sequence (SEQ ID N0:52) of GGF2BPP2.
FIGS. 18A-18B show the nucleotide sequence (SEQ ID N0:53) and deduced
amino acid sequence (SEQ ID N0:54) of GGF2BPP5.
5 DETAILED DESCRIPTION OF THE INVENTION
As discussed above, preferred neuregulins for use in the therapeutic methods
of the present invention include those disclosed in U.S. Patent 5,530,109 and
PCT/CJS93/07491, incorporated herein by reference. Particularly preferred
neuregulins comprise an amino acid sequence of the following formula:
10 WYBAZCX
wherein WYBAZCX is composed of amino acid sequences that include one or more
sequences shown in FIGS. 1 through 15 (which includes SEQ ID NOS:2, 4, 5, 8,
9,
12, 14, 15, 18, 19, 22, 23, 26, 27, 30, 33, 35, 38, 39, 41, 44, 45 and 48),
wherein W
comprises the polypeptide segment F, or is absent; wherein Y comprises the
15 polypeptide segment E, or is absent; wherein Z comprises the polypeptide
segment G
or is absent; and wherein X comprise a polypeptide segment selected from the
group
consisting of C/D HKL, C/D H, CID HL, C/D D, C/D' HL, CID' HKL, C/D' H, C/D'
D, C/D C/D' HKL, C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/d D'H, C/D D' HL,
C/D D' HKL, C/D' D' H, C/D' D' HL, C!D' D' HKL, C/D C/D' D' H, C/D C/D' D'
20 HL and C/D C/D' D' HKL, and preferably that either
a) at least one of F, Y, B, A, Z, C or X is of bovine origin; or
b) Y comprises the polypeptide segment E; or
c) X comprises the polypeptide segments C/D HKL, C/D D, C/D' HKI,,
C/D C/D' HKL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D'
25 D' HKL, CID C/D' D'H, C/D ClD' D HL, C/D ClD' D' HKL, C/D'H, C/D C/D' H or
C/D C/D' HL.
Particularly preferred neureguiins also include those polypeptides that
include
the segments FB polypeptides that include the segments FBA' (i.e. the groups
F, B
and A' as defined herein including in the drawings); polypeptides that include
the
30 segments EBA (i.e. the groups E, B and A as defined herein including in the
drawings); polypeptides that include the segments EBA' (i.e. the groups E, B
and A'
as defined herein including in the drawings); A (i.e. the group A as defined
herein
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_g_
including in the drawings); polypeptides that include the segments FEBA (i.e.
the
groups F, E, B and A as defined herein including in the drawings);
polypeptides that
include the segments FBA' (i.e. the groups F, B and A' as defined herein
including in
the drawings); and polypeptides that include the segments FEBA' (i.e. the
groups F,
E, B and A' as defined herein including in the drawings).
Also preferred are nucleic acids that code for the above preferred
polypeptides.
A "fragment" or "derivative" of a neuregulin refers to herein 1) a peptide in
which one or more amino acid residues are with a conserved or non-conserved
amino
acid residue (preferably a conserved amino acid residue) and such substituted
amino
acid residue may or may not be one encoded by the genetic code, or (ii) a
peptide in
which one or more of the amino acid residues includes a substituent group, or
(iii) a
peptide in which the mature protein is fused with another compound, such as a
compound to increase the half life of the polypeptide (for example,
polyethylene
glycol). Thus, a fragment or derivative for use in accordance with the methods
of the
invention includes a proprotein, which can be activated by cleavage of the
proprotein
portion to produce an active mature polypeptide.
The polypeptide fragments and derivatives of the invention are of a sufficient
length to uniquely identify a region of a neuregulin. Neuregulin fragments
thus
preferably comprise at least 8 amino acids, usually at least about 12 amino
acids, more
20 usually at least about 15 amino acids, still more typically at least about
30 amino
acids, even more typically at least about 50 or 70 amino acids. Preferred
fragments or
derivatives for use in the methods of the invention include those that have at
least
about 70 percent homology (sequence identity) to any of the preferred
sequences
mentioned above, more preferably about 80 percent or more homology to any of
the
25 preferred sequences mentioned above, still more preferably about 85 to 90
percent or
more homology to any of the preferred sequences mentioned above. Sequence
identity or homology with respect to a neuregulin as referred to herein is the
percentage of amino acid sequences of a neuregulin protein or fragment or
derivative
thereof that are identical with a specified sequence, after introducing any
gaps
30 necessary to achieve the maximum percent homology.
The neureguiin fragments and derivatives for use in the methods of the
invention preferably exhibit good activity in standard neuroprotective assays
such as
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the in vivo cerebral ischemia assay of Example 1, which follows. That assay
includes
the following steps: a) continuous intraventricular infusion of the protein
fragment or
derivative or vehicle alone to test rats for three days prior to inducing
focal ischemic
infarcts in right lateral cerebral cortex; and b) twenty-four hours after
inducing
S ischemic infarcts, infarct volume in each test animal is determined by image
analysis.
Preferably, a protein fragment or derivative of the invention provides at
least about a
10% reduction in infarct volume relative to vehicle-treated animals, more
preferably
about a 20% reduction in infarct volume, still more preferably about a 25%
reduction
in infarct volume relative to vehicle-treated animals in such an assay.
References
herein to in vivo cerebral ischemia assay are intended to refer to an assay of
the above
steps a) and b), which are more fully described in Example 1 which follows.
As discussed above, neuregulin nucleic acid fragments and derivatives are also
provided for use in the methods.of the invention. Those fragments and
derivatives
typically are of a length sufficient to bind to a sequence of any of the
nucleic acid
15 sequences shown in Figures 1-15 of the drawings, including SEQ ID NOS:1, 3,
6, 7,
10, 11, 13, 16, 17, 20, 21, 24, 25, 28, 29, 31, 32, 34, 36, 37, 40, 42 and 43
under the
following moderately stringent conditions (referred to herein as "normal
stringency"
conditions): use of a hybridization buffer comprising 20% formamide in 0.8M
saline/0.08M sodium citrate (SSC) buffer at a temperature of 37°C and
remaining
bound when subject to washing once with that SSC buffer at 37°C.
Preferred neuregulin nucleic acid fragments and derivatives of the invention
will bind to a sequence of any of the nucleic acid sequences shown in Figures
1-15 of
the drawings, including SEQ ID NOS:1, 3, 6, 7, 10, 11, 13, 16, 17, 20, 21, 24,
25, 28,
29, 31, 32, 34, 36, 37, 40, 42 and 43 under the following highly stringent
conditions
25 (referred to herein as "high stringency" conditions): use of a
hybridization buffer
comprising 20% formamide in 0.9M saline/0.09M sodium citrate (SSC) buffer at a
temperature of 42°C and remaining bound when subject to washing twice
with that
SSC buffer at 42°C.
The neuregulin nucleic acid fragments and derivatives preferably should
30 comprise at least 20 base pairs, more preferably at least about 50 base
pairs, and still
more preferably a nucleic acid fragment or derivative of the invention
comprises at
least about 100, 200, 300 or 400 base pairs. In some preferred embodiments,
the
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nucleic acid fragment or derivative is bound to some moiety which permits
ready
identification such as a radionucleotide, fluorescent or other chemical
identifier.
Isolated neuregulin and peptide fragments or derivatives of the invention are
preferably produced by recombinant methods, although suitable neuregulins also
can
be isolated from various sources. See the procedures disclosed U.S. Patent
5,530,109;
U.S. Patent 5,367,060; and PCT/LJS93/07491, incorporated herein by reference.
A
wide variety of molecular and biochemical methods are available for generating
and
expressing neuregulin; see e.g. the procedures disclosed in Molecular Cloning,
A
Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring
Harbor),
10 Current Protocols in Molecular Biology (Eds. Aufubel, Brent, Kingston,
More,
Feidman, Smith and Stuhl, Greene PubI. Assoc., Wiley-Interscience, NY, N.Y.
1992)
or other procedures that are otherwise known in the art. For example,
neuregulin or
fragments or derivatives thereof may be obtained by chemical synthesis, or
more
preferably by expression in bacteria such as E coli and eukaryotes such as
yeast,
baculovirus, or mammalian cell-based expression systems, etc., depending on
the size,
nature and quantity of neuregulin or fragment or derivative thereof. More
particularly, a recombinant DNA molecule comprising a vector and a DNA segment
encoding neuregulin, or a fragment or derivative thereof, can be constructed.
Suitable
vectors include e.g. baculovirus-derived vectors for expression in insect
cells (see
20 Pennock et al., Mol. Cell. Biol., 4:399-406 (1984)), T7-based expression
vector for
expression in bacteria (see Rosenberg et al., Gene, 56:125-I35 (1987)) and the
pMSXND expression vector for expression in mammalian cells (Lee and Nathans,
J.
Biol. Chem., 263:3521-3527 (1988)). The DNA segment can be present in the
vector
operably linked to regulatory elements, e.g., a promoter (e.g., polyhedron, T7
or
25 metallothionein (Mt-I) promoters), or a leader sequence to provide for
secretory
expression of the polypeptide. The recombinant DNA molecule containing the DNA
coding for a neuregulin or a fragment or derivative thereof can be introduced
into
appropriate host cells by known methods. Suitable host cells include e.g.
prokaryotes
such as E. coli, Bacillus subtilus, etc., and eukaryote such as animal cells
and yeast
30 strains, e.g., S. cerevisiae. Mammalian cells may be preferred such as
J558, NSO,
SP2-O or CHO. In general, conventional culturing conditions can be employed.
See
Sambrook, supra. Stable transformed or transfected cell lines can then be
selected.
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The expressed neuregulin or fragment or derivative thereof then can be
isolated and
purified by known methods. Typically the culture medium is centrifuged and the
supernatant purified by affinity or immunoaffinity chromatography, e.g.
Protein-A or
Protein-G affinity chromatography or an immunoaffinity protocol comprising use
of
5 monoclonal antibodies that bind neuregulins.
Neuregulin nucleic acids used in the methods of the invention are typically
isolated, meaning the nucleic acids comprise a sequence joined to a nucleotide
other
than that which it is joined to on a natural chromosome and usually constitute
at least
about 0.5%, preferably at least about 2%, and more preferably at least about
5% by
10 weight of total nucleic acid present in a given fraction. A partially pure
nucleic acid
constitutes at least about 10%, preferably at least about 30%, and more
preferably at
least about 60% by weight of total nucleic acid present in a given fraction. A
pure
nucleic acid constitutes at least about 80%, preferably at least about 90%,
and more
preferably at least about 95% by weight of total nucleic acid present in a
given
15 fraction.
As discussed above, the present invention includes methods for treating and
preventing certain neurological-related injuries and disorders, comprising the
administration of an effective amount of a neuregulin or fragment or
derivative
thereof, or nucleic acid encoding same, to a subject including a mammal,
particularly
20 a human, in need of such treatment.
In particular, the invention provides methods for treatment and/or prophylaxis
of nerve cell death (degeneration) resulting from hypoxia, hypoglycemia, brain
or
spinal cord ischemia, brain or spinal cord trauma, stroke, heart attack or
drowning.
Typical candidates for treatment include e.g. heart attack, stroke and/or
persons
25 suffering from cardiac arrest neurological deficits, brain or spinal cord
injury patients,
patients undergoing major surgery such as heart surgery where brain ischemia
is a
potential complication and patients such as divers suffering from
decompression
sickness due to gas emboli in the blood stream. Candidates for treatment also
will
include those patients undergoing a surgical procedure involving extra-
corporal
30 circulation such as e.g. a bypass procedure.
The invention also provides methods for treatment which comprise
administration of a neuregulin or fragment or derivative thereof, or nucleic
acid
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encoding same, to a patient that is undergoing surgery or other procedure
where brain
or spinal cord ischemia is a potential risk. For example, carotid
endarterectomy is a
surgical procedure employed to correct atherosclerosis of the carotid
arteries. Major
risks associated with the procedure include intraoperative embolization and
the danger
5 of hypertension in the brain following increased cerebral blood flow, which
may
result in aneurysm or hemorrhage. Thus, an effective amount of a neuregulin or
fragment or derivative thereof, or nucleic acid encoding same, could be
administered
pre-operatively or peri-operatively to reduce such risks associated with
carotid
endarterectomy, or other post-surgical neurological deficits.
10 The invention also is effective to promote and enhance recovery from acute
nerve cell death and neurological conditions. Thus, for example, a neuregulin
or
fragment or derivative thereof, or nucleic acid encoding same, could be
administered
to promote repair, remodeling or reprogramming to a patient that has suffered
from
stroke or other neuronal injury, suitably for an extended period as discussed
above. A
15 therapeutic agent of the invention also could be administered post-
operatively to
promote recovery from any neurological deficits that may have occurred to a
patient
that has undergone surgery.
The invention further includes methods for prophylaxis against neurological
deficits resulting from e.g. coronary artery bypass graft surgery and aortic
valve
20 replacement surgery, or other procedure involving extra-corporal
circulation. Those
methods will comprise administering to a patient undergoing such surgical
procedures
an effective amount of a neuregulin or fragment or derivative thereof, or
nucleic acid
encoding same, typically either pre-operatively or peri-operatively.
The invention also provides methods for prophylaxis and treatment against
25 neurological injury for patients undergoing myocardial infarction, a
procedure that can
result in ischemic insult to the patient. Such methods will comprise
administering to a
patient undergoing such surgical procedure an effective amount of a neuregulin
or
fragment or derivative thereof, or nucleic acid encoding same, typically
either pre-
operatively or peri-operatively.
30 Also provided are methods for treating or preventing neuropathic pain such
as
may be experienced by cancer patients, persons having diabetes, amputees and
other
persons who may experience neuropathic pain. These methods for treatment
comprise
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administration of an effective amount of a neuregulin or fragment or
derivative
thereof, or nucleic acid encoding same, to a patient in need of such
treatment.
The invention also provides methods for treatment and prophylaxis against
retinal ischemia or degeneration and resulting visual loss. For example, a
neuregulin
5 or fragment or derivative thereof, can be administered parenterally or by
other
procedure as described herein to a subject a suffering from or susceptible to
ischemic
insult that may adversely affect retinal function, e.g., significantly
elevated intraocular
pressures, diseases such as retinal artery or vein occlusion, diabetes or
other ischemic
ocular-related diseases. Post-ischemic administration also may limit retinal
damage.
10 The invention also includes methods for treating and prophylaxis against
decreased
blood flow or nutrient supply to retinal tissue or optic nerve, or treatment
or
prophylaxis against retinal trauma or optic nerve injury. Subjects for
treatment
according to such therapeutic methods of the invention may be suffering or
susceptible to retinal ischemia that is associated with atherosclerosis,
venous capillary
15 insufficiency, obstructive arterial or venous retinopathies, senile macular
degeneration, cystoid macular edema or glaucoma, or the retinal ischemia may
be
associated with a tumor or injury to the mammal. Intravitreal injection also
may be a
preferred administration mute to provide more direct treatment to the ischemic
retina.
The invention further provides a method of treating Korsakoff s disease, a
20 chronic alcoholism-induced condition, comprising administering to a subject
including a mammal, particularly a human, an effective amount of a neuregulin
or
fragment or derivative thereof, in an amount effective to treat the disease.
Compounds of the invention are anticipated to have utility for the attenuation
of cell
loss, hemorrhages and/or amino acid changes associated with Korsakoffs
disease.
25 The invention further includes methods for treating a person suffering from
or
susceptible to epilepsy, emesis, narcotic withdrawal symptoms and age-
dependent
dementia, comprising administering to a subject including a mammal,
particularly a
human, an effective amount of a neuregulin or fragment or derivative thereof,
in an
amount effective to treat the condition.
30 It will be appreciated that in some instances a neuregulin or a fragment or
derivative thereof will be preferably administered to a subject rather than a
neuregulin
nucleic acid, particularly where a patient is suffering from or susceptible to
an acute
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neurological injury that demands immediate therapy. For example,
administration of
a neuregulin polypeptide may be preferred to a patient suffering from stroke,
heart
attack, traumatic brain injury and the like where it is desired to deliver the
active
therapeutic as quickly as possible.
5 In the therapeutic methods of the invention, neuregulin peptides and nucleic
acids may be suitably administered to a subject such as a mammal, particularly
a
human, by any of a number of routes including parenteral (including
subcutaneous,
intramuscular, intravenous and intradermal), oral, rectal, nasal, vaginal and
optical
(including buccal and sublingual) administration. A neuregulin protein or
nucleic acid
10 or fragment or derivative thereof may be administered to a subject alone or
as part of a
pharmaceutical composition, comprising the peptide or nucleic acid together
with one
or more acceptable carriers and optionally other therapeutic ingredients. The
carriers
should be "acceptable" in the sense of being compatible with the other
ingredients of
the formulation and not deleterious to the recipient thereof.
15 Nucleic acids encoding a neuregulin or a neuregulin fragment or derivative
can
be administered to a patient by generally known gene therapy procedures. See,
for
example, WO 90/11092 and WO 93/00051. Thus, for instance, the nucleic acids
may
be introduced into target cells by any method which will result in the uptake
and
expression of the nucleic acid by the target cells. These methods can include
vectors,
20 liposomes, naked DNA, adjuvant-assisted DNA, catheters, etc. Preferably,
the
administered nucleic acid codes for an appropriate secretory sequence to
promote
expression upon administration. Suitable vectors for administering a nucleic
acid in
accordance with the invention include chemical conjugates such as described in
WO
93/04701, which has targeting moiety (e.g. a ligand to a cellular surface
receptor), and
25 a nucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNA
or RNA viral
vector), fusion proteins such as described in PCT/US 95/02140 (WO 95/22618)
which
is a fusion protein containing a target moiety (e.g. an antibody specific for
a target
cell) and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage,
etc. The
vectors can be chromosomal, non-chromosomal or synthetic.
30 Preferred vectors include viral vectors, fusion proteins and chemical
conjugates. Retroviral vectors include moloney marine leukemia viruses. DNA
viral
vectors are preferred. These vectors include pox vectors such as orthopox or
avipox
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vectors, herpes virus vectors such as a herpes simplex I virus (HSV) vector
[A.I.
Geller et al., .l. Neurochem, 64:487 (1995); F. Lim et al., in DNA Cloning:
Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995);
A.I. Geller et al., Proc Natl. Acad Sci. U.S.A.:90 7603 (1993); A.I. Geller et
al., Proc
5 Natl. Acad. Sci USA, 87:1149 (1990)], Adenovirus Vectors [LeGal LaSalle et
al.,
Science, 259:988 (1993); Davidson, et al., Nat. Genet., 3:219 (1993); Yang et
al., J.
Virol., 69:2004 (1995)] and Adeno-associated Virus Vectors [Kaplitt, M.G., et
al.,
Nat. Genet., 8:148 (1994)].
Pox viral vectors introduce the gene into the cell cytoplasm. Avipox virus
10 vectors result in only a short-term expression of the nucleic acid.
Adenovirus vectors,
adeno-associated virus vectors and herpes simplex virus (HSV) vectors are
preferred
for introducing the nucleic acid into neural cells. The adenovirus vector
results in a
shorter term expression (about 2 months) than adeno-associated virus (about 4
months), which in turn is shorter than HSV vectors. The particular vector
chosen will
15 depend upon the target cell and the specific condition being treated. The
introduction
can be by standard techniques, e.g. infection, transfection, transduction or
transformation. Examples of modes of gene transfer include e.g., naked DNA,
Ca,(PO4)2 precipitation, DEAF dextran, electroporation, protoplast fusion,
lipofecton,
cell microinjection, and viral vectors.
20 A vector can be employed to target essentially any desired target cell. For
example, stereotaxic injection can be used to direct the vectors (e.g.
adenovirus, HSV)
to a desired location. Additionally, the particles can be delivered by
intracerebroventricular (icv) infusion using a minipump infusion system, such
as a
SynchroMed Infusion System. A method based on bulk flow, termed convection,
has
25 also proven effective at delivering large molecules to extended areas of
the brain and
may be useful in delivering the vector to the target cell (Bobo et al., Proc.
Natl. Acad.
Sci. USA, 91:2076-2080 (I994); Morrison et al., Am. J. Physiol., 266:292-305
(1994)). Other methods that can be used include catheters, intravenous,
parenteral,
intraperitoneal and subcutaneous injection, and oral or other known routes of
30 administration.
Parenteral formulations for administration of a neuregulin or a fragment or
derivative thereof may be in the form of liquid solutions or suspensions; for
oral
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administration, formulations may be in the form of tablets or capsules; and
for
intranasal formulations, in the form of powders, nasal drops, or aerosols.
Methods well known in the art for making formulations are found in, for
example, "Remington's Pharmaceutical Sciences". Fozznulations for parenteral
5 administration may, for example, contain as excipients sterile water or
saline,
polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or
hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control the release
of
the present factors. Other potentially useful parenteral delivery systems for
a
10 neuregulin or fragments or derivatives thereof include ethylene-vinyl
acetate
copolymer particles, osmotic pumps, implantable infusion systems, and
liposomes.
Formulations for inhalation may contain as excipients, for example, lactose,
or may be
aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether,
glycocholate and deoxycholate, or may be oily solutions for administration in
the
15 form of nasal drops, or as a gel to be applied intranasally. Formulations
for parenteral
administration may also include glycocholate for buccal administration,
methoxysalicylate for rectal administration, or citric acid for vaginal
administration.
The concentration of a neuregulin or a fragment or derivative thereof, or
nucleic acid encoding such polypeptides, administered to a particular subject
will vary
20 depending upon a number of issues, including the condition being treated,
the mode
and site of administration, the age, weight sex and general health of the
subject, and
other such factors that are recognized by those skilled in the art. Optimal
administration rates for a given protocol of administration can be readily
determined
by those skilled in the art.
25 All documents mentioned herein are incorporated herein by reference in
their
entirety. The invention is further illustrated by the following non-limiting
Examples.
Example 1 -- In vivo neuroprotection assay
Neuregulins and neuregulin fragments and derivatives can be assessed for
neuroprotective efficacy pursuant to the following assay.
30 Mature male Long-Evans rats (Charles River, 250-350g) are allowed food and
water ad libitum. Animals are anesthetized with sodium pentobarbital (60
mg/kg, i.p.)
and placed in a stereotaxic head holder (David Kopf Instruments, Tujunga, CA).
The
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dorsal surface of the skull is exposed by midline incision, and a small burr
hole (2 mm
diameter) is drilled over the right lateral ventricle, 1.6 mm lateral and 0.9
mm
posterior to bregma. A stainless steel cannula (LD. 0.020", O.D. 0.028", 2 cm
long) is
then inserted stereotaxically into the ventricle to a depth of 4.4 mm beneath
the
5 surface of the skull. The tubing is suitably bent at a 90° angle 1-
1.6 cm from its tip
and connected to polyethylene tubing (LD. 0.76 mm, O.D. 1.22 mm, 10 cm long)
that
is connected (by glue) to a mini-osmotic pump (Alzet 1007D, 100 pl fill
volume,
pump rate = 0.5 p,l/hr; Alza Corp., Palo Alto, CA) implanted subcutaneously in
the
back. The cannula can be suitably fixed to the skull by orthodontic resin
(L.D. Culk
10 Co., Milford, DE) bonded to two small machine screws (1/8" stainless steel
slotted)
inserted in the skull. The pump, tubing, and cannula are primed before
insertion with
infusate solutions; a 3-0 nylon suture is inserted into the cannula during
implantation
to prevent obstruction by brain tissue. The wound is closed with 3-0 silk
suture and
cefazolin (10 mg, i.m.) is administered. After surgery animals are suitably
kept in
15 individual cages and fed soft food.
Pumps are filled with vehicle alone (containing 127 mM NaCI, 2.6 mM KCI,
1.2 mM CaCl2, 0.9 mM MgCh, 4.14 mM HEPES, 3 mM glycerin, 0.001 % bovine
serum albumin [BSA], and 0.01 % fast green), or vehicle neuregulin or fragment
or
derivative thereof (100 p.gm/ml). Heparin can be suitably used at relatively
low
20 doses, e.g. about 0.8 units/kg/day which is approximately 250-500 times
less than a
standard anticoagulant dose.
Three days after cannula implantation, animals are reanesthetized with 2%
halothane and given atropine (0.15 mglkg, i.p.). Animals are then intubated
and
connected to a ventilator (SAR-830; CWE Inc., Ardmore, PA) delivering 1%
25 halothane/70% nitrous oxide in oxygen. The right femoral artery and vein
are
cannulated for monitoring of mean arterial blood pressure (MABP; Gould RS3200
Blood Pressure Monitor, Gould Inc., Valley View, OH), and blood sampling.
Animals are then paralyzed with pancuronium bromide (0.5 mg/kg, i.v.).
Arterial
blood gasses (Coming 178 Blood Gas Analyzer, Ciba Coming Diagnostic Corp.,
30 Medford, MA), blood glucose (Accu-Check Blood Glucose Analyzer, Boehringer
Mannheim, Indianapolis, Il~, and hematocrit are measured at least twice during
surgery and the immediate post-operative period. The stroke volume and rate of
the
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ventilator are adjusted to maintain Pa02 between 100-200 mm Hg and PaCOz
between
30-40 mm Hg. Core body temperature may be monitored by rectal thermocouple
(e.g.
Model 73ATA, Yellow Springs Instrument Co., Yellow Springs, OH) and maintained
between 36-37°C with a homeothermic blanket control unit (Harvard
Bioscience,
South Natick, MA).
Focal ischemic infarcts are made in the right lateral cerebral cortex in the
territory of the middle cerebral artery (MCA) by the method of Chen, et al.,
Stroke,
17:738-743 (1986). Both common carotid arteries are exposed by midline
anterior
cervical incision. The animal is placed in a lateral position and a 1 cm skin
incision is
10 then made at the midpoint between the right lateral canthus and the
anterior pinna.
The temporal muscle is retracted, and a small (3 mm diameter) craniectomy is
made at
the junction of the zygoma and squamosal bone using a dental drill cooled with
saline.
Using a dissecting microscope, the dura can be opened with fine forceps, and
the
right MCA can be ligated with two 10-0 monofilament nylon ties just above the
rhinal
1 S fissure and transected between the ties. Both common carotid arteries then
can be
occluded by microaneurysm clips for 45 minutes. After removal of the clips,
return of
flow is visualized in the arteries. Anesthesia is maintained for 15 minutes,
and
animals are returned to individual cages and fed soft food after surgery.
Twenty four hours after cerebral infarction, animals are again weighed, and
20 then sacrificed by rapid decapitation. Brains are removed, inspected
visually for the
anatomy of the middle cerebral artery as well as for signs of hemorrhage or
infection,
immersed in cold saline for 10 minutes, and sectioned into six standard
coronal slices
(each 2 mm thick) using a rodent brain matrix slicer (Systems, Warren, MI).
Brains
are also examined visually for the presence of dye (fast green) in the
cerebral
25 ventricles. Slices are placed in the vital dye 2,3,5-triphenyl tetrazolium
chloride
(TTC, 2%; Chemical Dynamics Co., NH) at 37°C in the dark for 30
minutes, followed
by 10% formalin at room temperature overnight. The outline of right and left
cerebral
hemispheres as well as that of infarcted tissue, clearly visualizable by lack
of TTC
staining (Chen et al., Stroke, 17:738-743 (1986)), is outlined on the
posterior surface
30 of each slice using an image analyzer (MTI videocamera and Sony video
monitor
connected to a Bioquant IV Image Analysis System run on an EVEREX computer).
Infarct volume is calculated as the sum of infarcted area per slice multiplied
by slice
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thickness. Both the surgeon and image analyzer operator are blinded to the
treatment
given each animal.
Volumes of infarcts among vehicle vs. neuregulin-treated animals can be
compared by unpaired, two-tailed t-tests for each experiment, and by two-way
5 analysis of variance (ANOVA; Exp. X Treatment) for combined data. A
subsequent
slice-by-slice analysis of infarct area among pooled neuregulin- vs. vehicle-
treated
animals is suitably done by repeated measures two-way ANOVA (Treatment X
Slice).
Other anatomical and physiological measurements are compared among GDF-1- vs.
vehicle-treated animals by unpaired, two-tailed t-tests using the Bonferroni
correction
I O for multiple pairwise comparisons.
Example 2 -- In vivo behavioral assays
For behavioral outcome studies, such as to assess recovery, repair and
remodeling promoted by administration of a neuregulin or fragment or
derivative
thereof, or nucleic acid encoding same, a number of assays can be employed
such as
15 those described in G. Sinson et al., JNeurochem, 65(5):2209-2214 (1995);
T.K.
McIntosh et al., Neuroscience, 28:233-244 ( 1989); and T.K. McIntosh et al., J
Neurotrauma, 10:373-384 (1993).
Briefly, one suitable behavioral assay as described in G. Sinson et al.,
supra,
entails that test animals (male Sprague-Dawley rats) receive preinjury
training in a
20 Morris Water Maze, a circular tank 1 m in diameter that is filled with
I8°C water.
The water surface is made opaque with a covering of Styrofoam pieces. During
training of the animals a submerged platform is present in the maze. Each test
animal
undergoes 20 training trials over a two day period during which they learn to
locate
the platform using external visual cups. Immediately following the last
training trial,
25 animals are anesthetized and subjected to a lateral (parasagittal) fluid-
percussion (FP)
brain injury. Briefly, a 5-mm craniectomy is performed over the left parietal
cortex,
midway between lamda and bregma. A hollow Leur-loc fitting is cemented to the
craniectomy site. The injury is delivered after attaching the FP device. The
injury
should be of moderate severity (2.1-2.3 atm). After injury, the Leur-loc is
removed,
30 and the skin is sutured. Normothermia is maintained with warming pads until
the
animals being to ambulate.
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At 72 hours, 1 week or 2 weeks after injury, animals are assessed for their
ability to remember the learned task of locating the platform in the MWM. For
this
evaluation the platform is removed from the maze, and the animal's swimming
pattern
is suitably recorded with a computerized video system for 1 minute. The maze
is
5 separated in zones that are weighed according to the proximity to the
platform's
location. A memory score is generated by multiplying the weighted numbers by
the
time the animal spends in each zone and then adding the products.
Animals surviving for 1 or 2 weeks also can undergo evaluation of neurologic
motor function. Briefly, one suitable assay provides that animals are scored
from 0
(severely impaired) to 4 (normal) for each of the following: ( 1 ) left and
(2) right
forelimb during suspension by the tail; (3) left and (4) right hlndlimb
flexion when the
forelimbs remain on a surface and the hindlimbs are lifted up and back by the
tail; the
ability to resist lateral pulsion to the (5) left and {6) right; and the
ability to stand on
an inclined plane in the (7) left, (8) right, and (9) vertical positions.
Scores are
combined for each of the tests { 1 ) through (9). The observer for the tests
should be
blinded to the animal's previous treatment.
The invention has been described in detail with reference to preferred
embodiments thereof. However, it will be appreciated that those skilled in the
art,
upon consideration of this disclosure, may make modifications and improvements
within the spirit and scope of the invention.
