Note: Descriptions are shown in the official language in which they were submitted.
CA 02337964 2001-02-05
- WO 00/07611 PCT/US99/18022
TREATMENT OF CENTRAL NERVOUS SYSTEM TSCHEMIA OR TRAUMA
WITH EPIDERMAL GROWTH FACTOR-LIKE POLYPEPTIDES
Cross Reference to Related At~plication
This application claims the benefit of U.S. Serial
No. 60/095,830, filed August 7, 1998, which is
incorporated herein by reference.
Field of the Invention
The invention relates to the treatment of
neurological deficits caused by injuries to the central
nervous system.
Backctround of the Invention
Neurotrophic factors are polypeptides that are
required for proper development of the nervous system.
The first neurotrophic factor discovered, nerve growth
factor (NGF), is now known to be a part of a large family
of growth factors, as is epidermal growth factor (EGF),,
which was discovered a short time later. The EGF family
includes EGF, transforming growth factor-a (TGF-a),
vaccinia growth factor (VGF), amphiregulin (AR), heparin
binding-EGF (HB-EGF), betacellulin (BTC), epiregulin,
neuregulin-1 (NRG-1, which is also known as Neu
differentiation factor (NDF), heregulin (HRG),
acetylcholine receptor-inducing activity (ARIA) and glial
growth factor (GGF)), and neuregulin-2 (NRG-2). These
polypeptides contain a common amino acid motif, the
EGF-domain, which consists of about 40-45 amino acid
residues, including six highly conserved cysteine
residues that are arranged as follows : CX7CX4CXIOCXCXeC
(SEQ ID N0:3; where C is cysteine and X~ is any seven
amino acids, X4 is any four amino acids, and so on). The
six cysteine residues form three intramolecular disulfide
bonds (between the first and third, the second and
CA 02337964 2001-02-05
PCT/US99/18022
- W O 00/07611
- 2 -
fourth, and the fifth and sixth cysteine residues of SEQ
ID N0:3), which are essential for HB-EGF mitogenic
activity.
Another common feature of polypeptides in the EGF
family is that they are synthesized as transmembrane
precursor molecules and then proteolytically processed so
that the mature growth factor is released from the cell
surface in a soluble form.
While the ability of polypeptide growth factors to
support cell growth and differentiation in the developing
animal has become increasingly evident, their role in the
mature animal, particularly in the nervous system, is
much less clear. This is unfortunate because, when
neurons are injured or die in a mature animal, permanent
motor and cognitive deficits can result. Even when there
is some recovery over time, patients who suffer any form
of cerebral ischemic episode often remain mildly to
severely debilitated.
Summary of the Invention
The present invention features methods for
preventing, reducing, or eliminating a neurological
deficit (i.e., a cognitive or sensorimotor deficit)
caused by an injury to the central nervous system (CNS).
The methods can be carried out by administering a
polypeptide in the epidermal growth factor (EGF) family
to a patient who has or is at risk of incurring such a
deficit. Polypeptides in the EGF family are referred to
herein (and are defined further below) as "EGF-like
polypeptides."
The present methods can be applied to reduce or
eliminate a neurological deficit after an injury has
occurred (i.e., after an injury that causes or
precipitates the deficit). This is advantageous because
there is often no way to anticipate when such an injury
CA 02337964 2001-02-05
- WO 00/07611
- 3 -
PCT/US99/18022
will occur. For example, traumatic injuries, such as
those caused by automobile accidents or during sporting
events, as well as all forms of stroke, are
unpredictable. Surprisingly, EGF-like polypeptides can
reduce or eliminate a sensorimotor or cognitive deficit
even when administration commences long after an injury
has occurred. For example, administration can begin more
than 6, more than 12, more than 24, more than 48, or more
than 72 hours following an injury, and still provide a
significant benefit. Indeed, administration may
beneficially commence weeks, months, or years after an
injury. Of course, administration can also begin
immediately after an injury. A further surprising
advantage of the invention is that administration can be
carried out intravenously; the EGF-like polypeptides of
the invention are effective despite the blood-brain
barrier.
Thus, the invention features a method for reducing
a neurological deficit in a patient who has suffered an
injury to the central nervous system by administering to
the patient an amount of an EGF-like polypeptide
effective to reduce the neurological deficit in the
patient. The injury can be an ischemic episode (e-g., a
focal or global ischemic episode) or a traumatic injury.
The EGF-like polypeptide, as described herein, can be
EGF; TGFa; VGF; AR; HB-EGF; BTC; epiregulin; a
neuregulin; or an EGF receptor-binding fragment or analog
thereof (e. g., the EGF domain of any of these EGF-like
polypeptides). In addition to fragments, EGF-like
polypeptides containing one or more conservative amino
acid substitutions are within the scope of the invention.
Unless otherwise defined, all technical and
scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art
to which this invention belongs. Although methods and
CA 02337964 2001-02-05
- W O 00107611
- 4 -
PCT/US99/18022
materials similar or equivalent to those described herein
can be used in the practice or testing of the present
invention, suitable methods and materials are described
below. All publications, patent applications, patents,
and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the
present specification, including definitions, will
control. In addition, the materials, methods, and
examples are illustrative only and not intended to be
limiting.
Other features and advantages of the invention
will be apparent from the following detailed description,
and from the claims.
Brief Description of the Drawing_s
Fig. 1 is a representation of the nucleic acid
sequence of human heparin-binding epidermal growth factor
(HB-EGF) (SEQ ID NO:1) and the amino acid translation
(SEQ ID N0:2).
Detailed Description
Neurological deficits can be caused by injuries to
the brain or spinal cord. As described below, a patient
who has sustained such an injury can be treated with an
EGF-like polypeptide, which will prevent, reduce, or
eliminate the deficits that have occurred or would be
expected to occur. Suitable polypeptides, appropriate
modes of administration, injuries amenable to treatment,
and means of analyzing the results obtained from a
treatment regimen are described below.
A. Polyt~eptides
Polypeptides useful in the context of the present
invention are polypeptides in the EGF family ("EGF-like
polypeptides"). Those of ordinary skill in the art are
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 5 -
well able to identify a polypeptide in this family, as it
will have a recognizable EGF domain (the consensus
sequence being CX,CX4CXIOCXCXeC (SEQ ID N0:3) , as described
above) or an ability to activate an EGF receptor.
Four members of the EGF receptor family have been
identified and designated using the HER (Human EGF
Receptor) or erbB nomenclature. These receptors are:
the classical EGF receptor (EGFR), also known as HER1 or
erbBl; HER2 or erbB2, also known as p185 or neu; HERS or
erbB3; and HER4 or erbB4. EGF receptors contain a single
ectodomain to which polypeptides in the EGF family bind,
a single membrane spanning domain, and an intrinsic
kinase domain in the cytoplasm. Upon ligand binding, the
monomeric receptors form homodimers or heterodimers with
other members of the EGFR family, a process known as
transmodulation, and phosphorylate specific tyrosine
residues within the cytoplasmic domain. These
phosphorylated tyrosine residues act as docking sites for
effector molecules resulting in activation of signal
transduction pathways. The four EGF receptor sub-types
share a high degree of homology, most notably in the
290-amino acid cytoplasmic tyrosine kinase domain. Two
cysteine-rich regions in the extracellular ligand-binding
domain are also conserved. In contrast, very little
homology exists in the 100-amino acids at the C-terminal
that are essential for the activation of intracellular
signal transduction pathways after ligand binding.
EGF-like polypeptides exhibit different
specificities in binding and activating EGF receptors
(see Table 1). EGF, transforming growth factor-alpha
(TGF-a), and amphiregulin (AR) bind to HER1; neuregulin-1
(NRG-1) and neuregulin-2 (NRG-2) bind to HER3 and HER4,
but only activate HER4; HB-EGF and BTC bind and activate
both HER1 and HERO. No ligand has been identified so far
for the receptor HER2.
CA 02337964 2001-02-05
PCT/US99/18022
- WO 00/07611
- 6 -
For a review of EGF receptors, including specific
binding and sequence information, signalling, and
receptor topology, one can consult, for example, McInnes
and Sykes (Biopolymers 43:339-366, 1997), Boonstra et al.
(Cell Biol. Intl. 19:413-430, 1995), or Gill (Mol.
Reprod. Dev. 27:46-53, 1990).
As used herein, the terms "protein" and
"polypeptide" both mean any chain of amino acid residues,
regardless of length or post-translational modification
(e.g., glycosylation or phosphorylation). The
polypeptide growth factors useful in the invention are
"substantially pure," meaning that a composition
containing the polypeptide is at least 60% by weight (dry
weight) the polypeptide of interest, e.g., an EGF-like
polypeptide such as HB-EGF. Preferably, the polypeptide-
containing composition is at least 75%, more preferably
at least 90%, and most preferably at least 99%, by
weight, the polypeptide of interest. Purity can be
measured by any appropriate standard method, e.g., column
chromatography, polyacrylamide gel electrophoresis, or
HPLC analysis.
Any EGF-like polypeptide that binds to and
activates an EGF receptor can be used to treat (i.e.,
prevent, reduce, or eliminate) a sensorimotor deficit
caused by CNS ischemia or trauma. As used herein, the
term "bind(s) to and activate(s)" refers to a specific
interaction between an EGF-like polypeptide and an EGF
receptor that results in signal transduction sufficient
to elicit a biological response that contributes to the
prevention or reduction of a neurological deficit. An
EGF-like polypeptide of the invention will bind to an EGF
receptor with an affinity that is equivalent to at least
50%, more preferably at least 70%, and most preferably at
least 90% (e. g., 95%, 97%, or even 99%, 100%, or more
than 100%) of the binding affinity of a naturally
CA 02337964 2001-02-05
- WO 00/07611 PCT/US99/18022
occurring EGF family member for its cognate receptor
(ligand-receptor binding partners are indicated in
Table 1) (e. g., see Twardizik et al., Proc. Natl. Acad.
Sci. USA 82:5300-5304, 1985 for a comparison of the
binding affinity of VGF, TGFa, and EGF for the EGF
receptor) .
TABhE l:
Polypeptides in the EGF family and their
receptors
EGF ligand EGF receptor
subtype
Epidermal growth factor (EGF) HER1
Transforming growth factor-a (TGFa) HER1
Vaccinia growth factor (VGF) HER1
Amphiregulin (AR) HER1
Heparin-binding EGF-like
growth factor (HB-EGF) HER1, HER4
Betacellulin (BTC) HER1, HERO
Epiregulin HER1
Neuregulin-1 (NRG-1) HER3,HER4
Neu differentation factor (NDF)
Heregulin (HRG)
Glial growth factor
Acetylcholine receptor inducing
activity (AR.IA)
Neuregulin 2 (NRG 2) ~ HER3, HER4
One of ordinary skill in the art is readily able
to identify an EGF-like polypeptide. Assays for receptor
binding and activation are routinely practiced in the
art. In the context of the present invention, these
assays can be based, for example, on the ability of both
mouse and human EGF to compete with lzSl-labeled mEGF for
CA 02337964 2001-02-05
_ WO 00/07611 PCT/US99/18022
_ g _
binding sites on human foreskin fibroblasts. For
example, lzsl_labeled mEGF binding and competition assays
can be carried out on monolayer cultures of human
foreskin fibroblasts (e.g., approximately 1 x 106
cells/60 mm Falcon dish) by incubating a standard amount
of 1251-labeled mEGF in the simultaneous presence of
aliquots containing increasing amounts of a competing
peptide (e.g., EGF-like polypeptides and substitution and
deletion mutants thereof) for 1 hour at 37°C. The
binding medium can consist of 1.5 ml of an albumin-
containing modified Dulbecco medium. Unbound lzsl-labeled
mEGF can be removed by washing, and the cells can be
solubilized by the addition of 1 ml of 0.5 M NaOH.
Radioactivity can then be measured with a gamma
spectrometer (available, e.g., from Nuclear-Chicago).
One can then generate a standard curve by plotting the
percentage of lzsI-labeled mEGF displaced versus the
amount of the polypeptide applied (Cohen and Carpenter,
Proc. Natl. Acad. Sci. USA 72:1317-1321, 1975).
Similarly, a radioreceptor assay can be carried
out on monolayers of A431 cells that have been fixed on
24-well plates with 10~ formalin in phosphate buffered
saline (PBS). Formalin-fixed cells do not slough off
plates as easily as do unfixed cells, and replicate
values are thus more consistent. Under these assay
conditions, 1251-EGF (1 x 101° cpm/nmol) saturates the
binding assay at 3 nM; assays can be performed at 10~ of
the saturation value (Twardzik et al., Proc. Natl. Acad.
Sci. USA 82:5300-5304, 1985).
The invention encompasses "functional EGF-like
polypeptides," which possess one or more of the
biological functions or activities of the polypeptide
growth factors described herein (i.e., the EGF-like
polypeptides known in the art and listed in Table 1).
These functions or activities are those required to
CA 02337964 2001-02-05
_ WO 00/07611 PCT/I1S99/18022
- g _
elicit a reduction in a neurological deficit caused by an
injury to the CNS (i.e., those functions or activities
that reduce or eliminate a sensorimotor deficit, a speech
impediment, a visual impairment, the loss of a cognitive
skill, or the loss of one's ability to form accurate
memories). Accordingly, alternate molecular forms of
EGF-like polypeptides are within the scope of the
invention (e. g. fragments and other mutants, and analogs
with one or more conservative amino acid substitutions
compared to wild type EGF-like polypeptides).
EGF-like polypeptides containing one or more
conservative amino acid substitutions that do not destroy
the biological activity of the EGF-like polypeptide
(particularly the ability of the EGF-like polypeptide to
prevent, reduce, or eliminate sensorimotor deficits) are
useful. Conservative amino acid substitutions include
substitutions within the following groups: glycine and
alanine; valine, isoleucine, and leucine; aspartic acid
and glutamic acid; asparagine and glutamine; serine and
threonine; lysine and arginine; and phenylalanine and
tyrosine. Thus, EGF-like polypeptides containing one or
more conservative amino acid substitutions (preferably at
positions other than the six conserved cysteine residues
in the EGF domain) are within the scope of the invention.
An EGF-like polypeptide that consists of a mutant
of one of the full length polypeptides listed in Table 1
will fall within the scope of the invention when, at a
given concentration, the EGF-like polypeptide displaces
at least 50%, preferably at least 70%, and more
preferably at least 90% (e.g., 95%, 97%, or even 99%) as
much 125I-labeled mEGF as a wild-type human EGF
polypeptide in one of the binding assays described
herein. For example, this can be determined by
performing a standard receptor binding assay, such as
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- IO -
that described above (see also, Cohen and Carpenter,
Proc. Natl. Acad. Sci. USA 72:1317-1321, 1975).
A mutant EGF-like polypeptide of the invention can
be a fragment of a full-length polypeptide disclosed in
5 Table 1. For example, a mutant EGF-like polypeptide can
consist of the EGF domain of an EGF-like polypeptide
(i.e., the domain conforming to the consensus sequence
CX7CX4CXIOCXCXBC (SEQ ID N0:3), e.g., amino acid residues
108-143 of SEQ ID N0:2) or the EGF domain and one or more
10 of the amino acid residues that flank the EGF domain.
Truncated polypeptides (i.e., fragments of an EGF-like
polypeptide in which one or more of the amino acid
residues on one or more of the terminal ends of an EGF-
like polypeptide) can be used to practice the invention.
15 For example, a deletion of 1, 2, 5, 10, or more amino
acid residues from the amino and/or carboxy terminals of
EGF, TGF-a, AR, HB-EGF, BTC, epiregulin, or a neuregulin
would result in a mutant EGF-like polypeptide useful in
the present invention. For example, HB-EGF polypeptides
20 useful in the invention include those that include amino
acid residues 2-208; 6-208; 11-208; 100-208; 100-145; 1-
207; 1-202; 1-198; and the like (all of SEQ ID N0:2).
Thus, EGF-like polypeptides useful in the invention can
consist of active fragments of polypeptides in the EGF
25 family. By "active fragment" is meant any portion of an
EGF-like polypeptide that, when tested in a receptor
binding assay such as those described above, displaces at
least 50~ of the ligand that would otherwise bind to the
receptor (e. g., a fragment of HB-EGF is an active
30 fragment of HB-EGF if it displaces at least 50~ as much
l2sl_labeled mEGF as a wild-type human EGF polypeptide).
Active or functional fragments of an EGF-like polypeptide
can also be assessed by their ability to improve the
neurological deficits (i.e., the cognitive and
35 sensorimotor deficits) that can result following a
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 11 -
traumatic or ischemic injury. The ability of any given
EGF-like polypeptide to improve these deficits can be
assessed by tests of cognition and motor skill that are
well known to those of ordinary skill in the art. Prior
5 to clinical trials, the efficacy of an EGF-like
polypeptide in improving a neurological deficit can be
assessed in animal models (see the forelimb placing test,
the hindlimb placing test, the modified balance beam
test, and the postural reflex test described below). The
10 active fragment will produce at least 40%, preferably at
least 50%, more preferably at least 70%, and most
preferably at least 90% (including 100% or more) of the
benefit of the full-length polypeptide. The activity of
any given polypeptide or fragment thereof can be readily
15 determined in any number of ways. For example, a
fragment of an EGF-like polypeptide that, when
administered according to the methods of the invention
described herein, is shown to produce performance in
functional tests that is comparable to the performance
20 that is produced by administration of the full-length
EGF-like polypeptide, the fragment would be an "active
fragment" of the EGF-like polypeptide and encompassed by
the invention. It is well within the abilities of those
of ordinary skill in the art to determine whether an
25 EGF-like polypeptide, regardless of, e.g., size, retains
the functional activity of the full length, wild type
EGF-like polypeptide.
A description of particular EGF-like polypeptides
follows.
30 1. Heparin-binding epidermal crrowth factor
Heparin-binding EGF (HB-EGF) was initially
identified in the conditioned medium (CM) of macrophage-
like U-937 cells (Higashiyama et al., Science 251:936-
939, 1991). It is a growth factor with a strong affinity
35 for immobilized heparin that is mitogenic for Balb/c 3T3
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 12 -
cells and smooth muscle cells (SMC) but not for
endothelial cells (EC). HB-EGF has been purified from
the CM of macrophage-like human U-937 cells using
heparin-affinity chromatography and reverse phase liquid
chromatography and cloned from a U-937 cell cDNA library.
Analysis of HB-EGF cDNA revealed an open reading frame
that predicts a primary translation product of 208 amino
acids (SEQ ID N0:2) containing a putative signal peptide,
a propeptide, the mature HB-EGF, and transmembrane and
cytoplasmic domains (see, e.g., U.S. Patent No.
5,811,393). For a description of the structural
organization and chromosomal assignment of human HB-EGF,
see Fen et aI. (Biochem. 32:7932-7938, 1993).
Various forms of HB-EGF axe useful in the present
invention. For example, a 66 amino acid form (amino acid
residues 82-147 of SEQ ID N0:2), an 86 amino acid form
(amino acid residues 63-148 of SEQ ID N0:2), the full-
length, 208 amino acid form (SEQ ID N0:2), as well as any
EGF receptor-binding forms of HB-EGF that have undergone
post-translational modification or processing can be used
to practice the invention. Such post-translational
modification may include, without limitation, a processed
amino-terminus, for example, removal of all or part of a
signal sequence or all or part of a pro sequence; a
processed carboxy-terminus, for example, removal of all
or part of a membrane-spanning domain or all or part of a
cytoplasmic domain; O-linked glycosylation; or any
combination thereof.
One source of HB-EGF is the human histiocytic
lymphoma cell line U-937, which is available from the
American Type Culture Collection (Manassas, VA) under
Accession No. CRL 1593. Methods of purification are
known to those of ordinary skill in the art (for
particular guidance on purification of HB-EGF, see U.S.
Patent No. 5,811,393). HB-EGF and other EGF-like
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 13 -
polypeptides of the invention can also be recombinantly
or synthetically produced. Thus, polypeptide growth
factors useful in the invention can be naturally
occurring, synthetic, or recombinant molecules and can
5 consist of a hybrid or chimeric polypeptide with one
portion, for example, being an EGF-like polypeptide, and
a second portion being a distinct polypeptide (e.g., a
large molecule such as albumin or a portion of an
immunoglobulin). Methods by which EGF-like polypeptides
10 can be purified from a biological sample, chemically
synthesized, or produced recombinantly by standard
techniques are well known (see e.g., Ausubel et al.,
Current Protocols in Molecular Biology, New York, ,john
Wiley and Sons, 1993; Pouwels et al., Cloning vectors:
15 A Laboratory Manual, 1985, Supp. 1987).
The HB-EGF gene is normally expressed in a variety
of tissues, including the lung, heart, and skeletal
muscle. HB-EGF has been implicated as a participant in a
variety of normal and aberrant processes such as
20 blastocyst implantation, wound healing, SMC hyperplasia,
tumor growth, and atherosclerosis (see, e.g., Fen et al.,
supra ) .
HB-EGF is also expressed in the mammalian brain.
In particular, HB-EGF mRNA is found in the cortex,
25 hippocampus, and other deep structures of the rat
forebrain. While neurons and oligodendrocytes appear to
normally express HB-EGF polypeptides, the studies
described below suggest that HB-EGF mRNA is upregulated
within a few days after focal brain injury (or "stroke").
30 The upregulation is evident in tissue surrounding focal
brain wounds or infarcts. Accordingly, it would be
reasonable to expect that HB-EGF administered prior to or
shortly after an injury to the CNS would exert a
neuroprotective effect. However, the inventors have
35 discovered that HB-EGF is ineffective in reducing infarct
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- Z4 -
volume following a focal ischemic injury. As described
further below, an EGF-like polypeptide can be
administered to a patient who has suffered an ischemic or
traumatic injury to the CNS even after a substantial
5 period of time has elapsed. Surprisingly, administration
of an EGF-like polypeptide can commence more than six or
twelve or more hours after the onset of the injury. In
the event administration of HB-EGF (or any EGF-like
polypeptide of the invention) is begun at or after this
10 time (i.e., after neurons or glial cells in the brain are
adversely affected by ischemia) the polypeptide may act
by a different mechanism (i.e., a mechanism that differs
from infarct volume reduction). For example, EGF-like
polypeptides may reduce retrograde or secondary cell
15 death and/or stimulate neuronal sprouting and synapse
formation.
2. Betacellulin
Betacellulin (BTC) was originally purified from
the conditioned media of a mouse pancreatic tumor cell
20 line. The nucleotide sequence of BTC cDNA predicts a
peptide of 178 amino acids, which is proteolytically
processed so that a soluble 80-amino acid BTC protein is
released from BTC-expressing cells. Northern blot
analysis has revealed relatively high expression in lung,
25 uterus, kidney, thymus, heart, liver, spleen, small
intestine, pancreas, muscle, and testis.
3. Neurequlins
The neuregulins (heregulin, NDF, GGF, ARIA) are a
large group of secreted and membrane-attached EGF-like
30 growth factors that are expressed as alternatively
spliced isoforms of a single gene. They are produced by
neurons and mesenchymal cells and elicit various trophic
responses such as cellular proliferation, survival, and
differentiation. The secreted forms of these
35 polypeptides are about 34-44 kDa. In general, the
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 15 -
neuregulins are produced by neurons and exert their
effects on other types of receptor-bearing cells, such as
glia and skeletal muscle cells. Mice lacking neuregulin
exhibit defects in heart development and in the
5 developing rhombencephalon, a structure that develops
onto the brain stem. These animals are also devoid of
Schwann cell precursors.
B. Treatment Regimens
A treatment regimen suitable for practicing the
methods of the invention is any regimen that, when
carried out by administering a therapeutic dose of an
EGF-like polypeptide via an appropriate route and for an
appropriate period of time, prevents, reduces, or
eliminates a neurological deficit caused by an injury to
15 the patient's CNS. The treatment regimen may be assessed
by examining one or more of the patient's motor skills
(e. g., posture, balance, grasp, or gait), cognitive
skills, speech, or sensory perception (including visual
ability, taste, olfaction, and proprioception), and
20 determining whether the sensorimotor, cognitive, or other
skill improves following treatment with an EGF-like
polypeptide.
Administration of EGF-like polypeptides can be
carried out by any known route of administration,
25 including intravenously, orally, or intracerebrally
(e.g., intraventricularly, intrathecally, or
intracisternally). Intracisternal administration can be
carried out, e.g., using 0.1 to 100 ~,g/kg/injection of an
EGF-like polypeptide (e. g., HB-EGF or an active fragment
30 thereof), and administering a single injection or a
series of injections. As an alternative to determining
dosage based on body weight, one of skill in the art
could readily administer the required dosage to
cerebrospinal fluid based on the surface area of the
CA 02337964 2001-02-05
WO 00/07611 PCTNS99/18022
- 16 -
human brain. In this event, a human patient is typically
treated with 0.01-1000 mg (e. g., 0.1, 1, 10, 100, 250,
500, 700, 800, or 900 mg) of an EGF-like polypeptide.
Intracisternal administration can consist of a single
application given, for example, 24 hours after an injury,
a pair of applications, given, for example, 24 and
48 hours after an injury, or, if necessary, a series of
applications of, for example, 1.0 mg, given biweekly (for
example, every 3-4 days) in a treatment regimen that
commences 24 hours or longer following the ischemic
episode. The treatment regimen may last a number of
weeks. Alternatively, intracisternal administration can
consist of a series of applications, at, for example, 1.0
mg, given once, twice, or, for example, biweekly, in a
treatment regimen that begins six, twelve, or twenty-four
hours or longer following the ischemic episode.
Alternatively, the polypeptide growth factors can
be administered intravenously. Typically, the dosage for
intravenous administration will be greater than that for
intracisternal administration, e.g., 1 to 1,000 ~.g/kg of
an EGF-like polypeptide can be administered. Preferably,
an EGF-like polypeptide is administered intravenously at
concentrations ranging from 1 to 100 ~.g/kg/hour.
More specifically, EGF-like polypeptides can be
administered to a patient at therapeutically effective
doses (i.e., doses sufficient to result in functional
recovery, beyond that which would be expected without
administration of the polypeptide) that are determined as
follows.
1. Effective Dose
Toxicity and therapeutic efficacy of any given
EGF-like polypeptide can be determined by standard
pharmaceutical procedures, using either cells in culture,
experimental animals, or both, to determine the LDso (the
dose that proves lethal to 50% of the population) and the
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/I8022
- 17 -
EDso (the dose that proves therapeutically effective in
50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index, which is
expressed as the ratio LDSO:EDso. EGF-like polypeptides
5 that exhibit large therapeutic indices are preferred.
However, EGF-like polypeptides that exhibit toxic side
effects can still be used, so long as the system for
delivery targets such polypeptides to the site of
affected tissue in a limited manner (i.e., a manner in
10 which potential damage to unaffected cells is minimized
and adverse side effects are thereby reduced to an
acceptable level).
The data obtained from cell culture assays and
animal studies, notably the studies of rats described
15 below, can be used in formulating a range of dosage for
use in humans. The dosage of such polypeptides lies
preferably within a range of circulating concentrations
that include the EDso with little or no toxicity. The
dosage can vary within this range depending upon the
20 dosage form employed and the route of administration.
For any EGF-like polypeptide used in a method of the
invention, the therapeutically effective dose can be
estimated initially from the studies of surgically
induced ischemia in the mammalian brain that are
25 described below.
A dose can be formulated in animal models to
achieve a circulating plasma concentration range that
includes the ICso (that is, the concentration of the test
polypeptide that achieves a half-maximal induction of
30 recovery) as determined in the in vivo studies described
below. Such information can be used, if necessary, to
more accurately determine therapeutically effective doses
in humans. Moreover, it is well known in the
medical arts that dosages for any one patient depend on
35 many f actors, including the general health, sex, weight,
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 18 -
body surface area, and age of the patient, as well as the
particular compound to be administered, the time and
route of administration, and other drugs being
administered concurrently. Determining the most
5 appropriate dosage and route of administration is well
within the abilities of a physician of ordinary skill in
the art.
2. Formulations and Use
Pharmaceutical compositions for use in accordance
with the present invention (i.e. compositions that
include an EGF-like polypeptide) can be formulated in a
conventional manner using one or more physiologically
acceptable carriers or excipients, many of which are
known to those of ordinary skill in the art. Excipients
15 can be used, for example, when pharmaceutical
compositions of the invention are administered
intracerebrally or intravenously. It is known that
another polypeptide growth factor, bFGF, can cross the
blood brain barrier when administered intravenously and
20 enters ischemic brain tissue (Fisher et al., J. Cereb.
Blood Flow Metab. 15:953-959, 1995; Huang et al., Amer.
J. Physiol. in press). Suitable excipients include
buffers (e. g., citrate buffer, phosphate buffer, acetate
buffer, and bicarbonate buffer), amino acids, urea,
25 alcohols, ascorbic acid, phospholipids, proteins (e. g.,
serum albumin), EDTA, sodium chloride, liposomes,
mannitol, sorbitol, and glycerol.
In addition to the preferred routes of
administration described above, an EGF-like polypeptide
30 can be administered orally, intraarterially,
subcutaneously, intramuscularly, intraventricularly,
intracapsularly, intra-spinally, intracisternally, or
transmucosally. Thus, an EGF-like polypeptide can be
formulated for administration by inhalation or
35 insufflation (either through the mouth or the nose) or
CA 02337964 2001-02-05
WO 00/07611 PCT/US99118022
- 19 -
for oral, buccal, intranasal, intravaginal, intraocular,
parenteral, or rectal administration.
An EGF-like polypeptide can be formulated in
various ways, depending on the route of administration.
For example, liquid solutions can be made for ingestion
or injection; gels or powders can be made for ingestion,
inhalation, or topical application. For oral
administration, pharmaceutical compositions can take the
form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable
excipients such as binding agents (e. g., pregelatinised
maize starch, polyvinylpyrrolidone or hydroxypropyl
methylcellulose); fillers (e. g., lactose,
microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or
silica); disintegrants (e. g., potato starch or sodium
starch glycolate); or wetting agents (e. g., sodium lauryl
sulphate). The tablets can be coated by methods well
known in the art. Liquid preparations for oral
administration can take the form of, for example,
solutions, syrups or suspensions, or they can be
presented as a dry product for constitution with water or
other suitable vehicle before use. Such liquid
preparations can be prepared by conventional means with
pharmaceutically acceptable additives such as suspending
agents (e.g., sorbitol syrup, cellulose derivatives or
hydrogenated edible fats); emulsifying agents (e. g.,
lecithin or acacia); non-aqueous vehicles (e. g., almond
oil, oily esters, ethyl alcohol or fractionated vegetable
oils); and preservatives (e.g., methyl or propyl-p-
hydroxybenzoates or sorbic acid). The preparations can
also contain buffer salts, flavoring, coloring, and
sweetening agents as appropriate. Methods for making
such formulations are well known and can be found in, for
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 20 -
example, "Remington's Pharmaceutical Sciences"
(A. Gennaro, Ed., Mack Publ., 1990).
An EGF-like polypeptide can be formulated for
administration so that a given dosage can be presented
either all at once or gradually over time, for example,
by a continuous infusion or from an implantable slow-
release device. Accordingly, formulations for injection
can be presented in unit dosage form, for example, in
ampules or in mufti-dose containers, with an added
preservative. The compositions can take such forms as
suspensions, solutions, or emulsions in oily or aqueous
vehicles, and can contain formulatory agents such as
suspending, stabilizing, and/or dispersing agents.
Alternatively, the active ingredient can be in powder
form for constitution with a suitable vehicle, for
example,. sterile pyrogen-free water, before use. When
gradual release is desired, an EGF-like polypeptide can
be formulated as a depot preparation. Such long acting
formulations can be administered by implantation (e. g.,
subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds can be
formulated with suitable polymeric or hydrophobic
materials (e.g., as an emulsion in an acceptable oil) or
ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
The EGF-like polypeptides can, if desired, be
presented in a pack or dispenser device, which can
contain one or more unit dosage forms containing the
active ingredient. The pack can, for example, comprise
metal or plastic foil, such as a blister pack. The pack
or dispenser device can be accompanied by instructions
for administration.
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 21 -
C. Conditions Amenable to Treatment
The methods of the invention can be used to treat
the adverse consequences of central nervous system
injuries (i.e., a sensorimotor or cognitive deficit) that
result from any of a variety of conditions, so long as
those conditions are associated with an ischemic episode.
By "ischemic episode" is meant any circumstance that
results in a deficient supply of blood to a tissue.
Cerebral ischemic episodes result from a deficiency in
the blood supply to the brain. The spinal cord, which is
also a part of the central nervous system, is similarly
susceptible to ischemia resulting from diminished blood
flow. An ischemic episode may be caused by a
constriction or obstruction of a blood vessel, as occurs
in the case of a thrombus or embolus. Alternatively, the
ischemic episode can result from any form of compromised
cardiac function, including cardiac arrest, as described
above. It is expected that the invention will also be
useful for treating injuries to the central nervous
system that are caused by mechanical forces, such as a
blow to the head or spine. Trauma can involve a tissue
insult such as an abrasion, incision, contusion,
puncture, compression, and the like, such as can arise
from traumatic contact of a foreign object with any locus
of or appurtenant to the head, neck, or vertebral column.
Other forms of traumatic injury can arise from
constriction or compression of CNS tissue by an
inappropriate accumulation of fluid (e.g., a blockade or
dysfunction of normal cerebrospinal fluid or vitreous
humor fluid production, turnover, or volume regulation,
or a subdural or intracranial hematoma or edema).
Similarly, traumatic constriction or compression can
arise from the presence of a mass of abnormal tissue,
such as a metastatic or primary tumor.
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 22 -
Other injuries may be caused by hypertension,
hypertensive cerebral vascular disease, rupture of an
aneurysm, an angioma, blood dyscrasias, cardiac failure,
cardiac arrest, cardiogenic shock, septic shock, head
trauma, spinal cord trauma, seizure, bleeding from a
tumor, or other blood loss.
Where the ischemia is associated with a stroke, it
can be either global or focal ischemia. By "focal
ischemia," as used herein in reference to the central
nervous system, is meant the condition that results from
the blockage of a single artery that supplies blood to
the brain or spinal cord, resulting in the death of all
cellular elements (pan-necrosis} in the territory
supplied by that artery. By "global ischemia," as used
herein in reference to the central nervous system, is
meant the condition that results from a general
diminution of blood flow to the entire brain, forebrain,
or spinal cord, which causes the death of neurons in
selectively vulnerable regions throughout these tissues.
The pathology in each of these cases is quite different,
as are the clinical correlates. Models of focal ischemia
apply to patients with focal cerebral infarction, while
models of global ischemia are analogous to cardiac
arrest, and other causes of systemic hypotension.
D. Experimental Models
1. Occlusion of the Middle Cerebral Artery
Occlusion of the middle cerebral artery (MCA) is a
well accepted model of a focal ischemic episode and is
thought to mimic the events that occur in humans
following a stroke (Kawamata et al., J. Cereb. Blood Flow
Metab., 16:542-547, 1996; Gotti et al., Brain Res.
522:290-307, 1990). To generate this model in rodents,
one can use, for example, male Sprague-Dawley rats
weighing 250-300 grams. For surgical procedures, the
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 23 -
animals are anesthetized with 2% halothane in
70% NOz/30% Oz, and the tail artery is cannulated to
enable blood gas and blood glucose monitoring. Body
temperature can be monitored using a rectal probe and
should be maintained at 37 ~ 0.5°C, for example, by
placing the animal on a heating pad. The proximal right
middle cerebral artery (MCA) is occluded permanently
using a modification of the method of Tamura et al.
(J. Cereb. Blood Flow Metab. 1:53-60, 1981). Briefly,
the proximal MCA is exposed transcranially without
removing the zygomatic arch or transecting the facial
nerve. The artery is then electrocoagulated using a
bipolar microcoagulator from just proximal to the
olfactory tract to the inferior cerebral vein, and is
then transected (Bederson et al., Stroke 17:472-476,
1986). Animals should be observed until they regain
consciousness and can then be returned to their home
cages. Cefazolin sodium (40 mg/kg, i.p.), an antibiotic,
may be administered to all animals on the day before and
just after stroke surgery in order to prevent infection.
2. Occlusion of the Common Carotid Arteries
Using the procedures described above to
anesthetize rodents, one can, in lieu of occluding the
MCA, expose and occlude the common carotid arteries
(e.g., with a sterile suture) for variable lengths of
time (typically, the occlusion is applied for two to five
minutes). This procedure mimics a global ischemic
episode.
3. Traumatic Head Inlurv
Those of ordinary skill in the art are aware of
and able to induce traumatic head injuries by applying a
particular (i.e., defined) force to an animal's skull.
Similarly, the spinal cord may be injured by surgically
aspirating or otherwise severing it according to methods
known to those of ordinary skill in the art.
CA 02337964 2001-02-05
WO 00107611 PCT/US99/18022
- 24 -
E. Evaluation of Neurological Skills
To accustom experimental animals to handling,
which would be necessary for behavioral/functional
testing, they can be handled, for example, for three days
5 before surgery, for 10 minutes each day.
Following is a brief description of five
functional/behavioral tests that can be used to assess
sensorimotor and reflex function after an injury to the
CNS. The full details of these tests have been described
10 elsewhere (Bederson et al., Stroke 17:472-476, 1986;
DeRyck et al., Brain Res. 573:44-60, 1992; Markgraf et
al., Brain Res. 575:238-246, 1992; Alexis et al., Stroke
26:2338-2346, 1995). Those of ordinary skill in the art
are well able to assess sensorimotor deficits by other
15 tests known and used routinely in the art.
1. The Forelimb Placing Test
Briefly, the forelimb placing test is comprised of
three subtexts. Separate scores are obtained for each
forelimb. For the visual placing subtext, the animal is
20 held upright by the researcher and brought close to a
table top. Normal placing of the limb on the table is
scored as "0," delayed placing (< 2 sec) is scored as
"1," and no or very delayed placing (> 2 sec) is scored
as "2." Separate scores are obtained first as the animal
25 is brought forward and then again as the animal is
brought sideways to the table (maximum score per
limb = 4; in each case higher numbers denote greater
deficits). For the tactile placing subtext, the animal
is held so that it cannot see the table top or touch it
30 with its whiskers. The dorsal forepaw is touched lightly
to the table top as the animal is first brought forward
and then brought sideways to the table. Placing each
time is scored as above (maximum score per limb = 4).
For the proprioceptive placing subtext, the animal is
35 brought forward only and greater pressure is applied to
CA 02337964 2001-02-05
WO 00/07611 PC'T/US99/18022
- 25 -
the dorsal forepaw; placing is scored as above (maximum
score per limb = 2). These subscores are added to give
the total forelimb placing score per limb (range = 0-10).
2. The Hindlimb Placing Test
The hindlimb placing test is conducted in the same
manner as the forelimb placing test but involves only
tactile and proprioceptive subtests of the hindlimbs
(maximal scores 4 and 2, respectively; total score
range = 0-6).
3. The Modified Balance Beam Test
The modified balance beam test examines
vestibulomotor reflex activity as the animal balances on
a long, narrow beam (30 x 1.3 cm) for 60 seconds.
Ability to balance on the beam is scored as follows:
1 = animal balances with all four paws on top of beam;
2 - animal puts paws on side of beam or wavers on beam;
3 - one or two limbs slip off beam; 4 = three limbs slip
off beam; 5 = animal attempts to balance with paws on
beam but falls off; 6 = animal drapes over beam, then
falls off; 7 = animal falls off beam without an attempt
to balance. Animals can receive training trials (e. g.,
three training trials) before surgery.
4. The Postural Reflex Test
The postural reflex test measures both reflex and
sensorimotor function. Animals are first held by the
tail suspended above the floor. Animals that reach
symmetrically toward the floor with both forelimbs are
scored "0." Animals showing abnormal postures (flexing
of a limb, rotation of the body) are then placed on a
plastic-backed sheet of paper. Those animals able to
resist side-to-side movement with gentle lateral pressure
are scored "1," while those unable to resist such
movement are scored "2." All functional/behavioral tests
are administered just before surgery (or other means of
inducing injury) and then every other day from, e.g.,
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 26 -
day 1 to, e.g., day 31. At each session, animals should
be allowed to adapt to the testing room for 30 minutes
before testing is begun.
5. Morris Water Maze
Cognitive skills and memory are routinely
evaluated in rodents by the Morris Water Maze, which
tests an animal's ability to remember the position of a
submerged platform in a pool of water.
F. Histoloaical Analysis of the Iniured CNS
1. Determining Infarct Volume
Following an injury to the CNS (e. g., 31 days
after MCA occlusion), animals can be anesthetized deeply
with pentobarbital and perfused transcardially with
heparinized saline followed by 10~ buffered formalin.
Their brains are then removed, cut into three pieces, and
stored in 10~ buffered formalin before dehydration and
embedding in paraffin. Coronal sections (5 ~,m) are cut
on a sliding microtome, mounted onto glass slides, and
stained with hematoxylin and eosin. The area of cerebral
infarcts on each of seven slices (e. g., +4.7, +2.7, +0.7,
-1.3, -3.3, -5.3, and -7.3 compared to bregma) can be
determined using a computer-interfaced imaging system
(Bioquant, R&M Biometnix, Inc., Nashville, TN). Total
infarct area per slice is determined by the "indirect
method" as [the area of the intact contralateral
hemisphere] - [the area of the intact ipsilateral
hemisphere] to correct for brain shrinkage during
processing (Swanson et al., J. Cereb. Blood Flow Meta.b.
10:290-293, 1990). Infarct volume is then, if desired,
expressed as a percentage of the intact contralateral
hemispheric volume. The volumes of infarction in cortex
and striatum can also be determined separately using
these methods.
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 27 -
Ideally, the experimenter performing
intracisternal injections, behavioral testing, and
histological analysis is blinded to the treatments
assigned until all data have been collected. Data can be
expressed as means ~ SD or means ~ SEM and can be
analyzed by repeated measures analysis of variance
(ANOVA) followed by appropriate unpaired two-tailed
t-tests, with the Bonferroni correction for multiple
comparisons.
2. Immunostaining for Growth Associated Protein-
43
Growth Associated Protein-43 (GAP-43) is a
phosphoprotein component of the neuronal membrane and
growth cone that is selectively upregulated during new
axonal growth in both the peripheral and central nervous
systems (Skene, Ann. Rev. Neurosci. 12:127-156, 1989;
Aigner et al., Cell 83:269-278, 1995; Woolf et al.,
Neuroscience 34:465-478, 1990; Benowitz et al., Mol.
Brain Res. 8:17-23, 1990). GAP-43 has been used as a
reliable marker of new axonal growth during brain
development, and following brain injury or ischemia
(Stroemer et al., Stroke 26:2135-2144, 1995; Benowitz
et al. supra; Vaudano et al., J. Neurosci. 15:3594-3611,
1995). GAP-43 immunoreactivity (IR) can be examined in
animals with focal infarcts (produced by MCA occlusion as
described above) that either receive or do not receive an
EGF-like polypeptide.
For histological analysis, animals are killed, for
example, 3, 7, or 14 days post-stroke surgery (by MCA
occlusion) by transcardial perfusion fixation with normal
saline followed by 2~ formaldehyde, 0.01 M sodium-m-
periodate, and 0.075 M L-lysine monohydrochloride in
0.1 M sodium phosphate buffer (pH 7.4; PLP solution).
Their brains are removed, post-fixed, and cut into 40 ~,m
sections on a vibratome. The sections are cryoprotected.
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 28 -
Free-floating sections are successively incubated
in 20% normal goat serum, a mouse monoclonal antibody to
GAP-43 (1:500, clone 91E12, Boehringer-Mannheim,
Indianapolis, IN), and biotinylated horse anti-mouse IgG
adsorbed against rat IgG (45 x,1/10 ml; Vector,
Burlingame, CA). Sections are then mounted onto glass
slides, air dried, immersed in gradient ethanol, and
coverslipped. Brain sections from all animals at each
time point (i.e., animals sacrificed 3, 7, or 14 days
post-stroke surgery) were immunostained simultaneously.
Control sections were processed without primary antibody
and showed no specific staining.
Following immunostaining, two standard coronal
sections through the cerebral infarcts can be examined;
e.g., an "anterior" section at +0.2 mm compared to bregma
and a "posterior" section at -2.8 mm compared to bregma.
The relative changes in the intensity and extent of
GAP-43 immunoreactivity (IR) can be quantified using, for
example, a computer-interfaced imagining system
(Bioquant, Nashville, TN) by two different methods.
Adjacent brain sections, stained with hemotoxylin and
eosin by standard procedures, can be used to identify the
extent of the infarct. The optical density (O.D.) of a
region of reliably low GAP-43 IR (the corpus callosum)
may be considered the "background" value for each
section.
Measurements can be made in two ways. In one way,
all brain regions showing an O.D. of at least 1.5 times
the O.D. of the background are identified and
highlighted. The area (in mm2) of highlighted regions in
the dorsolateral sensorimotor cortex is determined for
each slice, and averaged among animals in each group. In
a second way, specific regions of dorsolateral
sensorimotor cortex are identified using a published
standard rat brain atlas (Paxinos and Watson, "The Rat
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 29 -
Brain in Stereotaxic Coordinates," Academic Press, San
Diego, CA). On "anterior" brain sections, these include
the medial peri-infarct cortex (s1 mm from the infarct
border) in the ipsilateral hemisphere, and frontal cortex
areas 1 and 2 (FR 1,2) and forelimb area of cortex (FL)
regions in both hemispheres. On "posterior" sections,
these include the medial peri-infarct region in the
ipsilateral hemisphere, as well as FR 1,2 and hindlimb
area of cortex (HL) regions bilaterally. The O.D. is
determined for each region on each section and normalized
to background. For each method, data in sham or vehicle-
treated and data in sham or bFGF-treated animals can also
be determined. Data in all groups are expressed as ratios
compared to stroke/vehicle-treated animals.
Exam~l es
Example 1: HB-EGF mRNA Expression
It is known that messenger RNA (mRNA) coding for
HB-EGF is expressed in the central nervous system (CNS).
In the present study, expression of HB-EGF mRNA and
protein was examined by Northern analysis, in situ
hybridization, and immunohistochemistry. Northern
analysis revealed transcripts for HB-EGF in all regions
of normal rat brain. In situ hybridization studies showed
that neurons in various regions, including cortex,
hippocampus, and deep structures, express HB-EGF mRNA.
Positively labeled cells were also present in white
matter, suggesting that both neurons and glia express HB-
EGF mRNA.
Immunohistvchemical studies with an antibody
specific to proHB-EGF, a transmembrane form of HB-EGF,
demonstrated ubiquitous immunoreactivity in neurons and
glial cells in white matter. In view of the wide
expression of its cognitive receptor, EGFR, in central
nervous system neurons, these results suggest that HB-EGF
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 30 -
is an endogenous ligand for EGFR in the central nervous
system and may play an important role in physiological
conditions.
Example 2: Receptor Binding Studies
Routine cross-linking analysis using l2sl-growth
factors bound to cortical neuronal cells demonstrated
that: (1) EGF binds to HERl, (2) NRG binds to HER4, and
(3) HB-EGF and BTC bind to both HER1 and HERO.
Example 3: HB-EGF treatment of serum-starved
aortic smooth muscle cells can induce basic
fibroblast growth factor (bFGF) gene expression
HB-EGF is a vascular smooth muscle cell (SMC)
mitogen and chemotactic factor that is expressed by
endothelial cells, SMCs, monocytes/macrophages, and
T lymphocytes. Both the membrane-anchored HB-EGF
precursor and the secreted mature HB-EGF protein are
biologically active; thus, HB-EGF may stimulate SMC
growth via autocrine, paracrine, and juxtacrine
mechanisms.
HB-EGF treatment of serum-starved aortic SMCs
induced fibroblast growth factor (FGF)-2 (basic FGF) gene
expression, but not FGF-1 (acidic FGF) gene expression.
Increased FGF-2 mRNA expression was first detectable at
1 hour after HB-EGF addition, and maximal FGF-2 mRNA
levels, corresponding to an approximately 46-fold
increase in expression, were present after 4 hours. The
effect of HB-EGF on FGF-2 mRNA levels appears to be
mediated primarily by a transcriptional mechanism and
requires proteins synthesized de novo. Western blot
analyses indicated that HB-EGF-treated SMCs also produced
an increased amount of bFGF protein.
HG-EGF induction of FGF-2 mRNA levels can be
inhibited by treating cells with the anti-inflammatory
glucocorticoid dexamethasone or the glycosaminoglycan
heparin. Taken together, these results indicate that HB-
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
- 31 -
EGF expressed at sites of vascular injury or inflammation
in vivo may upregulate FGF-2 production by SMCs.
Other Embodiments
It is to be understood that while the invention
has been described in conjunction with the detailed
description thereof, that the foregoing description is
intended to illustrate and not limit the scope of the
invention, which is defined by the scope of the appended
claims.
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
1/3
SEQUENCE LISTING
<110> The General Hospital Corporation
<120> TREATMENT OF CENTRAL NERVOUS SYSTEM ISCHEMIA OR TRAUMA WITH
EPIDERMAL GROWTH FACTOR-LIKE POLYPEPTIDES
<130> 00786/400W01
<150> US 60/095,830
<151> 1999-08-06
<160> 3
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 888
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (262)...(885)
<400> 1
gctacgcggg ccacgctgct gcggggtcgg 60
ggctggcctg gcggccgcgc
acctaggcgc
gggcgggctg agtgagcaag gagctgcgcc
220
acaagacact tgggtcccgg
caagaagagc
ccaggcttgc acgcagaggc cggaatctcc
180
gggcggcaga tgagctccgc
cggtgcccgg
cgcccagctc tggtgccagc aaagtgactg
240
gcccagtggc gtgcctcgcc
cgccgcttcg
gcctcctctc ggtgcgggac atgaagctg ctgccgtcg gtggtgctg aag 291
c
MetLysLeu LeuProSer ValValLeu Lys
1 5 10
ctcttt ctggetgca gttctctcggca ctggtgact ggcgagagc ctg 339
LeuPhe LeuAlaAla ValLeuSerAla LeuValThr GlyGluSer Leu
15 20 25
gagcgg cttcggaga gggctagetget ggaaccagc aacccggac cct 387
GluArg LeuArgArg GlyLeuAlaAla GlyThrSer AsnProAsp Pro
30 35 40
cccact gtatccacg gaccagctgcta cccctagga ggcggccgg gac 435
ProThr ValSerThr AspGlnLeuLeu ProLeuGly GlyGlyArg Asp
45 50 55
cggaaa gtccgtgac ttgcaagaggca gatctggac cttttgaga gtc 483
ArgLys ValArgAsp LeuGlnGluAla AspLeuAsp LeuLeuArg Val
60 65 70
acttta tcctccaag ccacaagcactg gccacacca aacaaggag gag 531
ThrLeu SerSerLys ProGlnAlaLeu AlaThrPro AsnLyeGlu Glu
75 80 85 90
cacggg aaaagaaag aagaaaggcaag gggctaggg aagaagagg gac 579
HisGly LyeArgLys LysLysGlyLys GlyLeuGly LysLysArg Asp
95 100 105
ccatgt cttcggaaa tacaaggacttc tgcatccat ggagaatgc aaa 627
ProCys LeuArgLys TyrLysAspPhe CysIleHis GiyGluCys Lys
110 115 120
tatgtg aaggagctc cgggetccctcc tgcatctgc cacccgggt tac 675
TyrVal LysGluLeu ArgAlaProSer CysIleCys HisProGly Tyr
125 130 135
CA 02337964 2001-02-05
WO 00/07611 PCT/US99/18022
2/3
catggagagagg tgtcatggg ctgagcctc ccagtggaa aatcgctta 723
HisGlyGluArg CysHisGly LeuSerLeu ProValGlu AsnArgLeu
140 145 150
tatacctatgac cacacaacc atcctggcc gtggtgget gtggtgctg 771
TyrThrTyrAsp HisThrThr IleLeuAla ValValAla ValValLeu
155 160 165 170
tcatctgtctgt ctgctggtc atcgtgggg cttctcatg tttaggtac 819
SerSerValCys LeuLeuVal IleValGly LeuLeuMet PheArgTyr
175 180 185
cataggagagga ggttatgat gtggaaaat gaagagaaa gtgaagttg 867
HisArgArgGly GlyTyrAsp ValGluAsn GluGluLys ValLysLeu
190 195 200
ggcatgactaat tcccactga 888
GlyMetThrAsn SerHis
205
<210> 2
<211> 208
<212> PRT
<213> Homo sapiens.
<400> 2
Met Lys Leu Leu Pro Ser Val Val Leu Lys Leu Phe Leu Ala Ala Val
1 5 10 15
Leu Ser Ala Leu Val Thr Gly Glu Ser Leu Glu Arg Leu Arg Arg Gly
20 25 30
Leu Ala Ala Gly Thr Ser Asn Pro Asp Pro Pro Thr Val Ser Thr Asp
35 40 45
Gln Leu Leu Pro Leu Gly Gly Gly Arg Asp Arg Lys Val Arg Asp Leu
50 55 b0
Gln Glu Ala Asp Leu Asp Leu Leu Arg Val Thr Leu Ser Ser Lys Pro
65 70 75 80
Gln Ala Leu Ala Thr Pro Asn Lys Glu Glu His Gly Lys Arg Lys Lye
85 90 95
Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys Leu Arg Lys Tyr
100 105 110
Lys Asp Phe Cys Ile His Gly Glu Cys Lys Tyr Val Lys Glu Leu Arg
115 120 125
Ala Pro Ser Cys Ile Cys His Pro Gly Tyr His Gly Glu Arg Cys His
130 135 140
Gly Leu Ser Leu Pro Val Glu Asn Arg Leu Tyr Thr Tyr Asp His Thr
145 150 155 160
Thr Ile Leu Ala Val Val Ala Val Val Leu Ser Ser Val Cys Leu Leu
165 170 175
Val Ile Val Gly Leu Leu Met Phe Arg Tyr His Arg Arg Gly Gly Tyr
180 185 190
Asp Val Glu Asn Glu Glu Lys Val Lys Leu Gly Met Thr Asn Ser His
195 200 205
<210> 3
<211> 36
<212> PRT
<213> Homo sapiens
<220>
<221> VARIANT
<222> (1)...(36)
<223> Xaa = Any Amino Acid
<400> 3
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa
1 5 10 15
CA 02337964 2001-02-05
WO 00/07611 PCTNS99/18022
3/3
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Cys
35
