U.S. patent application number 14/594821 was filed with the patent office on 2015-07-02 for chimeric neuregulins and method of making and use thereof.
The applicant listed for this patent is Brain-Gen Biotech. Invention is credited to Byron D. FORD.
Application Number | 20150183844 14/594821 |
Document ID | / |
Family ID | 50339066 |
Filed Date | 2015-07-02 |
United States Patent
Application |
20150183844 |
Kind Code |
A1 |
FORD; Byron D. |
July 2, 2015 |
CHIMERIC NEUREGULINS AND METHOD OF MAKING AND USE THEREOF
Abstract
Composition containing a chimeric neuregulin polypeptides and
method of making such polypeptides are disclosed. The chimeric
neuregulin comprises a first moiety of at least 10 amino acids,
wherein the first moiety is derived from a first polypeptide; and a
second moiety of at least 5 amino acids, wherein the second moiety
is derived from a second polypeptide; wherein the first polypeptide
is a neuregulin and the chimeric neuregulin exhibits an enhanced
binding affinity to integrin, Erb 3, or Erb 4 comparing to that of
the first neuregulin.
Inventors: |
FORD; Byron D.; (Duluth,
GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Brain-Gen Biotech |
Duluth |
GA |
US |
|
|
Family ID: |
50339066 |
Appl. No.: |
14/594821 |
Filed: |
January 12, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13865812 |
Apr 18, 2013 |
8933034 |
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14594821 |
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13627555 |
Sep 26, 2012 |
8748131 |
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13865812 |
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Current U.S.
Class: |
424/94.64 ;
514/7.6; 514/8.3; 514/9.4; 530/300; 530/345 |
Current CPC
Class: |
A61K 47/60 20170801;
A61K 38/482 20130101; A61K 38/49 20130101; C07K 14/4756 20130101;
C07K 2319/74 20130101; C12Y 304/21068 20130101; A61P 9/00 20180101;
A61K 45/06 20130101; A61K 38/1883 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61P 25/00 20180101; A61K 38/49 20130101;
A61K 38/1883 20130101 |
International
Class: |
C07K 14/475 20060101
C07K014/475; A61K 47/48 20060101 A61K047/48; A61K 45/06 20060101
A61K045/06; A61K 38/18 20060101 A61K038/18; A61K 38/48 20060101
A61K038/48 |
Claims
1. A chimeric neuregulin polypeptide having an integrin domain that
binds to an integrin and an Erb3/4 binding domain that binds to
Erb3 and/or Erb4, said chimeric neuregulin polypeptide comprising:
a neuregulin backbone derived from a native neuregulin polypeptide;
and a donor fragment of at least one amino acid, wherein said donor
fragment (1) replaces a target fragment in said native neuregulin
polypeptide, wherein said donor fragment differs from said target
fragment by at least one amino acid, or (2) is inserted into an
insertion site of said neuregulin polypeptide; and wherein said
donor fragment forms at least a portion of said integrin binding
domain and/or at least a portion of said Erb3/4 binding domain of
said chimeric neuregulin polypeptide.
2. The chimeric neuregulin polypeptide of claim 1, wherein said
chimeric neuregulin polypeptide has an enhanced binding affinity to
integrin, Erb 3, or Erb 4, comparing to said native neuregulin
peptide.
3. The chimeric neuregulin polypeptide of claim 1, wherein said
donor fragment comprises a polypeptide derived from another
neuregulin that is different from said native neuregulin.
4. The chimeric neuregulin polypeptide of claim 1, wherein said
donor fragment comprises a polypeptide derived from a
non-neuregulin.
5. The chimeric neuregulin polypeptide of claim 1, wherein said
donor fragment comprises polypeptides derived from two neuregulins
that are different from said native neuregulin.
6. The chimeric neuregulin polypeptide of claim 1, wherein said
original neuregulin polypeptide is NRG2 or a fragment of NRG2, and
wherein said donor fragment comprises a polypeptide derived from
NRG1.beta..
7. The chimeric neuregulin polypeptide of claim 1, wherein said
native neuregulin polypeptide is NRG4 or a fragment of NRG4, and
wherein said donor fragment comprises a polypeptide derived from
NRG1, or a polypeptide derived from NRG2, or both a polypeptide
derived from NRG1 and a polypeptide derived from NRG2.
8. The chimeric neuregulin polypeptide of claim 1, further
comprising a second donor fragment of at least one amino acid,
wherein said second donor fragment (1) replaces a second target
fragment in said native neuregulin polypeptide, wherein said second
donor fragment differs from said second target fragment by at least
one amino acid, or (2) is inserted into an insertion site of said
native neuregulin polypeptide,
9. The chimeric neuregulin polypeptide of claim 1, wherein said
chimeric neuregulin polypeptide is a PEGylated.
10. A pharmaceutical composition, comprising: an effective amount
of the chimeric neuregulin polypeptide of claim 1, and a
pharmaceutically acceptable carrier.
11. A chimeric neuregulin, comprising: a first moiety of at least
10 amino acids, wherein said first moiety is derived from a first
polypeptide; and a second moiety of at least 5 amino acids, wherein
said second moiety is derived from a second polypeptide; wherein
said first polypeptide is a first neuregulin, and wherein said
chimeric neuregulin exhibits an enhanced binding affinity to
integrin, Erb 3, or Erb 4 comparing to that of said first
neuregulin.
12. The chimeric neuregulin of claim 11, wherein said second
polypeptide is a neuregulin that is different from said first
neuregulin.
13. The chimeric neuregulin of claim 11, wherein said second
polypeptide is a non-neuregulin polypeptide.
14. The chimeric neuregulin of claim 11, further comprising a third
moiety derived from a third polypeptide.
15. The chimeric neuregulin of claim 14, wherein said third
polypeptide is a neuregulin that is different from said native
neuregulin.
16. The chimeric neuregulin of claim 14, wherein said second
polypeptide is a second neuregulin and said third polypeptide is a
third neuregulin, wherein said first neuregulin, said second
neuregulin and said third neuregulin are different from each
other.
17. The chimeric neuregulin polypeptide of claim 11, wherein said
first neuregulin is NRG2, and wherein said second polypeptide is
NRG1.beta..
18. The chimeric neuregulin of claim 11, wherein said first
neuregulin is NRG4, and wherein said second polypeptide is NRG1 or
NRG2.
19. The chimeric neuregulin of claim 11, wherein said first moiety
comprises a NRG integrin binding domain or a NRG Erb3/4 binding
domain.
20. The chimeric neuregulin of claim 11, wherein said first moiety
comprises a NRG integrin binding domain, and wherein said second
moiety comprises a NRG Erb3/4 binding domain.
21. The chimeric neuregulin of claim 11, wherein said first moiety
comprises a NRG Erb3/4 binding domain, and wherein said second
moiety comprises a NRG integrin binding domain.
22. A pharmaceutical composition, comprising: an effective amount
of the chimeric neuregulin polypeptide of claim 11, and a
pharmaceutically acceptable carrier.
23. A method for ameliorating neuronal damage in a subject,
comprising: administering to said subject an effective amount of
the pharmaceutical composition of claim 22.
24. The method of claim 23, wherein said neuronal damage is caused
by an occlusive stroke and wherein said pharmaceutical composition
is administered within 72 hours of said occlusive stroke.
25. The method of claim 24, wherein said pharmaceutical composition
is administered in conjunction with t-PA.
26. The method of claim 24, wherein said pharmaceutical composition
is administered in conjunction with a glutamate receptor
inhibitor.
27. The method of claim 24, wherein said pharmaceutical composition
is administered after the initial inflammation due to the occlusive
stroke has subsided to enhance migration of stem cells from a
ventricle into an area of the brain damaged by the occlusive
stroke.
28. The method of claim 23, wherein said neuronal damage is caused
by exposure to a neurotoxin and wherein said pharmaceutical
composition is administered within 72 hours of said exposure.
29. The method of claim 28, wherein said neurotoxin is an
organophosphate.
30. The method of claim 28, wherein the composition is administered
in conjunction with an antidotes to neurotoxin or an
anticonvulsant.
31. The method of claim 23, wherein said neuronal damage is caused
by acute CNS injuries, and wherein said composition is administered
within 72 hours of the cause of CNS injury.
32. The method of claim 23, wherein said neuronal damage is caused
by a neurodegenerative disorder.
33. The method of claim 32, wherein said neurodegenerative disorder
is Alzheimer's disease, Parkinson's disease, Huntington's disease
or amyotrophic lateral sclerosis.
34. A method for preventing or ameliorating secondary neuronal
injury and inflammation following traumatic brain injury (TBI),
comprising: administering into a subject in need of such treatment
an effective amount of the pharmaceutical composition of claim
22.
35. A method for ameliorating blood vessel damage caused by acute
mechanical or chemical assault in a subject, comprising:
administering to said subject an effective amount of the
pharmaceutical composition of claim 22.
36. The method of claim 35, wherein said blood vessel damage is
caused by placement of a balloon or stent in a blood vessel,
diagnostic cardiac catheterization, or cardiac surgery.
Description
[0001] This application is a continuation application of U.S.
patent application Ser. No. 13/865,812, filed on Apr. 18, 2013,
which is a continuation application of U.S. patent application Ser.
No. 13/627,555, filed on Sep. 26, 2012, now U.S. Pat. No.
8,748,131. The entirety of the aforementioned application is
incorporated herein by reference.
FIELD
[0002] This application generally relates to compositions
containing a chimeric neuregulin and methods for prevention and
treatment of neuronal and vascular damages with chimeric
neuregulins.
BACKGROUND
[0003] Neuregulins are a family of multipotent growth factors that
includes acetylcholine receptor inducing activities (ARIAs), growth
factors, heregulins, and neu differentiation factors. Neuregulins'
effects appear to be mediated by interaction with a class of
tyrosine kinase receptors related to the epidermal growth factor
receptor. Neuregulins stimulate the tyrosine phosphorylation of
these receptors and the subsequent activation of various signal
transduction mechanisms. Neuregulins are synthesized as
transmembrane precursors consisting of either an
immunoglobulin-like or cysteine-rich domain, and EGF-like domain a
transmembrane domain and acytoplastic tail. Neuregulins have been
known to be involved in the survival and function of neuronal
cells. Neuregulin is also expressed in vascular endothelial cells
and its receptors are localized in the underlying smooth muscle
cells.
SUMMARY
[0004] One aspect of the present application relates to a chimeric
neuregulin polypeptide having an integrin domain that binds to an
integrin and an Erb3/4 binding domain that binds to Erb3 and/or
Erb4. The chimeric neuregulin polypeptide comprises a neuregulin
backbone derived from a native neuregulin polypeptide, and a donor
fragment of at least one amino acid, wherein the donor fragment (1)
replaces a target fragment in the native neuregulin polypeptide,
wherein the donor fragment differs from the target fragment by at
least one amino acid, or (2) is inserted into an insertion site of
the native neuregulin polypeptide, and wherein the donor fragment
forms at least a portion of the integrin binding domain and/or at
least a portion of the Erb3/4 binding domain of the chimeric
neuregulin polypeptide.
[0005] Another aspect of the present application relates to a
chimeric neuregulin, comprising: a first moiety of at least 10
amino acids, wherein the first moiety is derived from a first
polypeptide; and a second moiety of at least 5 amino acids, wherein
the second moiety is derived from a second polypeptide; wherein the
first polypeptide is a neuregulin and wherein the chimeric
neuregulin exhibits an enhanced binding affinity to integrin, Erb
3, or Erb 4 comparing to that of the neuregulin.
[0006] Another aspect of the present application relates to a
pharmaceutical composition comprising the chimeric neuregulin of
the present application and a pharmaceutically acceptable
carrier.
[0007] Another aspect of the present application relates to a
method for ameliorating neuronal damage in a subject. The method
comprises administering to the subject an effective amount of the
pharmaceutical composition of the present application.
[0008] Another aspect of the present invention relates to a method
for preventing or ameliorating secondary neuronal injury and
inflammation following traumatic brain injury (TBI). The method
comprises administering into a subject in need of such treatment an
effective amount of the pharmaceutical composition of the present
application.
[0009] Another aspect of the present invention relates to a method
for ameliorating blood vessel damage caused by acute mechanical or
chemical assault in a subject. The method comprises administering
to said subject an effective amount of the pharmaceutical
composition of the present application.
[0010] Other aspects of the invention will become apparent to the
skilled artisan by the following description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The invention of this application is better understood in
conjunction with the following drawing, in which:
[0012] FIG. 1 is a protein sequence alignment of the integrin
binding domain and Erb3/4 binding domain of various
neuregulins.
[0013] FIG. 2 shows the consensus sequences of NRG1, NRG2, NRG3 and
NRG4.
[0014] FIG. 3 shows the amino acid sequences of certain chimeric
neuregulin.
[0015] FIGS. 4A-4F are pictures showing ErbB4 receptor expression
in apoptotic and degenerating neurons. After ErbB4
immunohistochemistry, brain sections from rats MCAO were stained
with Fluoro-Jade, a marker for degenerating neurons. Many neurons
in the cortex were Fluoro-Jade-positive (FIG. 4A). ErbB4 positive
cells (FIG. 4B) were co-localized in Fluoro-Jade-positive neurons
(FIG. 4C). Similarly, TUNEL staining (FIG. 4D) and ErbB4 (FIG. 4E)
were double-labeled (FIG. 4F) in a subpopulation of cells in the
ipsilateral brain. Arrows indicate examples of double-labeled
cells. Scale bar is 40 .mu.M in panels A-C and 20 .mu.M in FIGS.
4D-4F.
[0016] FIGS. 5A-5D are pictures showing ErbB4 expression in
macrophages/microglia but not astrocytes following MCAO. Sections
from the ipsilateral hemisphere were double labeled with antibodies
against ErbB4 (FIG. 5A) and GFAP (FIG. 5B). Cells in the
peri-infarct regions did not show co-localization of ErbB4 and GFAP
(FIG. 5C). Co-localization of ErbB4 and Mac-1/CD11b indicated that
ErbB4 is found in a subset of macrophages/microglia (FIG. 5D).
(Double arrows indicate examples of double labeled cells). Scale
bar is 40 .mu.M in panel A-C and 20 .mu.M in FIG. 5D.
[0017] FIGS. 6A-6D are pictures and graphs showing that NRG1.beta.
treatment reduce MCAO/reperfusion-induced brain infarction.
Representative 2,3,5-triphenyltetrazolium chloride (TTC) stained
brain sections are shown from rats injected with vehicle (FIG. 6A;
n=11), NRG-1.beta. (FIG. 6B; n=7) or NRG-1.alpha. (FIG. 6C; n=3)
before MCAO. Infarct volumes in brains from vehicle and NRG-1
treated animals are shown in the graph (FIG. 6D). Values are
presented as mean.+-.SEM; * denotes significantly different from
respective vehicle treated animals (P<0.01).
[0018] FIGS. 7A-7E are pictures showing that NRG1.beta. suppresses
MCAO/reperfusion-induced apoptotic damage in rat brain. Rats were
subjected to MCAO for 1.5 hours followed by reperfusion for 24
hours (representative views are shown for TUNEL labeling of rat
brain sections; n=5 for each condition). TUNEL staining is found in
the cortex (FIG. 7A) and striatum (FIG. 7B) following MCAO while no
TUNEL staining is seen in the cortex (FIG. 7C) and reduced levels
are seen in the striatum (FIG. 7D) in NRG1.beta.-treated rats. The
coronal brain image (.about.bregma+1.2 mm) indicates the areas
observed in the sections (FIG. 7E). Scale bar is 100 uM.
[0019] FIGS. 8A-8E are pictures and graphs showing that NRG1
treatment reduces MCAO/reperfusion-induced brain infarction.
Representative TTC stained coronal brain sections are shown where
rats were injected with vehicle (FIG. 8A) or NRG1 immediately after
MCAO (FIG. 8B) and 4 hours after reperfusion (FIG. 8C). Infarct
volumes in brains from rats treated with vehicle (n=10) or NRG1
immediately after MCAO (RO; n=8), 4 hours after reperfusion (R4;
n=6) or 12 hours after reperfusion (R12; n=8) are show in the graph
(FIG. 8D). Values are presented as mean.+-.SD of all infarct
volumes for each experimental condition; * denotes significantly
different from respective vehicle treated animals (P.sub.<0.01).
The time line (FIG. 8E) illustrates the MCAO protocol and NRG1
injections.
[0020] FIG. 9 is a graph showing that NRG1 administration resulted
in a significant improvement in neurological outcome (* denotes
P<0.01). NRG1 was administered after MCAO and 4 hours of
reperfusion. Neurological function was graded on a scale of 0-4
(normal score 0, maximal deficit score 4). All animals were tested
prior to surgery (controls; n=14) and after treatment with NRG1 or
vehicle. The NRG1 treated group (n=9) displayed a 33% improvement
in neurological score compared with vehicle treated rats (n=5).
[0021] FIGS. 10A-10F are pictures showing that NRG1.beta. prevents
microglial and astrocytic activation following MCAO. Rats were
subjected to MCAO followed by reperfusion for 24 hours (n=5 for
each condition). NRG1.beta. or vehicle was injected into the ECA.
Sections were labeled for immunohistochemistry with an antibody
against ED-1. While no staining was seen in the contralateral side
(FIG. 10A), ED-1 labeled cells are present in the ipsilateral
hemisphere (FIG. 10B) following MCAO in vehicle-treated animals.
Few ED-1 positive cells are found in animals treated with
NRG-1.beta. (FIG. 10C). Examples of ED-1 positive cells are
indicated by the arrows. Scale bar is 50 .mu.M. To assess
astrocytic activation, sections were labeled for
immunohistochemistry with an antibody against GFAP. Compared to the
contralateral control (FIG. 10D), heavy GFAP staining is found at
the border or infarct (FIG. 10E) following MCAO in vehicle-treated
animals. However, when rats were treated with NRG1.beta., GFAP
expression was dramatically reduced in the peri-infarct regions
(FIG. 10F). * denotes infarct core or the corresponding region in
the contralateral control; # denotes non-ischemic tissues or the
corresponding region in the contralateral control. Scale bar is 100
.mu.M.
[0022] FIG. 11 is a composite of pictures and graphs showing that
NRG1.beta. reduces MCAO/reperfusion-induced IL-1.beta. mRNA levels.
Rats were treated with NRG1.beta. or vehicle then subjected to
MCAO. RNA was isolated and IL-1.beta. mRNA expression was measured
by RT-PCR. The expression of IL-1 (panel a) and GAPDH (panel b)
mRNA is shown (n=4 for each condition). Panel c shows the average
percentage of change.+-.SEM in IL-1 mRNA levels from
NRG-.beta.-treated rat compared to vehicle-treated controls after
normalization to GAPDH (* denotes P<0.05). I=ipsilateral
hemisphere; C=contralateral hemisphere.
DETAILED DESCRIPTION
[0023] The present invention relates to chimeric neuregulins,
methods of making chimeric neuregulins, and methods and
compositions for prevention and treatment of neuronal and vascular
damages with chimeric neuregulins.
Definitions
[0024] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a pharmaceutical carrier" includes mixtures of two or
more such carriers, and the like.
[0025] The term "neuregulin" as used herein, refers to a family of
proteins that includes: neuregulin-1 (NRG1), neuregulin-2 (NRG2),
neuregulin-3 (NRG3), and neuregulin-4 (NRG4). Neuregulin-1, which
has also been described in the literature as acetylcholine receptor
inducing activity (ARIA), glial growth factor (GGF), glial growth
factor 2 (GGF2); heregulin and neu differentiation factor (NDF);
sensory and motor neuron-derived factor (SMDF), HGL; HRG; HRG1;
HRGA and MST131, further contains a number of isoforms that include
type I NRG1, type II NRG1, type III NRG1, type IV NRG1, type V NRG1
and type VI NRG1. The term "NRG1," as used herein, also includes
all other NRG1 isoforms. NRG1 isoforms are synthesized as
transmembrane precursors consisting of either an
immunoglobulin-like or cysteine-rich domain, an EGF-like domain, a
transmembrane domain and a cytoplasmic tail (Fischbach et al., Annu
Rev Neurosci, 1997. 20:429-58). NRG1 isoforms are generated from
one gene by alternative mRNA splicing, and most of them are
synthesized as part of a larger transmembrane precursor. The two
major classes of NRG1 include .alpha. and .beta. isoforms. The
NRG1.beta. isoforms predominate in the nervous system, while a
isoforms are prevalent in mesenchymal cells. The .beta. isoforms
are 100 to 1,000 fold more potent in stimulating AChR synthesis in
skeletal muscle and Schwann cell proliferation (Buonanno et al.,
Curr Opin Neurobiol, 2001. 11:287-96). The effects of NRG1 appear
to be mediated by interaction with a class of tyrosine kinase
receptors related to the epidermal growth factor (EGF) receptor
which includes ErbB2, ErbB3 and ErbB4 (Burden et al., Neuron, 1997.
18:847-55). The EGF-like domain of NRG1 appears to be sufficient
for activation of ErbB receptors and downstream signal transduction
pathways (Holmes et al., Science, 1992. 256:1205-1210). NRG1
stimulates the tyrosine phosphorylation of these receptors and the
subsequent activation of various signal transduction mechanisms
including Map kinase, PI3 kinase and CDK5 (Fu et al., Nat Neurosci,
2001.4:374-81).
[0026] Neuregulin 2 (NRG2) is a novel member of the neuregulin
family of growth and differentiation factors. Through interaction
with the ErbB family of receptors, NRG2 induces the growth and
differentiation of epithelial, neuronal, glial, and other types of
cells. The gene consists of 12 exons and the genomic structure is
similar to that of neuregulin 1 (NRG1). NRG1 and NRG2 mediate
distinct biological processes by acting at different sites in
tissues and eliciting different biological responses in cells. The
NRG2 gene is located close to the region for demyelinating
Charcot-Marie-Tooth disease locus, but is not responsible for this
disease. Alternative transcripts encoding distinct isoforms have
been described (Chang, H., et al. Nature (1997) 387: pp. 509-12;
Carraway, K L, et al. Nature (1997) 387: pp. 512-16).
[0027] Neuregulin 3 (NRG3) binds to the extracellular domain of the
ERBB4 receptor tyrosine kinase but not to the related family
members ERBB2 or ERBB3. NRG3 binding stimulates tyrosine
phosphorylation of ERBB4. Variants of the NRG3 gene have been
linked to a susceptibility to schizophrenia (Zhang D, et al. Proc.
Natl. Acad. Sci. U.S.A. (1997) 94: pp. 9562-7; Chen, P L, et al.
Am. J. Hum. Genet. (2009) 84: pp. 21-34).
[0028] Neuregulin 4 (NRG4) activates type-1 growth factor receptors
(EGFR) to initiating cell-to-cell signaling through tyrosine
phosphorylation. Loss of expression of NRG4 is frequently seen in
advanced bladder cancer while increased NRG4 expression correlates
to better survival (Harari, D., et al. Oncogene (1999) 18: pp.
2681-9); (Memon, A A., et al., Br. J. Cancer (2004) 91: pp.
2034-41)).
[0029] Examples of human neuregulin sequences include, but are not
limited to, those listed under GenBank Accession Nos: ADN85612.1,
AAF28851.1, AAF28850.1, AAF28849.1, AAF28848.1, ABR13844.1,
ABR13843.1, EAW63417.1, EAW63416.1, EAW63415.1, AAI50610.1,
ABQ53540.1, DAA00042.1, DAA00041.1, EAW62087.1, EAW62086.1,
EAW62085.1, EAW62084.1, EAW62082.1, EAW62081.1, ADK90032.1,
ADK90031.1, ADK90030.1, ADK90029.1, ADK90026.1, ADK90024.1,
ADK90022.1, AAF28853.1, AAF28852.1, BAG70289.1, BAG70145.1,
ABR13842.1, ABG77979.1, DAA00045.1, EAW63419.1, EAW63412.1,
EAW63410.1, EAW63409.1, EAW63408.1, ADN85613.1, AAI36812.1,
ABQ53539.1, DAA00048.1, DAA00047.1, EAW62083.1, EAW62078.1,
ADK90028.1, ADK90027.1, ADK90025.1, ADK90023.1, ADK90021.1,
ADK90020.1, AAH73871.1, ABY66350.1, EAW99228.1, EAW99227.1,
EAW62080.1, EAW62079.1, EAW63418.1, EAW63414.1, EAW63413.1,
EAW63411.1, EAW63407.1, EAW80374.1, AAH64587.1, AAH06492.1,
AA049724.1, AAH07675.1, AAP36053.1, AAH17568.1, CAI15622.1,
CAH73645.1, CAH70641.1, CAI17213.1, CAH71050.1, AAM71141.1,
AAM71140.1, AAM71139.1, AAM71138.1, AAM71137.1, AAM71136.1,
AAM71135.1, AAM71134.1, AAM71133.1, CAI22410.1, ABQ53543.1,
ABQ53542.1, ABQ53541.1, DAA00046.1, DAA00044.1, DAA00043.1,
DAA00040.1, CAL35830.1, CAL35831.1, CAL35829.1, BAD97155.1,
NP.sub.--001159445.1, NP.sub.--001159444.1, NP.sub.--001010848.2,
NP.sub.--004874.1, NP.sub.--053585.1, NP.sub.--001171864.1,
NP.sub.--053586.1, NP.sub.--053584.1, Q02297.3,
NP.sub.--001153467.1, NP.sub.--039258.1, NP.sub.--039251.2,
NP.sub.--001153473.1, NP.sub.--001153471.1, NP.sub.--039250.2,
NP.sub.--001153476.1, NP.sub.--612640.1, NP.sub.--039254.1,
NP.sub.--001153468.1, NP.sub.--039256.2, NP.sub.--001153480.1,
014511.1, Q9H013.3, Q7RTV8, NP.sub.--039252.2,
NP.sub.--001153479.1, NP.sub.--001153474.1, NP.sub.--001153477.1,
NP.sub.--004486.2, NP.sub.--039253.1, P56975.1, AAA19954.1,
AAA19953.1, AAA19951.1, AAA19950.1, AAB59358.1; AAB59622.1;
AAI03985.2, AAI03985.2, AAI14335.2, AAI03984.2, Q8WWG1.1,
AAA58640.1, AAA58639.1, AAA58638.1, AAC51756.1, AAY17216.1,
1910316A, ABY70644.1, ABY66349.1, ABY66348.1, ABC69293.1,
AAA19952.1; AAA19955.1; AAA58641.1; AAB59358.1; AAB59622.1;
AAC41764.1;AAH73871.1; AAI14334.2; AAI36812.1; AA049724.1;
AAP36053.1; ABC69293.1; ABQ53539.1; ABQ53540.1; ABR13843.1;
ABR18344.1; ABY66350.1; BAA23417.1; BAD97155.1; BAF83419.1;
BAF82616.1; BAG54044.1; BAG53780.1; BAG59183.1; BAH11473.1;
BAH11479.1; BAH12729.1; CAD98015.1; CAG29284.1; CAH18333.1
[0030] The term "integrin binding domain of a NRG" or "integrin
binding domain of an NRG consensus sequence," as used herein,
refers to the amino acid sequence in a native neuregulin that is
located between and including the first and the third cyctienes of
the NRG consensus sequence shown in FIGS. 1 and 2.
[0031] The term "Erb3/4 binding domain of a NRG" or "Erb3/4 binding
domain of an NRG consensus sequence," as used herein, refers to the
amino acid sequence in a native neuregulin that is located between
and including the last two cyctienes residues (i.e., the fifth and
sixth cyctienes) of the NRG consensus sequence shown in FIGS. 1 and
2.
[0032] The term "non-neuregulin polypeptide," as used herein,
refers to a polypeptide that is not a member of the neuregulin
family.
[0033] The term "polypeptide" refers to a polymer in which the
monomers are amino acid residues which are joined together through
amide bonds. The terms "polypeptide" or "protein" as used herein
are intended to encompass any amino acid sequence and include
modified sequences such as glycoproteins. The term "polypeptide" is
specifically intended to cover naturally occurring proteins, as
well as those which are recombinantly or synthetically produced.
The term "fragment," in reference to a polypeptide, refers to a
portion (that is, a subsequence) of a polypeptide. A fragment
contains at least three amino acids residues. In some embodiments,
the fragment contains at least 5 or 10 amino acids residues.
Orientation within a polypeptide is generally recited in an
N-terminal to C-terminal direction, defined by the orientation of
the amino and carboxy moieties of individual amino acids.
Polypeptides are translated from the N or amino-terminus towards
the C or carboxy-terminus.
[0034] The term "derived from" refers to the origin of a fragment
or a polypeptide. A fragment or polypeptide is deemed to be
"derived from" a protein if the amino acid sequence of the fragment
or the polypeptide is identical to the amino acid sequence of the
protein, or a portion or portions of the amino acid sequence of the
protein. For example, a polypeptide with a sequence of HHHHKKK is
deemed to be derived from a polypeptide with a sequence of
HHHHCCCKKK.
[0035] "Solubility" is a measure the amount of a substance, in the
context of this disclosure, a polypeptide, that will dissolve in a
given amount of another substance, usually a liquid. Thus, an
increase in solubility is an increase in the amount of a the
polypeptide that remains without aggregating or separating from the
substance (e.g., liquid) in which it is dissolved.
[0036] When referring to a polypeptide, "stability is a measure of
the polypeptide's resistance to degradation. Thus, an increase in
stability reflects an increase in the ability of the polypeptide to
withstand degradation, for example, measured as an increased
half-life in vivo, or an increased shelf life in vitro.
[0037] The term "enhanced binding affinity" is a relative term that
refers to an binding affinity that is at least 50%, 100% or 200%
above a baseline binding affinity. For example, if the binding
affinity between an integrin and a chimeric neuregulin is more than
150% of the binding affinity between the same integrin and a native
neuregulin (i.e., an over 50% increase in binding affinity), the
chimeric neuregulin has an "enhanced binding affinity" to the
integrin with regard to the native integrin.
[0038] The term "reduces" is a relative term, such that an agent
reduces a response or condition if the response or condition is
quantitatively diminished following administration of the agent, or
if it is diminished following administration of the agent, as
compared to a reference agent. Similarly, the term "prevents" does
not necessarily mean that an agent completely eliminates the
response or condition, so long as at least one characteristic of
the response or condition is eliminated. Thus, a composition that
reduces or prevents an infection or a response, such as a
pathological response, can, but does not necessarily completely
eliminate such an infection or response, so long as the infection
or response is measurably diminished, for example, by at least
about 50%, such as by at least about 70%, or about 80%, or even by
about 90% of (that is to 10% or less than) the infection or
response in the absence of the agent, or in comparison to a
reference agent.
[0039] The term "subject" refers to an individual. Preferably, the
subject is a mammal such as a primate, and, more preferably, a
human. The term "subject" can include domesticated animals, such as
cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep,
goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat,
guinea pig, etc.).
[0040] The term "treatment" or "treating" refers to administration
of a composition to a subject with an undesired condition or at
risk for the condition. The condition can be any pathogenic
disease, autoimmune disease, cancer or inflammatory condition. The
effect of the administration of the composition to the subject can
have the effect of but is not limited to reducing the symptoms of
the condition, a reduction in the severity of the condition, or the
complete ablation of the condition.
[0041] Ranges may be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed that "less than
or equal to" the value, "greater than or equal to the value" and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed the "less than or equal to 10" as well as "greater
than or equal to 10" is also disclosed.
Chimeric Neuregulins
[0042] One aspect of the present invention relates to chimeric
neuregulins that have improved biological activities or
pharmacokinetic characteristics. The term "chimeric neuregulin," as
used herein, refers to a polypeptide that comprises a sequence
derived from a neuregulin polypeptide and a sequence derived from
another polypeptide that can be a neuregulin or a non-neuregulin
polypeptide.
[0043] In some embodiments, the chimeric neuregulin polypeptide has
an integrin domain that binds to an integrin and an Erb3/4 binding
domain that binds to Erb3 and/or Erb4. The chimeric neuregulin
polypeptide comprises a neuregulin backbone derived from a native
neuregulin polypeptide, and a donor fragment of at least one amino
acid, wherein the donor fragment (1) replaces a target fragment in
the native neuregulin polypeptide, wherein the donor fragment
differs from the target fragment by at least one amino acid, or (2)
is inserted into an insertion site of the native neuregulin
polypeptide, and wherein the donor fragment forms at least a
portion of the integrin binding domain and/or at least a portion of
the Erb3/4 binding domain of the chimeric neuregulin polypeptide.
In one embodiment, the chimeric neuregulin polypeptide has an
enhanced binding affinity to integrin, Erb 3, or Erb 4, comparing
to said native neuregulin peptide. In another embodiment, the donor
fragment comprises a polypeptide derived from another neuregulin
that is different from the native neuregulin. In another
embodiment, the donor fragment comprises a polypeptide derived from
a non-neuregulin. In another embodiment, the donor fragment
comprises polypeptides derived from two neuregulins that are
different from said native neuregulin. In another embodiment, the
original neuregulin polypeptide is NRG2 or a fragment of NRG2, and
the donor fragment comprises a polypeptide derived from NRG1.beta..
In another embodiment, the native neuregulin polypeptide is NRG4 or
a fragment of NRG4, and the donor fragment comprises a polypeptide
derived from NRG1, or a polypeptide derived from NRG2, or both a
polypeptide derived from NRG1 and a polypeptide derived from NRG2.
In another embodiment, the chimeric neuregulin polypeptide further
comprises a second donor fragment of at least one amino acid,
wherein the second donor fragment (1) replaces a second target
fragment in the native neuregulin polypeptide, wherein the second
donor fragment differs from the second target fragment by at least
one amino acid, or (2) is inserted into an insertion site of the
native neuregulin polypeptide.
[0044] In other embodiments, the chimeric neuregulin comprises a
first moiety of at least 10 amino acids, wherein the first moiety
is derived from a first polypeptide; and a second moiety of at
least 5 amino acids, wherein the second moiety is derived from a
second polypeptide; wherein the first polypeptide is a neuregulin
and wherein the chimeric neuregulin exhibits an enhanced binding
affinity to integrin, Erb 3, or Erb 4 comparing to that of the
neuregulin. In one embodiment, the second polypeptide is a
neuregulin that is different from the first neuregulin. In another
embodiment, the second polypeptide is a non-neuregulin polypeptide.
In another embodiment, the chimeric neuregulin further comprises a
third moiety derived from a third polypeptide. In a relevant
embodiment, the third polypeptide is a neuregulin that is different
from the native neuregulin. In another relevant embodiment, the
second polypeptide is a second neuregulin and the third polypeptide
is a third neuregulin, wherein the first neuregulin, second and
third neuregulins are different from each other. In another
embodiment, the first neuregulin is NRG2, and the second
polypeptide is NRG1.beta.. In another embodiment, the first
neuregulin is NRG4 and the second polypeptide is NRG1 or NRG2. In
another embodiment, the first moiety comprises a NRG integrin
binding domain or a NRG Erb3/4 binding domain. In another
embodiment, the first moiety comprises a NRG integrin binding
domain, and the second moiety comprises a NRG Erb3/4 binding
domain. In another embodiment, the first moiety comprises a NRG
Erb3/4 binding domain and the second moiety comprises a NRG
integrin binding domain.
[0045] In yet other embodiments, the chimeric neuregulin is a
neuregulin polypeptide (the original neuregulin) in which a stretch
of at least 5, 6, 7, 8 or 9 consecutive amino acid residues (the
"original fragment") is replaced with a stretch of consecutive
amino acid residues (the "donor fragment") from a donor polypeptide
that is different from the original neuregulin polypeptide. The
original neuregulin can be a full length neuregulin or a fragment
of a neuregulin. The donor polypeptide can be another member of the
neuregulin family or a non-neuregulin polypeptide. The donor
fragment may have a length that is the same as, or is different
from, the length of the original fragment. A chimeric neuregulin
may contain more than one donor fragment. In one embodiment, one
original fragment is replaced with two different donor fragments.
In other embodiment, two original fragments are replaced with two
different donor fragments. In a preferred embodiment, the original
fragment comprises an integrin binding domain of a NRG consensus
sequence or an Erb3/4 binding domain of a NRG consensus
sequence.
[0046] In certain embodiments, the original neuregulin is NRG4 or a
fragment of NRG4 and the donor fragment(s) is derived from NRG1
and/or NRG2. In other embodiments, the original neuregulin is NRG3
or a fragment of NRG3 and the donor fragment(s) is derived from
NRG1 and/or NRG2. In other embodiments, the original neuregulin is
NRG2 or a fragment of NRG2 and the donor fragment(s) is derived
from NRG1 and/or NRG2. In yet other embodiments, the original
neuregulin is NRG1 or a fragment of NRG1 and the donor fragment(s)
is derived from NRG1 and/or NRG2. In one embodiment, the donor
fragment is derived from NRG1.beta..
[0047] In certain embodiments, the chimeric neuregulin is a more
effective Erb 3 or Erb 4 agonist than the original neuregulin
polypeptide. In other embodiments, the chimeric neuregulin has a
higher affinity to Erb 3 and/or Erb 4 than the original neuregulin.
In other embodiments, the chimeric neuregulin has a higher affinity
to integrin than the original neuregulin. Selection of the original
neuregulin, the original fragment, the donor polypeptide and the
donor fragment can be determined based on the sequence comparison
and known Erb 3 and/or Erb 4 binding activity of the original
neuregulin and the donor polypeptide.
[0048] For example, various neuregulins share the sequence homology
shown in FIG. 1. Specifically, the neuregulin molecules all have a
consensus sequence that contains six cystiene residues spread in a
region that is involved in binding to integrine and Erb3/4
molecules (see, e.g., FIGS. 1 and 2). As shown in FIG. 1, NRG1,
NRG2 and NRG3 all have either one or two positively charged amino
acids (R, K or H) at the amino-end of the first cyctiene in the
consensus sequence. NRGs with two positively charged amino acids
(e.g., NRG2, NRG2.2, NRG 2.3 and NRG2.6) are able to bind integrins
more effectively (i.e., having a higher affinity to integrins).
There integrins are up-regulated on inflamed tissue or tumors.
[0049] Further, the affinities of various NRGs to Erb3/4 have been
reported as follows:
[0050] NRG1.beta.>NRG1.alpha.>NRG4 ( 1/10 of NRG1.beta.)
[0051] NRG2>NRG3/NRG4 ( 1/10 the affinity of NRG2).
[0052] It thus appears that Erb3/4 interact with NRGs through the
amino acid residues between the last two cyctienes in the consensus
sequence, as well as the amino acid residues after the last
cyctiene residue in the consensus sequence.
[0053] Therefore, NRG2s have the highest affinities for integrins
but NRG1as have highest affinities for Erb 3/4. Therefore, a
chimeric neuregulin with the integrin binding domain of NRG2 and
the Erb 3/4 binding domain of NRG1.beta. would have similar
affinities to integrin or Erb 3/4 when compared to NRG2 or
NRG1.beta., respectively. However, such a chimeric neuregulin would
have a greater potency to bind to both integrin and Erb3/4 when
compared to NRG2 or NRG1.beta.. For example, a NRG1/NRG2/NRG4
chimeric neuregulin would have the ability to tightly bind
neurotropic or vascular integrins while preserving the chimeric's
ability to bind and inhibit ErbB3 and ErbB4 signaling. FIG. 3 shows
certain embodiments of chimeric neuregulins (SEQ ID NOS:20-29).
[0054] As used herein, a "variant of a chimeric neuregulin" is a
chimeric neuregulin polypeptide that contains conservative
substitutions. A "conservative substitution" is one in which an
amino acid is substituted for another amino acid that has similar
properties, such that one skilled in the art of peptide chemistry
would expect the secondary structure and hydropathic nature of the
polypeptide to be substantially unchanged. Amino acid substitutions
may generally be made on the basis of similarity in polarity,
charge, solubility, hydrophobicity, hydrophilicity and/or the
amphipathic nature of the residues. For example, negatively charged
amino acids include aspartic acid and glutamic acid; positively
charged amino acids include lysine and arginine; and amino acids
with uncharged polar head groups having similar hydrophilicity
values include leucine, isoleucine and valine; glycine and alanine;
asparagine and glutamine; and serine, threonine, phenylalanine and
tyrosine. Other groups of amino acids that may represent
conservative changes include: (1) ala, pro, gly, glu, asp, gin,
asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,
phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may
also, or alternatively, contain nonconservative changes. In a
preferred embodiment, variant polypeptides differ from a native
sequence by substitution, deletion or addition of five amino acids
or fewer. Variants may also (or alternatively) be modified by, for
example, the deletion or addition of amino acids that have minimal
influence on the desired bioactivity, secondary structure and
hydropathic nature of the polypeptide.
[0055] A variant of a chimeric neuregulin preferably exhibits at
least about 70%, more preferably at least about 80%, more
preferably at least about 90% and most preferably at least about
95% homology to the original chimeric neuregulin polypeptide.
Variants can be produced artificially using site directed or random
mutagenesis, or by recombination of two or more preexisting
variants. For example, a variant chimeric neuregulin polypeptide
can include 1, or 2, or 5 or 10, or 15, or up to about 50 amino
acid differences from the reference chimeric neuregulin.
[0056] Variants of chimeric neuregulin polypeptide also include
polypeptides that are modified from the original chimeric
neuregulin polypeptides by either natural process, such as
posttranslational processing, or by chemical modification
techniques which are well known in the art. Such modifications are
well described in basic texts and in more detailed monographs, as
well as in a voluminous research literature. Modifications can
occur anywhere in a polypeptide, including the peptide backbone,
the amino acid side-chains and the amino or carboxyl termini. It
will be appreciated that the same type of modification may be
present in the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched, for example, as a
result of ubiquitination, and they may be cyclic, with or without
branching. Cyclic, branched, and branched cyclic polypeptides may
result from post-translation natural processes or may be made by
synthetic methods. Modifications include pegylation, acetylation,
acylation, ADP-ribosylation, amidation, covalent attachment of
flavin, covalent attachment of a heme moiety, covalent attachment
of a nucleotide or nucleotide derivative, covalent attachment of a
lipid or lipid derivative, covalent attachment of
phosphotidylinositol, cross-linking, cyclization, disulfide bond
formation, demethylation, formation of covalent cross-links,
formation of cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation, and ubiquitination.
Methods of Producing Chimeric Neuregulins
[0057] The chimeric neuregulins or variants thereof may be produced
using methods well known in the art. In certain embodiments, the
chimeric neuregulins or variants thereof are produced by chemical
synthesis. Briefly, a chimeric neuregulin may be synthesized by
coupling the carboxyl group or C-terminus of one amino acid to the
amino group or N-terminus of another. Due to the possibility of
unintended reactions, protecting groups are usually necessary.
Chemical peptide synthesis starts at the C-terminal end of the
peptide and ends at the N-terminus. This is the opposite of protein
biosynthesis, which starts at the N-terminal end.
[0058] In some embodiments, the chimeric neuregulines may be
synthesized using traditional liquid- or solid-phase synthesis.
Fmoc and t-Boc solid phase peptide synthesis (SPPS) can be employed
to grow the peptides from carboxy to amino-terminus. In certain
embodiments, the last "amino acid" added to the reaction is
PEGylated. This last amino acid is often referred to as a
carboxyl-PEG-amine, carboxyl-PEO-amine, or amine-PEG-acid, whereby
the amine is blocked to protect against reaction and the acid is
free to react with the amine group from the previously added amino
acid in the reaction. PEG (polyethylene glycol) and PEO
(polyethylene oxide) are polymers composed of repeating subunits of
ethylene glycol and ethylene oxide monomers. In one embodiment, a
PEGylated GGF2/NRG2 chimeric (SEQ ID NO:28) would have the PEG
moiety connected to the histidine residue (H) at the amino-terminus
of the polypeptide. In one embodiment, the PEG moiety is 5 to 30
kDa in size. In another embodiment, the PEG moiety is 10 to 20 kDa
in size.
[0059] In addition to using PEGylated end amino acid during
synthesis, a chimeric neuregulin may be PEGylated by PEGylation.
PEGylation is the process of covalent attachment of polyethylene
glycol polymer chains to another molecule, normally a drug or
therapeutic protein. PEGylation can be achieved by incubation of a
reactive derivative of PEG with the target chimeric neuregulin. The
covalent attachment of PEG to a chimeric neuregulin can "mask" the
chimeric neuregulin from the host's immune system (reduced
immunogenicity and antigenicity), increase the hydrodynamic size
(size in solution) of the chimeric neuregulin which prolongs its
circulatory time by reducing renal clearance. PEGylation can also
provide water solubility to hydrophobic proteins.
[0060] The first step of the PEGylation is the suitable
functionalization of the PEG polymer at one or both terminals. PEGs
that are activated at each terminus with the same reactive moiety
are known as "homobifunctional", whereas if the functional groups
present are different, then the PEG derivative is referred as
"heterobifunctional" or "heterofunctional." The chemically active
or activated derivatives of the PEG polymer are prepared to attach
the PEG to the desired molecule.
[0061] The overall PEGylation processes used to date for protein
conjugation can be broadly classified into two types, namely a
solution phase batch process and an on-column fed-batch process.
The simple and commonly adopted batch process involves the mixing
of reagents together in a suitable buffer solution, preferably at a
temperature between 4 and 6.degree. C., followed by the separation
and purification of the desired product using a suitable technique
based on its physicochemical properties, including size exclusion
chromatography (SEC), ion exchange chromatography (IEX),
hydrophobic interaction chromatography (HIC) and membranes or
aqueous two phase systems.
[0062] The choice of the suitable functional group for the PEG
derivative is based on the type of available reactive group on the
molecule that will be coupled to the PEG. For proteins, typical
reactive amino acids include lysine, cysteine, histidine, arginine,
aspartic acid, glutamic acid, serine, threonine, tyrosine. The
N-terminal amino group and the C-terminal carboxylic acid can also
be used as a site specific site by conjugation with aldehyde
functional polymers.
[0063] In certain embodiments, the PEG derivatives are produced by
reacting the PEG polymer with a group that is reactive with
hydroxyl groups, typically anhydrides, acid chlorides,
chloroformates and carbonates. In other embodiments, more efficient
functional groups such as aldehyde, esters, amides, etc. are made
available for protein conjugation.
[0064] In certain embodiments, heterobifunctional PEGs are used for
conjugation. These heterobifunctional PEGs are very useful in
linking two entities, where a hydrophilic, flexible and
biocompatible spacer is needed. Preferred end groups for
heterobifunctional PEGs are maleimide, vinyl sulfones, pyridyl
disulfide, amine, carboxylic acids and NHS esters.
[0065] In other embodiments, the pegylation agents contain
branched, Y shaped or comb shaped polymers that show reduced
viscosity and lack of organ accumulation.
[0066] In other embodiments, chimeric neuregulins or variants
thereof are produced using recombinant DNA technologies. Procedures
for the expression and purification of recombinant proteins are
well established in the art.
[0067] In order to express a chimeric neuregulin is a biological
system, a polynucleotide that encodes the chimeric neuregulin is
constructed. In certain embodiments, the recombinant polynucleotide
is codon optimized for expression in a selected prokaryotic or
eukaryotic host cell, such as a mammalian, plant or insect cell. To
facilitate replication and expression, the polynucleotide can be
incorporated into a vector, such as a prokaryotic or a eukaryotic
expression vector. Although the polynucleotide disclosed herein can
be included in any one of a variety of vectors (including, for
example, bacterial plasmids; phage DNA; baculovirus; yeast
plasmids; vectors derived from combinations of plasmids and phage
DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus,
pseudorabies, adenovirus, adeno-associated virus, retroviruses and
many others), most commonly the vector will be an expression vector
suitable for generating polypeptide expression products. In an
expression vector, the polynucleotide encoding the neuregulin
chimera is typically arranged in proximity and orientation to an
appropriate transcription control sequence (promoter, and
optionally, one or more enhancers) to direct mRNA synthesis. That
is, the polynucleotide sequence of interest is operably linked to
an appropriate transcription control sequence. Examples of such
promoters include: the immediate early promoter of CMV, LTR or SV40
promoter, polyhedron promoter of baculovirus, E. coli lac or trp
promoter, phage T7 and lambda P.sub.L promoter, and other promoters
known to control expression of genes in prokaryotic or eukaryotic
cells or their viruses. The expression vector typically also
contains a ribosome binding site for translation initiation, and a
transcription terminator. The vector optionally includes
appropriate sequences for amplifying expression. In addition, the
expression vectors optionally comprise one or more selectable
marker genes to provide a phenotypic trait for selection of
transformed host cells, such as dihydrofolate reductase or neomycin
resistance for eukaryotic cell culture, or such as tetracycline or
ampicillin resistance in E. coli.
[0068] The expression vector can also include additional expression
elements, for example, to improve the efficiency of translation.
These signals can include, e.g., an ATG initiation codon and
adjacent sequences. In some cases, for example, a translation
initiation codon and associated sequence elements are inserted into
the appropriate expression vector simultaneously with the
polynucleotide sequence of interest (e.g., a native start codon).
In such cases, additional translational control signals are not
required. However, in cases where only a polypeptide coding
sequence, or a portion thereof, is inserted, exogenous
translational control signals, including an ATG initiation codon is
provided for expression of the chimeric neuregulin sequence. The
initiation codon is placed in the correct reading frame to ensure
translation of the polynucleotide sequence of interest. Exogenous
transcriptional elements and initiation codons can be of various
origins, both natural and synthetic. If desired, the efficiency of
expression can be further increased by the inclusion of enhancers
appropriate to the cell system in use.
[0069] Expression vectors carrying the chimeric neuregulins can be
introduced into host cells by any of a variety of well-known
procedures, such as electroporation, liposome mediated
transfection, calcium phosphate precipitation, infection,
transfection and the like, depending on the selection of vectors
and host cells.
[0070] Host cells that contain chimeric neuregulins-encoding
nucleic acids are, thus, also a feature of this disclosure.
Favorable host cells include prokaryotic (i.e., bacterial) host
cells, such as E. coli, as well as numerous eukaryotic host cells,
including fungal (e.g., yeast, such as Saccharomyces cerevisiae and
Picchia pastoris) cells, insect cells, plant cells, and mammalian
cells (such as CHO cells). Recombinant chimeric neuregulin nucleic
acids are introduced (e.g., transduced, transformed or transfected)
into host cells, for example, via a vector, such as an expression
vector. As described above, the vector is most typically a plasmid,
but such vectors can also be, for example, a viral particle, a
phage, etc. Examples of appropriate expression hosts include:
bacterial cells, such as E. coli, Streptomyces, and Salmonella
typhimurium; fungal cells, such as Saccharomyces cerevisiae, Pichia
pastoris, and Neurospora crassa; insect cells such as Drosophila
and Spodoptera frugiperda; mammalian cells such as 3T3, COS, CHO,
BHK, HEK 293 or Bowes melanoma; plant cells, including algae cells,
etc.
[0071] The host cells can be cultured in conventional nutrient
media modified as appropriate for activating promoters, selecting
transformants, or amplifying the inserted polynucleotide sequences.
The culture conditions, such as temperature, pH and the like, are
typically those previously used with the host cell selected for
expression, and will be apparent to those skilled in the art.
[0072] In bacterial systems, a number of expression vectors can be
selected depending upon the use intended for the expressed product.
For example, when large quantities of a polypeptide or fragments
thereof are needed for the production of antibodies, vectors which
direct high level expression of fusion proteins that are readily
purified are favorably employed. Such vectors include, but are not
limited to, multifunctional E. coli cloning and expression vectors
such as BLUESCRIPT (Stratagene), in which the coding sequence of
interest, e.g., a polynucleotide of the invention as described
above, can be ligated into the vector in-frame with sequences for
the amino-terminal translation initiating Methionine and the
subsequent 7 residues of beta-galactosidase producing a
catalytically active beta galactosidase fusion protein; pIN vectors
(Van Heeke & Schuster (1989) J Biol Chem 264:5503-5509); pET
vectors (Novagen, Madison Wis.), in which the amino-terminal
methionine is ligated in frame with a histidine tag, and the
like.
[0073] Similarly, in yeast, such as Saccharomyces cerevisiae, a
number of vectors containing constitutive or inducible promoters
such as alpha factor, alcohol oxidase and PGH can be used for
production of the desired expression products. In mammalian host
cells, a number expression systems, including both plasmids and
viral-based systems, can be utilized.
[0074] A host cell is optionally chosen for its ability to modulate
the expression of the inserted sequences or to process the
expressed protein in the desired fashion. Such modifications of the
protein include, but are not limited to, glycosylation, (as well
as, e.g., acetylation, carboxylation, phosphorylation, lipidation
and acylation). Post-translational processing for example, which
cleaves a precursor form into a mature form of the protein (for
example, by a furin protease) is optionally performed in the
context of the host cell. Different host cells such as 3T3, COS,
CHO, HeLa, BHK, MDCK, 293, W138, etc. have specific cellular
machinery and characteristic mechanisms for such post-translational
activities and can be chosen to ensure the correct modification and
processing of the introduced, foreign protein.
[0075] For long-term, high-yield production of recombinant chimeric
neuregulin polypeptide, stable expression systems are typically
used. For example, polynucleotides encoding a chimeric neuregulin
polypeptides are introduced into the host cell using expression
vectors which contain viral origins of replication or endogenous
expression elements and a selectable marker gene. Following the
introduction of the vector, cells are allowed to grow for 1-2 days
in an enriched media before they are switched to selective media.
The purpose of the selectable marker is to confer resistance to
selection, and its presence allows growth and recovery of cells
which successfully express the introduced sequences. For example,
resistant groups or colonies of stably transformed cells can be
proliferated using tissue culture techniques appropriate to the
cell type. Host cells transformed with a nucleic acid encoding a
chimeric neuregulin polypeptide are optionally cultured under
conditions suitable for the expression and recovery of the encoded
protein from cell culture.
[0076] Following transduction of a suitable host cell line and
growth of the host cells to an appropriate cell density, the
selected promoter is induced by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period. The secreted polypeptide product is then
recovered from the culture medium. Alternatively, cells can be
harvested by centrifugation, disrupted by physical or chemical
means, and the resulting crude extract retained for further
purification. Eukaryotic or microbial cells employed in expression
of proteins can be disrupted by any convenient method, including
freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing agents, or other methods, which are well know to those
skilled in the art.
[0077] Expressed chimeric neuregulin polypeptides can be recovered
and purified from recombinant cell cultures by any of a number of
methods well known in the art, including ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography (e.g., using
any of the tagging systems noted herein), hydroxylapatite
chromatography, and lectin chromatography. Protein refolding steps
can be used, as desired, in completing configuration of the mature
protein. Finally, high performance liquid chromatography (HPLC) can
be employed in the final purification steps.
[0078] In certain examples, the nucleic acids are introduced into
vectors suitable for introduction and expression in prokaryotic
cells, e.g., E. coli cells. For example, a nucleic acid including a
polynucleotide sequence that encodes a F2GF1 chimeric RSV antigen
can be introduced into any of a variety of commercially available
or proprietary vectors, such as the pET series of expression
vectors (e.g., pET19b and pET21d). Expression of the coding
sequence is inducible by IPTG, resulting in high levels of protein
expression. The polynucleotide sequence encoding the chimeric RSV
antigen is transcribed under the phage T7 promoter. Alternate
vectors, such as pURV22 that include a heat-inducible lambda pL
promoter are also suitable.
[0079] The expression vector is introduced (e.g., by
electroporation) into a suitable bacterial host. Numerous suitable
strains of E. coli are available and can be selected by one of
skill in the art (for example, the Rosetta and BL21 (DE3) strains
have proven favorable for expression of recombinant vectors
containing polynucleotide sequences that encode F2GF1 chimeric RSV
antigens.
[0080] In another example, a polynucleotide sequence that encodes a
chimeric neuregulin is introduced into insect cells using a
Baculovirus Expression Vector System (BEVS). Recombinant
baculovirus capable of infecting insect cells can be generated
using commercially available vectors, kits and/or systems, such as
the BD BaculoGold system from BD BioScience. Briefly, the
polynucleotide sequence encoding the chimeric neuregulin is
inserted into the pAcSG2 transfer vector. Then, host cells SF9
(Spodoptera frugiperda) are co-transfected by pAcSG2-chimer plasmid
and BD BaculoGold, containing the linearized genomic DNA of the
baculovirus Autographa californica nuclear polyhedrosis virus
(AcNPV). Following transfection, homologous recombination occurs
between the pACSG2 plasmid and the Baculovirus genome to generate
the recombinant virus. In one example, the chimeric neuregulin is
expressed under the regulatory control of the polyhedrin promoter
(pH). Similar transfer vectors can be produced using other
promoters, such as the basic (Ba) and p10 promoters. Similarly,
alternative insect cells can be employed, such as SF21 which is
closely related to the SF9, and the High Five (Hi5) cell line
derived from a cabbage looper, Trichoplusia ni.
[0081] Following transfection and induction of expression
(according to the selected promoter and/or enhancers or other
regulatory elements), the expressed chimeric polypeptides are
recovered (e.g., purified or enriched) and renatured to ensure
folding into a biologically active conformation.
[0082] In yet other embodiments, the chimeric neuregulins are
expressed in vivo by a plasmid vector or a viral vector.
Methods for Treating Neuronal and Vascular Damages with Chimeric
Neuregulin
[0083] One aspect of the present application relates to a method
for ameliorating neuronal damage in a subject. The method includes
administering to the subject an effective amount of a
pharmaceutical composition comprising a chimeric neuregulin or a
variant thereof, and/or an expression vector encoding a chimeric
neuregulin or a variant thereof. The neuronal damage may be cause
by an occlusive stroke, a neurotoxin, an acute CNS injury or
traumatic brain injury (TBI).
[0084] Another aspect of the present application relates to a
method for ameliorating secondary neuronal injury and inflammation
following TBI in a subject. The method includes administering to
the subject an effective amount of a pharmaceutical composition
comprising a chimeric neuregulin or a variant thereof, and/or an
expression vector encoding a chimeric neuregulin or a variant
thereof.
[0085] Another aspect of the present application relates to a
method for treating or ameliorating symptoms of neurodegenerative
disorders. The method includes administering to a subject suffering
from a neurodegenerative disorder an effective amount of a chimeric
neuregulin or a variant thereof, and/or an expression vector
encoding a chimeric neuregulin or a variant thereof. The term
"neurodegenerative disorders" refer to diseases caused by
progressive loss of structure or function of neurons, including
death of neurons. Examples of neurodegenerative disorders include,
but are not limited to, Alzheimer's disease, Parkinson's disease,
Huntington's disease and amyotrophic lateral sclerosis.
[0086] While not to be bound by the theory, it is believed that
administration of the chimeric neuregulin or variant thereof
results in the mobilization and migration of endogenous neural stem
cells (NSC) in vivo and can be used to stimulate adult
neurogenesis.
[0087] The chimeric neuregulin may be administered by various
routes, such as intrathecal administration, intravascular
administration, intramuscular administration, subcutaneous
administration, intraperitoneal administration, oral administration
and topical administration. Preferably, the neuregulin is
administered intrathecally.
[0088] In certain embodiments, the chimeric neuregulin is
administered intrathecally in an amount sufficient to enhance
migration of stem cells from the ventricle into damaged areas of
the brain. The chimeric neuregulin may be administered via a shunt
into the ventricle or subventricular zone or administered into the
cerebral spinal fluid by injection at the lumbar region.
[0089] The use of shunts into the ventricle is well established
practice in the medical community. Such shunts may be present for
several day or weeks. While usually used to drain excess cerebral
spinal fluid from the ventricle in cases of excessive production or
blockage of flow, such shunts would be appropriate means for
administration of chimeric neuregulin over a period of several
weeks. The care of the shunt would be an ongoing responsibility of
the medical team during the time the chimeric neuregulin is being
administered to facilitate migration of the stem cells to areas of
damage.
[0090] As used herein after "an effective amount of a chimeric
neuregulin" is a mount that is required to confer a prophylactic or
therapeutic effect on the treated subject, or an amount that is
required to meliorate at least one symptom of a target disease or
disorder in the treated subject. In certain embodiments, the
chimeric neuregulin is used at an intrathecal dose range of 0.01
.mu.g/kg body weight to 5 mg/kg body weight, 0.01 .mu.g/kg body
weight to 500 .mu.g/kg body weight, 0.01 .mu.g/kg body weight to 50
.mu.g/kg body weight, 0.01 .mu.g/kg body weight to 5 .mu.g/kg body
weight, 0.1 .mu.g/kg body weight to 5 mg/kg body weight, 0.1
.mu.g/kg body weight to 500 .mu.g/kg body weight, 0.1 .mu.g/kg body
weight to 50 .mu.g/kg body weight, 0.1 .mu.g/kg body weight to 5
.mu.g/kg body weight, 1 .mu.g/kg body weight to 5 mg/kg body
weight, 1 .mu.g/kg body weight to 500 .mu.g/kg body weight, 1
.mu.g/kg body weight to 50 .mu.g/kg body weight, 10 .mu.g/kg body
weight to 5 mg/kg body weight, 10 .mu.g/kg body weight to 500
.mu.g/kg body weight and 10 .mu.g/kg body weight to 50 .mu.g/kg
body weight.
[0091] In other embodiments, the chimeric neuregulin is used at an
intra-vascular or intramuscular dose range of 0.01 .mu.g/kg body
weight to 50 mg/kg body weight, 0.01 .mu.g/kg body weight to 5
mg/kg body weight, 0.01 .mu.g/kg body weight to 500 .mu.g/kg body
weight, 0.01 .mu.g/kg body weight to 50 .mu.g/kg body weight, 0.01
.mu.g/kg body weight to 5 .mu.g/kg body weight, 0.1 .mu.g/kg body
weight to 50 mg/kg body weight, 0.1 .mu.g/kg body weight to 5 mg/kg
body weight, 0.1 mg/kg body weight to 500 .mu.g/kg body weight, 0.1
mg/kg body weight to 50 .mu.g/kg body weight, 0.1 .mu.g/kg body
weight to 5 .mu.g/kg body weight, 1 .mu.g/kg body weight to 50
mg/kg body weight, 1 .mu.g/kg body weight to 5 mg/kg body weight, 1
.mu.g/kg body weight to 500 .mu.g/kg body weight, 1 .mu.g/kg body
weight to 50 .mu.g/kg body weight, 10 .mu.g/kg body weight to 50
mg/kg body weight, 10 .mu.g/kg body weight to 5 mg/kg body weight,
10 .mu.g/kg body weight to 500 .mu.g/kg body weight and 10 .mu.g/kg
body weight to 50 .mu.g/kg body weight, 100 .mu.g/kg body weight to
50 mg/kg body weight, 100 .mu.g/kg body weight to 5 mg/kg body
weight, 100 .mu.g/kg body weight to 500 mg/kg body weight and 1
mg/kg body weight to 50 mg/kg body weight. Administration can be a
single bolus dose or multiple bolus doses every day or every other
day for a desired period of time (e.g., 5 days, 10 days, 15 days,
20 days, 25 days, 30 days or longer). The desired treatment period
is determined by the health care provider based on the needs of
each individual subject.
[0092] In certain embodiments, the chimeric neuregulin comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOS:20-29. In certain other embodiments, the chimeric neuregulin is
a PEGylated chimeric neuregulin. In other embodiments, the chimeric
neuregulin is PEGlated at the N-terminal. In yet other embodiments,
the PEG moiety in the PEGylated chimeric neuregulin has a molecular
weight of 10-20 Kd.
[0093] In some embodiments, the chimeric neuregulin is used in a
method for ameliorating neuronal damage of occlusive stroke. The
method includes administering to a subject suffering from an
occlusive stroke an effective amount of a chimeric neuregulin or a
variant thereof, and/or an expression vector encoding a chimeric
neuregulin or a variant thereof. In one embodiment, the chimeric
neuregulin is administered after the onset of said occlusive
stroke. In another embodiment, the chimeric neuregulin is
administered within 24, 48 or 72 hours of the onset of said
occlusive stroke. Preferably, the chimeric neuregulin is used in an
amount that is effective in inhibiting inflammation in the subject.
In certain embodiments, the chimeric neuregulin is administered in
conjunction with a glutamate receptor inhibitor, a clot disrupting
agent, such as t-PA, and/or other active agents. Examples of
glutamate receptor inhibitors include, but are not limited to,
dizocilpin maleate (MK 801), R-2-amino-5-phosphonopentanoate (AP5),
2-amino-7-phosphonoheptanoic acid (AP7),
3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid
(CPPene), PEAQX, Selfotel, Amantadine, Dextrallorphan,
Dextromethorphan, Ethanol, Eticyclidine, Gacyclidine, Ibogaine,
Ketamine, Magnesium, Memantine: Methoxetamine, Nitrous oxide,
Phencyclidine, Rolicyclidine, Tenocyclidine, Methoxydine,
Tiletamine, Xenon, Neramexane, Eliprodil, Etoxadrol, Dexoxadrol,
WMS-2539, NEFA, Remacemide, Delucemine, 8A-PDHQ, Aptiganel
(Cerestat, CNS-1102), HU-211, HU-210, Remacemide, Atomoxetine, and
Rhynchophylline.
[0094] In certain other embodiments, the chimeric neuregulin is
administered after the 72 hour window in an amount sufficient to
enhance migration of stem cells from the ventricle into damaged
areas of the brain. In some embodiments, the chimeric neuregulin is
administered daily or every other day after the 72 hour window for
3-7, 3-14, 3-21 or 3-28 days.
[0095] The chimeric neuregulin may be administered by various
routes, such as intravascular administration, intrathecal
administration, intramuscular administration, subcutaneous
administration, intraperitoneal administration, oral administration
and topical administration. Preferably, the chimeric neuregulin is
administered intra-arterially.
[0096] In certain embodiments, the chimeric neuregulin is
administered intra-arterially in conjunction with a glutamate
receptor inhibitor, a clot disrupting agent, and/or other active
agents within 24, 48 or 72 hours of the onset of said occlusive
stroke to ameliorate acute damage and, then administered
intrathecally after the 72 hour window for 3-7, 3-14, 3-21 or 3-28
days in an amount sufficient to enhance migration of stem cells
from the ventricle into damaged areas of the brain.
[0097] For purposes of regeneration of neuronal tissue, the
administration of chimeric neuregulin should commence after the
initial inflammation due to the assault has subsided. In certain
embodiments, the administration of chimeric neuregulin is started
within 3, 4, 5, 6 or 7 days or within 1, 2, 3 or 4 weeks or within
1, 2 or 3 months of the initial assault, and is administered daily
or every other day or weekly for 1, 2, 3 4, 5 or 6 weeks. Because
it is necessary for the stem cells that have migrated to be
replenished in the ventricle, the intrathecal administration of
chimeric neuregulin should not be repeated at less than one week
intervals. Longer intervals may be appropriate in order to allow
greater replenishment of the stem cell supply in the ventricle.
[0098] In a preferred embodiment, the chimeric neuregulin is
administered using serum albumin as a carrier. In one embodiment,
the chimeric neuregulin is administered arterially in 1% serum
albumin. In certain embodiments, the chimeric neuregulin comprises
a sequence selected from the group consisting of SEQ ID NOS:20-29.
In certain other embodiments, the chimeric neuregulin is a
PEGylated chimeric neuregulin.
[0099] The glutamate receptor inhibitor is used in an amount that
is effective in blocking the excitotoxic events of ischemia and
decrease damage to neuronal tissue and injury arising from
reperfusion. In certain embodiments, the chimeric neuregulin and/or
glutamate receptor inhibitor are administered via the carotid
artery. In other embodiments, the chimeric neuregulin and/or
glutamate receptor inhibitor are administered to a particular area
via fluoroscopy guided catheter. Chimeric neuregulin may also be
administered intravenously in conjunction with reperfusion therapy
following occlusion of coronary arteries. In other embodiments, the
chimeric neuregulin and/or glutamate receptor inhibitor are
administered intrathecally. The chimeric neuregulin and glutamate
receptor inhibitor may be administrated simultaneously or
separately in any order.
[0100] In some other embodiments, the chimeric neuregulin is used
in a method for ameliorating neuronal damage caused by exposure to
neurotoxins, such as organophosphates. The method includes
administering to a subject in need of such treatment an effective
amount of a chimeric neuregulin or a variant thereof. In certain
embodiments, the chimeric neuregulin or variant thereof is
administered in conjunction with the administration of another
active agent to ameliorate permanent damage from the neurotoxins.
Examples of such active agents include, but are not limited to,
antidotes to neurotoxin and anticonvulsants.
[0101] Examples of neurotoxins include, but are not limited to,
organophosphates, ion channel inhibitors, insects and animal
venoms. Examples of antidotes to neurotoxin include, but are not
limited to, atropine, prostigmine glutamate antagonists, oximes and
benzodiazepies.
[0102] In certain embodiments, the chimeric neuregulin and/or other
active agent is given within 72 hours, preferably within 48 hours,
more preferably within 24 hours, from the initial exposure to the
neurotoxin. In other embodiments, the chimeric neuregulin and/or
other active agent are administered intrathecally or via the
carotid artery. In other embodiments, the chimeric neuregulin
and/or other active agent are administered to a particular area via
fluoroscopy guided catheter. Chimeric neuregulin may also be
administered intravenously or intramuscularly. The chimeric
neuregulin and other active agent may be administrated
simultaneously or separately in any order.
[0103] In some other embodiments, the chimeric neuregulin is used
in a method for ameliorating neuronal damage caused by neuronal
cell death associated with CNS injury due to: Alzeimer's Disease,
epilepsy, neonatal hypoxic ischemia, and seizures associated with
traumatic brain injury, nerve agent exposure, ischemic stroke,
hemorrhagic stroke, transient ischemic attacks, silent cerebral
infarct, blunt force trauma. The method includes administering to a
subject in need of such treatment an effective amount of a chimeric
neuregulin or a variant thereof.
[0104] Another aspect of the present invention relates a method for
preventing or ameliorating secondary neuronal injury and
inflammation following traumatic brain injury (TBI). The method
comprises the step of administering into a subject in need of such
treatment an effective amount of a chimeric neuregulin or a variant
thereof, or an expression vector encoding a chimeric neuregulin or
a variant thereof.
[0105] In certain embodiments, the chimeric neuregulin and/or other
active agent is given within 72 hours, preferably within 48 hours,
more preferably within 24 hours of TBI. In other embodiments, the
chimeric neuregulin and/or other active agent are administered
intrathecally or via the carotid artery. In other embodiments, the
chimeric neuregulin and/or other active agent are administered to a
particular area via fluoroscopy guided catheter. Chimeric
neuregulin may also be administered intravenously or
intramuscularly. The chimeric neuregulin and other active agent may
be administrated simultaneously or separately in any order.
[0106] In a preferred embodiment, the chimeric neuregulin is
administered using serum albumin as a carrier. In one embodiment,
the chimeric neuregulin is administered arterially in 1% serum
albumin. In certain embodiments, the chimeric neuregulin comprises
a sequence selected from the group consisting of SEQ ID NOS:20-29.
In another embodiment, the chimeric neuregulin is a PEGylated
chimeric neuregulin.
[0107] Another aspect of instant invention relates to a method for
ameliorating blood vessel damage caused by acute mechanical or
chemical assault. The method comprises the step of administering to
a subject in need of such treatment an effective amount of a
chimeric neuregulin or a variant thereof, or an expression vector
encoding a chimeric neuregulin or a variant thereof. While not to
be bound by the theory, it is believed that the chimeric neuregulin
or variant thereof prevents damages arising from responses to
invasive procedures by inhibiting mitogen-stimula VSMC
proliferation and migration.
[0108] In certain embodiments, the acute vascular conditions are
caused by physical assault to the blood vessels, such as placement
of a balloon or stent in the artery, diagnostic cardiac
catheterization, and cardiac surgery, especially surgery on the
heart valves. The neuregulin treatment provides protection from
permanent damage to blood vessels from restenosis and
artherosclerosis arising from such physical assault. In addition to
administration of neuregulin by intravenous or intra-arterial route
during or after the damaging occasion, a stent or catheter for use
in an invasive procedure may be coated with neuregulin. Other
routes, such as intramuscular injection, may also be used.
[0109] In certain embodiments, the chimeric neuregulin is used at
an intravascular dose range of 0.01 .mu.g/kg body weight to 50
mg/kg body weight, 0.01 .mu.g/kg body weight to 5 mg/kg body
weight, 0.01 .mu.g/kg body weight to 500 .mu.g/kg body weight, 0.01
.mu.g/kg body weight to 50 .mu.g/kg body weight, 0.01 .mu.g/kg body
weight to 5 .mu.g/kg body weight, 0.1 .mu.g/kg body weight to 50
mg/kg body weight, 0.1 .mu.g/kg body weight to 5 mg/kg body weight,
0.1 .mu.g/kg body weight to 500 .mu.g/kg body weight, 0.1 .mu.g/kg
body weight to 50 .mu.g/kg body weight, 0.1 .mu.g/kg body weight to
5 .mu.g/kg body weight, 1 .mu.g/kg body weight to 50 mg/kg body
weight, 1 .mu.g/kg body weight to 5 mg/kg body weight, 1 .mu.g/kg
body weight to 500 .mu.g/kg body weight, 1 .mu.g/kg body weight to
50 .mu.g/kg body weight, 10 .mu.g/kg body weight to 50 mg/kg body
weight, 10 .mu.g/kg body weight to 5 mg/kg body weight, 10 .mu.g/kg
body weight to 500 .mu.g/kg body weight and 10 .mu.g/kg body weight
to 50 .mu.g/kg body weight, 100 .mu.g/kg body weight to 50 mg/kg
body weight, 100 .mu.g/kg body weight to 5 mg/kg body weight, 100
.mu.g/kg body weight to 500 .mu.g/kg body weight and 1 mg/kg body
weight to 50 mg/kg body weight. Administration can be a single
bolus dose or multiple bolus doses every day or every other day for
a desired period of time (e.g., 5 days, 10 days, 15 days, 20 days,
25 days, 30 days or longer). The desired treatment period is
determined by the health care provider based on the needs of each
individual subject.
[0110] In a preferred embodiment, the chimeric neuregulin is
administered using serum albumin as a carrier. In one embodiment,
the chimeric neuregulin is administered arterially in 1% serum
albumin. In certain embodiments, the chimeric neuregulin comprises
a sequence selected from the group consisting of SEQ ID NOS:20-29.
In another embodiment, the chimeric neuregulin is a PEGylated
chimeric neuregulin. In other embodiments, the chimeric neuregulin
is PEGlated at the N-terminal. In yet other embodiments, the PEG
moiety in the PEGylated chimeric neuregulin has a molecular weight
of 10-20 Kd.
Pharmaceutical Compositions
[0111] Another aspect of the present invention relates to a
pharmaceutical composition for treating neurodegenrative disorder
and preventing or ameliorating neuronal damage caused by occlusive
stroke, neurotoxin or acute assault on vascular and neuronal
tissue. The pharmaceutical composition contains a chimeric
neuregulin or a variant thereof, and a pharmaceutically acceptable
carrier. In certain embodiments, the neuregulin comprises a
sequence selected from the group consisting of SEQ ID
NOS:20-29.
[0112] As used herein the language "pharmaceutically acceptable
carrier" is intended to include any and all solvents, solubilizers,
fillers, stabilizers, binders, absorbents, bases, buffering agents,
lubricants, controlled release vehicles, diluents, emulsifying
agents, humectants, lubricants, dispersion media, coatings,
antibacterial or antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with pharmaceutical
administration. The use of such media and agents for
pharmaceutically active substances is well-known in the art. See
e.g., A. H. Kibbe Handbook of Pharmaceutical Excipients, 3rd ed.
Pharmaceutical Press, London, UK (2000). Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the compositions is contemplated.
Supplementary agents can also be incorporated into the
compositions.
[0113] In certain embodiments, the pharmaceutically acceptable
carrier comprises serum albumin.
[0114] The pharmaceutical composition of the invention is
formulated to be compatible with its intended route of
administration. Examples of routes of administration include
parenteral, e.g., intrathecal, intra-arterial, intravenous,
intradermal, subcutaneous, oral, transdermal (topical) and
transmucosal administration. Solutions or suspensions used for
parenteral, intradermal, or subcutaneous application can include
the following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine; propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfate; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0115] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the injectable
composition should be sterile and should be fluid to the extent
that easy syringability exists. It must be stable under the
conditions of manufacture and storage and must be preserved against
the contaminating action of microorganisms such as bacteria and
fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyetheylene glycol, and
the like), and suitable mixtures thereof. The proper fluidity can
be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of the requited particle size in the
case of dispersion and by the use of surfactants. Prevention of the
action of microorganisms can be achieved by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, ascorbic acid, thimerosal, and the like. In many cases, it
will be preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, and sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0116] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a neuregulin) in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active, ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0117] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Stertes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0118] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser, which contains a suitable propellant, e.g., a gas
such as carbon dioxide, or a nebulizer.
[0119] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the
pharmaceutical compositions are formulated into ointments, salves,
gels, or creams as generally known in the art.
[0120] In certain embodiments, the pharmaceutical composition is
formulated for sustained or controlled release of the active
ingredient. Biodegradable, biocompatible polymers can be used, such
as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
e.g. Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0121] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein includes physically discrete units suited as unitary dosages
for the subject to be treated; each unit containing a predetermined
quantity of active compound calculated to produce the desired
therapeutic effect in association with the required pharmaceutical
carrier. The specification for the dosage unit forms of the
invention are dictated by and directly dependent on the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0122] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds which exhibit
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0123] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
1050 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. In certain embodiments, single dosage contains
0.01 ug to 50 mg of a chimeric neuregulin.
Kits
[0124] The invention also encompasses kits for treating or
preventing neuronal damage caused by an occlusive stroke or
exposure to neurotoxins, kits for treating neurodegenerative
disorders, kits for treating or preventing blood vessel damage
caused by acute mechanical or chemical assault, kits for treating
or preventing acute CNS injuries, and kits for preventing or
amelioratring secondary neuronal injury and inflammation following
traumatic brain injury (TBI). The kits comprise one or more
effective doses of a chimeric neuregulin, a variant of a chimeric
neuregulin, an expression vector encoding a chimeric neuregulin or
a variant of a chimeric neuregulin, or combinations thereof along
with a label or labeling with instructions on using the chimeric
neuregulin, the variant of chimeric neuregulin, or the expression
vector according to the methods of the invention. In certain
embodiments, the kits can comprise components useful for carrying
out the methods such as devices for delivering the chimeric
neuregulin, the variant of a chimeric neuregulin, or the expression
vector. In certain other embodiments, the kits can further contain
another active agent to be administered in conjunction with the
chimeric neuregulin, the variant of chimeric neuregulin, or the
expression vector.
EXAMPLES
Example 1
Prevention of Post-Trauma Damage to Blood Vessels With
Neuregulin-1
[0125] The effect of NRG1 on neointimal formation following balloon
injury to the carotid artery of the rat was examined. NRG1 (2.5
.mu.g/kg) was administered by tail-vein injection prior to injury
and every two days following injury. Two weeks after carotid artery
injury, NRG1-treated animals demonstrated a 50% reduction in lesion
size compared to controls receiving the vehicle. The effect of NRG1
on vascular smooth muscle cell (VSMC) function was studied. A7r5
rat VSMC cultures were pretreated with NRG1 for 24 hours, and then
stimulated with platelet derived growth factor (PDGF) for 48 hours.
NRG1 significantly decreased both baseline and PDGF-stimulated VSMC
proliferation in a dose-dependent manner. NRG1 also blocked VSMC
migration and prevented the downregulation of a-smooth muscle actin
by PDGF, indicating that it may prevent VSMC phenotypic reversion
following injury. These findings demonstrate NRG1 as a therapeutic
agent for the treatment of restenosis and atherosclerosis.
Experimental Injury, Harvest, and Tissue Preparation of Rat Carotid
Arteries
[0126] Male Sprague-Dawley rats (350-400 g) were balloon-injured
using methods as previously described in accordance with a protocol
approved by the Standing Committee on Animals, Morehouse School of
Medicine. Rats were anesthetized with an intraperitoneal injection
of xylazine (5 mg/kg body weight) and ketamine hydrochloride (90
mg/kg body weight). The left common carotid artery was exposed by a
6-cm midline cervical incision. Proximal and distal blood flow was
occluded by clamping. Polyethylene 10 tubing was inserted
retrogradely into the internal carotid artery and advanced into the
left common carotid artery. After gentle flushing of the artery
with normal saline, the tubing was removed and a 2-French (F)
Fogarty embolectomy balloon catheter was inserted. Balloon
inflation to 1.5 to 1.8 times the external diameter of the artery
was achieved by caliper measurement under stereomicroscopy. After
holding the inflation for 30 seconds, the catheter was removed. The
uninjured right carotid artery was used as the control. Rats were
treated with NRG1.beta. or NRG-1.alpha. (EGF-like domain, R&D
Systems, Minneapolis, Minn. dissolved in 1% BSA/PBS) by tail-vein
administration at a dose of 2.5 ug/kg body weight, starting at day
0 before injury, and continuing for every 2 days for the next 14
days. Control rats were treated with vehicle (1% BSA/PBS). The
animals were weighed before the procedure and at sacrifice to
evaluate the possible adverse effects of NRG1. Vessels were
harvested time points 0 and 14 days for mRNA analysis or histology.
Injured vessels were compared with their contralateral
controls.
Tissue Processing and Quantitative Histomorphometric Analysis
[0127] Animals were euthanized with CO.sub.2 14 days after injury.
Carotid arteries were washed with saline to clear blood, embedded
in Tissue-Tek OCT medium and frozen using liquid nitrogen. Carotid
sections were cut with a cryostat into cross sections of 12 .mu.m
taken from the center and distal portion of the vessels, and
stained with hematoxylin and eosin. The medial thickness was
determined by the area of the internal elastic lamina subtracted
from the external elastic lamina. Morphometry was performed using
at least six individual sections of each arterial segment and used
to determine the lesion size expressed as intima/media ratio. The
intimal and medial layer thicknesses were measured using a
computer-based image analyzing program (Image J, NIH).
A7r5 VSMC Cultures
[0128] A7r5 rat aortic vascular smooth muscle cells (VSMC) (ATCC
CRL-1444) were obtained from American Tissue Type Culture
(Manassas, Va.) and grown in Dulbecco's modified Eagle medium
supplemented with glutamine, 10% fetal calf serum (FCS), and 1%
Penicillin/Streptomycin at 37.degree. C. in a humidified incubator
with 5% CO.sub.2. Cells were passaged weekly. All studies were
performed on cells from passages 9-12.
Determination of VSMC Proliferation
[0129] VSMC were seeded at a density of 1.times.10.sup.3 cells in
triplicate wells of a 96 well plate. After 24 hours, cells were
serum starved in DMEM/F-12 (Gibco; Carlsbad, Calif.) containing
0.1% FCS (low serum medium; LSM) to induce quiescence. After 24
hours of serum deprivation, cells were pretreated with 0-200 nM of
NRG1.alpha. or NRG1.beta. for 24 hours. Cells were then treated
with 10 ng/mL of PDGF-BB for 48 hours to stimulate VSMC
proliferation. For direct measure of cell number, cells were
counted using a Coulter counter. VSMC cell proliferation and
viability was also measured using the CellTiter 96 AQueous Non
Radioactive Cell Proliferation Assay (Promega; Madison, Wis.)
according to the manufacturer's protocol. After incubation at
37.degree. C. in humidified 5% CO.sub.2 for 1 hour, the absorbance
was recorded at 490 nm using a plate reader. Measurement of DNA
synthesis was performed using the BrDU Cell Proliferation Assay
(Calbiochem, San Diego, Calif.) according to the manufacturer's
protocol.
Cell Migration Assay
[0130] Neuro Probe 48-well microchemotaxis chambers (Costar,
Corning Inc.) with PVP-free polycarbonate filter (8.0 .mu.m pore
size) were used to measure VSMC migration. Quiescent cells were
trypsinized and resuspended in LSM with or without NRG1 and
incubated for 24 hours at 37.degree. C. Cells were then treated
with PDGF which was added to the bottom well of the Boyden chamber
and incubated for 48 hours at 37.degree. C. Cells that migrated to
the lower side of the filters were fixed and stained with the Diff
Quick staining kit (VWR Laboratory, West Chester, Pa.). The filters
were mounted on glass slides and counted by light microscopy using
.times.100 magnification.
Protein Purification and Western Analysis
[0131] Reactions were terminated by placing the cells on ice,
aspirating the medium and adding ice-cold lysis buffer (50 mM Tris,
150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.5% Nonidet P-40, 1 mM
sodium orthovanadate, 1 mM phenyl methanesulfonyl fluoride, pH 8.0)
for 30 minutes at 4.degree. C. Harvested lysates were denatured
with loading buffer, resolved in SDS/5% polyacrylamide gels and
transferred to poly vinylidene difluoride (PVDF) membranes
(Millipore Corp., Bedford, Mass.). Membranes were be blocked with
3% nonfat dry milk in phosphate buffered saline-0.5% Tween 20
(PBST) and exposed to primary antibody, anti-smooth muscle
alpha-actin (SMA) (Santa Cruz, Calif.) diluted in blocking buffer
overnight at 4.degree. C. After incubation, membranes were washed
with PBST. After wash, membranes were exposed to an alkaline
phosphatase-conjugated anti-rabbit secondary antibody for 1 hour.
Membranes were subsequently washed with PBST, incubated with
chemiluminescence reagents and exposed to x-ray film. For ERK1/2
phosphorylation, VSMC were pre-treated with NRG1.beta. for 24 hours
and stimulated for 15 minutes with PDGF. Western blots were
performed using primary antibodies for phosphorylated and
unphosphorylated forms of ERK1/2 (Cell Signaling, Danvers, Mass.)
diluted 1:250 in blocking buffer. Immunoblotting using an
anti-tubulin antibody was used to normalize protein levels in each
sample.
Cell Viability Assay
[0132] Quantitative viability assessment was performed using 1%
Calcein-AM (Molecular Probes, Eugene, Oreg.), a fluorescent
membrane-integrity dye, diluted in HBSS according to the
manufacturer's protocol. Qualitative assessment of cell viability
in treated cells was performed using the trypan blue-exclusion
assay. Non-viable cells were quantified visually using a light
microscopy.
Statistical Analysis
[0133] Each experiment was repeated a minimum of three times. Data
are expressed as the mean.+-.standard deviation (SD). An unpaired
Student's t-test and ANOVA were performed to make comparisons
between groups. A value of p less than 0.05 was considered
significant.
NRG1 Attenuated Neointima Formation after Rat Carotid Balloon
Injury
[0134] Neointimal hyperplasia was histologically evident in the
carotid arteries 14 days after balloon injury compared to uninjured
contralateral controls. The neointima of the rats receiving
intravenous administration of NRG1 was significantly reduced
compared to balloon-injured animals. Morphometric analysis showed
that NRG1 reduced the size of the lesion by .about.50% compared to
vehicle-treated control animals. Treatment of animals with NRG1
showed no overt negative side effects and there was no significant
difference in body weight observed among the control and NRG1
treated rats.
NRG1 Inhibits Proliferation in VSMC
[0135] Serum-starved VSMC were pre-treated with NRG1 for 24 hours,
then stimulated with PDGF for an additional 48 hours. Stimulation
of cells with PDGF increased proliferation of VSMC 2-fold.
Pre-treatment with either NRG1.beta. or NRG-1.alpha. resulted in a
dose-dependent decrease in baseline and PDGF-stimulated
proliferation as measured by MTS activity. Direct cell counting
using Coulter counter demonstrated that NRG1 reduced
PDGF-stimulated VSMC proliferation, but not baseline cell numbers.
Analysis of BrDU incorporation revealed a similar pattern to the
Coulter counter demonstrating that NRG1.beta. significantly
inhibited PDGF-induced proliferation, but did not alter baseline
DNA synthesis.
[0136] Calcein-AM and trypan blue viability assays were carried out
in cells pre-treated with NRG1 with or without PDGF. The calcein-AM
assay demonstrated that treatment of VSMC with NRG1 does not alter
cell viability. These results were corroborated using the
trypan-exclusion assay, which revealed that less than 1.0% of the
cells took up the dye.
NRG1 Decreases VSMC Migration
[0137] The migration of VSMC was measured using a transwell
migration assay. VSMC were pretreated with 100 nM NRG1.alpha. or
NRG1.beta., and then stimulated with 10 ng/ml of PDGF-BB for 48
hours. The results show that NRG1 alone does not alter baseline
VSMC migration. VSMC treated with PDGF displayed a 2-3 fold
increase in migration. Both NRG-1.alpha. and NRG-1.beta. decreased
PDGF-stimulated VSMC migration by 80% and 90%, respectively.
NRG1 Regulates Smooth Muscle .alpha.-Actin Expression
[0138] The mRNA and protein expression on SMA, and a marker for
differentiated and contractile VSMC, after NRG1 treatment was
examined. Serum-starved, quiescent VSMC displayed SMA expression,
which was reduced after treatment with PDGF. NRG1.beta. alone did
not alter SMA mRNA or protein expression, however, pre-treatment of
PDGF-stimulated VSMC with NRG1.beta. resulted in SMA expression
that returned to near baseline levels.
NRG1 Inhibits PDGF-Induced Phosphorylation of ERK1/2
[0139] This study demonstrated that NRG1 attenuates neointimal
formation and vascular balloon injury. NRG1 reduced the size of the
lesion by .about.50% compared to vehicle-treated control animals.
This finding clearly shows that NRG1 is useful in the prevention of
vascular diseases such as restenosis and atherosclerosis. The NRG1
blocks PDGF-induced proliferation of VSMC in a dose-dependent
manner. The inhibitory effects of NRG1 on VSMC proliferation were
confirmed by direct cell counting and measuring DNA synthesis by
BrDU incorporation. An intriguing observation was the difference in
the effect of NRG1 on baseline VSMC proliferation using the
MTS-based assay compared to the other methods. In the cell counting
and BrDU approaches, PDGF increased VSMC proliferation was blocked
by NRG1, however, baseline VSMC numbers were not altered. Using the
MTS-based assay, a 50% decrease in baseline MTS activity was seen
after NRG1 administration. Since the MTS assay measures metabolic
activity, it is possible that NRG1 may prevent PDGF-stimulated
proliferation by promoting VSMC differentiation, which could result
in a decrease in metabolic activity and/or a reduction in the
capability of PDGF to stimulate VSMC proliferation. That this is
due to apoptosis resulting from treatment is unlikely since there
was no evidence of increased dead or non-viable cells after
neuregulin treatment.
Example 2
Combination Therapy for Preventing Permanent Neuronal Damage
[0140] In the case of prevention of damage resulting from exposure
to neurotoxins, such as organophosphates, or as a result of
obstructive stroke, such as that caused by an infarct, studies were
done studying effect on permanent middle cerebral artery occlusion
(pMCAO) using combination therapy. Studies were done giving
dizocilpin maleate (MK-801 from Sigma), a glutamate receptor
inhibitor which blocks the excitotoxic events of ischemia in
combination with neuregulin within a therapeutic window of about
13.5 hours in the rat to decrease permanent neuronal damage. The
therapeutic window in larger animals having a lower metabolism
would be in the range of 0 to 72 yours. In the case of expected
exposure to neurotoxins, the neuregulin could be administered in
conjunction with antidotes. Other active agents which may be used
in conjunction with neuregulin in the manner disclosed for use with
MK-801 are selfotel, aptiganel, magnesium, acetylcholine, GABA
agonists (clomethiazole, diazepam and other benzodiazepines) and
serotonin agonists.
[0141] In the case of damage arising from exposure to neurotoxins
such as organophosphate (OP) nerve agents, current post-exposure
medical countermeasures against nerve agents (e.g. atropine,
prostigmine glutamate antagonists, oximes (such as 20 pralidoxime
chloride) and benzodiazepines) are useful in preventing mortality,
but are not sufficiently effective in protecting the CNS from
seizures and permanent injury. Therefore, new and more effective
medical countermeasures against OP nerve agents are needed to
facilitate better treatment that will prevent extensive, permanent
nerve damage in survivors. Other agents that may be used to treat
patients that have been exposed to neurotoxin include
anticonvulsants.
[0142] In both instances of pMCAO and exposure to neurotoxins,
neuregulin may be administered concurrently with the other active
agents to ameliorate permanent damage from infarct disintegrators
or nerve agent counteractants. In certain embodiments, neuregulin
is given within a 72 hour widow after the initial exposure to the
causative agent or the onset of occlusion of the blood supply, more
preferably within 24 hours after the causal event.
[0143] In both instances where the neuregulin is given as
combination therapy to prevent cerebral neuronal damage the
neuregulin is administered into the carotid artery with an
appropriate carrier. In animal studies, the neuregulin is
administered in bovine serum albumin. In humans, a preferred
carrier would be human serum to be administered within the first 72
hours, preferably within the first 24 hours, of the assault,
whether chemical or physical. In the instance where the neuregulin
is to prevent damage resulting from mechanical damage to a blood
vessel, the neuregulin may be given intravenously in the usual
carriers used for intravenous administration. Addressing the use of
neuregulin simultaneously with other agents, studies were done on
rats that had been subjected to left middle cerebral artery
occlusion (MCAO).
Middle Cerebral Artery Occlusion
[0144] All surgical procedures were performed by sterile/aseptic
techniques in accordance with institutional guidelines. Adult male
Sprague-Dawley rats weighing 250-300 g were used for this study.
Animals were subjected to left MCA occlusion. Rats were
anesthetized with a ketamine/xylazine solution (10 mg/kg, i.p.).
MCA occlusion was induced by the intraluminal suture MCAO method as
previously described (Belayev et al. 1996; Belayev et al. 1995).
Briefly, the left common carotid artery (CCA) was exposed through a
midline incision and was carefully dissected free from surrounding
nerves and fascia. The occipital artery branches of the external
carotid artery (ECA) were then isolated, and the occipital artery
and superior thyroid artery branches of the ECA were coagulated.
The ECA was dissected further distally. The internal carotid artery
(ICA) was isolated and carefully separated from the adjacent vagus
nerve, and the pterygopalatine artery was ligated close to its
origin with a 6-0 silk suture. Then, a 40 mm 3-0 surgical mono
filament nylon suture (Harvard Apparatus, Holliston, Mass.) was
coated with poly-L-lysine with its tip rounded by heating near a
flame. The filament was inserted from the external carotid artery
(ECA) into the internal carotid artery (ICA) and then into the
circle of Willis to occlude the origin of the left middle cerebral
artery. The suture was inserted 18 to 20 mm from the bifurcation of
the CCA to occlude the MCA. In the permanent MCAO (pMCAO), the
suture was left in place for 24 hours prior to sacrificing the
animal. In the transient MCAO (tMCAO) model, the nylon suture was
withdrawn 1.5 hours following ischemia and the brain tissues were
reperfused for 24 hours before sacrificing. To determine the
effects of NRG1 on ischemic stroke, rats were injected
intra-arterially with a single bolus 10 ul dose of vehicle (1% BSA
in PBS) or NRG1.beta. (10 .mu.mol/L NRG1 (EGF-like domain, R&D
Systems, Minneapolis, Minn.) in 1% BSA in PBS) through a Hamilton
syringe.
[0145] NRG1 or vehicle was administered by bolus injection into the
ICA through ECA immediately before MCAO. MK-801 (0.5 mg/kg) was
either administered IP immediately prior to NRG1 administration or
co-administered IA simultaneously with NRG1. All NRG1 and vehicle
treatment studies were performed in a double-blinded manner. Core
body temperature was monitored with a rectal probe and maintained
at 37.degree. C. with a Homeothermic Blanket Control Unit (Harvard
Apparatus) during anesthesia. Neurological score was determined in
a double blinded fashion using a five-point neurological evaluation
scale (Menzies et al. 1992) in rats treated with vehicle or NRG1
four hours after reperfusion. All animals were tested prior to
surgery (controls) and after treatment with NRG1 or vehicle.
Neurological function was graded on a scale of 0-4 (normal score 0,
maximal deficit score 4). While intra-arterial injection into the
carotid artery was used, fluoroscopic guided catheter-based therapy
wherein the catheter is guided to the arteries which best access
the damaged tissue is appropriate.
Measurement of Infarct Formation
[0146] Twenty-four hours after reperfusion, the animals were killed
and the brain tissue was removed and sliced into 2.0 mm-thick
sections. Brain slices were incubated in a 2% triphenyltetrazolium
chloride (TTC) solution for 30 minutes at 3.degree. C. and then
transferred into a 4% formaldehyde solution for fixation. TTC, a
colorless salt, is reduced to form an insoluble red formazan
product in the presence of a functioning mitochondrial electron
transport chain. Thus, the infarcted region lacks staining and
appears white, whereas the normal non-infarcted tissue appears red.
Infarct area of four slices of 2 mm coronal sections of each brain
was calculated in a blinded manner by capturing the images with a
digital camera. Rats showing tremor and seizure (which rarely
occurred in this study) were excluded from studies of brain
infarction to eliminate cerebral hemorrhage or brain trauma as
potential variables in this study. Infarct volumes were analyzed by
ANOVA; P<0.05 was regarded as significant.
The Effect of NRG1 on Neural Stem Cells (NSCs) Isolated from Ell
Mouse Telencephalon
[0147] The telencephalon of El1 mouse embryos were isolated and the
dissociated cells cultured as neurosphere cultures. Cultures were
treated with the EGF-like domain of neuregulin-1.beta. (NRG1). The
EGF-like domain contains the receptor binding portion of the
molecule and has been shown to display all the known biological
activities of the full-length neuregulins. The cells formed
neurospheres and expressed nestin, an intermediate filament protein
present in NSCs and RPs in the developing CNS. The cultures were
examined to determine whether the addition of NRG1 to cell
suspensions obtained from El1 mouse cortical tissue would generate
neurospheres in the absence of bFGF. After 7 days in culture, there
was no significant difference in the total number or size of
neurospheres in the NRG1 treated group compared with the untreated
group. This result demonstrated that NRG1 alone, unlike bFGF, could
not generate neurospheres. When bFGF-generated neurospheres were
plated onto coated coverslips in the presence of bFGF, cells
continued to divide and migrate out of the sphere to form a
monolayer. Upon withdrawal of bFGF, migrating cells differentiated
into cells expressing neuronal, astrocyte and oligodendrocyte
markers. Neuronal cells were identified by labeling with the
anti-MAP2 antibody. Oligodendrocytes were identified with an
antibody directed against O4 and astrocytes were identified with an
antibody directed against GFAP. Morphologically, these
MAP2-positive cells appeared neuronal and showed: (i) a spherical,
ovoid, or pyramidal shaped soma; (ii) phase-bright appearance;
(iii) branching processes (presumably dendrites) arising from the
soma.
NRG1 Increases the Proliferation of MAP2-Positive Cells in
Neurosphere Cultures
[0148] The actions of NRG1 on .beta.FGF-generated neurospheres were
examined by plating neurospheres on coverslips as described above.
Neurospheres were cultured in the absence or presence of 5 nM NRG1
for 5 days, and then, co-labeled with BrDU and MAP2 or GFAP
antibodies. After 5 days of treatment with 5 nM NRG1, a dramatic
increase in the number of cells surrounding the core of the
neurosphere was observed in NRG1 treated cultures as compared to
control. A 44.+-.3.3% increase in [3H]thymidine incorporation was
seen in NRG1 treated cultures that paralleled the increase in the
total number of cells. More MAP2 and BrDU co-labeled cells were
found both in the central core and peripheral area of NRG1 treated
neurospheres, but few double-labeled cells were seen in the
control. There was a 2.5-fold increase in MAP2 positive cells, but
no increase in MAP2-negative cells, suggesting that the majority of
NRG1 treated cultures were neuronal.
[0149] To further characterize the effect of NRG1 on NSCs,
neurospheres were cultured in the absence or presence of 1 or 5 nM
NRG1, then co-labeled with BrDU and MAP2 or GFAP antibodies. After
5 days, there was a 4-fold increase in the number BrDU-labeled
cells in the neurosphere outgrowth area in 5 nM NRG1 treated group
compared to control. A smaller, but significant increase was also
observed with 1 nM NRG1 treatment demonstrating a dose-dependent
response of cells to NRG1. Most of the BrDU positive cells
co-labeled with the MAP2, but not the GFAP antibody. Therefore, the
increased proliferation was specific for neuronal cells and not in
GFAP-positive astrocytes. The increase in number of MAP2 positive
cells that co-labeled with BrDU was parallel to the increase of
BrDU positive cells, suggesting that most of the cells
proliferating in response to NRG1 were neuronal. In cultures
maintained for 8 days after withdrawal of bFGF, virtually no cells
showed BrDU incorporation in control cultures. By that time point,
most cells had differentiated and lost the ability to proliferate.
However, numerous BrDU-positive neurons were present in the NRG1
treated group, suggesting that NRG 1 prolongs the proliferation of
immature neuronal cells. Under our culture conditions, few cells
were labeled with GFAP and O4 in control cultures, or after
treatment with NRG1, therefore the cells that labeled with BrDU
alone were likely undifferentiated NSCs.
NRG1 Increases Proliferation Rather than Survival
[0150] The increased production of neurons could be altered by
affecting (1) the proliferation of NRPs (2) the differentiation of
NSCs into neurons, or (3) or by altering the survival of neuronal
cells. Increased proliferation might result in an increase in the
total number of cells as well as in the number of proliferating
MAP2-positive cells; increased differentiation might result in an
increase in the number of MAP2-positive cells within the same total
population of cells; increased survival might result in an increase
in MAP2-positive cells, but not necessarily cells co-labeled with
BrDU or nestin. To determine whether the increase in the number of
NRPs induced by NRG1 was due to the increase of cell survival, we
evaluated cell viability by using a Viability. Assay Kit (Molecular
probes). Results showed that the total number of cells increased
after 4 days in the cultures. The number of live cells was greater
in NRG1 treated group compared to control. Twice as many live cells
were present in the NRG1 group after 8 days. There was no
difference in the number of dead cells in control or NRG1 treated
cultures at most time points. This result shows that NRG1
stimulated proliferation rather than cell survival.
NRG1 Stimulates the Mobilization of NSCs in Adult Rat Brain In
Vivo
[0151] SVZ cells were labeled by stereotaxically injecting DiI into
the lateral ventricle of adult rats. Twenty-four hours later, NRG1
or vehicle were injected into the lateral ventricle and the animals
were sacrificed 1 day later. Intense labeling was visible in the
cells lining the ventricle (V) and in the choroids plexus (CP)
after injection of DiI. When the vehicle was injected 24 after the
DiI labeling, few cells had migrated out from the SVZ. However,
after NRG1 administration, numerous NSCs had migrated from the SVZ,
as far away as to the cerebral cortex. Similar results were seen
when Fluorogold was injected into the lateral ventricle.
Preliminary results indicate that a subpopulation of the labeled
cells co-label with an antibody for NeuN, a neuronal marker.
[0152] The poor regenerative capacity of the sensory of the
mammalian CNS has led to investigations of different approaches to
increase the function of these structures after neurodegeneration
or injury. One strategy to repair the injured CNS has been to
replace the lost neurons with embryonic stem cell-derived neuronal
stem cells (eNSCs). The use of eNSCs has shown promise in the
treatment of a variety of neurological diseases and they have
recently been shown to survive and differentiate into glia and
neurons after CNS transplantation. However, a number of biological
and ethical issues have slowed this area of research. NSCs have
been demonstrated in the adult brain and have been shown to have
the potential to differentiate into a variety of neuronal cell
types. Therefore, another strategy has focused on maximizing the
potential of this endogenous population of cells by stimulating
their mobilization, proliferation, migration and differentiation in
vivo following CNS injury and degeneration. Understanding the
technical and logistic considerations for employing adult NSCs is
essential to optimizing and maintaining cell survival before and
after activation, as well as for tracking the fate of mobilized
cells. It is now recognized that NSC strategies will be effective
only if the new cells have the same abilities and characteristics
as the original neurons. The administration of NRG1 into the
cerebral spinal fluid or through a shunt into the ventricle for
repeated administration (intrathecal administration) gives a method
of encouraging production of stem cells in the ventricle with
migration of stem cells from the ventricle to the damaged areas of
the brain. Administration may be used with spinal tap or may be
administered through a shunt into the ventricle. Appropriate
carriers include glucose, isotonic glucose and other carriers
usually used for intrathecal administration. However, the
neuregulin may be administered into the cerebral spinal fluid at
the lumbar region. For arterial administration, the carrier may,
advantageously, contain serum albumin.
[0153] To obtain maximum benefit, the neuregulin will usually
administer into the carotid artery or by some other means such as
fluoroscopy guided catheter-based means that will provide arterial
access to the brain.
[0154] While it had previously been demonstrated that a single
intra-arterial administration of NRG1 prior to MCAO prevented
neuronal death following ischemia and reperfusion, there was no
indication that use at or after time of assault, whether mechanical
(as with an infarct) or chemical, would be effective to ameliorate
damage arising from the assault.
[0155] Neuregulin also has use, with similar dosage for intravenous
administration in conjunction with reperfusion therapy such as
anticoagulant therapy to ameliorate damage to the artery. In such
instances, the neuregulin may be administered in carriers such as
glucose, saline, Ringer's lactate, etc.
[0156] Agents with other mechanisms of action that prevent or avoid
formation of obstructive occlusion such as those which cause clots
to dissolve can also be used with neuregulin. In one embodiment,
tissue plasminogen activator (t-PA) is used in conjunction with
neuregulin. At present, use of t-PA remains limited and must be
administered within three hours of the observed ischemic event.
However, t-PA patients are at high risk of hemorrhagic
transformation. Furthermore, t-PA causes inflammatory responses and
reperfusion injury in the brain. The t-PA is administered
intravenously in saline or similar carriers. In all instances, the
neuregulin is most effective if administered into the carotid
artery in a carrier containing serum albumin (in the case of
humans, human serum albumin). The agents may be administered
essentially simultaneously or the neuregulin may be administered
within the 0-72 hour time period, though it is preferred practice
to administer the neuregulin within 24 hours of administration of
the t-PA.
[0157] While treatments cited above may be effective in limiting
damage from a pathology-causing event, the recovery of function can
take place only with regeneration of neuronal tissue. The
administration of neuregulin into the ventricularzone provides
means for enhancing migration of stem cells which are formed in the
ventricle to the site of neuronal damage.
Example 3
ErbB4 Receptors are Expressed Apoptotic and Degenerating
Neurons
[0158] After erB4 immunohistochemistry, brain sections harvested
from rats subjected to middle cerebral artery occlusion (MCAO) were
stained with Fluoro-Jade, a marker for degenerating neurons, and
with antibodies against ErbB4. As shown in FIGS. 4A-4F, many
neurons in the cortex were Fluoro-Jade-positive (FIG. 4A). ErbB4
positive cells (FIG. 4B) were co-localized in Fluoro-Jade-positive
neurons (FIG. 4C). Similarly, TUNEL staining (FIG. 4D) and ErbB4
(FIG. 4E) were double-labeled (FIG. 4F) in a subpopulation of cells
in the ipsilateral brain.
Example 4
ErbB4 Expression is Upregulated in Macrophages/Microglia but not in
Astrocytes Following MCAO
[0159] Sections from the ipsilateral hemisphere of rats subjected
to MCAO were double labeled with antibodies against erbB4 (FIG. 5A)
and glial fibrillary acidic protein (GFAP) (FIG. 5B). Cells in the
peri-infarct regions did not show co-localization of erbB4 and GFAP
(FIG. 5C). Co-localization of erbB4 and Mac-1/CD11b indicated that
ErbB4 is found in a subset of macrophages/microglia (FIG. 5D).
Example 5
NRG-1B Treatment Reduces MCAO/Reperfusion-Induced Brain
Infarction
[0160] FIGS. 6A-6D show representative TTC stained brain sections
from rats injected with vehicle (panel a; n=11), NRG1.beta. (panel
b; n=7) or NRG1.alpha. (panel c; n=3) before MCAO. NRG1.beta. (2.5
.mu.g/kg) or NRG1.alpha. (2.5 mg/kg) was given by a single
intra-arterial injection immediately before MCAO. Adult male
Sprague-Dawley rats weighing 250-300 g were used for this study. A
total of 164 rats were used in this study. Rats were anesthetized
with a ketamine/xylazine solution (10 mg/kg, IP) and subjected to
left MCAO. MCAO was induced by the intraluminal suture method where
the left common carotid artery (CCA) was exposed through a midline
incision and was carefully dissected free from surrounding nerves
and fascia. The occipital artery branches of the external carotid
artery (ECA) were then isolated, and the occipital artery and
superior thyroid artery branches of the ECA were coagulated. The
ECA was dissected further distally. The internal carotid artery
(ICA) was isolated and carefully separated from the adjacent vagus
nerve, and the pterygopalatine artery was ligated close to its
origin with a 6-0 silk suture. Then, a 40 mm 3-0 surgical
monofilament nylon suture (Harvard Apparatus, Holliston, Mass.) was
coated with poly-L-lysine with its tip rounded by heating near a
flame. The filament was inserted from the ECA into the ICA and then
into the circle of Willis to occlude the origin of the left MCA.
The suture was inserted 18 to 20 mm from the bifurcation of the CCA
to occlude the MCA. After 1.5 hour of ischemia, the nylon suture
was withdrawn and the ischemic brain tissue was reperfused for 24
hours before sacrificing. Core body temperature was monitored with
a rectal probe and maintained at 37.degree. C. with a Homeothermic
Blanket Control Unit (Harvard Apparatus) during anesthesia. To
determine the effects of NRG-1 on ischemic stroke, rat were
injected intravascularly with a single bolus 10 .mu.l dose of
vehicle (1% BSA in PBS) or NRG-1.beta. (1-50 umol/L NRG-1 (EGF-like
domain, R&D Systems, Minneapolis, Minn.) dissolved in 1%
BSA/PBS) through a Hamilton syringe at a rate of 5 .mu.l/min. This
resulted in the administration of 0.5-2.5 .mu.g of NRG-1/kg body
weight. NRG-1 or vehicle was administered by bolus injection into
the ICA through ECA. Solutions were administered either before MCAO
or immediately following 1.5 hours of MCAO and either 0, 4 or 12
hours of reperfusion. Animals were sacrificed 24 hours after
reperfusion or after 14 days for the long-term studies. Animals
were killed 24 hours later and the brains were sliced into 2 mm
sections and stained with 2,3,5-triphenyltetrazolium chloride
(TTC). Infarct volumes in brains from vehicle and NRG1 treated
animals are shown in the graph (FIG. 6D). The data demonstrate that
NRG1.beta. treatment reduces MCAO/reperfusion-induced brain
infarction.
Example 6
NRG-1.beta. Suppresses MCAO/Reperfusion-Induced Apoptotic Damage in
Rat Brain
[0161] Rats were subjected to MCAO for 1.5 hours followed by
reperfusion for 24 hours. FIGS. 7A-7E show representative views of
TUNEL labeling of rat brain sections (n=5 for each condition).
TUNEL assay was performed with a DeadEND Fluorometric TUNEL System
(Promega, Madison, Wis.) according to the manufacturer's
instructions. Slides were then washed with PBS and mounted with
Vectashield Mounting Medium containing DAPI. All sections were
examined by fluorescence microscopy in three random middle cerebral
artery served areas in the inner border of the infarct in the
ischemic front-parietal cortex of each rat. In animals given
vehicle or neuregulin-1, cortex and striatum were examined in three
or more 20 .mu.m sections per animal. TUNEL staining was found in
the cortex (FIG. 7A) and striatum (FIG. 7B) following MCAO while no
TUNEL staining was seen in the cortex (FIG. 7C) and reduced levels
were seen in the striatum (FIG. 7D) in NRG1.beta.-treated rats. The
coronal brain image (.about.bregma+1.2 mm) indicates the areas
observed in the sections (FIG. 7E).
Example 7
NRG-1 Treatment Reduces MCAO/Reperfusion-Induced Brain
Infarction
[0162] FIGS. 8A-8E show] representative TTC stained coronal brain
sections from rats injected with vehicle (FIG. 8A) or NRG1
immediately after MCAO (FIG. 8B) and 4 hours after reperfusion
(FIG. 8C). Infarct volumes in brains from rats treated with vehicle
(n=10) or NRG1 immediately after MCAO (RO; n=8), 4 hours after
reperfusion (R4; n=6) or 12 hours after reperfusion (R12; n=8) are
show in the graph (FIG. 8D). The time line (FIG. 8E) illustrates
the MCAO protocol and NRG1 injections.
Example 8
NRG-1 Administration Resulted in a Significant Improvement in
Neurological Outcome
[0163] NRG1 was administered after MCAO and 4 hours of reperfusion.
As shown in FIG. 9, neurological function was graded on a scale of
0-4 (normal score 0, maximal deficit score 4). All animals were
tested prior to surgery (controls; n=14) and after treatment with
NRG-1 or vehicle. The NRG1 treated group (n=9) displayed a 33%
improvement in neurological score compared with vehicle treated
rats (n=5).
Example 9
NRG1.beta. Prevents Microglial and Astrocytic Activation Following
MCAO
[0164] Rats were subjected to MCAO followed by reperfusion for 24
hours (n=5 for each condition). NRG1I3 or vehicle was injected
intraarterially as described above. Sections were labeled for
immunohistochemistry with an antibody against ED-1. As shown in
FIGS. 10A-10F, while no staining was seen in the contralateral side
(FIG. 10A), ED-1 labeled cells are present in the ipsilateral
hemisphere (FIG. 10B) following MCAO in vehicle-treated animals.
Few ED-1 positive cells are found in animals treated with
NRG1.beta. (FIG. 10C). To assess astrocytic activation, sections
were labeled for immunohistochemistry with an antibody against
GFAP. Compared to the contralateral control (FIG. 10D), heavy GFAP
staining is found at the border or infarct (FIG. 10E) following
MCAO in vehicle-treated animals. However, when rats were treated
with NRG-1.beta., GFAP expression was dramatically reduced in the
peri-infarct regions (FIG. 10F).
Example 10
NRG1.beta. Reduces MCAO/Reperfusion-Induced IL-1.beta. mRNA
Levels
[0165] Rats were treated with NRG1.beta. or vehicle then subjected
to MCAO. RNA was isolated and IL1.beta. mRNA expression was
measured by RT-PCR. FIG. 11 shows the mRNA expression levels of
IL-1 (panel a) and GAPDH (panel b) (n=4 for each condition). Panel
c shows the average percentage of change.+-.SEM in IL-1 mRNA levels
from NRG1.beta.-treated rat compared to vehicle-treated controls
after normalization to GAPDH.
[0166] The above description is for the purpose of teaching the
person of ordinary skill in the art how to practice the present
invention, and it is not intended to detail all those obvious
modifications and variations of it which will become apparent to
the skilled worker upon reading the description. It is intended,
however, that all such obvious modifications and variations be
included within the scope of the present invention, which is
defined by the following claims. The claims are intended to cover
the claimed components and steps in any sequence which is effective
to meet the objectives there intended, unless the context
specifically indicates the contrary.
Sequence CWU 1
1
29170PRTHomo sapiens 1Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu
Lys Thr Phe Cys Val 1 5 10 15 Asn Gly Gly Glu Cys Phe Met Val Lys
Asp Leu Ser Asn Pro Ser Arg 20 25 30 Tyr Leu Cys Lys Cys Pro Asn
Glu Phe Thr Gly Asp Arg Cys Gln Asn 35 40 45 Tyr Val Met Ala Ser
Phe Tyr Lys His Leu Gly Ile Glu Phe Met Glu 50 55 60 Ala Glu Glu
Leu Tyr Gln 65 70 271PRTHomo sapiens 2Thr Ser His Leu Val Lys Cys
Ala Glu Lys Glu Lys Ala Phe Cys Val 1 5 10 15 Asn Gly Gly Glu Cys
Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg 20 25 30 Tyr Leu Cys
Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn 35 40 45 Tyr
Val Met Ala Ser Phe Tyr Lys His Leu Gly Ile Glu Phe Met Glu 50 55
60 Ala Glu Glu Leu Tyr Gln Lys 65 70 355PRTHomo sapiens 3Thr Ser
His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val 1 5 10 15
Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg 20
25 30 Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln
Asn 35 40 45 Tyr Val Met Ala Ser Phe Tyr 50 55 461PRTHomo sapiens
4His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly 1
5 10 15 Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr
Leu 20 25 30 Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln
Asn Tyr Val 35 40 45 Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr
Gln Lys 50 55 60 534PRTHomo sapiens 5His Leu Val Lys Cys Ala Glu
Lys Glu Lys Thr Phe Cys Val Asn Gly 1 5 10 15 Gly Glu Cys Phe Met
Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu 20 25 30 Cys Lys
636PRTHomo sapiens 6Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp
Leu Ser Asn Pro 1 5 10 15 Ser Arg Tyr Leu Cys Lys Cys Gln Pro Gly
Phe Thr Gly Ala Arg Cys 20 25 30 Thr Glu Asn Val 35 766PRTHomo
sapiens 7Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe
Cys Val 1 5 10 15 Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser
Asn Pro Ser Arg 20 25 30 Tyr Leu Cys Lys Cys Gln Pro Gly Phe Thr
Gly Ala Arg Cys Thr Glu 35 40 45 Asn Val Pro Met Lys Val Gln Asn
Gln Glu Lys Ala Glu Glu Leu Tyr 50 55 60 Gln Lys 65 865PRTHomo
sapiens 8Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys
Val Asn 1 5 10 15 Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn
Pro Ser Arg Tyr 20 25 30 Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly
Ala Arg Cys Thr Glu Asn 35 40 45 Val Pro Met Lys Val Gln Asn Gln
Glu Lys Ala Glu Glu Leu Tyr Gln 50 55 60 Lys 65 964PRTHomo sapiens
9His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly 1
5 10 15 Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr
Leu 20 25 30 Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr
Glu Asn Val 35 40 45 Pro Met Lys Val Gln Asn Gln Glu Lys Ala Glu
Glu Leu Tyr Gln Lys 50 55 60 1048PRTHomo sapiens 10His Leu Val Lys
Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly 1 5 10 15 Gly Glu
Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu 20 25 30
Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val 35
40 45 1168PRTHomo sapiens 11Ser Gly His Ala Arg Lys Cys Asn Glu Thr
Ala Lys Ser Tyr Cys Val 1 5 10 15 Asn Gly Gly Val Cys Tyr Tyr Ile
Glu Gly Ile Asn Gln Leu Ser Cys 20 25 30 Lys Cys Pro Val Gly Tyr
Thr Gly Asp Arg Cys Gln Gln Phe Ala Met 35 40 45 Val Asn Phe Ser
Lys His Leu Gly Phe Glu Leu Lys Glu Ala Glu Glu 50 55 60 Leu Tyr
Gln Lys 65 1252PRTHomo sapiens 12Ser Gly His Ala Arg Lys Cys Asn
Glu Thr Ala Lys Ser Tyr Cys Val 1 5 10 15 Asn Gly Gly Val Cys Tyr
Tyr Ile Glu Gly Ile Asn Gln Leu Ser Cys 20 25 30 Lys Cys Pro Asn
Gly Phe Phe Gly Gln Arg Cys Leu Glu Lys Leu Pro 35 40 45 Leu Arg
Leu Tyr 50 1374PRTHomo sapiens 13Ser Gly His Ala Arg Lys Cys Asn
Glu Thr Ala Lys Ser Tyr Cys Val 1 5 10 15 Asn Gly Gly Val Cys Tyr
Tyr Ile Glu Gly Ile Asn Gln Leu Ser Cys 20 25 30 Lys Cys Pro Asn
Gly Phe Phe Gly Gln Arg Cys Leu Glu Lys Leu Pro 35 40 45 Leu Arg
Leu Tyr Met Pro Asp Pro Lys Gln Lys His Leu Gly Phe Glu 50 55 60
Leu Lys Glu Ala Glu Glu Leu Tyr Gln Lys 65 70 1459PRTHomo sapiens
14Gly His Ala Arg Lys Cys Asn Glu Thr Ala Lys Ser Tyr Cys Val Asn 1
5 10 15 Gly Gly Val Cys Tyr Tyr Ile Glu Gly Ile Asn Gln Leu Ser Cys
Lys 20 25 30 Cys Pro Val Gly Tyr Thr Gly Asp Arg Cys Gln Gln Phe
Ala Met Val 35 40 45 Asn Phe Ser Lys Ala Glu Glu Leu Tyr Gln Lys 50
55 1556PRTHomo sapiens 15Gly Glu Cys Phe Met Val Lys Asp Leu Ser
Asn Pro Ser Arg Tyr Leu 1 5 10 15 Cys Lys Cys Gln Pro Gly Phe Thr
Gly Ala Arg Cys Thr Glu Asn Val 20 25 30 Pro Met Lys Val Gln Asn
Gln Glu Lys His Leu Gly Ile Glu Phe Ile 35 40 45 Glu Ala Glu Glu
Leu Tyr Gln Lys 50 55 1675PRTHomo sapiens 16Ser Glu His Phe Lys Pro
Cys Arg Asp Lys Asp Leu Ala Tyr Cys Leu 1 5 10 15 Asn Asp Gly Glu
Cys Phe Val Ile Glu Thr Leu Thr Gly Ser His Lys 20 25 30 His Cys
Arg Cys Lys Glu Gly Tyr Gln Gly Val Arg Cys Asp Gln Phe 35 40 45
Leu Pro Lys Thr Asp Ser Ile Leu Ser Asp Pro Asn His Leu Gly Ile 50
55 60 Glu Phe Met Glu Ser Glu Glu Val Tyr Gln Arg 65 70 75
1776PRTHomo sapiens 17Ser Glu His Phe Lys Pro Cys Arg Asp Lys Asp
Leu Ala Tyr Cys Leu 1 5 10 15 Asn Asp Gly Glu Cys Phe Val Ile Glu
Thr Leu Thr Gly Ser His Lys 20 25 30 His Cys Arg Cys Lys Glu Gly
Tyr Gln Gly Val Arg Cys Asp Gln Phe 35 40 45 Leu Pro Lys Thr Asp
Ser Ile Leu Ser Asp Pro Thr Asp His Leu Gly 50 55 60 Ile Glu Phe
Met Glu Ser Glu Glu Val Tyr Gln Arg 65 70 75 1849PRTHomo sapiens
18Ser Glu His Phe Lys Pro Cys Arg Asp Lys Asp Leu Ala Tyr Cys Leu 1
5 10 15 Asn Asp Gly Glu Cys Phe Val Ile Glu Thr Leu Thr Gly Ser His
Lys 20 25 30 His Cys Arg Cys Lys Glu Gly Tyr Gln Gly Val Arg Cys
Asp Gln Phe 35 40 45 Leu 1944PRTHomo sapiens 19Thr Asp His Glu Glu
Pro Cys Gly Pro Ser His Lys Ser Phe Cys Leu 1 5 10 15 Asn Gly Gly
Leu Cys Tyr Val Ile Pro Thr Ile Pro Ser Pro Phe Cys 20 25 30 Arg
Cys Val Glu Asn Tyr Thr Gly Ala Arg Cys Glu 35 40
2053PRTartificialhybrid polypeptide 20His Leu Val Lys Cys Ala Glu
Lys Glu Lys Thr Phe Cys Val Asn Gly 1 5 10 15 Gly Glu Cys Phe Met
Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu 20 25 30 Cys Lys Cys
Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val 35 40 45 Met
Ala Ser Phe Tyr 50 2153PRTartificialhybrid polypeptide 21His Leu
Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly 1 5 10 15
Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu 20
25 30 Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn
Val 35 40 45 Met Ala Ser Phe Tyr 50 2259PRTartificialhybrid
polypeptide 22Gly His Ala Arg Lys Cys Asn Glu Thr Ala Lys Ser Tyr
Cys Val Asn 1 5 10 15 Gly Gly Val Cys Tyr Tyr Ile Glu Gly Ile Asn
Gln Leu Ser Cys Lys 20 25 30 Cys Pro Asn Gly Phe Phe Gly Gln Arg
Cys Leu Glu Lys Leu Pro Leu 35 40 45 Arg Leu Tyr Lys Ala Glu Glu
Leu Tyr Gln Lys 50 55 2351PRTartificialhybrid polypeptide 23Gly His
Ala Arg Lys Cys Asn Glu Thr Ala Lys Ser Tyr Cys Val Asn 1 5 10 15
Gly Gly Val Cys Tyr Tyr Ile Glu Gly Ile Asn Gln Leu Ser Cys Lys 20
25 30 Cys Pro Asn Gly Phe Phe Gly Gln Arg Cys Gln Asn Tyr Val Met
Ala 35 40 45 Ser Phe Tyr 50 2453PRTartificialhybrid polypeptide
24His Ala Arg Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly 1
5 10 15 Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr
Leu 20 25 30 Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln
Asn Tyr Val 35 40 45 Met Ala Ser Phe Tyr 50 2551PRTartificialhybrid
polypeptide 25Gly His Ala Arg Lys Cys Ala Glu Lys Glu Lys Thr Phe
Cys Val Asn 1 5 10 15 Gly Gly Val Cys Tyr Tyr Ile Glu Gly Ile Asn
Gln Leu Ser Cys Lys 20 25 30 Cys Pro Asn Gly Phe Phe Gly Gln Arg
Cys Gln Asn Tyr Val Met Ala 35 40 45 Ser Phe Tyr 50
2642PRTartificialhybrid polypeptide 26His Leu Val Lys Cys Ala Glu
Lys Glu Lys Thr Phe Cys Leu Asn Gly 1 5 10 15 Gly Leu Cys Tyr Val
Ile Pro Thr Ile Pro Ser Pro Phe Cys Arg Cys 20 25 30 Val Glu Asn
Tyr Thr Gly Ala Arg Cys Glu 35 40 2742PRTartificialhtbrid
polypeptide 27His Ala Arg Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys
Leu Asn Gly 1 5 10 15 Gly Leu Cys Tyr Val Ile Pro Thr Ile Pro Ser
Pro Phe Cys Arg Cys 20 25 30 Val Glu Asn Tyr Thr Gly Ala Arg Cys
Glu 35 40 2850PRTartificialhybrid polypeptide 28His Ala Arg Lys Cys
Asn Glu Thr Ala Lys Ser Tyr Cys Val Asn Gly 1 5 10 15 Gly Val Cys
Tyr Tyr Ile Glu Gly Ile Asn Gln Leu Ser Cys Lys Cys 20 25 30 Pro
Asn Gly Phe Phe Gly Gln Arg Cys Gln Asn Tyr Val Met Ala Ser 35 40
45 Phe Tyr 50 2952PRTartificialhybrid polypeptide 29Thr Asp His Glu
Glu Pro Cys Gly Pro Ser His Lys Ser Phe Cys Leu 1 5 10 15 Asn Gly
Gly Leu Cys Tyr Val Ile Pro Thr Ile Pro Ser Pro Phe Cys 20 25 30
Lys Cys Pro Asn Gly Phe Phe Gly Gln Arg Cys Gln Asn Tyr Val Met 35
40 45 Ala Ser Phe Tyr 50
* * * * *