U.S. patent application number 11/648061 was filed with the patent office on 2007-08-16 for extended release of neuregulin for improved cardiac function.
Invention is credited to Mingdong Zhou.
Application Number | 20070190127 11/648061 |
Document ID | / |
Family ID | 38227911 |
Filed Date | 2007-08-16 |
United States Patent
Application |
20070190127 |
Kind Code |
A1 |
Zhou; Mingdong |
August 16, 2007 |
Extended release of neuregulin for improved cardiac function
Abstract
The present invention provides extended release compositions
comprising neuregulin for preventing, treating or delaying various
diseases or disorders. The present invention also provides methods
for preventing, treating or delaying various diseases or disorders
by extended release of neuregulin.
Inventors: |
Zhou; Mingdong; (Shanghai,
CN) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Family ID: |
38227911 |
Appl. No.: |
11/648061 |
Filed: |
December 29, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60755124 |
Dec 30, 2005 |
|
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60758626 |
Jan 13, 2006 |
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Current U.S.
Class: |
424/450 ;
424/490; 514/16.3; 514/16.4; 514/9.6 |
Current CPC
Class: |
A61K 48/00 20130101;
A61P 35/00 20180101; A61P 9/04 20180101; A61P 43/00 20180101; A61K
47/60 20170801; A61K 9/127 20130101; A61P 9/00 20180101; A61P 3/10
20180101; A61P 21/04 20180101; A61P 25/28 20180101; A61K 9/0019
20130101; A61P 27/02 20180101; A61P 25/18 20180101; A61P 21/00
20180101; A61K 38/1883 20130101; A61P 27/16 20180101; A61P 25/00
20180101 |
Class at
Publication: |
424/450 ;
514/012; 424/490 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 9/127 20060101 A61K009/127; A61K 9/50 20060101
A61K009/50 |
Claims
1-85. (canceled)
86. An extended release composition for preventing, treating or
delaying a disease or disorder in a mammal comprising an osmotic
pump and a therapeutically effective amount of neuregulin.
87. An extended release composition for preventing, treating or
delaying a disease or disorder in a mammal comprising poly(ethylene
glycol) and a therapeutically effective amount of neuregulin.
88. An extended release composition for preventing, treating or
delaying a disease or disorder in a mammal comprising a liposome
and a therapeutically effective amount of neuregulin.
89. An extended release composition for preventing, treating or
delaying a disease or disorder in a mammal comprising a microsphere
and a therapeutically effective amount of neuregulin.
90. A method for preventing, treating or delaying heart failure in
a mammal, the method comprising extended release of neuregulin into
a mammal.
91. The method of claim 90, wherein the extended release of NRG
into a mammal enhances the EF value of the mammal.
92. The method of claim 91 wherein the EF value of the mammal is
enhanced by a percentage selected from the group consisting of
greater than about 20%, greater than about 30%, greater than about
40%, greater than about 50% and greater than about 60%.
93. The method of claim 90, wherein the extended release of NRG
into a mammal enhances the FS value of the mammal.
94. The method of claim 93 wherein the FS value of the mammal is
enhanced by a percentage selected from the group consisting of
greater than about 20%, greater than about 30%, greater than about
40%, greater than about 50% and greater than about 60%.
95. The method of claim 90, wherein the extended release of NRG
into a mammal reduces the interior diameter of the left ventricle
(LVEDD or LVESD).
96. The method of claim 95, wherein the LVEDD value is reduced by a
percentage selected from the group consisting of greater than about
2%, greater than about 5%, greater than about 10% and greater than
about 15%.
97. The method of claim 95, wherein the LVESD value is reduced by a
percentage selected from the group consisting of greater than about
2%, greater than about 5%, greater than about 10%, greater than
about 15% and greater than about 20%.
98. The method of claim 90, wherein the extended release of
neuregulin into a mammal induces sustained activation of the ERK
signaling pathway in cardiac cells.
99. The method of claim 90, wherein the extended release of
neuregulin into a mammal induces sustained activation of the AKT
signaling pathway in cardiac cells.
100. The method of claim 90, herein the extended release of
neuregulin into a mammal is accomplished by attaching a polymer to
neuregulin.
101. The method of claim 100, wherein said polymer is poly(ethylene
glycol).
102. The method of claim 100, wherein said polymer is a
poly(ethylene glycol) derivative.
103. The method of claim 90, wherein the extended release of
neuregulin into a mammal is accomplished through the use of
liposomes.
104. The method of claim 90, wherein the extended release of
neuregulin into a mammal is accomplished through the use of
microspheres.
105. The method of claim 90, wherein the neuregulin is selected
from the group comprising NRG1, NRG2, NRG3 and NRG4.
106. The method of claim 90, wherein the neuregulin comprising the
epidermal growth factor-like (EGF-like) domain, wherein the
EGF-like domain is encoded by neuregulin 1 gene.
107. The method of claim 90, wherein the extended release of
neuregulin comprising continuously injection or infusion of
neuregulin for 1 min to 24 hours per day.
108. The method of claim 90, wherein the extended release of
neuregulin including continuously intravenously infusion or
injection of neuregulin for 4 or more hours per day.
109. The method of claim 90, wherein the extended release of
neuregulin including continuously hypodermically infusion or
injection of neuregulin for 6 or more hours per day.
110. A method for reducing a side effect in a mammal subjected to
neuregulin treatment, the method comprising extended release of
neuregulin into a mammal.
111. The method of claim 110, wherein the side effect is
gastrointestinal disorder or pericardial effusion.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
provisional application Nos. 60/755,124, filed Dec. 30, 2005, and
60/758,626, filed Jan. 13, 2006.
FIELD OF THE INVENTION
[0002] This invention relates generally to compositions and methods
for preventing, treating or delaying various cardiac diseases or
disorders by extended release of neuregulin to a mammal.
BACKGROUND OF THE INVENTION
[0003] Cardiac (ventricular) hypertrophy is an important adaptive
physiological response to increased stress or demands for cardiac
work. One of the early cellular changes that occurs after a
stimulus for hypertrophy is the synthesis of mitochondria and
expansion of myofibrillar mass (wall thickening) with a
proportional increase in the size of individual cells, but no (or
minimal ) increase in the number of cells.
[0004] When the ventricle is stressed, the initial response is an
increase in sarcomere length. This is followed by an increase in
the total muscle mass. When the overload is severe; myocardial
contractility becomes depressed. In its mildest form, this
depression is manifested by a reduction in the velocity of
shortening of unloaded myocardium or by a reduction in the rate of
force development during isometric contraction. As myocardial
contractility becomes further depressed, a more extensive reduction
in the velocity of shortening of unloaded myocardium occurs, now
accompanied by a decline in isometric force development and
shortening. At this point, circulatory compensation may still be
provided by cardiac dilation and an increase in muscle mass, which
tend to maintain wall stress at normal levels. As contractility
falls further, overt congestive heart failure, reflected in a
depression of cardiac output and work and/or an elevation of
ventricular end-diastolic volume and pressure at rest,
supervenes.
[0005] The transition from hypertrophy to heart failure is
characterized by several alterations in cellular organization. For
example, normal hypertrophic cells have a large size with increased
and well organized contractile units, as well as strong cell-cell
adhesions. In contrast, pathologically hypertrophic cells, which
also have large size and accumulation of proteins, display
disorganization of contractile proteins (disarray of sarcomeric
structures) and poor cell-cell adhesions (disarray of myofibers).
Thus, in pathological hypertrophy, the increased size and
accumulation of contractile proteins are associated with
disorganized assembly of sarcomeric structures and a lack of robust
cell-cell interactions.
[0006] Heart failure affects approximately five million Americans,
and more than 550,000 new patients are diagnosed with the condition
each year. Current drug therapy for heart failure is primarily
directed to angiotensin-converting enzyme (ACE) inhibitors, which
are vasodilators that cause blood vessels to expand, lowering blood
pressure and reducing the heart's workload. While the percent
reduction in mortality has been significant, the actual reduction
in mortality with ACE inhibitors has averaged only 3%-4%, and there
are several potential side effects.
[0007] ACE inhibitors have also been administered in combination
with other drugs such as digitalis, which increases the force of
the heart's contractions, and/or a diuretic, which helps relieve
the heart's workload by causing the kidneys to remove more sodium
and water from the bloodstream. However, at least one study
demonstrated no difference in survival associated with the use of
digitalis compared with placebo in patients with Class II-III heart
failure. Additionally, diuretics can improve some symptoms of heart
failure but it is not suitable as a sole treatment.
[0008] Additional limitations are associated with other options for
preventing or treating heart failure. For example, heart
transplantation is clearly more expensive and invasive than drug
treatment, and it is further limited by the availability of donor
hearts. Use of mechanical devices, such as biventricular
pacemakers, are similarly invasive and expensive. Thus, there has
been a need for new therapies given the deficiencies in current
therapies.
[0009] One promising new therapy involves administration of
neuregulin (hereinafter referred to as "NRG") to a patient
suffering from or at risk of developing heart failure. NRGs
comprise a family of structurally related growth and
differentiation factors that include NRG1, NRG2, NRG3 and NRG4 and
isoforms thereof. For example, over 15 distinct isoforms of NRG1
have been identified and divided into two large groups, known as
.alpha.- and .beta.-types, on the basis of differences in the
sequence of their essential epidermal growth factor (EGF)-like
domains.
[0010] NRGs bind to the EGF receptor family, which comprises EGFR,
ErbB2, ErbB3 and ErbB4, each of which plays an important role in
multiple cellular functions, including cell growth, differentiation
and survival. They are protein tyrosine kinase receptors,
consisting of an extracellular ligand-binding domain, transmembrane
domain and cytoplasmic tyrosine kinase domain. After NRG binds to
the extracellular domain of ErbB3 or ErbB4, it induces a
conformational change that leads to heterodimer formation between
ErbB3, ErbB4 and ErbB2 or homodimer formation between ErbB4 itself,
which results in phosphorylation of the receptors' C-terminal
domain inside the cell membrane. The phosphorylated intracellular
domain then binds additional signal proteins inside the cell,
activating the corresponding downstream AKT or ERK signaling
pathway, and inducing a series of cell reactions, such as
stimulation or depression of cell proliferation, cell
differentiation, cell apoptosis, cell migration or cell adhesion.
Among these receptors, mainly ErbB2 and ErbB4 are expressed in the
heart.
[0011] It has been shown that the EGF-like domains of NRG1, ranging
in size from 50 to 64-amino acids, are sufficient to bind to and
activate these receptors. Previous studies have shown that
neuregulin-1.beta. (NRG-1.beta.) can bind directly to ErbB3 and
ErbB4 with high affinity. The orphan receptor, ErbB2, can form
heterodimer with ErbB3 or ErbB4 with higher affinity than ErbB3 or
ErbB4 homodimers. Research in neural development has indicated that
the formation of the sympathetic nervous system requires an intact
NRG-1.beta., ErbB2 and ErbB3 signaling system. Targeted disruption
of the NRG-1.beta. or ErbB2 or ErbB4 led to embryonic lethality due
to cardiac development defects. Recent studies also highlighted the
roles of NRG-1.beta., ErbB2 and ErbB4 in the cardiovascular
development as well as in the maintenance of adult normal heart
function. NRG-1.beta. has been shown to enhance sarcomere
organization in adult cardiomyocytes. The short-term administration
of a recombinant NRG-1.beta. EGF-like domain significantly improves
or protects against deterioration in myocardial performance in
three distinct animal models of heart failure. More importantly,
NRG-1.beta. significantly prolongs survival of heart failure
animals. These effects make NRG-1.beta. promising as a broad
spectrum therapeutic or lead compound for heart failure due to a
variety of common diseases. However, there is still a need for more
effective methods of using NRG, which can be used in a clinical
setting for the prevention, treatment or delaying of heart failure
and/or cardiac hypertrophy.
SUMMARY OF THE INVENTION
[0012] Extended release of NRG greatly improves the effect of NRG
in the treatment of heart failure and cardiac hypertrophy compared
to NRG administered by other methods. Extended release of NRG also
has the benefit of reducing the adverse side effects of NRG
compared to NRG administered by other methods. Thus, the present
invention relates to compositions and methods for preventing,
treating or delaying various cardiac diseases or disorders in
mammals, particularly in humans, by extending the release of a NRG
protein, or a functional fragment thereof, or a nucleic acid
encoding a NRG protein, or a functional fragment thereof, or an
agent that enhances production and/or function of said NRG.
[0013] In a first aspect of the invention, a method is provided for
preventing, treating or delaying heart failure in a mammal, the
method comprising extended release of NRG into a mammal in need
thereof.
[0014] In one embodiment of the method for preventing, treating or
delaying heart failure in a mammal, the extended release of NRG
into a mammal leads to sustained activation of the ERK signaling
pathway in cardiac cells.
[0015] In another embodiment of the method for preventing, treating
or delaying heart failure in a mammal in need thereof, the extended
release of NRG into a mammal results in sustained activation of the
AKT signaling pathway in cardiac cells.
[0016] In another embodiment of the method for preventing, treating
or delaying heart failure in a mammal in need thereof, the extended
release of NRG into a mammal enhances the EF and/or FS values of
the left ventricle of mammal. In some embodiments, the EF value of
the mammal is enhanced by a percentage selected from the group
consisting of greater than about 20%, greater than about 30%,
greater than about 40%, greater than about 50% and greater than
about 60%. In some embodiments, the FS value of the mammal is
enhanced by a percentage selected from the group consisting of
greater than about 20%, greater than about 30%, greater than about
40%, greater than about 50% and greater than about 60%.
[0017] In another embodiment of the method for preventing, treating
or delaying heart failure in a mammal in need thereof, the extended
release of NRG into a mammal prevents cardiac hypertrophy.
[0018] Any extended release technology known in the art, including,
but not limited to, an osmotic pump or syringe pump, poly-ethylene
glycol ("PEG") coupling, and/or liposome or microsphere packaging,
can be used in the present invention.
[0019] In a second aspect of the invention, a method is provided
for reducing the interior diameter of the left ventricle, the
method comprising extended release of NRG into a mammal in need
thereof. In one preferred embodiment, extended release of NRG into
a mammal reduces the LVEDD value by greater than about 2%. More
preferably, extended release of NRG into a mammal reduces the LVEDD
value by greater than about 5%. Even more preferably, extended
release of NRG into a mammal reduces the LVEDD value by greater
than about 10%. More preferably, extended release of NRG into a
mammal reduces the LVEDD value by greater than about 15%. Most
preferably, extended release of NRG into a mammal reduces the LVEDD
value by greater than about 20%.
[0020] In another preferred embodiment, extended release of NRG
into a mammal reduces the LVESD value by greater than about 2%.
More preferably, extended release of NRG into a mammal reduces the
LVESD value by greater than about 5%. Even more preferably,
extended release of NRG into a mammal reduces the LVESD value by
greater than about 10%. Even more preferably, extended release of
NRG into a mammal reduces the LVESD value by greater than about
15%. Most preferably, extended release of NRG into a mammal
decreases the LVESD value by greater than about 20%.
[0021] In a third aspect of the invention, a method is provided for
causing cardiomyocyte growth and/or differentiation, the method
comprising extended release of NRG into a mammal in need thereof
thereby activating the MAP kinase pathway in cardiac cells and
causing growth and/or differentiation of the cardiomyocyte.
[0022] In a fourth aspect of the invention, a method is provided
for inducing remodeling of muscle cell sarcomeric and cytoskeleton
structures, or cell-cell adhesions, the method comprising extended
release of NRG into a mammal in need thereof thereby activating the
MAP kinase pathway in cardiac cells and causing remodeling of the
cell structures or the cell-cell adhesions.
[0023] In a fifth aspect of the invention, a method is provided for
treating or preventing disassociation of cardiac muscle cell-cell
adhesion and/or the disarray of sarcomeric structures in a mammal
in need thereof, the method comprising extended release of NRG into
a mammal.
[0024] Additionally, because NRG's interaction with ErbB receptors
has been implicated in other diseases and disorders, extended
release of NRG may also greatly improve the effect of NRG in the
treatment of such other diseases and disorders compared to NRG
administered by other methods. Thus, the present invention also
relates to compositions and methods for preventing, treating or
delaying various diseases or disorders in mammals, particularly in
humans, by extending the release of a NRG protein, or a functional
fragment thereof, or a nucleic acid encoding a NRG protein, or a
functional fragment thereof, or an agent that enhances production
and/or function of said NRG. Such diseases and disorders include
generally those of the central and peripheral nervous system.
Examples of other diseases and disorders, include, various
cardiovascular diseases, cancer, neural system disease and/or
muscle diseases, including muscular dystrophy (e.g., Duchenne,
Limb-girdle) and multiple sclerosis, spinal injury, eye and ear
diseases, diabetes, schizophrenia, and Alzheimer's.
[0025] The invention also provides an extended release composition
or formulation of NRG for preventing, treating or delaying heart
failure in a mammal. In one embodiment, the composition or
formulation sustains activation of the ERK signaling pathway in
cardiac cells. In another embodiment, the composition or
formulation sustains activation of the AKT signaling pathway in
cardiac cells. In another embodiment, the composition or
formulation enhances the EF and/or FS values of the mammal. In yet
another embodiment, the composition or formulation prevents cardiac
hypertrophy. The composition or formulation may incorporate the use
of any extended release technology known in the art, including, but
not limited to, an osmotic pump or syringe pump, poly-ethylene
glycol (PEG) coupling, and/or liposome or microsphere
packaging.
[0026] The invention also provides a kit comprising a NRG
composition or formulation and an extended release technology known
in the art, including, but not limited to, an osmotic pump or
syringe pump, poly-ethylene glycol (PEG) coupling, and/or liposome
or microsphere packaging. In some embodiments, the kit further
comprises an instruction for using the NRG composition or
formulation and or extended release technology in preventing,
treating or delaying heart failure in a mammal; preventing,
treating or delaying cardiac hypertrophy in a mammal; or reducing
the interior diameter of the left ventricle in a mammal.
[0027] Those and other aspects, objects, advantages and features of
the invention will be apparent to those persons skilled in the art
upon reading the disclosure of the invention as more fully
described below.
BRIEF DESCRIPTION OF THE DRAWING
[0028] FIG. 1 shows the phosphorylation of AKT and ERK in the left
ventricle of rats over time after NRG was infused by intramuscular
injection, intravenous injection and intravenous glucose tolerance
test infusion. "P-AKT," "P-ERK" and "NRG" mean phosphorylated AKT,
phosphorylated ERK and neuregulin. "im," "iv," and "ivgtt" mean
intramuscular injection, intravenous injection and intravenous
glucose tolerance test, respectively.
[0029] FIG. 2 shows gel stained by BaI.sub.2 to detect PEG. In the
figure, "mixture" means the solution of PEG and NRG mixture after
their reaction. "M", "peak1", "peak2" and "peak3" stand for protein
marker and elution peak fraction 1, 2 and 3 of the mixture from the
S100 column. "NRG-mono-PEG", "NRG-di-PEG" and "NRG-poly-PEG" mean
NRG coupled to one PEG, two PEG and multiple (at least 3) PEG,
respectively.
[0030] FIG. 3 shows gel coomassie stained to detect NRG protein.
The abbreviations are the same as in FIG. 2. In the M lane, the
molecular weight for each band (from bottom to above) is 14.4 kD,
20.1 kD, 31.0 kD, 43.0 kD, 66.2 kD and 97.4 kD respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0031] Although any methods similar or equivalent to those
described herein can be used in the practice of the present
invention, the preferred methods and materials are now
described.
[0032] The present invention provides methods for treating or
preventing heart failure or cardiac hypertrophy in a mammal by
extended release of a sustained or varied amount of NRG.
Preferably, the mammal is a human patient suffering from or at risk
of developing heart failure.
[0033] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention hereinafter is divided into
the subsections that follow. All publications mentioned herein are
incorporated by reference to disclose and describe the methods
and/or materials in connection with which the publications are
cited.
A. Definitions
[0034] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which this invention belongs. All
patents, applications, published applications and other
publications referred to herein are incorporated by reference in
their entirety. If a definition set forth in this section is
contrary to or otherwise inconsistent with a definition set forth
in the patents, applications, published applications and other
publications that are herein incorporated by reference, the
definition set forth in this section prevails over the definition
that is incorporated herein by reference.
[0035] As used herein, the singular forms "a", "an", and "the" mean
"at least one"or "one or more" unless the context clearly dictates
otherwise.
[0036] As used herein, "neuregulin" or "NRG" used in the present
invention refers to proteins or peptides that can bind and activate
ErbB2, ErbB3, ErbB4 or combinations thereof, including but not
limited to all neuregulin isoforms, neuregulin EGF domain alone,
polypeptides comprising neuregulin EGF-like domain, neuregulin
mutants or derivatives, and any kind of neuregulin-like gene
products that also activate the above receptors as described in
detail below. In preferred embodiments, neuregulin used in the
present invention binds to and activates ErbB2/ErbB4 or ErbB2/ErbB3
heterodimers. Neuregulin also includes NRG-1, NRG-2, NRG-3, and
NRG-4 proteins, peptides, fragments and compounds that mimic the
activities of neuregulin. Neuregulin used in the present invention
can activate the above ErbB receptors and modulate their biological
reactions, e.g., stimulate breast cancer cell differentiation and
milk protein secretion; induce the differentiation of neural crest
cell into Schwann cell; stimulate acetylcholine receptor synthesis
in skeletal muscle cell; and/or improve cardiocyte differntiation,
survival and DNA synthesis. Neuregulin also includes those variants
with conservative amino acid substitutions that do not
substantially alter their biological activity. Suitable
conservative substitutions of amino acids are known to those of
skill in this art and may be made generally without altering the
biological activity of the resulting molecule. Those of skill in
this art recognize that, in general, single amino acid
substitutions in non-essential regions of a polypeptide do not
substantially alter biological activity (see, e.g., Watson et al.
Molecular Biology of the Gene, 4th Edition, 1987, The
Bejacmin/Cummings Pub. co., p. 224).
[0037] Neuregulin protein encompasses neuregulin protein and
peptide. Neuregulin nucleic acid encompasses neuregulin nucleic
acid and neuregulin oligonucleotide.
[0038] As used herein, "epidermal growth factor-like domain" or
"EGF-like domain" refers to a polypeptide motif encoded by the
neuregulin gene that binds to and activates ErbB2, ErbB3, ErbB4, or
combinations thereof, and bears a structural similarity to the EGF
receptor-binding domain as disclosed in WO 00/64400, Holmes et al.,
Science, 256:1205-1210 (1992); U.S. Pat. Nos. 5,530,109 and
5,716,930; Hijazi et al., Int. J. Oncol., 13:1061-1067 (1998);
Chang et al., Nature, 387:509-512 (1997); Carraway et al., Nature,
387:512-516 (1997); Higashiyama et al., J. Biochem., 122:675-680
(1997); and WO 97/09425, the contents of which are all incorporated
herein by reference. In certain embodiments, EGF-like domain binds
to and activates ErbB2/ErbB4 or ErbB2/ErbB3 heterodimers. In
certain embodiments, EGF-like domain comprises the amino acid
sequence of the receptor binding domain of NRG-1. In some
embodiments, EGF-like domain comprises the amino acid sequence
corresponding to amino acid residues 177-226, 177-237, or 177-240
of NRG-1. In certain embodiments, EGF-like domain comprises the
amino acid sequence of the receptor binding domain of NRG-2. In
certain embodiments, EGF-like domain comprises the amino acid
sequence of the receptor binding domain of NRG-3. In certain
embodiments, EGF-like domain comprises the amino acid sequence of
the receptor binding domain of NRG-4. In certain embodiments,
EGF-like domain comprises the amino acid sequence of Ala Glu Lys
Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu
Ser Asn Pro, as described in U.S. Pat. No. 5,834,229.
[0039] As used herein, an "effective amount" of an active agent for
treating a particular disease is an amount that is sufficient to
ameliorate, or in some manner reduce the symptoms associated with
the disease. The amount may cure the disease but, typically, is
administered in order to ameliorate the symptoms of the
disease.
[0040] As used herein, "active agent" means any substance intended
for the diagnosis, cure, mitigation, treatment, or prevention of
disease in humans and other animals, or to otherwise enhance
physical and mental well being.
[0041] As used herein, "amelioration" of the symptoms of a
particular disorder by administration of a particular active agent
refers to any lessening, whether permanent or temporary, lasting or
transient that can be attributed to or associated with
administration of the agent.
[0042] As used herein, "treat", "treatment" and "treating" refer to
any manner in which the symptoms of a condition, disorder or
disease are ameliorated or otherwise beneficially altered. The
effect may be prophylactic in terms of completely or partially
preventing a disease or symptom thereof and/or may be therapeutic
in terms of a partial or complete cure for a disease and/or adverse
effect attributable to the disease. Treatment also encompasses any
pharmaceutical use of the compositions herein.
[0043] As used herein, "vector (or plasmid)" refers to discrete
elements that are used to introduce heterologous DNA into cells for
either expression or replication thereof. Selection and use of such
vehicles are well known within the skill of the artisan. An
expression vector includes vectors capable of expressing DNA that
are operatively linked with regulatory sequences, such as promoter
regions, that are capable of effecting expression of such DNA
fragments. Thus, an expression vector refers to a recombinant DNA
or RNA construct, such as a plasmid, a phage, recombinant virus or
other vector that, upon introduction into an appropriate host cell,
results in expression of the cloned DNA. Appropriate expression
vectors are well known to those of skill in the art and include
those that are replicable in eukaryotic cells and/or prokaryotic
cells and those that remain episomal or those which integrate into
the host cell genome.
[0044] As used herein, "cardiac muscle cell differentiation" means
a condition characterized by the decrease in DNA synthesis by more
than 10%, inhibition of other factor-stimulated DNA synthesis more
than 10%, well organized sarcomeric structures and cell-cell
adhesions, sustained activation of MAP kinases, and enhanced
expression of p21.sup.C1P1. Further discussion is provided in
WO00/37095, the contents of which are incorporated herein by
reference in their entireties.
[0045] As used herein, "ejection fraction" or "EF" means the
portion of blood that is pumped out of a filled ventricle as the
result of a heartbeat. It may be defined by the following formula:
(LV diastolic volume--LV systolic volume)/LV diastolic volume.
[0046] As used herein, "fractional shortening" or "FS" means a
ratio of the change in the diameter of the left ventricle between
the contracted and relaxed states. It may be defined by the
following formula: (LV end diastolic diameter--LV end systolic
diameter)/LV end diastolic diameter.
[0047] As used herein, "heart failure" means an abnormality of
cardiac function where the heart does not pump blood at the rate
needed for the requirements of metabolizing tissues. Heart failure
includes a wide range of disease states such as congestive heart
failure, myocardial infarction, tachyarrhythmia, familial
hypertrophic cardiomyopathy, ischemic heart disease, idiopathic
dilated cardiomyopathy, myocarditis and the like. The heart failure
can be caused by any number of factors, including, without
limitation, ischemic, congenital, rheumatic, or idiopathic forms.
Chronic cardiac hypertrophy is a significantly diseased state which
is a precursor to congestive heart failure and cardiac arrest.
[0048] As used herein, "myocardial infarction" refers to a blockade
of a coronary artery or blood flow interruption leading to focal
necrosis of part of the myocardium caused by severe and persistent
ischemia. As used herein, "extended release" refer s to providing
continuous therapeutic level of an active agent (e.g., neuregulin)
over a period of time. The extended release includes, without
limitation various forms of release, such as continuous release,
controlled release, delayed release, depot, gradual release,
long-term release, programmed release, prolonged release,
proportionate release, protracted release, repository, retard, slow
release, spaced release, sustained release, time coat, timed
release, delayed action, extended action, layered-time action, long
acting, prolonged action, repeated action, slow acting, sustained
action, sustained-action medications, and controlled release. The
ability to obtain extended release, controlled release, timed
release, sustained release, delayed release, long acting, pulsatile
delivery or immediate release is performed using well-known
procedures and techniques available to the ordinarily skilled
artisan.
[0049] The amount of time over which the active agent continues to
be released depends on the characteristics of the active agent and
the extended release technology or technologies used, but in all
cases is longer than that of administration of the active agent
without the extended release technology or technologies.
[0050] As used herein, "microsphere" is synonymous with
"microparticle", "microcapsule", "nanosphere", "nanoparticle" and
"nanocapsule" unless the context clearly dictates otherwise.
[0051] As used herein, "pegylate" means to attach at least one Poly
(ethylene glycol) molecule or at least one derivative of Poly
(ethylene glycol) to an active agent or other molecule.
[0052] As used herein, "organized, or enhanced organization of
sarcomeres or sarcomeric structures" means a condition
characterized by the straight array of contractile proteins
revealed by immunofluorescent staining of a-actinin in cardiac
muscle cells. The straight array of .alpha.-actinin proteins in
cells can be distinguished by microscopy and its connected
photography. As used herein, "disorganized or disarray of
sarcomeres or sarcomeric structures" means the opposite of the
"organized, or enhanced organization of sarcomeres or sarcomeric
structures"
[0053] As used herein, "organized, or enhanced organization of
cytoskeleton structures" means a condition characterized by the
straight actin fibers revealed by phalloidin staining of cardiac
muscle cells. The straight actin fibers in cells can be
distinguished by microscopy and its connected photography as
exampled in figures of this specification. As used herein,
"disorganized or disarray of cytoskeleton structures" means the
opposite of "organized, or enhanced organization of cytoskeleton
structures".
[0054] As used herein, "protein" is synonymous with "polypeptide"
or "peptide" unless the context clearly dictates otherwise.
[0055] As used herein, "sustained activation of MAP kinases" means
that the phosphorylated state of MAP kinases, p 42/44, is
maintained for at least 21 hr in cells. Further discussion is
provided in WO00/37095, the contents of which are incorporated
herein by reference.
[0056] The terms "synergistic, "synergistic effect" and like are
used herein to describe improved treatment effects obtained by
combining one or more therapeutic agents with one or more retinoic
acid compounds. Although a synergistic effect in some fields is
meant an effect which is more than additive (e.g., 1+1=3), in the
field of medical therapy an additive (1+1=2) or less than additive
(1+1=1.6) effect may be synergistic. For example, if each of two
drugs were to inhibit the development of ventricular muscle cell
hypertrophy by 50% if given individually, it would not be expected
that the two drugs would be combined to completely stop the
development of ventricular muscle cell hypertrophy. In many
instances, due to unacceptable side effects, the two drugs cannot
be administered together. In other instances, the drugs counteract
each other and slow the development of ventricular muscle cell
hypertrophy by less than 50% when administered together. Thus, a
synergistic effect is said to be obtained if the two drugs slow the
development of ventricular muscle cell hypertrophy by more than 50%
while not causing an unacceptable increase in adverse side
effects.
[0057] As used herein "cardiac hypertrophy" means a condition
characterized by an increase in the size of individual ventricular
muscle cells, the increase in cell size being sufficient to result
in a clinical diagnosis of the patient or sufficient as to allow
the cells to be determined as larger (e.g., 2-fold or more larger
than non-hypertrophic cells). It may be accompanied by accumulation
of contractile proteins within the individual cardiac cells and
activation of embryonic gene expression.
[0058] In vitro and in vivo methods for determining the presence of
ventricular muscle cell hypertrophy are known. In vitro assays for
ventricular muscle cell hypertrophy include those methods described
WO00/37095, e.g., increased cell size and increased expression of
atrial natriuretic factor (ANP). Changes in cell size are used in a
scoring system to determine the extent of hypertrophy. These
changes can be viewed with an inverted phase microscope, and the
degree of hypertrophy scored with an arbitrary scale of 7 to 0,
with 7 being fully hypertrophied cells, and 3 being non-stimulated
cells. The 3 and 7 states may be seen in Simpson et al. (1982)
Circulation Res. 51: 787-801, FIG. 2, A and B, respectively. The
correlation between hypertrophy score and cell surface area
(.mu.m2) has been determined to be linear (correlation
coefficient=0.99). In phenylephrine-induced hypertrophy,
non-exposed (normal) cells have a hypertrophy score of 3 and a
surface area/cell of 581 .mu.m2 and fully hypertrophied cells have
a hypertrophy score of 7 and a surface area/cell of 1811 .mu.m2, or
approximately 200% of normal. Cells with a hypertrophy score of 4
have a surface area/cell of 771 .mu.m2, or approximately 30%
greater size than non-exposed cells; cells with a hypertrophy score
of 5 have a surface area/cell of 1109 .mu.m2, or approximately 90%
greater size than non-exposed cells; and cells with a hypertrophy
score of 6 have a surface area/cell of 1366 .mu.m2, or
approximately 135% greater size than non-exposed cells. The
presence of ventricular muscle cell hypertrophy preferably includes
cells exhibiting an increased size of about 15% (hypertrophy score
3.5) or more. Inducers of hypertrophy vary in their ability to
induce a maximal hypertrophic response as scored by the
above-described assay. For example, the maximal increase in cell
size induced by endothelin is approximately a hypertrophy score of
5.
[0059] As used herein, "suppression of cardiac hypertrophy" means a
reduction in one of the parameters indicating hypertrophy relative
to the hypertrophic condition, or a prevention of an increase in
one of the parameters indicating hypertrophy relative to the normal
condition. For example, suppression of ventricular muscle cell
hypertrophy can be measured as a reduction in cell size relative to
the hypertrophic condition. Suppression of ventricular muscle cell
hypertrophy means a decrease of cell size of 10% or greater
relative to that observed in the hypertrophic condition. More
preferably, suppression of hypertrophy means a decrease in cell
size of 30% or greater; most preferably, suppression of hypertrophy
means a decrease of cell size of 50% or more. Relative to the
hypertrophy score assay when phenylephrine is used as the inducing
agent, these decreases would correlate with hypertrophy scores of
about 6.5 or less, 5.0-5.5, and 4.0-5.0, respectively. When a
different agent is used as the inducing agent, suppression is
examined relative to the maximum cell size (or hypertrophic score)
measured in the presence of that inducer.
[0060] Prevention of ventricular muscle cell hypertrophy is
determined by preventing an increase in cell size relative to
normal cells, in the presence of a concentration of inducer
sufficient to fully induce hypertrophy. For example, prevention of
hypertrophy means a cell size increase less than 200% greater than
non-induced cells in the presence of maximally stimulating
concentration of inducer. More preferably, prevention of
hypertrophy means a cell size increase less than 135% greater than
noninduced cells; and most preferably, prevention of hypertrophy
means a cell size increase less than 90% greater than non-induced
cells. Relative to the hypertrophy score assay when phenylephrine
is used as the inducing agent, prevention of hypertrophy in the
presence of a maximally-stimulating concentration of phenylephrine
means a hypertrophic score of about 6.0-6.5, 5.0-5.5, and 4.0-4.5,
respectively.
[0061] In vivo determination of hypertrophy may include measurement
of cardiovascular parameters such as blood pressure, heart rate,
systemic vascular resistance, contractility, force of heartbeat,
concentric or dilated hypertrophy, left ventricular systolic
pressure, left ventricular mean pressure, left ventricular
end-diastolic pressure, cardiac output, stroke index, histological
parameters, and ventricular size and wall thickness. Animal models
available for determination of development and suppression of
ventricular muscle cell hypertrophy in vivo include the
pressure-overload mouse model, RV murine dysfunctional model,
transgenic mouse model, and post-myocardial infarction rat model.
Medical methods for assessing the presence, development, and
suppression of ventricular muscle cell hypertrophy in human
patients are known, and include, for example, measurements of
diastolic and systolic parameters, estimates of ventricular mass
and pulmonary vein flows.
[0062] Hypertrophy may be from any cause which is responsive to
retinoic acid, including congenital viral, idiopathic,
cardiotrophic, or myotrophic causes, or as a result of ischemia or
ischemic insults such as myocardial infarction. Typically, the
treatment is performed to stop or slow the progression of
hypertrophy, especially after heart damage, such as from ischemia,
has occurred. Preferably, for treatment of myocardial infarctions,
the agent(s) is given immediately after the myocardial infarction,
to prevent or lessen hypertrophy.
[0063] As used herein, "activity unit" or "1 U" means the quantity
of standard product that can induce 50% maximal reaction. In other
words, to determine the activity unit for a given active agent, the
EC50 must be measured. For example, if the EC50 for a batch of
product was 0.067 .mu.g/ml then that would be one unit. Further, if
1 .mu.g of that product is being used then 14.93 U (1/0.067) is
being used. The EC50 can be determined by any method known in the
art, including the method employed by the inventors in the Examples
below. This determination of the activity unit is important for
quality control of genetically engineered products and clinically
used drugs, permits product from different pharmaceuticals and/or
different batch numbers to be quantified with uniform criteria.
[0064] In certain embodiments, unit of neuregulin is determined by
measuring the activity of neuregulin through kinase receptor
activation enzyme-linked immunosorbant assay (KIRA-ELISA) as
described in detail in Example 6 below and in WO03/099300, and
Sadick et al., 1996, Analytical Biochemistry, 235:207-14, the
contents of which are incorporated by reference in their
entireties. Briefly, the assay measures neuregulin induced ErbB2
activation and phosphorylation on the adherent breast carcinoma
cell line, MCF-7. Membrane proteins are solubilized via Triton
X-100 lysis and the receptor is captured in ELISA wells coated with
ErbB2-specific antibodies (e.g., H4) with no cross-reaction to
ErbB3 or ErbB4. The degree of receptor phosphorylation is then
quantified by antiphosphotyrosine ELISA.
B. Neuregulin
[0065] The present invention provides methods for treating or
preventing heart failure or cardiac hypertrophy in a mammal by
extended release of a sustained or varied amount of NRG. Any NRG
(e.g., NRG-1, NRG-2, NRG-3 and NRG-4 and isoforms thereof) protein,
peptide or fragment can be used in the practice of this
invention.
[0066] Neuregulin or NRG refers to proteins or peptides that can
bind and activate ErbB2, ErbB3, ErbB4 or combinations thereof,
including but not limited to all neuregulin isoforms, neuregulin
EGF domain alone, polypeptides comprising neuregulin EGF-like
domain, neuregulin mutants or derivatives, and any kind of
neuregulin-like gene products that also activate the above
receptors as described in detail below. In preferred embodiments,
neuregulin used in the present invention binds to and activate
ErbB2/ErbB4 or ErbB2/ErbB3 heterodimers. Neuregulin used in the
present invention can activate the above ErbB receptors and
modulate their biological reactions, e.g., stimulate breast cancer
cell differentiation and milk protein secretion; induce the
differentiation of neural crest cell into Schwann cell; stimulate
acetylcholine receptor synthesis in skeletal muscle cell; and/or
improve cardiocyte differentiation, survival and DNA synthesis.
Assays for measuring the receptor binding activity are known in the
art. For example, cells transfected with ErbB-2 and ErbB-4 receptor
can be used. After receptor expressing cells are incubated with
excess amount of radiolabeled neuregulin, the cells are pelleted
and the solution containing unbound radiolabeled neuregulin is
removed before unlabeled neuregulin solution is added to compete
with radiolabeled neuregulin. EC50 is measured by methods known in
the art. EC50 is the concentration of ligands which can compete 50%
of bound radiolabeled ligands off the receptor complex. The higher
the EC50 value is, the lower the receptor binding affinity is.
[0067] Neuregulin used in the present invention includes any
neuregulin and isoforms thereof known in the art, including but not
limited to all isoforms of neuregulin-1 ("NRG-1"), neuregulin-1
("NRG-2"), neuregulin-1 ("NRG-3") and neuregulin-4 ("NRG-43").
NRG-1 is described, for example, in U.S. Pat. Nos. 5,530,109,
5,716,930, and 7,037,888; Lemke, Mol. Cell. Neurosci. 1996,
7:247-262; Peles and Yarden, 1993, BioEssays 15:815-824, 1993;
Peles et al., 1992, Cell 69, 205-216; Wen et al., 1992, Cell 69,
559-572, 1992, Holmes et al., 1992, Science 256:1205-1210, Falls et
al., 1993, Cell 72:801-815, Marchionni et al. 1993, Nature
362:312-8, the contents of which are incorported by reference in
their entireties. NRG-2 is described, for example, in Chang et al.,
1997, Nature 387:509-512; Carraway et al., 1997, Nature
387:512-516; Higashiyama et al., 1997, J. Biochem. 122:675-680,
Busfield et al., 1997, Mol. Cell. Biol. 17:4007-4014 and
International Pat. Pub. No. WO 97/09425), the contents of which are
incorported by reference in their entireties. NRG-3 is described,
for example, in Hijazi et al., 1998, Int. J. Oncol. 13:1061-1067,
the contents of which are incorported by reference in their
entireties. NRG-4 is described, for example, in Harari et al., 1999
Oncogene. 18:2681-89, the contents of which are incorported by
reference in their entireties.
[0068] Neuregulin used in the present invention includes neuregulin
mutants or derivatives that comprise one or more amino acid
substitutions, deletions, and/or additions that are not present in
the naturally occurring neuregulin. Preferably, the number of amino
acids substituted, deleted, or added is 1, 2, 3, 4, 5, 6, 7, 8. 9,
or 10 amino acids. In one embodiment, such a derivative contains
one or more amino acid deletions, substitutions, or additions at
the amino and/or carboxy terminal end of the peptide. In another
embodiment, such a derivative contains one or more amino acid
deletions, substitutions, or additions at any residue within the
length of the peptide.
[0069] In certain embodiments, the amino acid substitutions may be
conservative or non-conservative amino acid substitutions.
Conservative amino acid substitutions are made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the amino acid
residues involved. For example, nonpolar (hydrophobic) amino acids
include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan, and methionine; polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine; positively charged (basic) amino acids
include arginine, lysine, and histidine; and negatively charged
(acidic) amino acids include aspartic acid and glutamic acid. In
addition, glycine and proline are residues that can influence chain
orientation. Non-conservative substitutions will entail exchanging
a member of one of these classes for another class.
[0070] In certain embodiments, neuregulin used in the present
invention is a neuregulin derivative with conservative amino acid
substitutions that do not substantially alter their biological
activity. Suitable conservative substitutions of amino acids are
known to those of skill in this art and may be made generally
without altering the biological activity of the resulting molecule.
Those of skill in this art recognize that, in general, single amino
acid substitutions in non-essential regions of a polypeptide do not
substantially alter biological activity (see, e.g., Watson et al.
Molecular Biology of the Gene, 4th Edition, 1987, The
Bejacmin/Cummings Pub. co., p. 224).
[0071] In certain embodiments, neuregulin used in the present
invention includes neuregulin mutants or derivatives having an
amino acid substitution with a non-classical amino acid or chemical
amino acid analog. Non-classical amino acids include, but are not
limited to, the D-isomers of the common amino acids, .alpha.-amino
isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,
.gamma.-Abu, .epsilon.-Ahx, 6-amino hexanoic acid, Aib, 2-amino
isobutyric acid, 3-amino propionic acid, omithine, norleucine,
norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
.beta.-alanine, fluoro-amino acids, designer amino acids such as
.beta.-methyl amino acids, C.alpha.-methyl amino acids,
N.alpha.-methyl amino acids, and amino acid analogs in general.
[0072] Neuregulin used in the present invention includes neuregulin
homologue, that is, a polypeptide that exhibits an amino acid
sequence homology and/or structural resemblance to neuregulin, or
to one of the interacting domains of neuregulin such that it is
capable of bind and activate ErbB2/ErbB4 or ErbB2/ErbB3
heterodimers protein kinases. Typically, a protein homologue of a
native protein may have an amino acid sequence that is at least
50%, preferably at least 75%, more preferably at least 80%, 85%,
86%, 87%, 88% or 89%, even more preferably at least 90%, 91%, 92%,
93% or 94%, and most preferably 95%, 96%, 97%, 98% or 99% identical
to the native protein.
[0073] Percent homology in this context means the percentage of
amino acid residues in the candidate sequence that are identical
(i.e., the amino acid residues at a given position in the alignment
are the same residue) or similar (i.e., the amino acid substitution
at a given position in the alignment is a conservative
substitution, as discussed above), to the corresponding amino acid
residue in the peptide after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
homology. In certain embodiments, neuregulin homologue is
characterized by its percent sequence identity or percent sequence
similarity with the naturally occurring neuregulin sequence.
Sequence homology, including percentages of sequence identity and
similarity, are determined using sequence alignment techniques
well-known in the art, preferably computer algorithms designed for
this purpose, using the default parameters of said computer
algorithms or the software packages containing them.
[0074] Nonlimiting examples of computer algorithms and software
packages incorporating such algorithms include the following. The
BLAST family of programs exemplify a preferred, non-limiting
example of a mathematical algorithm utilized for the comparison of
two sequences (e.g., Karlin & Altschul, 1990, Proc. Natl. Acad.
Sci. USA 87:2264-2268 (modified as in Karlin & Altschul, 1993,
Proc. Natl. Acad. Sci. USA 90:5873-5877), Altschul et al., 1990, J.
Mol. Biol. 215:403-410, (describing NBLAST and XBLAST), Altschul et
al., 1997, Nucleic Acids Res. 25:3389-3402 (describing Gapped
BLAST, and PSI-Blast). Another preferred example is the algorithm
of Myers and Miller (1988 CABIOS 4:11-17) which is incorporated
into the ALIGN program (version 2.0) and is available as part of
the GCG sequence alignment software package. Also preferred is the
FASTA program (Pearson W. R. and Lipman D. J., Proc. Nat. Acad.
Sci. USA, 85:2444-2448, 1988), available as part of the Wisconsin
Sequence Analysis Package. Additional examples include BESTFIT,
which uses the "local homology" algorithm of Smith and Waterman
(Advances in Applied Mathematics, 2:482-489, 1981) to find best
single region of similarity between two sequences, and which is
preferable where the two sequences being compared are dissimilar in
length; and GAP, which aligns two sequences by finding a "maximum
similarity" according to the algorithm of Neddleman and Wunsch (J.
Mol. Biol. 48:443-354, 1970), and is preferable where the two
sequences are approximately the same length and an alignment is
expected over the entire length.
[0075] Examples of homologues may be the ortholog proteins of other
species including animals, plants, yeast, bacteria, and the like.
Homologues may also be selected by, e.g., mutagenesis in a native
protein. For example, homologues may be identified by site-specific
mutagenesis in combination with assays for detecting
protein-protein interactions. Additional methods, e.g., protein
affinity chromatography, affinity blotting, in vitro binding
assays, and the like, will be apparent to skilled artisans apprised
of the present invention.
[0076] For the purpose of comparing two different nucleic acid or
polypeptide sequences, one sequence (test sequence) may be
described to be a specific "percent identical to" another sequence
(reference sequence) in the present disclosure. In this respect,
when the length of the test sequence is less than 90% of the length
of the reference sequence, the percentage identity is determined by
the algorithm of Myers and Miller, Bull. Math. Biol., 51:5-37
(1989) and Myers and Miller, Comput. Appl. Biosci., 4(1):11-17
(1988). Specifically, the identity is determined by the ALIGN
program. The default parameters can be used.
[0077] Where the length of the test sequence is at least 90% of the
length of the reference sequence, the percentage identity is
determined by the algorithm of Karlin and Altschul, Proc. Natl.
Acad. Sci. USA, 90:5873-77 (1993), which is incorporated into
various BLAST programs. Specifically, the percentage identity is
determined by the "BLAST 2 Sequences" tool. See Tatusova and
Madden, FEMS Microbiol. Lett., 174(2):247-250 (1999). For pairwise
DNA-DNA comparison, the BLASTN 2.1.2 program is used with default
parameters (Match: 1; Mismatch: -2; Open gap: 5 penalties;
extension gap: 2 penalties; gap x_dropoff: 50; expect: 10; and word
size: 11, with filter). For pairwise protein-protein sequence
comparison, the BLASTP 2.1.2 program is employed using default
parameters (Matrix: BLOSUM62; gap open: 11; gap extension: 1;
x_dropoff: 15; expect: 10.0; and wordsize: 3, with filter).
[0078] Neuregulin used in the present invention also include
neuregulin EGF domain alone, polypeptides comprising neuregulin EGF
domain or neuregulin-like gene products that mimic the activities
of neuregulin and binds and activates ErbB2, ErbB3, ErbB4 or
combinations thereof. As used herein, "epidermal growth factor-like
domain" or "EGF-like domain" refers to a polypeptide motif encoded
by the neuregulin gene that binds to and activates ErbB2, ErbB3,
ErbB4, or combinations thereof, and bears a structural similarity
to the EGF receptor-binding domain as disclosed in WO 00/64400,
Holmes et al., Science, 256:1205-1210 (1992); U.S. Pat. Nos.
5,530,109 and 5,716,930; Hijazi et al., Int. J. Oncol.,
13:1061-1067 (1998); Chang et al., Nature, 387:509-512 (1997);
Carraway et al., Nature, 387:512-516 (1997); Higashiyama et al., J.
Biochem., 122:675-680 (1997); and WO 97/09425, the contents of
which are all incorporated herein by reference.
[0079] In certain embodiments, neuregulin used in the present
invention comprises the EGF-like domain encoded by NRG-1. In some
embodiments, EGF-like domain comprises the amino acid sequence of
the receptor binding domain of NRG-1. In some embodiments, EGF-like
domain comprises the amino acid sequence corresponding to amino
acid residues 177-226, 177-237, or 177-240 of NRG-1.
[0080] In preferred embodiments, neuregulin used in the present
invention comprises the amino acid sequence of:
[0081] Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val
Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr
Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val
Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln (SEQ ID NO:1),
which corresponds to amino acids 177-237 of human NRG-1. The human
nucleic acid sequence encoding the fragment is:
[0082] agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg
aggggagtgc ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc
aaatgagttt actggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaaggcgga
ggagctgtac cag (SEQ ID NO:2).
[0083] In certain embodiments, neuregulin used in the present
invention comprises the EGF-like domain encoded by NRG-2. In
certain embodiments, neuregulin used in the present invention
comprises the EGF-like domain encoded by NRG-3. In certain
embodiments, neuregulin used in the present invention comprises the
EGF-like domain encoded by NRG-4. In certain embodiments,
neuregulin used in the present invention comprises the amino acid
sequence of Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys
Phe Met Val Lys Asp Leu Ser Asn Pro, as described in U.S. Pat. No.
5,834,229.
C. Extended Release Technology in General
[0084] The present invention provides compositions for extended
release of neuregulin and methods for preventing, treating or
delaying various disease, such as heart failure using such.
Extended release of neuregulin allows for simplification of
administration scheme, improves clinical efficacy and attenuates
adverse events, e.g., related to high blood level of neuregulin. It
is contemplated that extended release of neuregulin over a certain
period could induce or maintain expression of certain genes for
cardiomyocyte growth and/or differentiation, remodeling of muscle
cell sarcomeric and cytoskeleton structures, or cell-cell
adhesions.
[0085] Extended release of neuregulin can be administered by any
route according to the judgment of those of skill in the art,
including but not limited to orally, inhalationally, parenterally
(e.g., intravenously, intramuscularly, subcutaneously, or
intradermally). In certain embodiments, neuregulin is administered
orally. In certain embodiments, neuregulin is administered
intravenously. In certain embodiments, neuregulin is administered
intramuscularly. In preferred embodiments, neuregulin is extendedly
released to the bloodstream of a mammal.
[0086] Neuregulin can be administered by any extended release means
or by any delivery devices that are known to those of ordinary
skill in the art. Specifically, any extended means or delivery
devices for deliverying peptides known in the art can be used in
the present invention. Examples include, but are not limited to,
those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809;
3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767,
5,120,548, 5,073,543, 5,639,476, 5,354,556, 5,639,480, 5,733,566,
5,739,108, 5,891,474, 5,922,356, 5,972,891, 5,980,945, 5,993,855,
6,045,830, 6,087,324, 6,113,943, 6,197,350, 6,248,363, 6,264,970,
6,267,981, 6,376,461,6,419,961, 6,589,548, 6,613,358, 6,699,500,
6,740,634, 6,838,076, 6,866,866, 7,087,246, each of which is
incorporated herein by reference. Such dosage forms can be used to
provide extended release of neuregulin using, for example,
hydropropylmethyl cellulose, other polymer matrices, gels,
permeable membranes, osmotic systems, multilayer coatings,
microparticles, liposomes, microspheres, or a combination thereof
to provide the desired release profile in varying proportions. The
invention also encompasses single unit dosage forms suitable for
oral administration such as, but not limited to, tablets, capsules,
gelcaps, and caplets that are adapted for controlled-release.
[0087] Extended release of neuregulin provides continuous
therapeutic level of neuregulin over a period of time. In some
embodiments, neuregulin is released over a period of 1 hour, 2
hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16
hours, 20 hours, 24 hours or longer. In some embodiments,
neuregulin is released over a period of 1 day, 2 days, 3 days, 4
days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or longer. In
yet another embodiments, neuregulin is released over a period of 1
week, 2 weeks, 3 weeks, 4 weeks or longer. In another embodiments,
neuregulin is released over a period of 1 month, 2 months, 4
months, 8 months, 12 months or longer. In yet another embodiments,
neuregulin is released over a period of 1 years, 2 years, 3 years,
4 years or longer. In some embodiments, neuregulin is released over
a period of between 1 minutes to 24 hours, 1 hour and 2 week,
between 2 hours and 2 week, between 4 hours to 24 hours, between 4
days and 10 days. The amount of time over which neuregulin is
released may depend on various factors such as the extended release
technology or technologies used.
[0088] Extended release of neuregulin maintains neuregulin in the
blood within a desirable range, particularly at a level which is at
or above the minimum effective therapeutic level and is below the
minimum toxic level over a period of time. The serum concentration
of neuregulin in patients who received an extended release
neuregulin composition can be compared with serum concentrations of
patients receiving a non-extended release neuregulin composition
(e.g., intravenous administration) at a time when the maximum blood
level concentration occurs (C.sub.max). In a preferred embodiment,
the patients receiving an extended release neuregulin composition
have a lower maximum serum concentration (C.sub.max) of neuregulin
than the patients receiving a non-extended neuregulin composition.
Preferably, the patients receiving an extended release neuregulin
composition have a C.sub.max less than about 90%, 80%, 70% or 60%
of the C.sub.max in patients receiving a non-extended release
neuregulin composition. More preferably, the patients receiving an
extended release neuregulin composition have a C.sub.max less than
about 50%, 40% or 30% of the C.sub.max in patients receiving a
non-extended release neuregulin composition. Most preferably, the
patients receiving an extended release neuregulin composition have
a C.sub.max less than about 20%, 10% or less of the C.sub.max in
patients receiving a non-extended release neuregulin composition.
Methods for measuring the concentration of neuregulin in the serum
are known in the art. For instance, cells expressing ErbB-2 and
ErbB-3 receptors, such as SKBR-3 breast cancer cell line, can be
used. 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039, 0.019
and 0.0079 ng of neuregulin is added to different tubes containing
cells separately on ice, then radiolabeled neuregulin (50,000 cpm)
is added. The sample solution is mixed and left at 4.degree. C.
overnight. Next morning, cells are pelleted and the supernatant is
sucked away before the radioactivity is counted. A standard curve
is drawn by radioactivity versus unlabeled neuregulin amount. When
measuring the concentration of neuregulin in the serum, certain
amount of serum is added to tube containing cells on ice,
radiolabeled neuregulin (50,000 cpm) is then added, and the sample
solution is mixed and left at 4.degree. C. overnight. Next morning,
cells are pelleted and the supernatant is sucked away before the
radioactivity is counted. The radioactivity is counted and the
amount of neuregulin in the serum can be calculated according to
the standard curve.
[0089] Various extended release profiles can be provided in
accordance with the present invention. "Extended release profile"
means a release profile in which less than 50% of the total release
of neuregulin that occurs over the course of implantation/insertion
or other method of administering neuregulin in the body occurs
within the first 24 hours of administration. In a preferred
embodiment of the present invention, the extended release profile
is selected from the group consisting of: (a) the 50% release point
occurring at a time that is between 24 and 48 hours after
implantation/insertion or other method of administration, (b) the
50% release point occurring at a time that is between 48 and 96
hours after implantation/insertion or other method of
administration, (c) the 50% release point occurring at a time that
is between 96 and 168 hours (1 week) after implantation/insertion
or other method of administration, (d) the 50% release point
occurring at a time that is between 1 and 2 weeks after
implantation/insertion or other method of administration, (e) the
50% release point occurring at a time that is between 2 and 4 weeks
after implantation/insertion or other method of administration, (f)
the 50% release point occurring at a time that is between 4 and 8
weeks after implantation/insertion or other method of
administration, (g) the 50% release point occurring at a time that
is between 8 and 16 weeks after implantation/insertion or other
method of administration, (h) the 50% release point occurring at a
time that is between 16 and 52 weeks (1 year) after
implantation/insertion or other method of administration, and (i)
the 50% release point occurring at a time that is between 52 and
104 weeks after implantation/insertion or other method of
administration.
[0090] Additionally, use of the present invention can reduce the
degree of fluctuation ("DFL") of an agent's plasma concentration.
DFL is a measurement of how much plasma levels of a drug vary over
the course of a dosing interval. The closer the DFL is to zero (0),
the less variance there is over the course of a dosing period. Thus
a reduced DFL signifies that the difference in peak and trough
plasma levels has been reduced. Preferably, the patients receiving
an extended release composition have a DFL approximately 90%, 80%,
70% or 60% of the DFL in patients receiving a non-extended release
composition. More preferably, the patients receiving an extended
release composition have a DFL approximately 50%, 40%, or 30% of
the DFL in patients receiving a non-extended release composition.
Most preferably, the patients receiving an extended release
neuregulin composition have a DFL approximately 20%, 10% or less of
the DFL in patients receiving a non-extended release neuregulin
composition.
[0091] Any technologies known in the art for extended release of a
biomolecule can be used in the prevent invention. Generally, the
size and frequency of dosing is determined by the pharmacodynamic
and pharmacokinetic properties of the active agent. The slower the
rate of absorption, the less the blood concentrations fluctuate
within a dosing interval. This enables higher doses to be given
less frequently. However, many active agents that are readily
soluble in the body are usually absorbed rapidly and provide a
sudden burst of available drug. An example is hypotension patients
taking rapid-release nifedipine products. The use of an
extended-release product avoids the high initial blood
concentrations which cause the sudden reduction in blood pressure
and other significant haemodynamic changes such as reflex
tachycardia.
[0092] Additionally, some active agents are targeted and removed or
destroyed by the body, e.g., immune system, proteases. Drugs with
short half-lives for this and other reasons often need to be given
the active agent at frequent intervals to maintain blood
concentrations within the therapeutic range. There is an inverse
correlation between the frequency of dosing and patient compliance.
For such agents with relatively short half-lives, the use of
extended-release products may maintain therapeutic concentrations
over prolonged periods. Thus, a reduction in the number of daily
doses offered by extended-release products has the potential to
improve compliance. Although specific extended release technologies
are disclosed herein, the invention is more general than any
specific extended release technology. This includes the discovery
that extended release of NRG at low doses unexpectedly improves the
function of infarct heart. Further, there are numerous extended
release drug delivery technologies currently known in the art.
Several are generally discussed below as preferred extended release
technologies, but they are offered solely for purposes of
illustration and not limitation. Many other related and unrelated
technologies are well known in the art and may be employed in the
practice of the invention disclosed herein. Additionally,
combinations of the extended release technologies discussed herein
and/or other extended release technologies known in the art may be
employed in the practice of this invention. For example, many
companies with specific expertise in extended release drug delivery
technologies--e.g., Alza Corp., Durect Corp., Gilead Sciences,
Baxter Pharmaceuticals, Brookwood Pharmaceuticals and
OctoPlus--offer products and services that can be employed in the
practice of this invention. Additionally, a search of patents,
published patent applications and related publications will provide
those skilled in the art reading this disclosure with significant
possible extended release technologies. Thus, one skilled in the
art will be able to select the desired extended release technology
or technologies for use in the practice of this invention.
C. 1. Osmotic Pumps
[0093] In one embodiment of the present invention, the extended
release of NRG into the blood comprises the use of an osmotic pump.
Osmotic devices have demonstrated utility in delivering beneficial
active agents to a target area in a controlled manner over
prolonged periods of time. Known devices include tablets, pills,
capsules and implantable devices. Tablets and pills can be taken
orally, whereas other pumps are implanted subcutaneously or
intraperitoneally, or attached to a catheter for intravenous,
intracerebral or intra-arterial infusion.
[0094] Generally, in an osmotic pump system, a core is encased by a
semipermeable membrane having at least one orifice. The
semipermeable membrane is permeable to water, but impermeable to
the active agent. When the system is exposed to body fluids, water
penetrates through the semipermeable membrane into the core
containing osmotic excipients and the active agent. Osmotic
pressure increases within the core and the agent is displaced
through the orifice at a controlled, predetermined rate.
[0095] In many osmotic pumps, the core contains more than one
internal compartment. For example, a first compartment may contain
the active agent. A second compartment contains an osmotic agent
and/or "driving member." See, e.g., U.S. Pat. No. 5,573,776, the
contents of which are incorporated herein by reference. This
compartment may have a high osmolality, which causes water to flux
into the pump through the semipermeable membrane. The influx of
water compresses the first compartment. This can be accomplished,
for example, by using a polymer in the second compartment, which
swells on contact with the fluid. Accordingly, the agent is
displaced at a predetermined rate.
[0096] In another embodiments, the osmotic pump may comprise more
than one active agent-containing compartment, with each compartment
containing the same agent or a different agent. The concentrations
of the agent in each compartment, as well as the rate of release,
may also be the same or different.
[0097] The rate of delivery is generally controlled by the water
permeability of the semipermeable membrane. Thus, the delivery
profile of the pump is independent of the agent dispensed, and the
molecular weight of an agent, or its physical and chemical
properties, generally have no bearing on its rate of delivery.
Further discussion regarding the principle of operation, the design
criteria, and the delivery rate for osmotic pumps is provided in
Thecuwes and Yum, Annals of Biomedical Engineering, Vol. 4, No. 4
(1976) and Urquhart el. al., Ann. Rev. Pharmacol. Toxicol.
24:199-236 (1984), the contents of which are incorporated by
reference.
[0098] Osmotic pumps are well known in the art and readily
available to one of ordinary skill in the art from companies
experienced in providing osmotic pumps for extended release drug
delivery. For example, ALZA's DUROS.RTM. technology is an
implantable, nonbiodegradable, osmotically driven system that
enables delivery of small drugs, peptides, proteins, DNA and other
bioactive macromolecules for up to one year; ALZA's OROS.RTM.
technology embodies tablets that employ osmosis to provide precise,
controlled drug delivery for up to 24 hours; Osmotica
Pharmaceutical's Osmodex.RTM. system includes a tablet, which may
have more than one layer of the drug(s) with the same or different
release profiles; Shire Laboratories' EnSoTrol.RTM. system
solubilizes drugs within the core and delivers the solubilized drug
through a laser-drilled hole by osmosis; and Alzet.RTM. Osmotic
pumps are miniature, implantable pumps used for research in mice,
rats and other laboratory animals.
[0099] A search of patents, published patent applications and
related publications will also provide those skilled in the art
reading this disclosure with significant possible osmotic pump
technologies. For example, U.S. Pat. Nos. 6,890,918; 6,838,093;
6,814,979; 6,713,086; 6,534,090; 6,514,532; 6,361,796; 6,352,721;
6,294,201; 6,284,276; 6,110,498; 5,573,776; 4,200,0984; and
4,088,864, the contents of which are incorporated herein by
reference, describe osmotic pumps and methods for their
manufacture. One skilled in the art, considering both the
disclosure of this invention and the disclosures of these other
patents could produce an osmotic pump for the extended release of
NRG.
[0100] Typical materials for the semipermeable membrane include
semipermeable polymers known to the art as osmosis and reverse
osmosis membranes, such as cellulose acylate, cellulose diacylate,
cellulose triacylate, cellulose acetate, cellulose diacetate,
cellulose triacetate, agar acetate, amylase triacetate, beta glucan
acetate, acetaldehyde dimethyl acetate, cellulose acetate ethyl
carbamate, polyamides, plyurethanes, sulfonated polystyrenes,
cellulose acetate pphthalate, cellulose acetate methyl carbamate,
cellulose acetate succinate, cellulose acetate dimethyl
aminoacetate, cellulose acetate ethyl carbamate, cellulose acetate
chloracetate, cellulose dipalmitate, cellulose dioctanoate,
cellulose dicaprylate, cellulose dipentanlate, cellulose acetate
valerate, cellulose acetate succinate, cellulose propionate,
succinate, methyl cellulose, cellulose acetate p-toluene sulfonate,
cellulose acetate butyrate, cross-linked selectively semipermeable
polymers formed by the coprecipitation of a polyanion and a
polycation, semipermeable polymers, lightly cross-linked
polystyrene derivatives, cross-linked poly(sodium styrene
sulfonate), poly(vinylbenzyltrimethyl ammonium chloride), cellulose
acetate having a degree of substitution up to 1 and an acetyl
content up to 50%, cellulose diacetate having a degree of
substitution of 1 to 2 and an acetyl content of 21 to 35%,
cellulose triacetate having a degree of substitution of 2 to 3 and
an acetyl content of 35 to 44.8%, as disclosed in U.S. Pat. No.
6,713,086, the contents of which are incorporated herein by
reference.
[0101] The osmotic agent(s) present in the pump may comprise any
osmotically effective compound(s) that exhibit an osmotic pressure
gradient across the semipermeable wall against the exterior fluid.
Effective agents include, without limitation, magnesium sulfate,
calcium sulfate, magnesium chloride, sodium chloride, lithium
chloride, potassium sulfate, sodium carbonate, sodium sulfite,
lithium sulfate, potassium chloride, sodium sulfate, d-mannitol,
urea, sorbitol, inositol, raffinose, sucrose, flucose, hydrophilic
polymers such as cellulose polymers, mixtures thereof, and the
like, as disclosed in U.S. Pat. No. 6,713,086, the contents of
which are incorporated herein by reference.
[0102] The "driving member" is typically a hydrophilic polymer
which interacts with biological fluids and swells or expands. The
polymer exhibits the ability to swell in water and retain a
significant portion of the imbibed water within the polymer
structure. The polymers swell or expand to a very high degree,
usually exhibiting a 2 to 50 fold volume increase. The polymers can
be non-crosslinked or crosslinked. Hydrophilic polymers suitable
for the present purpose are well known in the art.
[0103] The orifice may comprise any means and methods suitable for
releasing the active agent from the system. The osmotic pump may
include one or more apertures or orifices which have been bored
through the semipermeable membrane by mechanical procedures known
in the art, including, but not limited to, the use of lasers as
disclosed in U.S. Pat. No 4,088,864. Alternatively, it may be
formed by incorporating an erodible element, such as a gelatin
plug, in the semipermeable membrane.
[0104] Although specific embodiments of osmotic pumps are discussed
above, the invention is more general than any specific extended
release technology. This includes the discovery that extended
release of NRG improves the ftunction of infarct heart and reduces
the interior diameter of the left ventricle. There are numerous
variations and different types of osmotic pumps currently known in
the art and may be employed in the practice of the invention
disclosed herein.
C. 2. Poly(ethylene glycol) Coupling
[0105] In one embodiment of the present invention, the extended
release of NRG into the blood comprises the coupling of the active
agent to a polymer, such as Poly(ethylene glycol) (hereinafter
referred to as "PEG"). Coupling PEG to biologically active agents
has demonstrated utility in delivering active agents to a target
area in a controlled manner over prolonged periods of time.
Particularly, modification of proteins with PEG has been
extensively used within the biotechnology industry to reduce the
antigenicity of therapeutically active agents and to extend their
in vivo availability. For example, coupling PEG to bovine adenosine
deaminase using cyanuric chloride results in a loss of
immunogenicity. Similarly, the PEG adduct of both human growth
hormone and E. coli L-asparaginase has been shown to have an
extended circulatory half-life.
[0106] Coupling PEG to an active agent or other molecules, e.g.,
outer surface of liposomes, can improve the efficacy and half-life
of the active agent or other molecule, and also reduce its
toxicity. Particularly, in an aqueous medium, the PEG molecule is
hydrated and in rapid motion. This rapid motion causes the PEG to
sweep out a large volume and prevents the approach and interference
of other molecules, e.g., immune cells or proteases. Thus, when
coupled to PEG, the PEG polymer chains can protect the attached
molecule from immune response and other clearance mechanisms,
sustaining availability of the active agent.
[0107] Generally, polyethylene glycol molecules are connected to
the protein via a reactive group found on the protein. Commonly
amino groups, such as those on lysine residues or at the
N-terminus, are used for attachment. U.S. Pat. Nos. 5,824,784 and
4,002,531 disclose such methods for attaching PEG to an enzyme by
reductive alkylation. Lysine residues may be strategically
substituted for other amino acids or inserted into a polypeptide
sequence to provide additional points of attachment as disclosed in
U.S. Pat. No. 4,904,584. Additional methods are known in the art
for attaching branched or "multi-armed" PEG-derivatives to proteins
as disclosed in U.S. Pat. No. 5,932,462. There are many other
methods of attachment known in the art for attaching polymers to
cysteine residues, carboxy groups, carbohydrates and other
moieties. For example, U.S. Pat. No. 5,900,461 discloses
derivatives of PEG and other polymers having one more active
sulfone moieties that are highly selective for coupling with thiol
moieties instead of amino moieties on molecules.
[0108] PEGs can also be used to link macromolecules to a targeting
ligand or moiety, which directs the macromolecules to particular
areas of interest. U.S. Pat. No. 6,436,386 discloses active
agent-polymer conjugates attached to a hydroxyapatite-targeting
moiety for delivery of the active agent, such as bone growth
factors, to hydroxyapatite surfaces, such as bone.
[0109] A wide variety of PEG derivatives are both available and
suitable for use in the preparation of PEG-conjugates. For example,
NOF Corp.'s SUNBRIGHT.RTM. Series provides numerous PEG
derivatives, including methoxypolyethylene glycols and activated
PEG derivatives, such as methoxy-PEG amines, maleimides and
carboxylic acids, for coupling by various methods to drugs,
enzymes, phospholipids and other biomaterials and Nektar
Therapeutics' Advanced PEGylation also offers diverse PEG-coupling
technologies to improve the safety and efficacy of
therapeutics.
[0110] A search of patents, published patent applications and
related publications will also provide those skilled in the art
reading this disclosure with significant possible PEG-coupling
technologies and PEG-derivatives. For example, U.S. Pat. Nos.
6,436,386; 5,932,462; 5,900,461; 5,824,784; 4,904,584 and
4,002,531, the contents of which are incorporated by reference in
their entirety, describe such technologies and derivatives, and
methods for their manufacture. Thus, one skilled in the art,
considering both the disclosure of this invention and the
disclosures of these other patents could couple PEG, a
PEG-derivative or some other polymer to NRG for its extended
release.
[0111] PEG is a well known polymer having the properties of
solubility in water and in many organic solvents, lack of toxicity,
lack of immunogenecity, and also clear, colorless, odorless and
stable. One use of PEG is to covalently attach the polymer to
insoluble molecules to make the resulting PEG-molecule conjugate
soluble. For these reasons and others, PEG has been selected as the
preferred polymer for attachment, but it has been employed solely
for purposes of illustration and not limitation. Similar products
may be obtained with other water soluble polymers, including
without limitation, poly(vinyl alcohol), other poly(alkylene
oxides) such as poly(propylene glycol) and the like,
poly(oxyethylated polyols) such as poly(oxyethylated glycerol) and
the like, carboxymethylcellulose, dextran, polyvinyl alcohol,
polyvinyl purrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride, and polyaminoacids. One skilled in the
art will be able to select the desired polymer based on the desired
dosage, circulation time, resistance to proteolysis, and other
considerations.
C. 3. Liposome Packaging
[0112] In another embodiment of the present invention, the extended
release of NRG into the blood comprises packaging NRG in a
liposome, which has demonstrated utility in delivering beneficial
active agents in a controlled manner over prolonged periods of
time. Liposomes are completely closed bilayer membranes containing
an entrapped aqueous volume. Liposomes may be unilamellar vesicles
possessing a single membrane bilayer or multilamellar vesicles with
multiple membrane bilayers, each separated from the next by an
aqueous layer. The structure of the resulting membrane bilayer is
such that the hydrophobic (non-polar) tails of the lipid orient
toward the center of the bilayer while the hydrophilic (polar)
heads orient towards the aqueous phase.
[0113] Generally, in a liposome-drug delivery system, the active
agent is entrapped in the liposome and then administered to the
patient to be treated. However, if the active agent is lipophilic,
it may associate with the lipid bilayer.
[0114] The immune system may recognize conventional liposomes as
foreign bodies and destroy them before significant amounts of the
active agent reaches the intended disease site. Thus, in one
embodiment, the liposome may be coated with a flexible water
soluble polymer that avoids uptake by the organs of the mononuclear
phagocyte system, primarily the liver and spleen. Suitable
hydrophilic polymers for surrounding the liposomes include, without
limitation, PEG, polyvinylpyrrolidone, polyvinylmethylether,
polymethyloxazoline, polyethyloxazoline,
polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide,
polymethacrylamide, polydimethylacrylamide,
polyhydroxypropylmethacrylate, polyhydroxethylacrylate,
hydroxymethylcellulose hydroxyethylcellulose, polyethyleneglycol,
polyaspartamide and hydrophilic peptide sequences as described in
U.S. Pat. Nos. 6,316,024; 6,126,966; 6,056,973; 6,043,094, the
contents of which are incorporated by reference in their
entirety.
[0115] Liposomes may be comprised of any lipid or lipid combination
known in the art. For example, the vesicle-forming lipids may be
naturally-occurring or synthetic lipids, including phospholipids,
such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic
acid, phosphatidylserine, phasphatidylglycerol,
phosphatidylinositol, and sphingomyelin as disclosed in U.S. Pat.
Nos. 6,056,973 and 5,874,104. The vesicle-forming lipids may also
be glycolipids, cerebrosides, or cationic lipids, such as
1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP);
N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium
bromide (DMRIE); N-[1
[(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium
bromide (DORIE);
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA); 3 [N-(N',N'-dimethylaminoethane) carbamoly] cholesterol
(DC-Chol); or dimethyldioctadecylammonium (DDAB) also as disclosed
in U.S. Pat. No. 6,056,973. Cholesterol may also be present in the
proper range to impart stability to the vesicle as disclosed in
U.S. Pat. Nos. 5,916,588 and 5,874,104.
[0116] In another embodiment, the liposome is targeted to specific
sites within the body of a mammal by the attachment of a targeting
ligand or moiety. The targeting ligands are believed to be
recognized by receptors or other compounds on the surface of target
cells. Typical target ligands include antibodies or antibody
fragments, cell-receptor ligands, lectins and the like. For further
discussion see U.S. Pat. Nos. 6,316,024 and 6,294,191, the contents
of which are incorporated by reference in their entirety.
[0117] Such targeting ligands can be attached to liposomes by any
means known in the art for the covalent or noncovalent attachment
of such ligands to lipsomes. For example, polymer coated liposomes
have been modified to achieve site specific delivery of active
agents, by attaching a targeting ligand to either the polar head
group residues of liposomal lipid components or the free ends of
the polymer chains forming the surface coat on the liposomes as
described in U.S. Pat. Nos. 6,316,024 and 6,043,094, the contents
of which are incorporated by reference in their entirety. Such
attachments may be accomplished by, for example, the coupling of
proteins to liposomes through the use of a crosslinking agent
having at least one maleimido group and an amine reductive function
as described in U.S. Pat. No. 5,399,331; linking proteins to
liposomes through the use of the glycoprotein streptavidin as
described in U.S. Pat. Nos. 4,885,172; 5,059,421 and 5,171,578; the
coating of targeted liposomes with polysaccharides; or a vesicle
forming lipid may derivatized with a hydrophilic polymer chain,
which is end-fuinctionalized for coupling antibodies through the
use of a hydrazide or hydrazine group that is reactive toward
aldehyde groups as described in U.S. Pat. No. 6,126,966. The end
functionalized group may also be 2-pyridyldithio-propionamide, for
coupling an antibody or other molecule to the liposome through a
disulfide linkage.
[0118] The liposomes of this invention can be manufactured by
standard techniques known to those of skill in the art. For
example, in one embodiment, as disclosed in U.S. Pat. No.
5,916,588, a buffered solution of the active agent is prepared.
Then a suitable lipid, such as hydrogenated soy
phosphatidylcholine, and cholesterol, both in powdered form, are
dissolved in chloroform or the like and dried by rotoevaporation.
The lipid film thus formed is resupsended in diethyl ether or the
like and placed in a flask, and sonicated in a water bath during
addition of the buffered solution of the active agent. Once the
ether has evaporated, sonication is discontinued and a stream of
nitrogen is applied until residual ether is removed. Other standard
manufacturing procedures are described in U.S. Pat. Nos. 6,352,716;
6,294,191; 6,126,966; 6,056,973; 5,965,156; and 5,874,104. The
liposomes of this invention can be produced by any method generally
accepted in the art for making liposomes, including, without
limitation, the methods of the above-cited documents (the contents
of which are incorporated herein by reference).
[0119] Liposomes are also well known in the art and readily
available from companies experienced in providing liposomes for
extended release drug delivery. For example, ALZA's (formerly
Sequus Pharmaceuticals') STEALTH.RTM. liposomal technology for
intravenous drug delivery uses a polyethylene glycol coating on
liposomes to evade recognition by the immune system; Gilead
Sciences (formerly Nexstar's) liposomal technology was incorporated
into AmBisome.RTM., and FDA approved treatment for fungal
infections; and NOF Corp. offers a wide variety of GMP-grade
phospholipids, phospholipids derivatives, and PEG-phospholipids
under the tradenames COATSOME.RTM. and SUNBRIGHT.RTM..
[0120] A search of patents, published patent applications and
related publications will also provide those skilled in the art
reading this disclosure with significant possible liposomal
technologies. U.S. Pat. Nos. 6,759,057; 6,406,713; 6,352,716;
6,316,024; 6,294,191; 6,126,966; 6,056,973; 6,043,094; 5,965,156;
5,916,588; 5,874,104; 5,215,680; and 4,684,479, the contents of
which are incorporated herein by reference, describe liposomes and
lipid-coated microbubbles, and methods for their manufacture. Thus,
one skilled in the art, considering both the disclosure of this
invention and the disclosures of these other patents could produce
a liposome for the extended release of NRG.
[0121] Although specific embodiments of liposomes are discussed
above, the invention is more general than any specific extended
release technology. This includes the discovery that extended
release of NRG improves the function of infarct heart and reduces
the interior diameter of the left ventricle. There are numerous
variations and different types of liposomes currently known in the
art and may be employed in the practice of the invention disclosed
herein.
C. 4. Microsphere Packaging
[0122] In another embodiment of the present invention, the extended
release of NRG into the blood comprises packaging NRG in a
microsphere. Microspheres have demonstrated utility in delivering
beneficial active agents to a target area in a controlled manner
over prolonged periods of time. Microspheres are generally
biodegradable and can be used for subcutaneous, intramuscular and
intravenous administration.
[0123] Generally, each microsphere is composed of an active agent
and polymer molecules. As disclosed in U.S. Pat. No. 6,268,053, the
active agent may be centrally located within a membrane formed by
the polymer molecules, or, alternatively dispersed throughout the
microsphere because the internal structure comprises a matrix of
the active agent and a polymer excipient. Typically, the outer
surface of the microsphere is permeable to water, which allows
aqueous fluids to enter the microsphere, as well as solubilized
active agent and polymer to exit the microsphere.
[0124] In one embodiment, the polymer membrane comprises
crosslinked polymers as disclosed in U.S. Pat. No. 6,395,302. When
the pore sizes of the crosslinked polymer are equal or smaller than
the hydrodynamic diameter of the active agent, the active agent is
essentially released when the polymer is degraded. On the other
hand, if the pore size of the crosslinked polymers are larger than
the size of the active agent, the active agent is at least
partially released by diffusion.
[0125] Additional methods for making microsphere membranes are
known and used in the art and can be used in the practice of the
invention disclosed herein. Typical materials for the outer
membrane include the following categories of polymers: (1)
carbohydrate-based polymers, such as methylcellulose, carboxymethyl
cellulose-based polymers, dextran, polydextrose, chitins, chitosan,
and starch (including hetastarch), and derivatives thereof; (2)
polyaliphatic alcohols such as polyethylene oxide and derivatives
thereof including polyethylene glycol (PEG), PEG-acrylates,
polyethyleneimine, polyvinyl acetate, and derivatives thereof; (3)
poly(vinyl) polymers such as poly(vinyl) alcohol,
poly(vinyl)pyrrolidone, poly(vinyl)phosphate, poly(vinyl)phosphonic
acid, and derivatives thereof; (4) polyacrylic acids and
derivatives thereof; (5) polyorganic acids, such as polymaleic
acid, and derivatives thereof; (6) polyamino acids, such as
polylysine, and poly-imino acids, such as polyimino tyrosine, and
derivatives thereof; (7) co-polymers and block co-polymers, such as
poloxamer 407 or Pluronic L-101.TM. polymer, and derivatives
thereof; (8) tert-polymers and derivatives thereof; (9) polyethers,
such as poly(tetramethylene ether glycol), and derivatives thereof;
(10) naturally occurring polymers, such as zein, chitosan and
pullulan, and derivatives thereof; (11) polyimids, such as poly
n-tris(hydroxymethyl) methylmethacrylate, and derivatives thereof;
(12) surfactants, such as polyoxyethylene sorbitan, and derivatives
thereof; (13) polyesters such poly(ethylene glycol) (n)monomethyl
ether mono(succinimidyl succinate)ester, and derivatives thereof;
(14) branched and cyclo-polymers, such as branched PEG and
cyclodextrins, and derivatives thereof; and (15) polyaldehydes,
such as poly(perfluoropropylene oxide-b-perfluoroformaldehyde), and
derivatives thereof as disclosed in U.S. Pat. No. 6,268,053, the
contents of which are incorporated herein by reference. Other
typical polymers known to those of ordinary skill in the art
include poly(lactide-co-glycolide, polylactide homopolymer;
polyglycolide homopolymer; polycaprolactone;
polyhydroxybutyrate-polyhydroxyvalerate copolymer;
poly(lactide-co-caprolactone); polyesteramides; polyorthoesters;
poly 13-hydroxybutyric acid; and polyanhydrides as disclosed in
U.S. Pat. No. 6,517,859, the contents of which are incorporated
herein by reference.
[0126] In one embodiment, the microsphere of the present invention
are attached to or coated with additional molecules. Such molecules
can facilitate targeting, enhance receptor mediation, and provide
escape from endocytosis or destruction. Typical molecules include
phospholipids, receptors, antibodies, hormones and polysaccharides.
Additionally, one or more cleavable molecules may be attached to
the outer surface of microspheres to target it to a predetermined
site. Then, under appropriate biological conditions, the molecule
is cleaved causing release of the microsphere from the target.
[0127] The microspheres of this invention are manufactured by
standard techniques. For example, in one embodiment, volume
exclusion is performed by mixing the active agent in solution with
a polymer or mixture of polymers in solution in the presence of an
energy source for a sufficient amount of time to form particles as
disclosed in U.S. Pat. No. 6,268,053. The pH of the solution is
adjusted to a pH near the isoelectric point (PI) of the
macromolecule. Next, the solution is exposed to an energy source,
such as heat, radiation, or ionization, alone or in combination
with sonication, vortexing, mixing or stirring, to form
microparticles. The resulting microparticles are then separated
from any unincorporated components present in the solution by
physical separation methods well known to those skilled in the art
and may then be washed. Other standard manufacturing procedures are
described in U.S. Pat. Nos. 6,669,961; 6,517,859; 6,458,387;
6,395,302; 6,303,148; 6,268,053; 6,090,925; 6,024,983; 5,942,252;
5,981,719; 5,578,709; 5,554,730; 5,407,609; 4,897,268; and
4,542,025, the contents of which are incorporated by reference in
their entirety.
[0128] Microspheres are well known and readily available to one of
ordinary skill in the art from companies experienced in providing
such technologies for extended release drug delivery. For example,
Epic Therapeutics, a subsidiary of Baxter Healthcare Corp.,
developed PROMAXX.RTM., a protein-matrix drug delivery system that
produces bioerodible protein microspheres in a totally water-based
process; OctoPlus developed OctoDEX.RTM., crosslinked dextran
microspheres that release active ingredients based on bulk
degradation of matrix rather than based on surface erosion; and
Brookwood Pharmaceuticals advertises the availability of its
microparticle technologies for drug delivery.
[0129] A search of patents, published patent applications and
related publications will also provide those skilled in the art
reading this disclosure with significant possible microsphere
technologies. For example, U.S. Pat. Nos. 6,669,961; 6,517,859;
6,458,387; 6,395,302; 6,303,148; 6,268,053; 6,090,925; 6,024,983;
5,942,252; 5,981,719; 5,578,709; 5,554,730; 5,407,609; 4,897,268;
and 4,542,025, the contents of which are incorporated by reference
in their entirety, describe microspheres and methods for their
manufacture. One skilled in the art, considering both the
disclosure of this invention and the disclosures of these other
patents could make and use microspheres for the extended release of
NRG.
D. Dosage and Frequency of Administration
[0130] The amount of neuregulin used in the present invention will
vary with the nature and severity of the disease or condition, and
the route by which the active ingredient is administered. The
frequency and dosage will also vary according to factors specific
for each patient depending on the specific therapy (e.g.,
therapeutic or prophylactic agents) administered, the severity of
the disorder, disease, or condition, the route of administration,
as well as age, body, weight, response, and the past medical
history of the patient. Effective doses may be extrapolated from
dose-response curves derived from in vitro or animal model test
systems.
[0131] Exemplary doses of neuregulin include milligram or microgram
amounts of neuregulin per kilogram of subject or sample weight
(e.g., about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram). For extened release of neuregulin used in
the invention, the dosage administered to a patient is typically
0.001 mg/kg to 15 mg/kg of the patient's body weight, based on
weight of the active peptide. Preferably, the dosage administered
to a patient is between 0.001 mg/kg and 15 mg/kg, 0.005 mg/kg and
10 mg/kg, 0.01 mg/kg and 5 mg/kg, 0.001 mg/kg and 4 mg/kg, 0.005
mg/kg and 3 mg/kg, 0.01 mg/kg and 2 mg/kg, 0.001 mg/kg and 1 mg/kg,
0.005 mg/kg and 0.5 mg/kg, 0.010 mg/kg and 0.2 mg/kg, 0.005 mg/kg
and 0.050 mg/kg of the patient's body weight.
[0132] Exemplary doses of neuregulin also include unit (U) or unit
amounts of neuregulin per kilogram of subject or sample weight
(e.g., about 1 U per kilogram to about 5000 U per kilogram, about
10 U micrograms per kilogram to about 1000 per kilogram, or about
100 U per kilogram to about 500 U per kilogram). For extened
release of neuregulin used in the invention, the dosage
administered to a patient is typically 10 U/kg to 1000 U/kg of the
patient's body weight, based on weight of the active peptide.
Preferably, the dosage administered to a patient is between 1 U/kg
and 10,000 U/kg, 1 U/kg and 5000 U/kg, 10 U/kg and 5000 U/kg, 10
U/kg and 1000 U/kg, 50 U/kg and 2000 U/kg, 50 U/kg and 1000/kg, 50
U/kg and 500 U/kg, 100 U/kg and 1000 U/kg, 100 U/kg and 500 U/kg,
100 U/kg and 200 U/kg, of the patient's body weight.
[0133] In general, the recommended daily dose range of neuregulin
in the methods of the invention for the conditions described herein
lie within the range of from about 0.001 mg to about 1000 mg per
day. Specifically, a total daily dose range should be between 0.001
mg per day and 15 mg per day, 0.005 mg per day and 10 mg per day,
0.01 mg per day and 5 mg per day, 0.001 mg per day and 4 mg per
day, 0.005 mg per day and 3 mg per day, 0.01 mg per day and 2 mg
per day, 0.001 mg per day and 1 mg per day, 0.005 mg per day and
0.5 mg per day, 0.010 mg per day and 0.2 mg per day. In managing
the patient, the therapy can be initiated at a lower dose, perhaps
about 0.1 .mu.g to about 1 .mu.g, and increased if necessary up to
about 20 .mu.g mg to about 1000 .mu.g per day as either a single
dose or divided doses, depending on the patient's global response.
It may be necessary to use dosages of the active ingredient outside
the ranges disclosed herein in some cases, as will be apparent to
those of ordinary skill in the art. Furthermore, it is noted that
the clinician or treating physician will know how and when to
interrupt, adjust, or terminate therapy in conjunction with
individual patient response. In certain embodiments, neuregulin is
administered in an amount of about 1 U/day to about 10,000 U/day.
In some embodiments, it is administered in an amount of about 1
U/day to about 5000 U/day. In some embodiments, it is administered
in an amount of about 10 U/day to about 2000 U/day. In some
embodiments, it is administered in an amount of about 10 U/day to
about 1000 U/day. In some embodiments, it is administered in an
amount of about 100 U/day to about 200 U/day.
[0134] Neuregulin can also be administered in a dosing schedule or
"therapeutic cycle." Daily dosage of neuregulin in the therapeutic
cycle is described in detail above. The therapeutic cycle can last
2 days, 5 days, 7 days, 10 days, two weeks, three weeks, four
weeks, five weeks, or six weeks.
[0135] In certaine embodiments, neuregulin is administered daily
for each day of the therapeutic cycle. In certain embodiments,
neuregulin is administered consecutively for three, four, five,
six, seven, eight, nine, ten, eleven or twelve days in a
therapeutic cycle.
[0136] In certain embodiments, in a therapeutic cycle neuregulin is
administered on day 1 of the cycle and the cycle concludes with one
or more days of no neuregulin administration In some embodiments,
neuregulin is administered daily for 3, 5, 7, or 10 days followed
by a resting period in a therapeutic cycle.
E. Combinational Therapy
[0137] In one embodiment, the present invention is useful in
preventing heart failure and cardiomyopathy in patients being
treated with a drug that causes cardiac hypertrophy or heart
failure, e.g., fludrocortisone acetate or herceptin. NRG may be
administered prior to, simultaneously with, or subsequent to a drug
which causes such cardiac diseases.
[0138] In another embodiment of the invention, NRG is administered
in combination with an effective amount of a compound that acts to
suppress a different hypertrophy induction pathway than NRG. In an
alternative embodiment, NRG is administered with such hypertrophy
suppressors and/or additional components, without limitation, a
cardiotrophic inhibitor such as a Ct-1 (cardiotrophin-1)
antagonist, an ACE inhibitor, such as captopril (Capoten.RTM.),
and/or human growth hormone and/or IGF-I (Insulin like growth
factor I) in the case of congestive heart failure, or with another
anti-hypertrophic, myocardiotrophic factor, anti-arrhythmic, or
inotropic factor in the case of other types of heart failure or
cardiac disorder.
[0139] In another embodiment of the invention, NRG is administered
in combination with current therapeutic approaches for treatment of
heart failure, including, without limitation, ACE inhibitors and
other vasodilators, diuretics, digitalis preparations, beta
blockers, blood thinners, angiotensin II receptor blockers, calcium
channel blockers or potassium.
[0140] ACE inhibitors, which prevent the conversion of angiotensin
I to angiotensin II, are vasodilators that cause the blood vessels
to expand, lowering the blood pressure and reducing the heart's
workload. Vasodilators suitable for use in embodiments of the
present invention include, without limitation, the following drugs:
quinapril (Accupril.RTM.), ramipril (Altace.RTM.), captopril
(Capoten.RTM.), benazepril (Lotensin.RTM.), fosinopril
(Monopril.RTM.), lisinopril (Prinivil.RTM. or Zestril.RTM.),
enalapril (Vasotec.RTM.), moexipril (Univasc.RTM.), trandolapril,
and perindopril. Additional vasodilators useful in the present
invention, include, without limitation, isosorbide dinitrate
(Isordil.RTM.), nesiritide (Natrecor.RTM.), hydralazine
(Apresoline.RTM.), nitrates and minoxidil.
[0141] Diuretics cause the kidneys to remove sodium and water from
the blood stream, reducing the heart's workload, and include,
without limitation, the following drugs: hydrochlorothiazide
(HydroDIURIL.RTM.), chlorothiazide (Diuril.RTM.), furosemide
(Lasix.RTM.), bumetanide (Bumex.RTM.), spironolactone
(Aldactone.RTM.), triamterene (Dyrenium.RTM.), metolazone
(Zaroxolyn.RTM.), torsemide, indapamide, polythiazide, amiloride,
and combination agents (Dyazide.RTM.).
[0142] Digitalis preparations increase the force of the heart's
contractions and include, without limitation, digoxin
(Lanoxin.RTM.) and digitoxin.
[0143] Beta blockers reduce the heart's tendency to beat faster and
include, without limitation, the following drugs: carvedilol
(Coreg.RTM.) metoprolol (Lopressor.RTM. or Toprol XL.RTM.,
atenolol, bisoprolol, labetalol, propranolol, sotalol, pindolol,
penbutolol, acebutolol, timolol, nadolol, and betaxolol.
[0144] Blood thinners for use in embodiments of the present
invention, include, without limitation, warfarin (Coumadin.RTM.)
and heparin.
[0145] Embodiments of the present invention may also use
angiotensin II receptor blockers, which, rather than lowering the
levels of angiotensin II (as ACE inhibitors do), prevents
angiotensin II from effecting the heart and blood vessels.
Angiotensin II receptor blockers suitable for use in the present
invention, include, without limitation, iosartan (Cozaar.RTM.),
valsartan (Diovan.RTM.), irbesartan (Avapro.RTM.), candesartan,
eprosartan, telmisartan, and olmesartan.
[0146] Calcium channel blockers are generally used to treat high
blood pressure often associated with heart failure. Calcium channel
blockers suitable for use in the present invention include, without
limitation, amlodipine (Norvasc.RTM.).
[0147] In alternative embodiments of the present invention,
extended release of NRG can also be combined with the
administration of drug therapies for the treatment of heart
diseases such as hypertension. For example, NRG can be administered
with endothelin receptor antagonists, such as antibodies to the
endothelin receptor, and peptides or other such small molecule
antagonists; 3-adrenoreceptor antagonists such as carvedilol;
x,-adrenoreceptor antagonists; anti-oxidants; compounds having
multiple activities (e.g., 3-blocker/a-blocker/anti-oxidant);
carvedilol-like compounds or combinations of compounds providing
multiple functions found in carvedilol; growth hormone, etc.
[0148] Neuregulin agonists alone or in combination with other
hypertrophy suppressor pathway agonists or with molecules that
antagonize known hypertrophy induction pathways, are useful as
drugs for in vivo treatment of mammals experiencing heart failure,
so as to prevent or lessen heart failure effects.
[0149] Therapeutic formulations of agonist(s) for treating heart
disorders are prepared for storage by mixing the agonist(s) having
the desired degree of purity with optional physiologically
acceptable carriers, excipients, or stabilizers (Remington's
Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., 1980), in the
form of lyophilized cake or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are non-toxic to recipients at the
dosages and concentrations employed, and include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobins; hydrophilic polymers such as olyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counter ions such as sodium; and/or non-ionic surfactants such as
Tween, Pluronics, or polyethylene glycol (PEG). The antagonist(s)
are also suitably linked to one of a variety of nonproteinaceous
polymers, e.g., polyethylene glycol, polypropylene glycol, or
polyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;
4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The amount
of carrier used in a formulation may range from about 1 to 99%,
preferably from about 80 to 99%, optimally between 90 and 99% by
weight.
[0150] The agonist(s) to be used for in vivo administration should
be sterile. This is readily accomplished by methods known in the
art, for example, by filtration through sterile filtration
membranes, prior to or following lyophilization and reconstitution.
The agonist(s) ordinarily will be stored in lyophilized form or in
solution.
[0151] Therapeutic agonist compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle. The agonist(s) administration is in a chronic
fashion only, for example, one of the following routes: injection
or infusion by intravenous, intraperitoneal, intracerebral,
intramuscular, intraocular, intraarterial, or intralesional routes,
orally or using sustained-release systems as noted above.
[0152] As discussed above, suitable examples of sustained-release
preparations include semipermeable matrices of solid hydrophobic
polymers containing the protein, which matrices are in form of
shaped articles, e.g., films, or microcapsules. Examples of
sustained-release matrices include polyesters, hydrogels (e.g.,
poly(2-hydroxyethyl-methacrylate) as described by Langer et al.
(1981) J. Biomed. Mater. Res. 15: 167-277 and Langer (1982) Chem.
Tech. 12: 98-105, or poly(vinyl alcohol)), polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma
ethyl-L-. glutamate (Sidman et al. (1983) Biopolymers 22: 547-556),
non-degradable ethylene-vinyl acetate (Langer et al. (1981) supra)
degradable lactic acidglycolic acid copolymers such as the Lupron
Depot.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[0153] The agonist(s) also may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization (for example, hydroxymethylcellulose or
gelatin-microcapsules and poly[methylmethacylate] microcapsules,
respectively), in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules), or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, supra.
[0154] While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release molecules for shorter time periods. When
encapsulated molecules remain in the body for a long time, they may
denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be
devised for stabilization depending on the mechanism involved,
e.g., using appropriate additives, and developing specific polymer
matrix compositions.
[0155] Sustained-release agonist(s) compositions also include
liposomally entrapped agonists(s). Liposomes containing agonists(s)
are prepared by methods known in the art, for example, those
disclosed in DE 3,218,121; Epstein et al. (1985) Proc. Natl. Acad.
Sci. USA 82: 3688-3692; Hwang et al. (1980) Proc. Natl. Acad. Sci.
USA 77:4030-4034: EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP
142,641; Japanese patent application 83-118008; U.S. Pat. Nos.
4,485,045 and 4,544,545; and EP 102, 324. A specific example of
suitable sustained-release formulation is in EP 647,449.
[0156] In another embodiment of the present invention, NRG is
combined with or administered in concert with other agents for
treating congestive heart failure, including ACE inhibitors (as
discussed above), CT-1 inhibitors, human growth hormone, and/or
IGF-I. The effective amounts of such agents, if employed will be at
the clinician's discretion. Dosage administration and adjustment
are determined by methods known to those skilled in the art to
achieve the best management of congestive heart failure and ideally
takes into account use of diuretics or digitalis, and conditions
such as hypotension and renal impairment. The dose will
additionally depend on such factors as the type of drug used and
the specific patient being treated. Typically the amount employed
will be the same dose as that used if the drug were to be
administered without agonist; however, lower doses may be employed
depending on such factors as the presence of side-effects the
condition being treated, the type of patient, and the type of
agonists and drug, provided the total amount of agents provides an
effective dose for the condition being treated.
[0157] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
F. Kits
[0158] The invention also provides kits for carrying out the
therapeutic regiments of the invention. Such kits comprise in one
or more containers therapeutically effective amounts of NRG
described herein, alone or in combination with other agents, in
pharmaceutically acceptable form and in combination with an
extended release technology as described herein. Instructions are
optionally included for administration of the extended release NRG
composition by a physician or by the patient.
G. Examples
[0159] As shown in the Examples, the invention resides in the
discovery that extended release of NRG activates the AKT or ERK
signaling pathway as effectively as NRG delivered by other methods,
and improves the function of infarct heart much more than NRG
delivered by other methods. However, the invention also has broader
application to other diseases and disorders given that NRG's
interactions with ErbB receptors have been implicated in other
diseases and disorders, e.g., diseases of the central and
peripheral nervous system. Examples of other diseases and
disorders, include, various cardiovascular diseases, cancer, neural
system disease and/or muscle diseases, including muscular dystrophy
(e.g., Duchenne, Limb-girdle) and multiple sclerosis, spinal
injury, eye and ear diseases, diabetes, schizophrenia, and
Alzheimer's.
[0160] The invention will be further illustrated by reference to
the following non-limiting Examples. The examples are put forth so
as to provide those of ordinary skill in the art with a complete
disclosure and description of how to make and use the present
invention, and are not intended to limit the scope of what the
inventors regard as their invention nor are they intended to
represent that the experiments below are all or the only
experiments performed. Efforts have been made to ensure accuracy
with respect to numbers used but some experimental errors and
deviations should be accounted for.
EXAMPLE 1
[0161] Phosphorylation of AKT and ERK in the Left Ventricle of
Normal Rats after NRG is Infused by Different Methods.
[0162] To compare the effect of NRG with various treating methods
on the signal transduction inside the cardiac myocytes in the left
ventricle, we infused NRG by intravenous (hereinafter referred to
as "IV"), intramuscular (hereinafter referred to as "IM") and IV
glucose tolerance test (hereinafter referred to as "IVGTT").
[0163] Wistar male rats (Shanghai Animal Center of Chinese Academy
of Science), which weighed 180.+-.20 grams, were numbered, weighed,
and divided into groups. Each group contained three rats. One group
received IV injection of 4 ml/kg (volume/body weight) of vehicle
(10 mM Na.sub.2HPO.sub.4-NaH.sub.2PO4, 150 mM NaCl, 0.2% human
serum albumin (HSA), 5% mannitol, pH 6.0) as a control. Four other
groups of rats received IM injection of 4 ml/kg (volume/body
weight) of NRG (37.3 U/ml recombinant human NRG fragment (from the
177th to 237th amino acid sequence of human NRG1.beta.2 produced by
Zensun Science & Technology--batch number 200503002)) dissolved
in vehicle (as described above). Another four groups of rats
received IV injection of 4 ml/kg (volume/body weight) of NRG (as
described above). Another five groups of rats received 20 .mu.l/min
of glucose tolerance test infusion of NRG (as described above) by
IV injection (IVGTT) for two hours. Thus, the total amount of NRG
administered to each rat (except for the vehicle group) was 149.3
U/kg of body weight.
[0164] Rats were killed separately at 20 min, 1 hr, 2 hr, 4 hr and
6 hr. The left ventricles of each group of rats were cut into
pieces in cold lysis buffer (50 mM Tris pH 7.4, 5 mM EDTA, 150 mM
NaCl, 1% Triton X-100, 2 mM Na.sub.2VO.sub.4, 50 mM NaF, 2 mM PMSF,
protease inhibitor cocktail (no EDTA, Roche)) after pooled, and
washed with cold PBS. The ventricles were then homogenized in ice
water and centrifuged (Kendro Biofuge) at 12,000 rpm for 5 min at
4.degree. C. in 1.5 ml Eppendorf tubes. The supernatant was
collected and spun one more time, then stored at -80.degree. C. The
samples were thawed and spun again before use. The protein
concentration of each sample was determined by BCA protein assay
(Pierce BCA protein assay kit). A certain amount of each sample was
mixed with 2.times. sample buffer (0.125 M Tris ph 6.8, 20%
glycerol, 4% SDS, 0.2 M DTT, 0.012% bromophenol blue) and boiled
for electrophoresis before transfer to PVDF membrane (Millipore).
The phosphorylation of AKT and ERK, as well as the amount of AKT
and ERK in each sample was detected with antibodies (ERK antibody
and phosphorylated ERK antibody (Santa Cruz Biotechnology); AKT
antibody and phosphorylated AKT antibody (Cell Signaling)).
[0165] The time course of phosphorylation of AKT and ERK in the
left ventricle of normal rats when NRG was infused by each of these
different methods is shown in FIG. 1. Compared to the vehicle, NRG
infused by IM, IV and IVGTT all activated sustained phosphorylation
of ERK. AKT phosphorylation induced by each method peaked at 20 min
and decreased at lhr, but increased again at 2 hr, where it
maintained a high level from 4 hr to 6 hr. Thus, there is no
obvious difference among the different methods of injecting NRG
with respect to their ability to sustain phosphorylation of ERK and
AKT. This indicates that NRG infused constantly is as effective as
injection of NRG. Thus, IVGTT infusion is a potential method for
treating poor cardiac conditions.
EXAMPLE 2
[0166] The Function of Left Ventricle Coronary Artery Ligated Rat
Heart after Neuregulin Treatment by Different Methods
[0167] As osmotic pump is a way to deliver NRG constantly (as
IVGTT), we examined whether NRG infused by osmotic pump was as
effective as conventional IV injection in restoring the function of
myocardial infarct (MI) heart.
A. Rat Left Ventricle Coronary Artery Ligation and
Echocardiography
[0168] Wistar male rats (Shanghai Animal Center of Chinese Academy
of Science), which weighed 200.+-.20 g, were anesthetized by
intraperitoneally injecting 100 mg/kg (drug/body weight) of
ketamine. The neck and chest were depilated and sanitized. An
incision was made in the middle front neck to expose the tracheae.
An 18 G catheter overneedle was inserted into the tracheae between
the 3rd and 5th cartilage of tracheae. After the needle was drawn
out, a plastic cannula was pushed into the trachea 1-2 cm and fixed
to connect the Rodent Ventilator (SAR-830/P ventilator--Inspiratory
flow rate, 1 ml/100 g/breath; Respiratory rate, 60 breaths/min).
Another incision was made on the left front chest. The skin was
blunt dissected to expose the fourth and fifth rib, then the fourth
rib was cut by elbowed mosquito forceps. The ventilator (as
described above) was linked to the cannula and turned on, and the
heart was exposed to check the status of lung and heart. The
pericardium was rived off to identify the left atria and the
pulmonary arterious cone after the heart was exteriorized through
the incision. The left ventricle anterior descending coronary
artery between them was ligated tight with 6/0 medical suture
before the heart was replaced into the thorax. The thoracic wall
was stitched. The ventilator was blocked to full fill the lung. The
chest muscle and skin was stitched after the air in the thoracic
cavity was gently squeezed out. The ventilators were removed from
the rats until constant spontaneous respiration resumed.
[0169] The cardiac function of the rats was then examined by
echocardiography (Philips Sonos 7500 S4 probe) on the 14th day
after ligation. The rats with ejection fraction (hereinafter "EF")
values from 30 to 50 percent were separated and grouped (15 rats
per group).
B. Treating the Ligated Rats with Neuregulin.
[0170] The rats were weighed on the 15th day after left ventricle
coronary ligation to determine the amount of NRG needed. Rats in
the vehicle group received 0.4 ml/100 g (volume/body weight) of
vehicle by IV injection. The vehicle was injected once a day for
five days, stopped for two days, and then injected for another five
days.
[0171] The IM and IV groups of rats received IM and IV injection of
NRG, respectively (the amount of NRG was 149.3 U/kg (protein/body
weight), the volume was 0.4 ml/100 g). The NRG was injected once a
day for five days, stopped for two days, and then injected for
another five days.
[0172] As discussed further below, the IVGTT group had osmotic
pumps (ALZET osmotic pump 2ML1) implanted on the fifth day after
grouping. Each pump contained 2 ml of NRG solution, which contained
933.1 U of NRG (as a rat now weighed about 250 g) and the infusion
speed was about 18.7 U/kg/h. Thus, the maximum drug concentration
compared to about 2.67 U/kg by IV injection.
[0173] After 7 days, cardiac function of all rats was checked again
by echocardiography (Philips Sonos 7500 S4 probe). The next day,
hemodynamic parameter check and anatomy check were also undertaken
to further confirm the cardiac function of the rats.
B. 1. Transplantation of Osmotic Pump into Rats (all Steps Must be
Sterile)
[0174] 1 ml of sterile water and lml of sterile 0.9% saline was
injected into a vial of NRG (993.1 U, 62.5 .mu.g) in the hood
successively. The NRG solution was drawn into a sterile syringe. A
blunt-tipped needle was exchanged for the syringe and the bubble in
the syringe was removed. The pump was held upright and the needle
was inserted through the small opening at the top of the upright
pump until it could go no further. The plunger was pushed slowly to
add NRG solution into the pump until the solution began to overflow
the pump. The needle was removed and the pump was wiped clean. The
transparent cap of the flow moderator was taken off to expose a
short stainless steel tube. The steel tube was then inserted into
one end of a 5 cm PE60 tube. The syringe needle was inserted into
another end of the PE60 tube. The plunger of syringe was pushed to
add NRG solution to the flow moderator until it was full. The long
tube of the flow moderator was then inserted into the pump until
its white flange attached to the pump. The needle was drawn out of
the flow moderator before soaking the pump in sterile 0.9% saline
at 37.degree. C. overnight.
[0175] The rats were anesthetized by Ketamine (as described above).
The area between neck and shoulder of the rats was depilated and
sanitized. The body was covered with a piece of sterile wet cloth.
An incision was then carefully made in the skin between the
scapulae to locate and separate the external jugular vein. The
distal end of the vein from the heart was ligated. A small hole was
made by eye scissors on the wall of the external jugular vein and
enlarged by microforceps. The PE60 tube connected to the osmotic
pump was inserted 2 cm into the vein through the hole. The proximal
end of the vein from the heart was then bound with PE60 tube to fix
the tube. The distal end of the vein surrounding the PE60 tube was
tied tight to further fix the tube. Using a hemostat, a tunnel was
formed by blunt separation of the skin from the incision to
scapula. A pocket was finally made on the back of the rat in the
midscapular region by spreading the skin further. The pump was slid
through the tunnel into the pocket with the flow moderator pointing
away from the incision. The skin incision was then closed with a
suture. The rats were put back into the animal room after revival
and were fed as usual.
C. Experimental Results
[0176] The function of MI heart following NRG infusion by IVGTT and
IV is shown in Table 1 below. In Table 1 "IVS", "LVEDD", "PW",
"LVESD", "EF", "FS" and "CC" stand for interventricular septum,
left ventricle end diastolic dimension, posterior wall thickness,
left ventricle end systolic dimension, ejection fraction,
fractional shortening and cardiac cycle, respectively. Here EF and
FS reflect the contractility of heart, especially for left
ventricle.
[0177] EF=(end diastolic volume-end systolic volume)/end diastolic
volume
[0178] FS=(end diastolic dimension-end systolic dimension)/end
diastolic dimension
[0179] In Table 1, P<0.01 for LVEDD, LVESD, EF and FS in IVGTT
or IV group compared with their counterparts in the vehicle group,
indicating highly significant difference. TABLE-US-00001 TABLE 1
cardiac function of MI rats after NRG infusion by IVGTT and IV IVS
LVEDD PW LVESD EF FS CC cm cm cm cm % % ms Vehicle 0.168 .+-. 0.005
0.952 .+-. 0.082 0.173 .+-. 0.009 0.819 .+-. 0.107 34.3 .+-. 5.0
14.5 .+-. 2.4 162.5 .+-. 23.1 IVGTT 0.169 .+-. 0.007 0.857 .+-.
0.093 0.190 .+-. 0.013 0.644 .+-. 0.061 54.6 .+-. 5.4 25.2 .+-. 3.0
173.1 .+-. 22.5 IV 0.177 .+-. 0.027 0.912 .+-. 0.081 0.189 .+-.
0.013 0.759 .+-. 0.099 40.5 .+-. 8.9 17.5 .+-. 4.6 164.5 .+-.
18.2
[0180] NRG infused by osmotic pump dramatically increased the
cardiac function of MI rats compared to the IV group. Particularly,
the EF value--a measurement of the heart's blood pumping efficiency
that can be used to estimate the function of the left ventricle--in
the IVGTT group was 59.18% higher than that of the vehicle group,
and 34.81% higher than the IV group. Additionally, the FS
value--also a way of measuring left ventricle performance--of the
IVGTT group was 73.79% higher than that of the vehicle group, and
44.0% higher than the IV group. These results show that extended
release of NRG is more effective than conventional IV injection for
improving cardiac function.
[0181] Surprisingly, NRG infused by osmotic pump not only greatly
increased the cardiac function of MI rats compare with the IV
group, but also reduced the interior diameter of the left
ventricle. Specifically, the average Left Ventricle End Diastolic
Dimension (hereinafter referred to as "LVEDD") of the IVGTT group
was 9.98% smaller than that of the vehicle group, and 6.03% smaller
than the IV group. Additionally, the Left Ventricle End Systolic
Dimension (hereinafter referred to as "LVESD") of the IVGTT group
was 21.37% smaller than that of the vehicle group, and 15.15%
smaller than the IV group. These results show that administering
NRG constantly can reduce left ventricular volume and mass, thereby
improving left ventricular health and performance.
EXAMPLE 3
[0182] Heart Function of Myocardial Infarcted Rats after Neuregulin
was Constantly Intravenously Infused by Syringe Pump (Zhejiang
University Medical Instrument Co. LTD, WZS 50-F2)
[0183] In this example, syringe pump is used for extended release
of neuregulin in human patients. Syringe pump can pump the solution
continuously at certain speed into the bloodstream through a needle
injected into the vein in rat tail. For syringe pump, it's easy to
control the infusion time and speed. Neuregulin was intravenously
infused by syringe pump at different speed for different time per
day into MI rats to better time period and speed for treatment.
[0184] Grouped MI rats was treated by intravenous injection of 4
ml/kg (volume/body weight) vehicle everyday for 10 days (group A);
or intravenous injection of 10 .mu.g/kg neuregulin (2.5 .mu.g/ml)
everyday for 10 days (group B); or intravenous syringe pump
infusion of neuregulin (0.625 .mu.g/ml) at 1.25 .mu.g/kg/h with 4
hours per day for 10 days (group C); or intravenous syringe pump
infusion of neuregulin (1.25 .mu.g/ml) at 2.5 .mu.g/kg/h with 4
hours per day for 10 days (group D); or intravenous syringe pump
infusion of neuregulin (0.625 .mu.g/ml) at 0.625 .mu.g/kg/h with 8
hours per day for 10 days (group E); or intravenous syringe pump
infusion of neuregulin (1.25 .mu.g/ml) at 1.25 .mu.g/kg/h with 8
hours per day for 10 days (group F). Echocardiography was then
performed for all groups to examine the function of heart.
TABLE-US-00002 TABLE 2 echocardiography data for MI rats after
intravenous syringe pump infusion (ISPI) or IV injection of NRG IVS
LVEDD PW LVESD EF FS HR/ cm cm cm cm % % min A vehicle 0.057 .+-.
0.003 0.947 .+-. 0.041 0.142 .+-. 0.013 0.811 .+-. 0.047 34.5 .+-.
3.3 14.4 .+-. 1.6 418 .+-. 51 B IV 0.060 .+-. 0.005 0.924 .+-.
0.060 0.164 .+-. 0.016 0.770 .+-. 0.057 41.5 .+-. 2.6 17.8 .+-. 1.6
382 .+-. 52 C ISPI 0.059 .+-. 0.005 0.935 .+-. 0.050 0.156 .+-.
0.013 0.779 .+-. 0.067 41.2 .+-. 5.7 17.7 .+-. 2.8 395 .+-. 30 1.25
.mu.g/kg/h 4 h/day D ISPI 0.061 .+-. 0.004 0.943 .+-. 0.058 0.160
.+-. 0.015 0.762 .+-. 0.055 43.7 .+-. 5.4 19.0 .+-. 2.9 391 .+-. 41
2.5 .mu.g/kg/h 4 h/day E ISPI 0.062 .+-. 0.006 0.941 .+-. 0.061
0.164 .+-. 0.011 0.742 .+-. 0.079 47.4 .+-. 8.6 21.1 .+-. 4.5 391
.+-. 48 0.625 .mu.g/kg/h 8 h/day F ISPI 0.061 .+-. 0.004 0.966 .+-.
0.038 0.166 .+-. 0.019 0.766 .+-. 0.045 47.2 .+-. 4.2 20.8 .+-. 2.5
364 .+-. 33 1.25 .mu.g/kg/h 8 h/day
[0185] P<0.01 for LVEDD, LVESD, EF and FS in any of ISPI or IV
group compared with their counterparts in the vehicle group,
indicating highly significant difference. HR means heart rate.
[0186] As shown in table 2, compared with the vehicle group,
neuregulin by IV (B group) enhanced the EF value of MI rats by
20.29%, intravenous syringe pump infusion for 4 h/day (C, D group)
was just as effective as IV, while neuregulin by intravenous
syringe pump infusion for 8 h/day (E, F group) enhanced the EF
value by around 37.10%. At the same time, compared with the vehicle
group, neuregulin by IV injection (B group) enhanced the FS value
of MI rats by 23.61%, intravenous syringe pump infusion for 4 h/day
(C, D group) was as effective as IV, while neuregulin by
intravenous syringe pump infusion for 8 h/day (E, F group) enhanced
the FS value by around 45.49%. Surprisingly, although MI rats in
group E received only half amount of neuregulin for group F, the EF
or FS value is nearly the same. The results showed that after
neuregulin was continuously intravenously infused by syringe pump
for 8 or more hours per day it could enhance the cardiac
function.
EXAMPLE 4
[0187] The Cardiac Function of MI Rats after Extended Hypodermic
Infusion of NRG by Osmotic Pump
[0188] Left ventricle coronary artery ligation and transplantation
of osmotic pump into rats was performed in the same way as in
example 2, except the amount of NRG injected into the pump was
1791.3 U (125 .mu.g), and the pump was embedded without a tube
connected to the vein to make NRG infusion hypodermic. The infusion
speed is 37.33 U/kg/h.
[0189] IV infusion was started at the same time as extended
hypodermic infusion so the IV group was treated with NRG for 7
days. The amount of NRG for the IV group was also changed to 223.95
U/kg.
[0190] The function of MI heart following NRG infusion by extended
hypodermic and IV is shown in Table 3. In Table 3, P<0.01 for
LVEDD, LVESD, EF and FS in the IVGTT and the IV group compared with
their counterparts in the vehicle group, indicating a highly
significant difference. TABLE-US-00003 TABLE 3 cardiac function of
MI rats after extended hypodermic (EHI) and IV infusion of NRG IVS
LVEDD PW LVESD EF FS CC cm cm cm cm % % ms Vehicle 0.174 .+-. 1.02
.+-. 0.185 .+-. 0.876 .+-. 33.9 .+-. 14.3 .+-. 153 .+-. 0.005 0.077
0.012 0.098 7.9 3.8 19 EHI 0.177 .+-. 0.908 .+-. 0.209 .+-. 0.712
.+-. 48.4 .+-. 21.7 .+-. 153 .+-. 0.006 0.079 0.023 0.091 9.3 5.1
11 IV 0.171 .+-. 1.013 .+-. 0.188 .+-. 0.874 .+-. 33.9 .+-. 14.3
.+-. 157 .+-. 0.007 0.111 0.010 0.124 6.8 3.3 15
[0191] Table 3 shows that extended hypodermic infusion of NRG
significantly increased the cardiac function of MI rats compared to
the IV and vehicle groups. Compared to vehicle group, extended
hypodermic infusion of NRG enhanced the EF value of MI hearts
42.77%, the FS value 51.75%. As discussed above, the EF and FS
values are ways of measuring the heart's blood pumping efficiency
and can be used to estimate the function of the left ventricle.
Thus, these results show that extended release of NRG is much more
effective than conventional IV injection for improving cardiac
function.
[0192] Extended hypodermic infusion of NRG also reduced interior
diameter of the left ventricle. Specifically, the LVEDD of MI
hearts decreased 10.98% and the LVESD decreased 18.72% compare to
vehicle group. IV injection of NRG in this experiment did not have
an obvious effect on the cardiac function of MI heart compare to
vehicle. The results show that extended hypodermic infusion of NRG
can reduce left ventricular volume and mass, thereby improving left
ventricular health and performance, which suggests that it may also
be used as a treatment for heart failure.
EXAMPLE 5
[0193] Heart Function of Myocardial Infarcted Rats after Neuregulin
was Constantly Hypodermically Infused by Syringe Pump
[0194] Neuregulin was further infused by syringe pump at different
speed for different time per day into MI rats.
[0195] Grouped MI rats was treated by intravenous injection of 4
ml/kg (volume/body weight) vehicle everyday for 10 days (group A);
or intravenous injection of 10 .mu.g/kg neuregulin (2.5 .mu.g/ml)
everyday for 10 days (group B); or hypodermic injection (HI) of 10
.mu.g/kg neuregulin (2.5 .mu.g/ml) everyday for 10 days (group C);
hypodermic syringe pump infusion of neuregulin (1.25 .mu.g/ml) at
2.5 .mu.g/kg/h with 4 hours per day for 10 days (group D); or
hypodermic syringe pump infusion of neuregulin (1.11 .mu.g/ml) at
1.67 .mu.g/kg/h with 6 hours per day for 10 days (group E); or
hypodermic syringe pump infusion of neuregulin (1.25 .mu.g/ml) at
1.25 .mu.g/kg/h with 8 hours per day for 10 days (group F).
Echocardiography was then performed for all groups to examine the
function of heart. TABLE-US-00004 TABLE 4 echocardiography data for
MI rats after hypodermic syringe pump infusion (HSPI) or IV
injection of NRG IVS LVEDD PW LVESD EF FS HR/ cm cm cm cm % % min A
vehicle 0.060 .+-. 0.007 0.906 .+-. 0.107 0.151 .+-. 0.027 0.757
.+-. 0.130 39.3 .+-. 10.8 16.9 .+-. 6.1 388 .+-. 33 B IV 0.063 .+-.
0.004 0.812 .+-. 0.045 0.159 .+-. 0.010 0.726 .+-. 0.047 43.4 .+-.
2.8 18.8 .+-. 1.4 385 .+-. 33 C HI 0.063 .+-. 0.003 0.909 .+-.
0.054 0.163 .+-. 0.011 0.744 .+-. 0.048 42.1 .+-. 3.7 18.1 .+-. 1.9
390 .+-. 40 D HSPI 0.065 .+-. 0.007 0.933 .+-. 0.055 0.160 .+-.
0.016 0.754 .+-. 0.069 44.2 .+-. 6.5 19.3 .+-. 3.4 385 .+-. 32 2.5
.mu.g/kg/h 4 h/day E HSPI 0.067 .+-. 0.003 0.880 .+-. 0.073 0.168
.+-. 0.019 0.693 .+-. 0.076 48.3 .+-. 6.0 21.4 .+-. 3.5 404 .+-. 38
1.67 .mu.g/kg/h 6 h/day F HSPI 0.066 .+-. 0.005 0.899 .+-. 0.056
0.168 .+-. 0.014 0.709 .+-. 0.098 47.2 .+-. 11.8 21.3 .+-. 8.2 377
.+-. 44 1.25 .mu.g/kg/h 8 h/day
[0196] P<0.01 for LVEDD, LVESD, EF and FS in any of HSPI, HI or
IV group compared with their counterparts in the vehicle group,
indicating highly significant difference. HR means heart rate.
[0197] As shown in table 4, compared with the vehicle group,
neuregulin by IV (B group) enhanced the EF value of MI rats by
10.43%, neuregulin by hypodermic injection (C group) enhanced the
EF value of MI rats by 7.12%, while neuregulin by hypodermic
syringe pump infusion for 4 h/day (D group) enhanced the EF value
by 12.47%, neuregulin by hypodermic syringe pump infusion for 6
h/day (E group) made the EF value jump to 22.90%, neuregulin by
hypodermic syringe pump infusion for 8 h/day (E group) also raised
the EF value by 20.10%. At the same time, compared with the vehicle
group, neuregulin by IV (B group) enhanced the FS value of MI rats
by 11.24%, neuregulin by hypodermic injection (C group) enhanced
the FS value of MI rats by 7.10%, while neuregulin by hypodermic
syringe pump infusion for 4 h/day (D group) enhanced the FS value
by 14.20%, neuregulin by hypodermic syringe pump infusion for 6
h/day (E group) made the FS value jump to 26.63%, neuregulin by
hypodermic syringe pump infusion for 8 h/day (E group) also raised
the FS value by 26.04%. The results showed that after neuregulin
was continuously hypodermically infused by syringe pump for 6 or
more hours per day could it increased the cardiac function
dramatically.
EXAMPLE 6
[0198] PEG Coupling of NRG and Activity of PEG Coupled NRG
A, PEG Coupling and Isolation of PEG Coupled NRG
[0199] PEG (mPEG-SPA-5000, NEKTAR) was added into 10 ml 20 mM PBS
(pH 8.0) containing 1 mg/ml NRG (PEG:NRG=1:1, molar ratio) and
mixed quickly, and the mixture was gently stirred at room
temperature for 30 min, then certain amount of glacial acetic acid
was added to stop coupling reaction. The mixture was then loaded
onto a gel filtration column (S100, Pharmacia) to separate the
components. Each peak fraction was collected and its sample was
prepared for SDS-PAGE. After electrophoresis, the gel was stained
by BaI.sub.2 and Coomassie brilliant blue sequentially to detect
PEG and NRG separately.
[0200] As shown in FIG. 2 for BaI.sub.2 stained gel, the mixture
contains PEG monomer, NRG-monoPEG, NRG-diPEG and NRG-polyPEG. After
the mixture was loaded onto a S100 gel filtration column, the
components were well separated into NRG-polyPEG and NRG-diPEG
(peak1), NRG-monoPEG and PEG (peak2).
[0201] Coomassie stained gel in FIG. 3 further confirmed that peak1
and peak2 contain NRG which was coupled to PEG, while peak3
contains only NRG.
B, Measuring Activity of PEG Coupled NRG
[0202] MCF-7 cells was harvested, counted, pelleted and resuspended
into DMEM (with 10% serum and 9 .mu.g/ml insulin) at
5.times.10.sup.4 cells/ml. 100 .mu.l cell suspension was added to
each well of 96 well plate and the plate was incubated at
37.degree. C. overnight. The cells were then washed 3 times with
PBS and grew in serum free DMEM for another 24 hours.
[0203] ErbB2 antibody H4 (Zensun, anti-ErbB2 monoclonal antibody)
was diluted to 6 .mu.g/ml by coating buffer (50 mM
Na.sub.2CO.sub.3-NaHCO.sub.3, pH9.6), and added to 96 well plate
501 .mu.l/well. The plate was left at 4.degree. C. overnight to
coat with antibody.
[0204] DMEM was sucked away from the starved MCF-7 cells, and 100
.mu.l serial dilutions of NRG, NRG-monoPEG or NRG-diPEG in DMEM was
added to each well separately. DMEM was added to two wells as
blank. The plate was incubated at 37.degree. C. for 20 min. The
cells were washed once with PBS before adding 100 .mu.l/well lysis
buffer (50 mM Hepes, pH 8.0, 150 mM NaCl, 2 mM sodium
orthovanadate, 0.01% thimerosal, 1% Triton X-100 and one protease
inhibitor cocktail tablet per 25 ml solution) and lysing at
4.degree. C. for 30 min. The plate was then shaken gently to
completely lyse and remove cells from the plate and centrifugated
at 15000 rpm for 15 min.
[0205] The plate with coating antibody was washed five times with
washing buffer (10 mM PBS, pH7.4, 0.05% Tween 20) before adding 200
.mu.l/well of 5% nonfat milk in washing buffer. The plate was
incubated at 37.degree. C. for 2 hours before washed again 3 times
with washing buffer.
[0206] A 90 .mu.l solution of lysed cells was drawn from each well
in culture plate and transferred to corresponding well in coated
plate. Following incubation at 37.degree. C. for 1 hour, the coated
plate with cell lysis was washed again 5 times with washing buffer
and treated with 100 .mu.l suitable concentration of horseradish
peroxidase (HRP) conjugated anti-phosphotyrosine monoclonal
antibody (Santa Cruz Biotechnology) at 37.degree. C. for 1 hour.
After the plate was washed again 5 times with washing buffer, 100
.mu.l freshly prepared HRP substrate solution [50 mM citric acid,
100 mM Na.sub.2PO.sub.4, pH 5.0, 0.2 mg/ml
3,3',5,5'-tetramethylbenzidine (TMB), 0.003% H.sub.2O.sub.2] was
added to each well before the plate was incubated at 37.degree. C.
for 10 min. Finally 50 .mu.l of 2N H.sub.2SO.sub.4 was added to
each well to destroy HRP activity. The OD value at 450 nm for each
well was read on a microplate reader (BIO-RAD Model 550), and EC50
was the concentration of NRG which achieved 50% of maximum OD
value. The lower the EC50, the higher the activity.
[0207] The EC50 of NRG, NRG-monoPEG and NRG-diPEG was shown in
Table 5. TABLE-US-00005 TABLE 5 EC50 of NRG, NRG-monoPEG and
NRG-diPEG sample EC50.quadrature. .mu.g/ml.quadrature. NRG 0.070
NRG-monoPEG 0.070 NRG-diPEG 0.098
[0208] From table 5, we can see clearly that EC50 of NRG-monoPEG is
the same as that of NRG, while EC50 of NRG-diPEG is 40% higher.
This means that NRG-monoPEG has the same activity as NRG in vitro,
but the activity of NRG-diPEG is 40% lower.
EXAMPLE 7
[0209] Extended Release of Neuregulin Reduces Side Effects of
Neuregulin Administration
[0210] This examples shows that compared with long time or high
dose administration, extended release of neuregulin can reduce side
effects, such as gastrointestinal disorder or pericardial effusion,
associated with neuregulin administration.
[0211] NRG-1.beta. was administered intravenously by syringe pump
to two groups of monkeys, each consisting of twenty four healthy
rhesus monkeys (twelve male and twelve female, weighing about 5-7
kg). Group I was infused with NRG-1.beta. for twelve hours a day
for fourteen days, at the speed of 1 ug/kg/hr. No Side effect was
observed in this group. Group II was infused for twenty four hours
a day for fourteen days, at the speed of 1 ug/kg/hr. In Group II,
about 3-5 ml pericardial effusion in the heart of monkeys was
observed.
[0212] Two groups of healthy individuals were administered the same
amount of NRG-1.beta. per day for 10 days. Eight individuals in
Group I, were infused with NRG-1.beta. for four hours each day for
ten days at speed of 0.3 .mu.g/kg/hr. In this group, each
individual on average experienced gastrointestinal disorder about
two times during the ten-day period. Six individuals were infused
with NRG-1.beta. for two hours each day for ten days at speed of
0.6 .mu.g/kg/hr. In Group II, each individual on average
experienced gastrointestinal disorder about five times during the
ten-day period.
[0213] These results show that extended release of neuregulin can
reduce adverse side effects associated with long time or high dose
neuregulin administration. These results suggest that intravenously
or hypodermically infusion for hsoert time or lower doage per day
could reduce the side effects of 24-hour neuregulin infusion.
EXAMPLE 8
Gene Expression by Extended Released NRG in the Left Ventricle of
Myocardial Infarcted Rat
[0214] In this example, myocardial infarcted rats were infused with
NRG-1.beta. and gene expression pattern in the left ventricle of
these rats was analyzed by microarray. Compare with myocardial
infarcted rats infused with vehicle, rats infused with NRG have
different gene expression pattern. After extended release of NRG,
thymosin beta like protein mRNA level increased 3.10 times;
defensin beta 1 mRNA level increased 2.87 times; growth associated
protein mRNA level increased 2.16 times; mRNA level of thymosin
beta 4, Laminin gamma 1, myocardin, PI3K gamma regulatory subunit
almost all doubled, while mRNA level of Elastin and PI3K gamma was
nearly the same as before. It shows that neuregulin changes the
expression level of various proteins in heart.
[0215] The scope of the invention is not limited by the description
of the examples. Modifications and alterations of the present
invention will be apparent to those skilled in the art without
departing from the scope and spirit of the present invention.
Therefore, it will be appreciated that the scope of this invention
is to be defined by the appended claims, rather than by the
specific examples which have been presented by way of example.
Sequence CWU 1
1
2 1 61 PRT Homo sapiens 1 Ser 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 Pro Asn
Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr 35 40 45 Val Met Ala Ser
Phe Tyr Lys Ala Glu Glu Leu Tyr Gln 50 55 60 2 183 DNA Homo sapiens
2 agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgc
60 ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc
aaatgagttt 120 actggtgatc gctgccaaaa ctacgtaatg gcgagcttct
acaaggcgga ggagctgtac 180 cag 183
* * * * *