U.S. patent application number 15/139157 was filed with the patent office on 2016-11-03 for use of neuregulin-4 for treatment of inflammatory bowel disease and necrotizing enterocolitis.
This patent application is currently assigned to Children's Hospital Los Angeles. The applicant listed for this patent is Children's Hospital Los Angeles. Invention is credited to Mark R. Frey.
Application Number | 20160317619 15/139157 |
Document ID | / |
Family ID | 47715459 |
Filed Date | 2016-11-03 |
United States Patent
Application |
20160317619 |
Kind Code |
A1 |
Frey; Mark R. |
November 3, 2016 |
USE OF NEUREGULIN-4 FOR TREATMENT OF INFLAMMATORY BOWEL DISEASE AND
NECROTIZING ENTEROCOLITIS
Abstract
The invention provides methods, pharmaceutical compositions and
kits for treating, inhibiting and/or reducing the severity of
inflammatory bowel disease and necrotizing enterocolitis in a
subject in need thereof by administering an effective amount of a
composition comprising an activator of ErbB4.
Inventors: |
Frey; Mark R.; (Glendale,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Hospital Los Angeles |
Los Angeles |
CA |
US |
|
|
Assignee: |
Children's Hospital Los
Angeles
Los Angeles
CA
|
Family ID: |
47715459 |
Appl. No.: |
15/139157 |
Filed: |
April 26, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14236831 |
Feb 3, 2014 |
9352023 |
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PCT/US2012/050970 |
Aug 15, 2012 |
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15139157 |
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61523733 |
Aug 15, 2011 |
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61548645 |
Oct 18, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2500/10 20130101;
G01N 2800/065 20130101; A61P 1/00 20180101; A61K 38/00 20130101;
G01N 33/6872 20130101; G01N 2440/14 20130101; A61K 38/1883
20130101; G01N 33/573 20130101; G01N 2333/71 20130101; C12Q 1/485
20130101; A61P 29/00 20180101 |
International
Class: |
A61K 38/18 20060101
A61K038/18; G01N 33/573 20060101 G01N033/573 |
Goverment Interests
GOVERNMENT RIGHTS
[0001] The invention was made with government support under Grant
Nos. DK077956 and DK090295 awarded by the National Institutes of
Health. The government has certain rights to the invention.
Claims
1-22. (canceled)
23. A method for screening for an activator of ErbB4 comprising:
(i) contacting the ErbB4 in a ErbB4 positive cells with a molecule
of interest, and (ii) determining whether the contact results in
increased phosphorylation of ErbB4, an increase in phosphorylation
of ErbB4 being indicative that the molecule of interest is an
activator of ErbB4.
24. The method of claim 23, wherein the ErbB4 activator is selected
from the group consisting of a small molecule, a polypeptide, a
peptide, an antibody or a fragment thereof and a nucleic acid
molecule.
25. The method of claim 23, wherein the activator does not
phosphorylate ErbB1 (EGFR), ErbB2, and ErbB3.
26. The method of claim 23, wherein determining phosphorylation
comprises phospho-specific immunoassay of ErbB4.
27. The method of claim 23, wherein the ErbB4 positive cell is a
colonic epithelial cell or a paneth cell.
28. The method of claim 23, wherein the increase in phosphorylation
by the molecule of interest is at least the same as the level of
phosphorylation of ErbB4 in the presence of Neuregulin-4.
29. A screening method according to claim 23, which comprises
separately contacting each of a plurality of samples to be
tested.
30. The screening method of claim 29, wherein the plurality of
samples comprises more than about 10.sup.4 samples.
31. The screening method of claim 29, wherein the plurality of
samples comprises more than about 5.times.10.sup.4 samples.
32. A method for treating, inhibiting or reducing the severity of a
disease in a subject in need thereof comprising: providing a
composition comprising an activator of ErbB4; and administering a
therapeutically effective amount of the composition to the subject
so as to treat the disease, wherein the activator of ErbB4
activates ErbB4 and does not activate ErbB1, ErbB2 and ErbB3.
33. The method of claim 32, wherein the disease is any one or more
of inflammatory bowel disease, inflammation-associated death of
small intestinal and colonic epithelial cells, necrotizing
enterocolitis, or combination thereof.
34. The method of claim 32, wherein the ErbB4 activator is
Neuregulin-4 or a pharmaceutical equivalent, analog, derivative or
a salt thereof.
35. A pharmaceutical composition comprising an activator of ErbB4
and a pharmaceutically acceptable carrier.
36. The pharmaceutical composition of claim 35, wherein the ErbB4
activator is Neuregulin-4 or a pharmaceutical equivalent, analog,
derivative or a salt thereof.
37. The pharmaceutical composition of claim 36, wherein
Neuregulin-4 is administered orally.
38. The pharmaceutical composition of claim 37, wherein the
composition is administered by gavage.
39. A kit for the treatment of inflammatory bowel disease,
inhibition of inflammatory bowel disease, treatment of necrotizing
enterocolitis and/or inhibition of necrotizing enterocolitis in a
subject in need thereof, comprising: (i) a composition comprising
an ErbB4 activator; and (ii) instructions for use of the
composition for the treatment of inflammatory bowel disease,
inhibition of inflammatory bowel disease, treatment of necrotizing
enterocolitis and/or inhibition of necrotizing enterocolitis.
40. The kit of claim 39, wherein the ErbB4 activator is
Neuregulin-4 or a pharmaceutical equivalent, analog, derivative or
a salt thereof.
41. The kit of claim 40, wherein the composition is administered
orally by gavage.
Description
FIELD OF INVENTION
[0002] The invention provides methods for treating inflammatory
bowel disease and necrotizing enterocolitis in a subject in need
thereof using activators of ErbB4.
BACKGROUND
[0003] All publications herein are incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference. The following description includes information that may
be useful in understanding the present invention. It is not an
admission that any of the information provided herein is prior art
or relevant to the presently claimed invention, or that any
publication specifically or implicitly referenced is prior art.
[0004] ErbB4 is the least well-understood member of the family of
receptor tyrosine kinases which also includes EGF receptor
(EGFR/ErbB1), ErbB2/HER2, and ErbB3 (Wieduwilt, M. J., and Moasser,
M. M. (2008) Cellular and molecular life sciences: CMLS 65,
1566-1584). ErbBs recognize and are activated by a suite of ligands
including heparin-binding EGF-like growth factor (HB-EGF),
betacellulin, and the heregulin/neuregulin family (Wilson, K. J.,
Gilmore, J. L., Foley, J., Lemmon, M. A., and Riese, D. J., 2nd.
(2009) Pharmacology & therapeutics 122, 1-8). Ligand binding is
associated with receptor dimerization, increased tyrosine kinase
activity, and auto-phosphorylation on c-terminal tyrosine residues,
which then provide docking sites for downstream effectors (Bublil,
E. M., and Yarden, Y. (2007) Current opinion in cell biology 19,
124-134). Different ligands show distinct specificities and
affinities for different ErbB receptors, and stimulate diverse
dimerization patterns, signaling, and cellular responses (Saito,
T., Okada, S., Ohshima. K., Yamada, E., Sato, M., Uehara, Y.,
Shimizu, H., Pessin, J. E., and Mori, M. (2004) Endocrinology 145,
4232-4243; Sweeney, C., Lai, C., Riese, D. J., 2nd, Diamonti, A.
J., Cantley, L. C., and Carraway, K. L., 3rd. (2000) J Biol Chem
275, 19803-19807).
[0005] ErbB4 has several features which distinguish it from other
tyrosine kinases, making it a unique target both in terms of
signaling and potential role in human disease. It can bind both
heregulin/neuregulin growth factors and a subset of EGF-family
factors (Jones, J. T., Akita, R. W., and Sliwkowski, M. X. (1999)
FEBS letters 447, 227-231), but at least one peptide
ligand--NRG4--is exclusive to ErbB4 and does not bind ErbB1-3
(Harari, D., Tzahar, E., Romano, J., Shelly, M., Pierce, J. H.,
Andrews, G. C., and Yarden, Y. (1999) Oncogene 18, 2681-2689).
Furthermore, ErbB4 associates with a divergent and more restricted
suite of SH2-containing targets than EGFR, ErbB2, or ErbB3
(Kaushansky, A., Gordus, A., Budnik, B. A., Lane, W. S., Rush, J.,
and MacBeath, G. (2008) Chem Biol 15, 808-817). Thus, selective
ErbB4 activation with NRG4 may elicit different cellular outcomes
than stimulation with other EGF-like or heregulin family
molecules.
[0006] ErbB4 is induced in colonic epithelial cells by inflammatory
cytokines, and is present at elevated levels in the inflamed
colonic mucosa of IBD patients (Frey, M. R., Edelblum, K. L.,
Mullane, M. T., Liang, D., and Polk, D. B. (2009) Gastroenterology
136, 217-226). This appears to be a compensatory protective
response rather than a pathological process, as ectopic ErbB4
overexpression protects cultured mouse colon epithelial cells from
cytokine-induced apoptosis in a ligand-dependent manner (Frey, M.
R., Edelblum, K. L., Mullane, M. T., Liang, D., and Polk, D. B.
(2009) Gastroenterology 136, 217-226; Hilliard, V. C., Frey, M. R.,
Dempsey, P. J., Peek, R. M., Jr., and Polk, D. B. (2011) American
journal of physiology. Gastrointestinal and liver physiology 301,
G338-346; Frey, M. R., Hilliard, V. C., Mullane, M. T., and Polk,
D. B. (2010) Laboratory Investigation 90, 1415-1424). However,
these studies, like most investigation of ErbB4 function, used
shared ErbB ligands heregulin(HRG)-1.beta. or HB-EGF, raising the
question of signal specificity.
[0007] Crohn's disease and ulcerative colitis, collectively known
as IBD, together affect more than 1.4 million American patients
(Strober, W., Fuss, I., and Mannon, P. (2007) J Clin Invest 117,
514-521). The causes and cures of IBD remain elusive, but it is
clear that a general feature of the pathology of these disorders is
elevated apoptosis in the intestinal epithelium (Qiu, W., Wu, B.,
Wang, X., Buchanan, M. E., Regueiro, M. D., Hartman, D. J., Schoen,
R. E., Yu, J., and Zhang, L. (2011) J Clin Invest 121, 1722-1732;
Di Sabatino, A., Ciccocioppo, R., Luinetti, O., Ricevuti, L.,
Morera, R., Cifone, M. G., Solcia, E., and Corazza, G. R. (2003)
Diseases of the colon and rectum 46, 1498-1507), driven by
inflammatory cytokines such as TNF and IFN-.gamma.. Thus,
identifying signal transduction pathways which protect colon
epithelial cells from cytokine- or injury-induced apoptosis will
lead to new methods to control disease flares.
[0008] Current therapies for inflammatory bowel disease include
anti-TNF therapies and steroid anti-inflammatories and are
generally aimed at interrupting inflammation rather than
specifically promoting mucosal healing. These therapies have shown
limited effectiveness. In the instant invention, the inventor
proposes alternative therapies for treating inflammatory bowel
disease.
[0009] Necrotizing enterocolitis is a disease condition in which
portions of the bowel undergo tissue necrosis. It is predominant in
premature infants, wherein the timing of its onset is generally
inversely proportional to the gestational age of the baby at birth.
Current treatments include using an IV catheter to provide
nutrients, antibiotic therapy to treat infections, surgery etc.
Herein, the inventor provides an alternative therapy for treating
necrotizing enterocolitis.
BRIEF DESCRIPTION OF FIGURES
[0010] FIG. 1 depicts, in accordance with an embodiment of the
invention that (A) ErbB4 and NRG4 are expressed throughout the
intestinal tract and (B) NRG4 blocks cytokine-induced apoptosis in
cultured colon epithelial cells
[0011] FIG. 2 depicts, in accordance with an embodiment of the
invention that ErbB4, but not ErbB1-3, is stimulated by NRG4 in
colonocytes.
[0012] FIG. 3 depicts, in accordance with an embodiment of the
invention that NRG4 blocks cytokine-induced apoptosis in vivo. Mice
were injected i.p. with TNF (250 .mu.g/kg) plus IFN-.gamma. (250
units/g), with or without NRG4 (100 .mu.g/kg). After 24 h, colons
were excised. Apoptosis was assessed by (A, B) western blot for
cleaved caspase-3 on mucosal scrapings (graph in B is
quantification of blots from 4 mice per condition) and (C, D) ISOL
stain (images in C show representative labeled cells) on sections
of fixed, paraffin-embedded tissue.
[0013] FIG. 4 depicts, in accordance with an embodiment of the
invention that NRG4 does not stimulate colonocyte proliferation or
migration. (A) Young adult mouse colon epithelial cells expressing
ErbB4 (YAMC-B4 cells) were given NRG4 or epidermal growth factor
(EGF, positive control for cell stimulation) for 24 h, then labeled
with EdU to determine proliferative index. Graph depicts results
from 3 independent experiments. *, p<0.01 vs. all other columns.
(B) Fixed colon sections from PBS- or NRG4-injected mice were
immunostained for the proliferative marker Ki-67, and number of
labeled cells per crypt counted. Data points are average
cells/crypt in individual mice. (C) YAMC-B4 cells were subjected to
an 8 h migration/restitution assay in the presence of NRG4 or EGF.
*, p<0.01 vs. all other columns. (D) Exogenous NRG4 is
protective in acute DSS colitis.
[0014] FIG. 5 depicts, in accordance with an embodiment of the
invention that Akt phosphorylation is stimulated by NRG4 in vitro
and in vivo. (A) YAMC-B4 cells were stimulated with NRG4 for 10'
and whole cell lysates were prepared. (B) Mice were injected with
NRG4 with or without TNF+IFN-.gamma.; after 24 h epithelial
homogenates were prepared. Expression and phosphorylation of
indicated molecules were determined by western blot analysis. (C)
Akt phosphorylation in fixed, paraffin-embedded colonic tissue was
assessed by immunofluorescence analysis. Data are representative of
at least 3 independent experiments or mice per condition.
[0015] FIG. 6 depicts, in accordance with an embodiment of the
invention that PI3K inhibitor blocks NRG4 antiapoptotic effects.
(A) YAMC-B4 cells were exposed to TNF+IFN-.gamma., with or without
NRG4 and/or inhibitor to PI3K (LY294002). After 6 h, cells were
fixed and apoptosis was assessed by immunofluorescent staining for
cleaved caspase-3. Data are representative of 4 independent
experiments. (B) Mice were injected with TNF+IFN-.gamma., with or
without NRG4 and/or inhibitor to PI3K (LY294002). After 24 h,
colons were fixed and apoptosis was quantified by ISOL stain.
[0016] FIG. 7 depicts, in accordance with an embodiment of the
invention that NRG4 levels are decreased in human inflammatory
bowel disease. (A,B) qPCR analysis for (A) NRG4 and (B) HRG-1.beta.
gene expression was performed on TissueScan Crohn's/Colitis qPCR
Arrays. Relative mRNA levels were calculated using the
2.sup.-.DELTA..DELTA.CT method with 13-actin as the reference. (C)
Colonic homogenates from wild type controls (WT) or IL-10.sup.-/-
mice were subjected to western blot analysis for ErbB4,
phospho-ErbB4, and NRG4. Results shown are representative of 4 mice
per genotype.
[0017] FIG. 8 depicts, in accordance with an embodiment of the
invention that in rat ileal epithelial cells, Cronobacter
sakazakii-induced apoptosis is attenuated by NRG4.
[0018] FIG. 9 depicts, in accordance with an embodiment of the
invention that rat pups are resistant to experimental NEC when
treated with NRG4.
DETAILED DESCRIPTION OF THE INVENTION
[0019] All references cited herein are incorporated by reference in
their entirety as though fully set forth. Unless defined otherwise,
technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which
this invention belongs. Singleton et al., Dictionary of
Microbiology and Molecular Biology 3.sup.rd ed., J. Wiley &
Sons (New York, N.Y. 2001): March, Advanced Organic Chemistry
Reactions, Mechanisms and Structure 5.sup.th ed., J. Wiley &
Sons (New York, N.Y. 2001); and Sambrook and Russel, Molecular
Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory
Press (Cold Spring Harbor, N.Y. 2001), provide one skilled in the
art with a general guide to many of the terms used in the present
application.
[0020] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described. For purposes of the present invention, the following
terms are defined below.
[0021] "Beneficial results" may include, but are in no way limited
to, lessening or alleviating the severity of the disease condition,
preventing the disease condition from worsening, curing the disease
condition, preventing the disease condition from developing,
lowering the chances of a patient developing the disease condition
and prolonging a patient's life or life expectancy. In some
embodiments, the disease condition is cancer.
[0022] "Mammal" as used herein refers to any member of the class
Mammalia, including, without limitation, humans and nonhuman
primates such as chimpanzees and other apes and monkey species;
farm animals such as cattle, sheep, pigs, goats and horses;
domestic mammals such as dogs and cats; laboratory animals
including rodents such as mice, rats and guinea pigs, and the like.
The term does not denote a particular age or sex. Thus, adult and
newborn subjects, as well as fetuses, whether male or female, are
intended to be included within the scope of this term.
[0023] "Treatment" and "treating," as used herein refer to both
therapeutic treatment and prophylactic or preventative measures,
wherein the object is to prevent or slow down (lessen) the targeted
pathologic condition, prevent the pathologic condition, pursue or
obtain beneficial results, or lower the chances of the individual
developing the condition even if the treatment is ultimately
unsuccessful. Those in need of treatment include those already with
the condition as well as those prone to have the condition or those
in whom the condition is to be prevented.
[0024] "Inflammatory Bowel Disease" or "IBD" as used herein refers
to the inflammatory conditions including but not limited to Crohn's
disease, ulcerative colitis, collagenous colitis, lymphocytic
colitis, ischaemic colitis, diversion colitis, Behcet's disease and
indeterminate colitis.
[0025] The inventor proposes an alternative to existing therapies
for treating inflammatory bowel disease comprising administering an
effective amount of an ErbB4 activator to the subject. As described
herein, ErbB4 is activated by Neuregulin-4 (NRG4). NRG4 is the only
known growth factor found in the intestine which is entirely
specific for ErbB4 receptor tyrosine kinase. Inventor's data
indicates that NRG4-ErbB4 signaling is an anti-apoptotic pathway in
colon epithelial cells which is compromised in colitis by loss of
NRG4 ligand. While not wishing to be bound by a specific theory,
the inventor hypothesizes that ErbB4 activation by NRG4 promotes
colon epithelial cell survival and protects the epithelium from
inflammation-induced damage.
Therapeutic Methods of the Invention
[0026] The invention provides methods for treating. inhibiting,
reducing the symptoms of and/or promoting prophylaxis of diseases
associated with apoptosis induced by cytokines such as TNF and
IFN-.alpha.. In some embodiments, the methods include administering
to the subject an activator of ErbB4. Examples of diseases include
but are not limited to inflammatory bowel disease, necrotizing
enterocolitis, rheumatoid arthritis and asthma. In some
embodiments, the activator of ErbB4 is selected from the group
consisting of a small molecule, a peptide, an antibody or a
fragment thereof and a nucleic acid molecule. In one embodiment,
the ErbB4 activator is Neuregulin-4 or a pharmaceutical equivalent,
analog, derivative or a salt thereof.
[0027] The invention provides therapeutic methods for treating.
inhibiting, reducing the symptoms of and/or promoting prophylaxis
of inflammatory bowel disease in a subject in need thereof using
activators of ErbB4.
[0028] The invention also provides therapeutic methods for
treating, inhibiting, reducing the symptoms of and/or promoting
prophylaxis of necrotizing enterocolitis in a subject in need
thereof using activators of ErbB4.
[0029] Inflammatory Bowel Disease and ErbB4 Activator
[0030] The invention provides methods for treating inflammatory
bowel disease in a subject in need thereof. The methods comprise
providing a composition comprising an activator of ErbB4 and
administering an effective amount of the composition to the subject
so as to treat inflammatory bowel disease in the subject. In some
embodiments, the activator of ErbB4 is selected from the group
consisting of a small molecule, a peptide, an antibody or a
fragment thereof and a nucleic acid molecule. In one embodiment,
the ErbB4 activator is Neuregulin-4 or a pharmaceutical equivalent,
analog, derivative or a salt thereof.
[0031] The invention also provides methods for inhibiting and/or
reducing symptoms of inflammatory bowel disease in a subject in
need thereof. The methods comprise providing a composition
comprising an activator of ErbB4 and administering an effective
amount of the composition to the subject so as to inhibit and/or
reduce symptoms of, inflammatory bowel disease in the subject. In
some embodiments, the activator of ErbB4 is selected from the group
consisting of a small molecule, a peptide, an antibody or a
fragment thereof and a nucleic acid molecule. In one embodiment,
the ErbB4 activator is Neuregulin-4 or a pharmaceutical equivalent,
analog, derivative or a salt thereof.
[0032] The invention also provides methods for inhibiting
inflammation-associated death of small intestinal and colonic
epithelial cells in a subject in need thereof. The methods comprise
providing a composition comprising an activator of ErbB4 and
administering an effective amount of the composition to the subject
so as to inhibit inflammation-associated death of small intestinal
and colonic epithelial cells in a subject. In some embodiments, the
activator of ErbB4 is selected from the group consisting of a small
molecule, a peptide, an antibody or a fragment thereof and a
nucleic acid molecule. In one embodiment, the ErbB4 activator is
Neuregulin-4 or a pharmaceutical equivalent, analog, derivative or
a salt thereof.
[0033] The invention also provides methods for promoting the
prophylaxis of inflammatory bowel disease, mitigating the effect of
inflammatory bowel disease, reducing the severity of inflammatory
bowel disease, reducing the likelihood of developing inflammatory
bowel disease and/or slowing the progression of inflammatory bowel
disease in subjects in need thereof. The methods comprise providing
a composition comprising an activator of ErbB4 and administering a
therapeutically effective amount of the composition to the subject
so as to promote the prophylaxis of inflammatory bowel disease,
mitigate the effect of inflammatory bowel disease, reduce the
severity of inflammatory bowel disease, reduce the likelihood of
developing inflammatory bowel disease and/or slow the progression
of inflammatory bowel disease in subjects in need thereof. In some
embodiments, the activator of ErbB4 is selected from the group
consisting of a small molecule, a peptide, an antibody or a
fragment thereof and a nucleic acid molecule. In one embodiment,
the ErbB4 activator is Neuregulin-4 or a pharmaceutical equivalent,
analog, derivative or a salt thereof.
[0034] In various embodiments, the ErbB4 activator may be used in
conjunction with existing treatments for inflammatory bowel
disease. For example, ErbB4 activators may be used in conjunction
with existing therapies such as diet modifications and
administrations of therapeutic drugs including but not limited to
sulfasalazine (Azulfadine), mesalamine (Asacol, Pentasa),
azathioprine (Imuran), 6-MP (Purincthol), cyclosporine,
methotrexate, infliximab (Remicade), Budesonide (Entocort EC) and
corticosteroids (prednisone), so as to treat inflammatory bowel
disease. Dosages of existing therapies that may be used with the
ErbB4 activators will be apparent to one skilled in the art. In one
embodiment, the ErbB4 activator is Neuregulin-4 or a pharmaceutical
equivalent, analog, derivative or a salt thereof.
[0035] Colorectal cancer represents the major cause for excess
morbidity and mortality by malignant disease in ulcerative colitis
as well as in Crohn's disease. In an embodiment, treatment,
inhibition or reduction of symptoms of inflammatory bowel disease
in a subject may prevent and/or treat colorectal cancer.
Accordingly, the invention provides methods for treating, reducing
the severity of and/or preventing colorectal cancer in a subject in
need thereof. The methods comprise providing a composition
comprising an activator of ErbB4 and administering an effective
amount of the composition to the subject so as to inhibit
inflammation-associated death of small intestinal and colonic
epithelial cells in a subject. In some embodiments, the activator
of ErbB4 is selected from the group consisting of a small molecule,
a peptide, an antibody or a fragment thereof and a nucleic acid
molecule. In one embodiment, the ErbB4 activator is Neuregulin-4 or
a pharmaceutical equivalent, analog, derivative or a salt
thereof.
[0036] In some embodiments, the activator of ErbB4 is a direct
activator of ErbB4 such that the activator binds ErbB4 and
activates it by, for example, inducing or increasing
phosphorylation of ErbB4. In some embodiments, the activator of
ErbB4 is an indirect activator of ErbB4 such that the activator
inhibits the inhibitor of ErbB4 so that ErbB4 is activated.
[0037] Necrotizing Enterocolitis and ErbB4 Activator
[0038] The invention provides a method for treating necrotizing
enterocolitis in a subject in need thereof. The method comprises
providing a composition comprising an activator of ErbB4 and
administering a therapeutically effective amount of the composition
to the subject so as to treat necrotizing enterocolitis. In some
embodiments, the activator of ErbB4 is selected from the group
consisting of a small molecule, a peptide, an antibody or a
fragment thereof and a nucleic acid molecule. In one embodiment,
the ErbB4 activator is Neuregulin-4 or a pharmaceutical equivalent,
analog, derivative or a salt thereof.
[0039] The invention also provides a method for inhibiting
necrotizing enterocolitis in a subject in need thereof. The method
comprises providing a composition comprising an activator of ErbB4
and administering a therapeutically effective amount of the
composition to the subject so as to inhibit necrotizing
enterocolitis. In one embodiment, the ErbB4 activator is NRG4, a
salt thereof, or a pharmaceutical equivalent thereof. In some
embodiments, the activator of ErbB4 is selected from the group
consisting of a small molecule, a peptide, an antibody or a
fragment thereof and a nucleic acid molecule. In one embodiment,
the ErbB4 activator is Neuregulin-4 or a pharmaceutical equivalent,
analog, derivative or a salt thereof.
[0040] The invention also provides methods for promoting the
prophylaxis of necrotizing enterocolitis, mitigating the effect of
necrotizing enterocolitis, reducing the severity of necrotizing
enterocolitis, reducing the likelihood of developing necrotizing
enterocolitis and/or slowing the progression of necrotizing
enterocolitis in subjects in need thereof. The methods comprise
providing a composition comprising an activator of ErbB4 and
administering a therapeutically effective amount of the composition
to the subject so as to inhibit necrotizing enterocolitis. In some
embodiments, the activator of ErbB4 is selected from the group
consisting of a small molecule, a peptide, an antibody or a
fragment thereof and a nucleic acid molecule. In one embodiment,
the ErbB4 activator is Neuregulin-4 or a pharmaceutical equivalent,
analog, derivative or a salt thereof.
[0041] In some embodiments, the activator of ErbB4 is a direct
activator of ErbB4 such that the activator binds ErbB4 and
activates it by, for example, inducing or increasing
phosphorylation of ErbB4. In some embodiments, the activator of
ErbB4 is an indirect activator of ErbB4 such that the activator
inhibits the inhibitor of ErbB4 so that ErbB4 is activated.
[0042] Additionally, in case of necrotizing enterocolitis, ErbB4
activators may be used in conjunction with existing treatments such
as stopping enteral feedings, performing nasogastric decompression,
and initiating broad-spectrum antibiotics. In one embodiment, the
ErbB4 activator is Neuregulin-4 or a pharmaceutical equivalent,
analog, derivative or a salt thereof.
[0043] Various methods may be utilized to administer the
composition of the claimed methods, including but not limited to
aerosol, nasal, oral, transmucosal, transdermal, parenteral,
implantable pump, continuous infusion, topical application,
capsules and/or injections.
[0044] The subjects treated by the present invention include
mammalian subjects, including, human, monkey, ape, dog, cat, cow,
horse, goat, pig, rabbit, mouse and rat.
[0045] Dosages of the Invention
[0046] As described above, in various embodiments of the invention,
the ErbB4 activator may be used in conjunction with existing
treatments for inflammatory bowel disease and necrotizing
enterocolitis. In some embodiments, the ErbB4 activator is
administered concurrently with the existing treatments for
inflammatory bowel disease and necrotizing enterocolitis. In
various embodiments, the ErbB4 activator is administered
sequentially with the existing treatments for inflammatory bowel
disease and necrotizing enterocolitis. The ErbB4 activator alone or
in conjunction with the existing treatments for inflammatory bowel
disease and necrotizing enterocolitis, may be administered at
various stages of inflammatory bowel disease and necrotizing
enterocolitis, such as early stages, middle stages and/or late
stages of inflammatory bowel disease and necrotizing enterocolitis.
In one embodiment, the ErbB4 activator is Neuregulin-4 or a
pharmaceutical equivalent, analog, derivative or a salt
thereof.
[0047] In some embodiments of the invention, the effective amount
of ErbB4 activator in the composition can be in the range of about
10-50 mg/day, 50-100 mg/day, 100-150 mg/day, 150-200 mg/day,
100-200 mg/day, 200-300 mg/day, 300-400 mg/day, 400-500 mg/day,
500-600 mg/day, 600-700 mg/day, 700-800 mg/day, 800-900 mg/day,
900-1000 mg/day, 1000-1100 mg/day, 1100-1200 mg/day, 1200-1300
mg/day, 1300-1400 mg/day, 1400-1500 mg/day, 1500-1600 mg/day,
1600-1700 mg/day, 1700-1800 mg/day, 1800-1900 mg/day, 1900-2000
mg/day, 2000-2100 mg/day, 2100-2200 mg/day, 2200-2300 mg/day,
2300-2400 mg/day, 2400-2500 mg/day, 2500-2600 mg/day, 2600-2700
mg/day, 2700-2800 mg/day, 2800-2900 mg/day or 2900-3000 mg/day. In
one embodiment of the invention, the ErbB4 is Neuregulin-4 or a
pharmaceutical equivalent, analog, derivative or a salt
thereof.
[0048] In further embodiments of the invention, the effective
amount of activator of ErbB4 for use with the claimed methods may
be in the range of 0.001-0.005 mg/kg, 0.005-0.01 mg/kg, 0.01-0.02
mg/kg, 0.02-0.04 mg/kg, 0.04-0.06 mg/kg, 0.06-0.08 mg/kg, 0.08-1
mg/kg, 1-5 mg/kg, 5-10 mg/kg, 10-15 mg/kg, 15-20 mg/kg, 20-25
mg/kg, 25-30 mg/kg, 30-35 mg/kg, 35-40 mg/kg, 40-45 mg/kg, 45-50
mg/kg, 10-50 mg/kg, 50-100 mg/kg, 100-150 mg/kg, 150-200 mg/kg,
100-200 mg/kg, 200-300 mg/kg, 300-400 mg/kg, 400-500 mg/kg, 500-600
mg/kg, 600-700 mg/kg, 700-800 mg/kg, 800-900 mg/kg, 900-1000 mg/kg,
1000-1100 mg/kg, 1100-1200 mg/kg, 1200-1300 mg/kg, 1300-1400 mg/kg,
1400-1500 mg/kg, 1500-1600 mg/kg, 1600-1700 mg/kg, 1700-1800 mg/kg,
1800-1900 mg/kg, 1900-2000 mg/kg, 2000-2100 mg/kg, 2100-2200 mg/kg,
2200-2300 mg/kg, 2300-2400 mg/kg, 2400-2500 mg/kg, 2500-2600 mg/kg,
2600-2700 mg/kg, 2700-2800 mg/kg, 2800-2900 mg/kg or 2900-3000
mg/kg. In one embodiment of the invention, the ErbB4 activator is
Neuregulin-4 or a pharmaceutical equivalent, analog, derivative or
a salt thereof.
[0049] Typical dosages of an effective amount of an ErbB4
activator, such as Neuregulin-4, can be in the ranges recommended
by the manufacturer where known therapeutic compounds are used, and
also as indicated to the skilled artisan by the in vitro responses
or responses in animal models. The actual dosage can depend upon
the judgment of the physician, the condition of the patient, and
the effectiveness of the therapeutic method based, for example, on
the in vitro responsiveness of relevant cultured cells or
histocultured tissue sample, or the responses observed in the
appropriate animal models.
[0050] Screening Methods of the Invention
[0051] Another aspect of the invention relates to assays and
methods for identifying compounds that activate ErbB4. In one
embodiment, the method comprises contacting ErbB4 in an ErbB4
positive cell with the compound of interest and subsequently
determining whether the contact results in altered phosphorylation
of ErbB4. In an embodiment, an alteration in the amount
phosphorylation of ErbB4 is an increase in the amount of
phosphorylation of ErbB4. In one embodiment, an increase in the
amount of phosphorylation of ErbB4 indicates that the molecule of
interest is an activator of ErbB4.
[0052] Screening methods of the invention further provide methods
for identifying compounds that activate ErbB4 wherein the method
includes contacting ErbB4 in an ErbB4 positive cell with the
compound of interest, contacting the ErbB4 positive cell and the
compound of interest with a target cell and subsequently
determining whether the contact results in altered apoptosis of
target cells. In an embodiment, a decrease in apoptosis of target
cells indicates that the molecule of interest is an activator of
ErbB4.
[0053] The compound of interest that activates ErbB4 may be any one
or more of a small molecule, a peptide, a polypeptide, an antibody
or a fragment thereof and a nucleic acid molecule.
[0054] Assays that may be employed to identify compounds that
activate ErbB4 include but are not limited to any one or more of
microarray assay, quantitative PCR, Northern blot assay, Southern
blot assay, Western blot assay immunohistochemical assays, binding
assays, gel retardation assays, assays using yeast two-hybrid
systems, assays that measure cell apoptosis, or a combination
thereof. A person skilled in the art can readily employ numerous
techniques known in the art to determine whether a particular agent
activates ErbB4.
[0055] Pharmaceutical Compositions
[0056] In various embodiments, the present invention provides
pharmaceutical compositions including a pharmaceutically acceptable
excipient along with a therapeutically effective amount of an ErbB4
activator, such as Neuregulin-4 or a pharmaceutical equivalent,
analog, derivative or a salt thereof. "Pharmaceutically acceptable
excipient" means an excipient that is useful in preparing a
pharmaceutical composition that is generally safe, non-toxic, and
desirable, and includes excipients that are acceptable for
veterinary use as well as for human pharmaceutical use. Such
excipients may be solid, liquid, semisolid, or, in the case of an
aerosol composition, gaseous.
[0057] In various embodiments, the pharmaceutical compositions
according to the invention may be formulated for delivery via any
route of administration. "Route of administration" may refer to any
administration pathway known in the art, including but not limited
to aerosol, nasal, oral, transmucosal, transdermal or
parenteral.
[0058] The pharmaceutical compositions according to the invention
can also contain any pharmaceutically acceptable carrier.
"Pharmaceutically acceptable carrier" as used herein refers to a
pharmaceutically acceptable material, composition, or vehicle that
is involved in carrying or transporting a compound of interest from
one tissue, organ, or portion of the body to another tissue, organ,
or portion of the body. For example, the carrier may be a liquid or
solid filler, diluent, excipient, solvent, or encapsulating
material, or a combination thereof. Each component of the carrier
must be "pharmaceutically acceptable" in that it must be compatible
with the other ingredients of the formulation. It must also be
suitable for use in contact with any tissues or organs with which
it may come in contact, meaning that it must not carry a risk of
toxicity, irritation, allergic response, immunogenicity, or any
other complication that excessively outweighs its therapeutic
benefits.
[0059] The pharmaceutical compositions according to the invention
can also be encapsulated, tableted or prepared in an emulsion or
syrup for oral administration. Pharmaceutically acceptable solid or
liquid carriers may be added to enhance or stabilize the
composition, or to facilitate preparation of the composition.
Liquid carriers include syrup, peanut oil, olive oil, glycerin,
saline, alcohols and water. Solid carriers include starch, lactose,
calcium sulfate, dihydrate, terra alba, magnesium stearate or
stearic acid, talc, pectin, acacia, agar or gelatin. The carrier
may also include a sustained release material such as glyceryl
monostearate or glyceryl distearate, alone or with a wax.
[0060] The pharmaceutical composition according to the invention
can also be bead system for delivery for the ErbB4 activator to the
target cells. For example, pectin/zein hydrogel bead system may be
used to deliver Neuregulin-4 or a pharmaceutical equivalent,
analog, derivative or a salt thereof, to the target cells in the
subject (Yan F. et al., J Clin Invest. 2011 June;
121(6):2242-53).
[0061] The pharmaceutical preparations are made following the
conventional techniques of pharmacy involving milling, mixing,
granulation, and compressing, when necessary, for tablet forms; or
milling, mixing and filling for hard gelatin capsule forms. When a
liquid carrier is used, the preparation will be in the form of
syrup, elixir, emulsion or an aqueous or non-aqueous suspension.
Such a liquid formulation may be administered directly p.o. or
filled into a soft gelatin capsule.
[0062] The pharmaceutical compositions according to the invention
may be delivered in a therapeutically effective amount. The precise
therapeutically effective amount is that amount of the composition
that will yield the most effective results in terms of efficacy of
treatment in a given subject. This amount will vary depending upon
a variety of factors, including but not limited to the
characteristics of the therapeutic compound (including activity,
pharmacokinetics, pharmacodynamics, and bioavailability), the
physiological condition of the subject (including age, sex, disease
type and stage, general physical condition, responsiveness to a
given dosage, and type of medication), the nature of the
pharmaceutically acceptable carrier or carriers in the formulation,
and the route of administration. One skilled in the clinical and
pharmacological arts will be able to determine a therapeutically
effective amount through routine experimentation, for instance, by
monitoring a subject's response to administration of a compound and
adjusting the dosage accordingly. For additional guidance, see
Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th
edition, Williams & Wilkins PA, USA) (2000).
[0063] Kits of the Invention
[0064] The present invention is also directed to kits to treat
inflammatory bowel disease and/or necrotizing colitis. The kit is
an assemblage of materials or components, including at least one of
the inventive compositions. Thus, in some embodiments the kit
contains a composition including an ErbB4 activator, such as
Neuregulin-4 or a pharmaceutical equivalent, analog, derivative or
a salt thereof, as described above.
[0065] The exact nature of the components configured in the
inventive kit depends on its intended purpose. In one embodiment,
the kit is configured particularly for human subjects. In further
embodiments, the kit is configured for veterinary applications,
treating subjects such as, but not limited to, farm animals,
domestic animals, and laboratory animals.
[0066] Instructions for use may be included in the kit.
"Instructions for use" typically include a tangible expression
describing the technique to be employed in using the components of
the kit to effect a desired outcome, such as to treat or prevent
inflammatory bowel disease and/or necrotizing colitis in a subject.
Optionally, the kit also contains other useful components, such as,
measuring tools, diluents, buffers, pharmaceutically acceptable
carriers, syringes or other useful paraphernalia as will be readily
recognized by those of skill in the art.
[0067] The materials or components assembled in the kit can be
provided to the practitioner stored in any convenient and suitable
ways that preserve their operability and utility. For example the
components can be in dissolved, dehydrated, or lyophilized form;
they can be provided at room, refrigerated or frozen temperatures.
The components are typically contained in suitable packaging
material(s). As employed herein, the phrase "packaging material"
refers to one or more physical structures used to house the
contents of the kit, such as inventive compositions and the like.
The packaging material is constructed by well-known methods,
preferably to provide a sterile, contaminant-free environment. As
used herein, the term "package" refers to a suitable solid matrix
or material such as glass, plastic, paper, foil, and the like,
capable of holding the individual kit components. Thus, for
example, a package can be a bottle used to contain suitable
quantities of an inventive composition containing an ErbB4
activator, such as Neuregulin-4 or a pharmaceutical equivalent,
analog, derivative or a salt thereof. The packaging material
generally has an external label which indicates the contents and/or
purpose of the kit and/or its components.
EXAMPLES
[0068] The following example is provided to better illustrate the
claimed invention and is not to be interpreted as limiting the
scope of the invention. To the extent that specific materials are
mentioned, it is merely for purposes of illustration and is not
intended to limit the invention. One skilled in the art may develop
equivalent means or reactants without the exercise of inventive
capacity and without departing from the scope of the invention.
Expression of the ErbB4 tyrosine kinase is elevated in colonic
epithelial cells during inflammatory bowel disease (IBD), while
ErbB4 overexpression in cultured colonocytes blocks TNF-induced
apoptosis in a ligand-dependent manner. Together these observations
suggest that ErbB4 induction may be a protective response. However,
the effects of ErbB4 signaling in the colonic epithelium in vivo
were not known. Furthermore, previous work on ErbB4 used ligands
shared with other receptors, raising the question of whether
observed responses arc explicitly due to ErbB4. Herein, the
inventor used the ErbB4-specific ligand neuregulin-4 (NRG4) to
activate ErbB4 and define its role in colonocyte biology. Treatment
with NRG4, either in cultured cells or in mice, blocked colonic
epithelial apoptosis induced by TNF and IFN-.gamma.. NRG4
stimulated phosphorylation of ErbB4 but not of other ErbB
receptors, indicating that this is a specific response.
Furthermore, in contrast to related ligands, NRG4 enhanced cell
survival but not proliferation or migration, and stimulated
phosphorylation of the anti-apoptotic mediator Akt but not ERK
MAPK. Pharmacological inhibition of PI3K/Akt signaling reversed
NRG4's anti-apoptotic effects, confirming the role of this cascade
in NRG4-induced cell survival. With regard to the potential
clinical importance of this pathway, NRG4 expression was decreased
in human IBD samples and mouse models of colitis, suggesting that
activation of ErbB4 is altered in disease. Thus, exogenous NRG4 may
be beneficial for disorders in which epithelial apoptosis is part
of the pathology.
Example 1
Experimental Methods
[0069] Cell Culture--
[0070] Conditionally immortalized, non-transformed young adult
mouse colon (YAMC) epithelial cells were provided by Dr. Robert
Whitehead (Whitehead, R. H., VanEeden, P. E., Noble, M. D.,
Ataliotis, P., and Jat, P. S. (1993) Proc Natl Acad Sci USA 90,
587-591). These cells express low levels of endogenous ErbB4;
YAMC-B4 cells expressing a human ErbB4 construct were generated as
previously described (Frey, M. R., Edelblum, K. L., Mullane, M. T.,
Liang, D., and Polk, D. B. (2009) Gastroenterology 136, 217-226).
Cell pools showing no autocrine ErbB4 activation were selected for
use and maintained under permissive conditions [33.degree. C. in
RPMI 1640 with 5% FBS, 5 units/ml mouse interferon-.gamma.
(Peprotech, Rocky Hill, N.J.), 100 U/ml penicillin and
streptomycin, 5 .mu.g/ml insulin, 5 .mu.g/ml transferrin, and 5
ng/ml selenous acid (BD Biosciences, San Jose, Calif.)], then
shifted to nonpermissive conditions (RPMI 1640 containing 0.5% FBS,
streptomycin and penicillin without IFN-.gamma., insulin,
transferrin, or selenous acid, at 37.degree. C.) overnight before
use in experiments.
[0071] Antibodies, Cytokines, Growth Factors, and Inhibitors--
[0072] Antibodies were purchased from: monoclonal anti-actin, Sigma
Corp. (St. Louis, Mo.); polyclonal anti-phospho-Y1284 ErbB4,
phospho-Y1068 EGFR, phospho-Y1248 ErbB2, phospho-Y1289 ErbB3, EGFR,
ErbB2, ErbB3, phospho-S473 Akt, cleaved caspase-3, cleaved poly
(ADP-ribose) polymerase, phospho-p38, and phospho-ERK, Cell
Signaling (Danvers, Mass.); polyclonal anti-ErbB4 (c-18) and goat
polyclonal anti-COX-2, Santa Cruz Biotechnology (Santa Cruz,
Calif.); anti-Ki67, Dako Corp (Carpinteria, Calif.); IRDye
anti-mouse, anti-rabbit, and anti-goat, Li-Cor Corp (Lincoln,
Nebr.): AlexaFluor-555-conjugated anti-rabbit, Invitrogen
(Carlsbad, Calif.). Recombinant murine TNF, IFN-.gamma., and EGF
were from Peprotech. HRG-1.beta. and anti-caspase antibody were
from R&D Systems (Minneapolis, Minn.). Recombinant NRG4 [A1/A2
ectodomain sequence (Hayes, N. V., Newsam, R. J., Baines, A. J.,
and Gullick, W. J. (2008) Oncogene 27, 715-720)] was synthesized by
Genscript Corp (Piscataway, N.J.). LY294002 (PI 3-kinase inhibitor)
was purchased from Cell Signaling.
[0073] Animals--
[0074] All animal use was approved and monitored by the Children's
Hospital Los Angeles Institutional Animal Care and Use Committee.
Experiments used 6-8 week old male C57B1/6J mice (The Jackson
Laboratory, Bar Harbor, Me.). Cytokines, growth factors, and
inhibitors were given by intraperitoneal injection, 24 h after
injections, mice were sacrificed and colons collected either for
fixation and embedding or for epithelial lysates. For the latter,
colons were incubated in Cell Recovery Solution (BD Biosciences,
San Jose, Calif.) for 1 h at 4.degree. C.; crypts were released by
vigorous shaking and collected by centrifugation, similar to as
previously described (Whitehead, R. H., and Robinson, P. S. (2009)
American journal of physiology. Gastrointestinal and liver
physiology 296, G455-460). Collected crypts were lysed and
processed similar to cultured cells as below. For murine colitis,
IL-10.sup.-/- mice on C57B1/6 background were maintained
unchallenged until 32-36 weeks of age, at which point knockout mice
but not wild-type animals have extensive inflammatory
cytokine-driven colitis (Kuhn, R., Lohler, J., Rennick, D.,
Rajewsky, K., and Muller, W. (1993) Cell 75, 263-274).
[0075] Immunofluorescence and Histochemical Analysis--
[0076] Tissues and cells were fixed with 4% paraformaldehyde
(Electron Microscopy Sciences, Hatfield, Pa.). Immunostaining was
performed using standard techniques on 4-6 .mu.M paraffin-embedded
sections or fixed YAMC-B4 cells, using the manufacturers'
suggestions for antibody dilutions. For immunofluorescence, slides
were mounted with Vectashield medium containing DAPI (Vector
Laboratories, Burlingame, Calif.). For immunohistochemistry,
diaminobenzidine substrate was from Sigma Corp (St. Louis, Mo.) and
sections were counterstained with methyl green (Dako Corp).
[0077] Cell Lysates and Western Blot Analysis--
[0078] Cell lysates were extracted in modified RIPA buffer as
previously described (Frey, M. R., Disc, R. S., Edelblum, K. L.,
and Polk, D. B. (2006) Embo J 25, 5683-5692), cleared by
centrifugation, and boiled in Laemmli sample buffer. Samples were
separated on SDS-polyacrylamide gels (6-10% as appropriate),
blotted on PVDF membranes, and subjected to quantitative immunoblot
using the Li-Cor Odyssey infrared detection system. Equal loading
was monitored by immunoblots for actin and at least one additional
protein.
[0079] Apoptosis Assays--
[0080] Apoptosis was stimulated in cell culture by 6 h exposure to
a cytokine cocktail containing TNF (100 ng/ml) plus IFN-.gamma.
(150 units/ml), with or without recombinant NRG4 (100 ng/ml). In
some experiments, LY294002 (PI3K inhibitor, 5 .mu.M) was also used.
Cells were fixed in 4% paraformaldehyde and immunostained for
cleaved caspase-3.
[0081] For apoptosis analysis in vivo, mice were injected i.p. with
PBS/vehicle, TNF (250 .mu.g/kg) plus IFN-.gamma. (250 units/g),
NRG4 (100 .mu.g/kg), or NRG4 plus TNF and IFN-.gamma.. In some
experiments, LY294002 (50 mg/kg) was included. After 24 h, mice
were euthanized and colons were removed and either fixed or used to
make mucosal homogenates. Apoptosis was detected by in situ oligo
ligation (ISOL; EMD Millipore, Billerica, Mass.) staining for DNA
fragmentation on sections of paraffin-embedded tissue, and by
cleaved caspase-3 western blot on homogenates.
[0082] Proliferation Assays--
[0083] Proliferative index in fixed colon specimens was assessed by
immunohistochemical stain for Ki-67. In vitro, cells were exposed
to vehicle or growth factors for 24 h, then labeled with
5-ethynyl-2-deoxyuridine (EdU) for 2 h and fixed. Nuclei were
marked with DAPI, and incorporated EdU was detected using a
Click-iT EdU kit (Invitrogen).
[0084] Cell Migration/Restitution--
[0085] Cells grown on fibronectin-coated plates were subjected to
multiple circular wounds with a rotating silicone probe as
previously described (Corredor, J., Yan. F., Shen, C. C., Tong, W.,
John, S. K., Wilson, G., Whitehead, R., and Polk, D. B. (2003)
American journal of physiology. Cell physiology 284, C953-961).
Cultures were photographed immediately and 8 h after wounding; %
closure was determined by measuring wounds in Image.
[0086] RN/4 Isolation and RT-PCR Detection of ErbB Ligands--
[0087] Total RNA from flash-frozen mouse colon or isolated
epithelial cells was purified with RNeasy columns (Qiagen,
Valencia, Calif.) including on-column DNase treatment, cDNA was
synthesized from 1 .mu.g RNA with iScript (Bio-Rad, Hercules,
Calif.), amplified by standard PCR techniques using previously
described primers (Huotari, M. A., Miettinen, P. J., Palgi, J.,
Koivisto, T., Ustinov, J., Harari, D., Yarden, Y., and Otonkoski,
T. (2002) Endocrinology 143, 4437-4446), and visualized by gel
electrophoresis.
[0088] Real-Time Quantitative PCR (qPCR) Analysis of NRG4 in
IBD--
[0089] NRG4 and HRG-1.beta. expression levels were determined in
human IBD using TaqMan gene expression assays (Life Technologies,
Grand Island, N.Y.) on TissueScan Crohn's/Colitis qPCR Arrays
[OriGene Technologies, Rockville Md.; these specimens were fully
de-identified before receipt, and the study was reviewed by the
CHLA Institutional Review Board and determined to not qualify as
"human subject" research per .sctn.46.102(f)(Wilson, K. J.,
Gilmore, J. L., Foley, J., Lemmon, M. A., and Riese, D. J., 2nd.
(2009) Pharmacology & therapeutics 122, 1-8)]. Relative mRNA
levels were calculated using the 2.sup.-.DELTA..DELTA.CT method
with .beta.-actin as the reference; validity of the reference was
confirmed by comparing to a second reference gene
(.beta.-glucuronidase).
[0090] Statistics and Replicates--
[0091] All data are representative of at least three independent
experiments. Statistical analyses were performed with Prism
software (GraphPad Inc., La Jolla, Calif.). Statistical
significance was assessed by ANOVA analysis with Tukey post-test.
Error bars indicate standard error of means.
Example 2
NRG4 Blocks Inflammatory Cytokine Induced Colonocyte Apoptosis Both
In Vitro and In Vivo
[0092] Multiple ErbB family ligands are expressed in the colon
(Table 1). RNA was prepared from flash-frozen whole mouse colon or
isolated colon epithelium, then subjected to RT-PCR analysis for
presence of the indicated ligands (receptor binding specificities
indicated in parentheses of Table 1).
TABLE-US-00001 TABLE 1 ErbB ligands expressed in mouse colon Ligand
Whole Colon Isolated epithelial cells EGF (EGFR) + - HB-EGF (EGFR,
ErbB4) + + Betacellulin (EGFR, ErbB4) + - NRG4 (ErbB4) + -
HRG-1.beta. (ErbB3, ErbB4) + - TGF-.alpha. (EGFR) + +
[0093] Of these, only NRG4 is thought to be specific for ErbB4
versus other family members (Harari, D., Tzahar, E., Romano, J.,
Shelly, M., Pierce, J. H., Andrews, G. C., and Yarden, Y. (1999)
Oncogene 18, 2681-2689); thus, we used this ligand to define the
role of ErbB4 activation in colonic epithelial biology. ErbB4 is
expressed throughout the intestinal tract (FIG. 1A) and is induced
in colon epithelial cells by inflammation. Homegenates of 6-weeks
old male C57/BL6 duodenum (Duo), jejunum (Jej), ileum (Ile),
proximal colon (PC) and distal colon (CD) were analyzed by Western
blot for expression of ErbB4 and its specific ligand, NRG4. Both
ErbB4 and NRG4 were expressed from duodenum to distal colon. Blots
are representative of data from 3 mice.
[0094] Cultured mouse colonic epithelial cells expressing ErbB4
(YAMC-B4) cells were exposed to TNF (100 ng/ml) plus interferon
(IFN)-.gamma. (150 units/ml), with or without NRG4 (100 ng/ml).
After 6 h, the cells were fixed and apoptosis was assessed by
immunofluorescence analysis for cleaved caspase-3. When the YAMC-B4
cells were given 100 ng/ml NRG4, cells were protected from
apoptosis induced by the 6 h exposure to the cytokine cocktail
containing TNF (100 ng/ml) and IFN-.gamma. (150 U/ml), as measured
by immunofluorescence analysis for cleaved caspase-3 (FIG. 1B;
2.7-fold decrease with cytokines plus NRG4 vs. cytokines alone,
p<0.03). Similar results were obtained by TUNEL assay.
[0095] To confirm that this result was specifically due to ErbB4
stimulation, YAMC-B4 cells were exposed to HRG-1.beta. (100 ng/ml),
NRG4, or EGF (10 ng/ml) for 10 min. Phosphorylation of EGFR, ErbB2,
ErbB3, and ErbB4 was determined by western blot analysis of cell
lysates using phospho-specific antibodies. Blots are representative
of results at least 3 independent experiments. While the positive
controls EGF and HRG-1 stimulated phosphorylation of multiple ErbBs
(FIG. 2), NRG4 activated only ErbB4, confirming that NRG4 is an
ErbB4-specific ligand.
[0096] To translate these results into an in vivo setting, we
injected 6-8 week old C57B1/6 mice intraperitoncally with 250
.mu.g/kg TNF plus 250 U/g of IFN-.gamma., with or without NRG4 (100
.mu.g/kg). 24 hours after injections, colons were harvested.
Apoptosis assessed by Western blot analysis for cleaved caspase-3
on isolated epithelial cells determined that NRG4 blocked
cytokine-induced apoptosis in vivo (FIG. 3A, B; 3.6-fold decrease,
p=0.012). Similar results were obtained with western blot analysis
for cleaved poly (ADP-ribose) polymerase. ISOL staining on sections
of fixed, paraffin-embedded colons also showed a significant
reduction in apoptosis with NRG4 (FIG. 3C, D; 3.1-fold decrease,
p<0.0001). Together, the data herein show that NRG4 activation
of ErbB4 in colon epithelial cells protects them from
cytokine-induced apoptosis both in cell culture and in vivo.
Example 3
Colonocyte Proliferation and Migration are not Affected by NRG4
[0097] Most ErbB ligands stimulate multiple cellular processes,
including proliferation and migration, in colon epithelial cells
(Frey, M. R., Golovin, A., and Polk, D. B. (2004) J Biol Chem 279,
44513-44521). To determine if NRG4 is similarly broad in its
cellular effects, we subjected YAMC-B4 cells to proliferation
assays in the presence of NRG4. After 24 h stimulation with NRG4,
cellular uptake of the modified nucleoside EdU was not different
from control, while in contrast EGF stimulated proliferation by
31.5% over control (FIG. 4A). In vivo, the number of Ki67-positive
nuclei per crypt in PBS vs. NRG4-injected mice was not altered 24 h
post-injection (FIG. 4B).
[0098] To test the potential effect of NRG4-ErbB4 signaling on cell
motility, we used a modified scratch assay for
restitution/migration (Corredor, J., Yan, F., Shen, C. C., Tong,
W., John, S. K., Wilson, G., Whitehead, R., and Polk, D. B. (2003)
American journal of physiology. Cell physiology 284, C953-961).
Over an 8 h period, NRG4 had no effect on the rate at which YAMC-B4
cells moved into the denuded area of a culture plate (FIG. 4C),
while in contrast EGF caused a 54% increase in migration over
control. Together, these results indicate that, unlike many other
growth factors, NRG4 selectively promotes colon epithelial cell
survival without affecting cell proliferation or migration.
[0099] In the dextran sodium sulfate-induced model of acute murine
colitis, NRG4 treatment reduces weight loss, colon shortening, and
histologically detectable colonic damage (FIG. 4D). 6-8 week-old
male C57/BL6 mice were given 3% DSS in drinking water for 7 days
and injected daily with either PBS or NRG4 (100 g/kg). Weight-loss
was monitored over the course of the experiment (FIG. 4D(i)). On
day 7, mice were sacrificed; colons were excised, rolled and fixed.
H&E sections were scored by a pathologist blind to the
experimental conditions using a scoring system adapted from that of
Dielman et al. (Dieleman, L. A., et al., Chronic experimental
colitis induced by dextran sulphate sodium (DSS) is characterized
by Th1 and Th2 cytokines. Clinical and experimental immunology,
1998. 114(3): p. 385-91) (FIG. 4D(ii)). NRG4 is also protective in
the small intestine in the formula feeding/hypoxia model of
necrotizing enterocolitis. Together, these data indicate that NRG4
is protective in multiple intestinal injury models, possibly
through inhibition of epithelial cell apoptosis.
Example 4
NRG4-ErbB4 Signaling Stimulates Akt Activation
[0100] To identify signaling pathways which could be involved in
NRG4-stimulated cell survival, we exposed YAMC-B4 cells to NRG4 or
EGF and prepared whole cell lysates for western blot analysis. NRG4
exposure (100 ng/ml, 10 min) resulted in phosphorylation of Akt but
not ERK MAPK (FIG. 5A), in contrast to HRG-13 and EGF which
activated both cascades. Furthermore, COX-2 expression, which is
induced by either HRG-13 or EGF after 6 h exposure, and is required
for cell survival induced by ErbB4-EGFR dimers (Frey, M. R.,
Hilliard, V. C., Mullane, M. T., and Polk, D. B. (2010) Laboratory
Investigation 90, 1415-1424), was not induced by NRG4. Thus, NRG4
promotes cell survival through pathways at least partly distinct
from those activated by other ligands. Similar to the cell culture
data, western blot analysis of colon epithelial cells isolated from
mice injected with NRG4 (with or without TNF plus IFN-.gamma.)
showed increased Akt phosphorylation versus controls (FIG. 5B);
increased Akt phosphorylation was also detectable by
immunofluorescence analysis on fixed colons from NRG4-injected mice
(FIG. 5C).
Example 5
PI 3-Kinase/Akt Signaling is Necessary for NRG4-Induced Cell
Survival In Vitro and In Vivo
[0101] To test the role of PI 3-kinase/Akt signaling in
NRG4-stimulated YAMC-B4 cell survival, we added the PI3K inhibitor
LY294002 (5 .mu.M) to cultures incubated with TNF plus IFN-.gamma.
or TNF plus IFN-.gamma. and NRG4. Immunofluorescence analysis for
cleaved caspase-3 showed that the PI3K inhibitor reversed NRG4's
protective effect in YAMC-B4 cells (FIG. 6A). In vivo. LY294002
injection (50 .mu.g/kg) reversed NRG4's blockade of
cytokine-induced colon epithelial apoptosis as measured by ISOL
stain (FIG. 6B).
Example 6
NRG4 Expression is Reduced in Crohn's Disease and Ulcerative
Colitis
[0102] To investigate the potential clinical relevance of a colon
epithelial cell survival pathway driven by NRG4, we studied the
ligand's expression in human inflammatory bowel disease. qPCR
analysis of biopsies from Crohn's disease & ulcerative colitis
patients showed 5.6 (p<0.05) and 7.1 (p<0.01) fold
reductions, respectively, in NRG4 expression versus uninflamed
controls (FIG. 7A). In contrast, we detected no change in
expression of the shared ErbB3/ErbB4 ligand HRG-1.beta.. (FIG. 7B),
suggesting a selective loss of the specific ErbB4 ligand only. In
the IL-10.sup.-/- mouse model of chronic colitis (Kuhn, R., Lohler,
J., Rcnnick, D., Rajcwsky, K., and Muller, W. (1993) Cell 75,
263-274), NRG4 protein levels were reduced in colitic animals as
determined by western blot analysis of intestinal homogenates (FIG.
7C). Furthermore, while ErbB4 expression was elevated in the
inflamed colons, similar to what we previously reported for human
IBD or murine DSS colitis (Frey, M. R., Edelblum, K. L., Mullane,
M. T., Liang, D., and Polk, D. B. (2009) Gastroenterology 136,
217-226), ErbB4 phosphorylation was not increased (FIG. 7C). Thus,
loss of the NRG4 ligand is associated with failure to activate
ErbB4 despite up-regulation of the receptor.
Example 7
[0103] Cronobacter sakazakii are used as the damaging agent in an
infectious necrotizing enterocolitis (NEC) rat model, and has been
implicated in NEC outbreaks driven by contaminated formula. In
vitro, it causes apoptosis of colon epithelial cells. NRG4 inhibits
CS-induced cell death. FIG. 8 shows that NRG4 attenuates
Cronobacter sakazakii induced apoptosis in cultured rat intestinal
epithelial cells. FIG. 9 shows that rat pups are resistant to
experimental NEC when treated with NRG4. This survival curve
demonstrates that in the hypoxia/hypothermia model of NEC in rat
pups (modeled after Barlow et al., Journal of Pediatric Surgery
Volume 9, Issue 5, October 1974, Pages 587-595), the pups survive
longer if NRG is administered by gavage with each feed.
Example 8
[0104] Previous investigation into the role of the ErbB4 receptor
tyrosine kinase in epithelial tissues has largely relied on results
using ligands such as HB-EGF or HRG-11 (Frey, M. R., Hilliard, V.
C., Mullane, M. T., and Polk. D. B. (2010) Laboratory Investigation
90, 1415-1424; Ni, C. Y., Murphy, M. P., Golde, T. E., and
Carpenter, G. (2001) Science 294, 2179-2181), which also activate
other ErbB family members (e.g., see FIG. 2). Herein, the inventor
used NRG4 to specifically activate ErbB4 in colonocytes. The
results show that ErbB4 activation in the absence of detectable
phosphorylation of EGFR, ErbB2, or ErbB3 results in an increase in
anti-apoptotic signaling both in vitro (FIG. 1) and in vivo (FIG.
3), with no change in cell proliferation or migration (FIG. 4).
NRG4-induced suppression of apoptosis was dependent on the PI3K/Akt
pathway (FIGS. 5 & 6). Thus, selective activation of ErbB4 with
NRG4 may be a specific cell survival stimulus.
[0105] The data show phosphorylation of ErbB4, but not other ErbBs,
in response to NRG4. Taken together with previous observations that
NRG4 does not directly bind other receptors (Harari, D., Tzahar,
E., Romano, J., Shelly, M., Pierce, J. H., Andrews, G. C., and
Yardcn, Y. (1999) Oncogene 18, 2681-2689), this suggests that ErbB4
homodimers are the effective mediator of its signaling. It is
formally possible that asymmetric heterodimers (Monsey, J., Shen,
W., Schlesinger, P., and Bose, R. (2010) J Biol Chem 285,
7035-7044; Macdonald-Obermann, J. L., Piwnica-Worms, D., and Pike,
L. J. (2012) Proc Natl Acad Sci USA 109, 137-142) play a role in
NRG4-induced effects, but we have not been able to detect
NRG4-drivcn association of ErbB4 with other ErbBs. Regardless, in
the absence of their phosphorylation it seems unlikely that ErbB1-3
play a significant role in downstream signaling in response to
NRG4.
[0106] In this context, it is interesting that NRG4, unlike other
ErbB ligands, promotes survival but not proliferation or migration
of colon epithelial cells. In proteomic peptide-binding studies,
ErbB4 associates with a more restricted suite of SH2-containing
targets than EGFR, ErbB2, or ErbB3 (Kaushansky, A., Gordus, A.,
Budnik, B. A., Lane, W. S., Rush, J., and MacBeath, G. (2008) Chem
Biol 15, 808-817). Furthermore, in the current report we show that
specific ErbB4 activation elicits only a subset of the downstream
signaling, including activation of Akt, but not ERK MAPK, compared
to other ErbB ligands including HRG-1.beta. [FIG. 5 and (12)],
HB-EGF (26), and TGF-.alpha. (McCole, D. F., Keely, S. J., Coffey,
R. J., and Barrett, K. E. (2002) J Biol Chem 277, 42603-42612).
These observations are consistent with our data showing that NRG4
is selectively a survival factor, thus positioning ErbB4 as the
only family member that can promote cell survival without affecting
proliferation or migration. Interestingly, some pathways (e.g.,
COX-2) required for the cell survival response to HRG-1.beta. are
apparently not necessary in the case of NRG4, whether the absence
of proliferative signaling (e.g. ERK) with NRG4 narrows the
requirement for cell protection from apoptosis, or in contrast an
overlapping but distinct set of alternative pathways are activated
but the different ligands, is an area of ongoing study in our
laboratory.
[0107] Specificity for cell survival but not cell division is in
agreement with the apparent lower oncogenic potential reported in
the literature for ErbB4 versus other receptor tyrosine kinases.
While increased levels or activity of EGFR, ErbB2, or ErbB3 are in
general associated with increased tumor growth, the role of ErbB4
is less certain. It is overexpressed in endometrial (Srinivasan,
R., Benton, E., McCormick, F., Thomas, H., and Gullick, W. J.
(1999) Clin Cancer Res 5, 2877-2883) and non-small cell lung
cancers (Starr, A., Greif, J., Vexler, A., Ashkenazy-Voghera, M.,
Gladesh, V., Rubin, C., Kerber, G., Marmor, S., Lev-Ari, S., Inbar,
M., Yarden, Y., and Ben-Yosef, R. (2006) Int J Cancer 119,
269-274), while in contrast transitional cell carcinoma of the
bladder (Memon, A. A., Sorensen, B. S., Melgard, P., Fokdal, L.,
Thykjaer, T., and Nexo, E. (2004) Br J Cancer 91, 2034-2041;
Rotterud, R., Nesland, J. M., Berner. A., and Fossa. S. D. (2005)
BJU Int 95, 1344-1350) and prostate cancer (Edwards, J., Traynor,
P., Munro, A. F., Pirret, C. F., Dunne, B., and Bartlett, J. M.
(2006) Clin Cancer Res 12, 123-130; Robinson, D., He, F., Pretlow,
T., and Kung, H. J. (1996) Proc Natl Acad Sci USA 93, 5958-5962)
show either no correlation between ErbB4 levels and tumor behavior
or an association between expression and good prognosis. Studies on
in breast cancer have yielded a contradictory literature, with
different papers suggesting ErbB4 expression is associated with
either poor (Srinivasan, R., Gillett, C. E., Barnes, D. M., and
Gullick, W. J. (2000) Cancer Res 60, 1483-1487; Junttila, T. T.,
Sundvall, M., Lundin, M., Lundin, J., Tanner, M., Harkonen, P.,
Jocnsuu, H., Isola, J., and Elenius, K. (2005) Cancer Res 65,
1384-1393) or favorable (Tovey, S. M., Witton, C. J., Bartlett. J.
M., Stanton, P. D., Reeves, J. R., and Cooke, T. G. (2004) Breast
Cancer Res 6, R246-251; Witton, C. J., Reeves, J. R., Going, J. J.,
Cooke, T. G., and Bartlett, J. M. (2003) J Pathol 200, 290-297)
outcome. These apparently inconsistent findings may be in part
explained by our results, if ErbB4 signaling per se does not
necessarily promote cell proliferation. ErbB4 activation by HRG-11,
which stimulates multiple receptors, does activate
cancer-associated pathways such as COX-2, but this response is
dependent on partnering with EGFR (Frey, M. R., Hilliard, V. C.,
Mullane, M. T., and Polk, D. B. (2010) Laboratory Investigation 90,
1415-1424). It may be that ErbB4 only promotes tumorigenesis in
partnership with other, more frankly oncogenic, ErbBs, as in for
example ErbB2/4 heterodimerization observed in late-stage
colorectal cancers (Lee, J. C., Wang. S. T., Chow, N. H., and Yang,
H. B. (2002) Eur J Cancer 38, 1065-1071) or the results of Lee and
colleagues identifying ErbB3/4 dimers as tumor promoters (Lee, D.,
Yu. M., Lee, E., Kim, H., Yang, Y., Kim, K., Pannicia, C., Kurie,
J. M., and Threadgill, D. W. (2009). J Clin Invest 119,
2702-2713).
[0108] The ability to block cytokine-stimulated colonocyte
apoptosis, combined with the decreased risk for proliferative
disorders compared with other growth factors, makes NRG4 an
attractive potential therapy for conditions such as IBD which
involve elevated apoptosis in the epithelium of the small intestine
or colon (Qiu, W., Wu, B., Wang, X., Buchanan, M. E., Regueiro, M.
D., Hartman, D. J., Schoen, R. E., Yu, J., and Zhang, L. (2011) J
Clin Invest 121, 1722-1732; Di Sabatino, A., Ciccocioppo, R.,
Luinetti, O., Ricevuti, L., Morera, R., Cifone, M. G., Solcia, E.,
and Corazza, G. R. (2003) Diseases of the colon and rectum 46,
1498-1507). As ErbB4 has a number of biochemical features
(including being the sole receptor for NRG4) that distinguish it
from other ErbB family members (Carpenter, G. (2003) Exp Cell Res
284, 66-77), it is a unique and specific signaling target. In this
regard, the observations that (a) NRG4 is deficient in both human
IBD and murine colitis (FIG. 7A), and (b) although ErbB4 expression
is elevated in the IL-10.sup.-/- murine colitis model, it is not
phosphorylated/activated (FIG. 7C), raise the possibility that NRG4
down-regulation may lead to deficient epithelial cell survival
signaling despite ErbB4 up-regulation. Consistent with this
possibility, recent work from Feng and colleagues showed loss of
NRG4 in a model of total parenteral nutrition (Feng. Y., and
Teitelbaum, D. H. (2012) American journal of physiology,
gastrointestinal and liver physiology 302, G236-249), which is
associated with increased inflammatory cytokines and decreased Akt
phosphorylation (Feng, Y., Rails, M. W., Xiao, W., Miyasaka, E.,
Herman, R. S., and Teitelbaum, D. H. (2012) Annals of the New York
Academy of Sciences 1258, 71-77). A dysregulated NRG4/ErbB4 balance
and an altered ratio between NRG4 and other ErbB ligands may be
important features of IBD which can be addressed with exogenous
ligand.
[0109] The data herein show that selective activation of the ErbB4
receptor tyrosine kinase with its specific ligand NRG4 is a
survival signal in colon epithelial cells. This pathway activates
PI3K/Akt signaling and blocks inflammatory cytokine-induced
apoptosis without affecting cell proliferation or migration.
However, this pathway is deficient in IBD due to a loss of the
ligand. Our observations underscore the unique properties of ErbB4
compared with other ErbB family members, and suggest that selective
ErbB4 activation represents a divergent branch of receptor tyrosine
kinase signaling with potential therapeutic use in injury or
inflammatory diseases.
[0110] Various embodiments of the invention are described above.
While these descriptions directly describe the above embodiments,
it is understood that those skilled in the art may conceive
modifications and/or variations to the specific embodiments shown
and described herein. Any such modifications or variations that
fall within the purview of this description are intended to be
included therein as well. Unless specifically noted, it is the
intention of the inventors that the words and phrases in the
specification and claims be given the ordinary and accustomed
meanings to those of ordinary skill in the applicable art(s).
[0111] The foregoing description of various embodiments of the
invention known to the applicant at this time of filing the
application has been presented and is intended for the purposes of
illustration and description. The present description is not
intended to be exhaustive nor limit the invention to the precise
form disclosed and many modifications and variations are possible
in the light of the above teachings. The embodiments described
serve to explain the principles of the invention and its practical
application and to enable others skilled in the art to utilize the
invention in various embodiments and with various modifications as
are suited to the particular use contemplated. Therefore, it is
intended that the invention not be limited to the particular
embodiments disclosed for carrying out the invention.
[0112] While particular embodiments of the present invention have
been shown and described, it will be obvious to those skilled in
the art that, based upon the teachings herein, changes and
modifications may be made without departing from this invention and
its broader aspects. It will be understood by those within the art
that, in general, terms used herein are generally intended as
"open" terms (e.g., the term "including" should be interpreted as
"including but not limited to," the term "having" should be
interpreted as "having at least," the term "includes" should be
interpreted as "includes but is not limited to," etc.).
* * * * *