U.S. patent application number 10/096327 was filed with the patent office on 2003-02-06 for methods of treating intestinal ischemia using heparin-binding epidermal growth factor.
This patent application is currently assigned to Children's Hospital, Inc.. Invention is credited to Besner, Gail E., Pillai, Srikumar B..
Application Number | 20030027758 10/096327 |
Document ID | / |
Family ID | 26743883 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030027758 |
Kind Code |
A1 |
Besner, Gail E. ; et
al. |
February 6, 2003 |
Methods of treating intestinal ischemia using heparin-binding
epidermal growth factor
Abstract
The present invention provides methods of treating pathologic
conditions associated with intestinal ischemia. In the methods,
patients at risk for or suffering from intestinal ischemia are
treated with a heparin-binding epidermal growth factor product.
Inventors: |
Besner, Gail E.; (Dublin,
OH) ; Pillai, Srikumar B.; (Chicago, IL) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN
6300 SEARS TOWER
233 SOUTH WACKER
CHICAGO
IL
60606-6357
US
|
Assignee: |
Children's Hospital, Inc.
|
Family ID: |
26743883 |
Appl. No.: |
10/096327 |
Filed: |
March 12, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10096327 |
Mar 12, 2002 |
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09518950 |
Mar 6, 2000 |
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6387878 |
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09518950 |
Mar 6, 2000 |
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09181974 |
Oct 29, 1998 |
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6191109 |
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60063858 |
Oct 31, 1997 |
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Current U.S.
Class: |
514/9.6 ;
514/15.1; 514/8.9; 530/350 |
Current CPC
Class: |
A61K 38/1808
20130101 |
Class at
Publication: |
514/12 ;
530/350 |
International
Class: |
C07K 017/00; C07K
014/00; C07K 001/00; A61K 038/00 |
Claims
We claim:
1. A method of treating pathological conditions associated with
intestinal ischemia comprising administering an HB-EGF product to
patients in an amount effective to reduce intestinal cell necrosis.
Description
[0001] This application claims the benefit of the filing date of
U.S. provisional application Serial No. 60/063,858.
FIELD OF THE INVENTION
[0002] The present invention generally relates to prevention and/or
treatment of ischemia-induced intestinal injury. More particularly
the invention relates to prevention and/or treatment of intestinal
injury using heparin-binding epidermal growth factor (HB-EGF)
products.
BACKGROUND OF THE INVENTION
[0003] Hemorrhagic disorders and ischemic states are the two major
classes of gastrointestinal circulatory disorders. A sudden
reduction in the blood supply to a tissue is considered to be an
ischemic event. Intestinal ischemic events continue to play a major
role in the morbidity and mortality of numerous patients. Ischemic
injury to the small intestine results in mucosal destruction,
bacterial translocation, and perforation. Parks et al., Am. J.
Physiol., 250: G749-753 (1986) attributes much of the injury
associated with ischemic episodes to the reperfusion phenomena that
begin when blood flow is restored. Immediately after an ischemic
event, the intestinal epithelium undergoes desquamation with
destruction of the lamina propria. At a cellular level, ischemia
leads to depletion of ATP and loss of cytoskeletal integrity. With
return of the blood supply (i.e., reperfusion), there is continued
destruction of the villus structures. These injuries manifest
themselves in disease states such as necrotizing enterocolitis and
can lead to overwhelming sepsis and multisystem organ failure.
Recovery from an ischemic event depends on rapid proliferation and
migration of intestinal epithelial cells to regenerate damaged
villi. Restitution requires the presence of multiple substances,
including cytokines, hormones, and growth factors. Dignass and
Podolsky, Gastroenterology, 105: 1323-1332 (1993) reports that
transforming growth factor-.alpha. (TGF-.alpha.),
interleukin-1.beta. (IL-1.beta.), interferon-.gamma. (IFN-.gamma.),
and epidermal growth factor (EGF) have been shown to enhance
restitution, possibly through increased production of transforming
growth factor-.beta. (TGF-.beta.). These substances act to remodel
the intestine after injury and to modulate the inflammatory
response.
[0004] HB-EGF was originally identified in 1990 as a
macrophage-secreted heparin binding growth factor. Like other
members of the EGF family, HB-EGF exerts its biological effects by
binding to the erb class of EGF receptor (EGF-R) molecules.
However, unlike most members of the EGF family including EGF,
HB-EGF binds heparin with a high affinity. Heparin appears to
potentiate binding of HB-EGF to the signal-transducing EGF-R, and
may also modulate the biologic effects of the growth factor on
target cells, including cellular migration and proliferation.
HB-EGF is mitogenic for fibroblasts, smooth muscle cells and
epithelial cells, but not for endothelial cells. In addition,
HB-EGF is produced by epithelial cells and acts as an autocrine
growth factor for these cells. It is a heat-resistant, cationic
protein, with a molecular weight of approximately 22,000 kDa that
elutes from heparin-affinity chromatography columns with 1.0 M
NaCl.
[0005] The cloning of a cDNA encoding human HB-EGF (or HB-EHM) is
described in Higashiyama et al., Science, 251: 936-939 (1991) and
in a corresponding international patent application published under
the Patent Cooperation Treaty as International Publication No. WO
92/06705 on Apr. 30, 1992. Both publications are hereby
incorporated by reference herein. The sequence of the protein
coding portion of the cDNA is set out in SEQ ID NO: 1 herein, while
the deduced amino acid sequence is set out in SEQ ID NO: 2. Mature
HB-EGF is a secreted protein that is processed from a transmembrane
precursor molecule (pro-HB-EGF) via extracellular cleavage. The
predicted amino acid sequence of the full length HB-EGF precursor
represents a 208 amino acid protein. A span of hydrophobic residues
following the translation-initiating methionine is consistent with
a secretion signal sequence. Two threonine residues (Thr.sup.75 and
Thr.sup.85 in the precursor protein) are sites for O-glycosylation.
Mature HB-EGF consists of at least 86 amino acids (which span
residues 63-148 of the precursor molecule), and several
microheterogeneous forms of HB-EGF, differing by truncations of 10,
11, 14 and 19 amino acids at the N-terminus have been identified.
HB-EGF contains a C-terminal EGF-like domain (amino acid residues
30 to 86 of the mature protein) in which the six cysteine residues
characteristic of the EGF family members are conserved and which is
probably involved in receptor binding. HB-EGF has an N-terminal
extension (amino acid residues 1 to 29 of the mature protein)
containing a highly hydrophilic stretch of amino acids to which
much of its ability to bind heparin is attributed. Besner et al.,
Growth Factors, 7: 289-296 (1992), which is hereby incorporated by
reference herein, identifies residues 20 to 25 and 36 to 41 of the
mature HB-EGF protein as involved in binding cell surface heparin
sulfate and indicates that such binding mediates interaction of
HB-EGF with the EGF receptor.
[0006] The EGF family comprises at least five polypeptides: EGF,
HB-EGF, TGF-.alpha., amphiregulin (AR), and betacellulin. For
reviews of the family, see Barnard et al., Gastroenterology, 108:
564-580 (1995) and Prigent and Lemoine, Prog. Growth Factor Res.,
4: 1-24 (1992). The amino acid sequence homology of HB-EGF to the
EGF family members is 40 (compared to EGF) to 53% (compared to AR)
between the first and sixth cysteine residues in the EGF-like
domains, but HB-EGF exhibits lower homology when the full length
sequences are compared. Overall, HB-EGF most closely resembles AR
in that the two polypeptides exhibit the highest homology, appear
to have a similar number of amino acids, and include the N-terminal
extension of highly hydrophilic amino acids upstream of the
EGF-like domain.
[0007] Administration of EGF to prevent tissue damage after an
ischemic event in the brains of gerbils has been reported in U.S.
Pat. No. 5,057,494 issued Oct. 15, 1991 to Sheffield. The patent
projects that EGF "analogs" having greater than 50% homology to EGF
may also be useful in preventing tissue damage and that treatment
of damage in myocardial tissue, renal tissue, spleen tissue,
intestinal tissue, and lung tissue with EGF or EGF analogs may be
indicated. However, the patent includes no experimental data
supporting such projections.
[0008] The small intestine receives the majority of its blood
supply from the SMA, but also has a rich collateral network such
that only extensive perturbations of blood flow lead to pathologic
states. Villa et al., Gastroenterology, 110(4 Suppl): A372 (1996)
reports that in a rat model of intestinal ischemia in which thirty
minutes of ischemia are caused by occlusion of the superior
mesenteric artery (SMA), pre-treatment of the intestines with EGF
attenuated the increase in intestinal permeability compared to that
in untreated rats. The intestinal permeability increase is an early
event in intestinal tissue changes during ischemia. Multiple animal
models, like that described in Villa et al., supra have been used
to study the effects of ischemic injury to the small bowel. Since
the small intestine has such a rich vascular supply, researchers
have used complete SMA occlusion to study ischemic injury of the
bowel. Animals who experience total SMA occlusion suffer from
extreme fluid loss and uniformly die from hypovolenia and sepsis,
making models of this type useless for evaluating the recovery from
intestinal ischemia. Nevertheless, the sequence of morphologic and
physiologic changes in the intestines resulting from ischemic
injury has remained an area of intense examination.
[0009] Miyazaki et al., Biochem Biophys Res Comm, 226: 542-546
(1996) discusses the increased expression in a rat gastric mucosal
cell line of HB-EGF and AR resulting from oxidative stress. The
authors speculate that the two growth factors may trigger the
series of reparative events following acute injury (apparently
ulceration) of the gastrointestinal tract. To date, there has been
no published report of administration of HB-EGF in vivo for any
purpose, much less to test its ability to protect the
gastrointestinal tract from injury from an ischemic event.
[0010] The prevention and treatment of ischemic damage in the
clinical setting therefore continues to be a challenge in medicine.
There thus exists a need in the art for models for testing the
effects of potential modulators of ischemic events and for methods
of preventing and/or treating ischemic damage, particularly
ischemic damage to the intestines.
SUMMARY OF THE INVENTION
[0011] In a first aspect, the invention provides methods of
treating pathological conditions associated with intestinal
ischemia by administering an HB-EGF product to patients.
[0012] As used herein, "HB-EGF product" includes HB-EGF proteins
comprising about amino acid 63 to about amino acid 148 of SEQ ID
NO: 2; HB-EGF proteins comprising about amino acid 73 to about
amino acid 148 of SEQ ID NO: 2; HB-EGF proteins comprising about
amino acid 74 to about amino acid 148 of SEQ ID NO: 2; HB-EGF
proteins comprising about amino acid 77 to about amino acid 148 of
SEQ ID NO: 2; HB-EGF proteins comprising about amino acid 82 to
about amino acid 148 of SEQ ID NO: 2; HB-EGF proteins comprising a
continuous series of amino acids of SEQ ID NO: 2 which exhibit less
than 50% homology to EGF and which are efficacious in the rat model
specified below; fusion proteins comprising the foregoing HB-EGF
proteins; and the foregoing HB-EGF proteins including conservative
amino acid substitutions. Conservative amino acid substitions are
understood by those skilled in the art. The HB-EGF products may be
isolated from natural sources known in the art [e.g., the U-937
cell line (ATCC CRL 1593)], chemically synthesized, or produced by
recombinant techniques such as disclosed in WO92/06705, supra, the
disclosure of which is hereby incorporated by reference. In order
to obtain HB-EGF products of the invention, HB-EGF precursor
proteins may be proteolytically processed in situ. The HB-EGF
products may be post-translationally modified depending on the cell
chosen as a source for the products.
[0013] The administration of HB-EGF products is preferably
accomplished with a pharmaceutical composition comprising an HB-EGF
product and a pharmaceutically acceptable carrier. The carrier may
be in a wide variety of forms depending on the route of
administration. The route of administration may be oral, rectal,
parenteral, or through a nasogastric tube. Examples of parenteral
routes of adminstration are intravenous, intraperitoneal,
intramuscular, or subcutaneous injection. The presently preferred
route of administration is the oral route as the present invention
contemplates that the acid stability of HB-EGF is a unique factor
as compared to, for example, EGF. The HB-EGF pharmaceutical
composition may also include other ingrediants to aid solubility,
or for buffering or preservation purposes. Pharmaceutical
composition containing HB-EGF products comprises HB-EGF at a
concentration of about 0.5 to 10 mg/ml and preferably at a
concentration of 1 mg/ml in saline. Addition of other bioactive
compounds [e.g., antibiotics, free radical scavenging or conversion
materials (e.g., vitamin E, beta-carotene, BHT, ascorbic acid, and
superoxide dimutase), fibrolynic agents (e.g., plasminogen
activators), and slow-release polymers] to the HB-EGF compounds or
separate administration of the other bioactive compounds is also
contemplated.
[0014] As used herein, "pathological conditions associated with
intestinal ischemia" includes conditions which directly or
indirectly cause intestinal ischemia (e.g., premature birth; birth
asphyxia; congenital heart disease; cardiac disease; polycythemia;
hypoxia; exchange transfusions; low-flow states; atherosclerosis,
embolisms or arterial spasms; ischemia resulting from vessel
occlusions in other segments of the bowel; ischemic colitis; and
intestinal torsion such as occurs in infants and particularly in
animals) and conditions which are directly or indirectly caused by
intestinal ischemia (e.g., necrotizing enterocolitis, shock,
sepsis, and intestinal angina). Thus, the present invention
contemplates administration of HB-EGF products to patients in need
of such treatment including patients at risk for intestinal
ischemia, patients suffering from intestinal ischemia, and patients
recovering from intestinal ischemia. The administration of HB-EGF
to patients is contemplated in both the pediatric and adult
populations.
[0015] More particularly, the invention contemplates a method of
reducing necrosis associated with intestinal ischemia comprising
administering an HB-EGF product to a patient at risk for, suffering
from, or recovering from intestinal ischemia. Also contemplated is
a method of protecting intestinal epithelial cells from hypoxia
comprising exposing the cells to an HB-EGF product. Administration
of, or exposure to, HB-EGF products reduces lactate dehyrogenase
efflux from intestinal epithelial cells, maintains F-actin
structure in intestinal epithelial cells, increases ATP levels in
intestinal epithelial cells, and induces proliferation of
intestinal epithelial cells.
[0016] In view of the efficacy of HB-EGF in protecting intestinal
tissue from ischemic events, it is contemplated that HB-EGF has a
similar protective effect on myocardial, renal, spleen, lung, and
liver tissue.
[0017] In another aspect, the invention provides a novel animal
model of intestinal ischemia, designated herein a model of
"segmental" intestinal ischemia, that is useful for evaluating the
efficacy of putative therapeutics. Mammals, preferably rats, are
subjected to reversible arterial occlusion, wherein a first order
branch of the SMA and terminal collateral branches are occluded.
Preferably, the first order branch of the SMA is selected from the
group consisting of the middle ileum and the distal ilieum. Also
preferably, six to seven terminal collateral branches are occluded.
Reversible occlusion may be accomplished by means such as a
micro-vascular clip or sutures.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Practice of the methods of the present invention is
illustrated in the following examples wherein Example 1 describes
production of HB-EGF by recombinant techniques and demonstrations
of activity of the recombinant protein in various assays including
cytoprotection of intestinal epithelial cells from hypoxia in
vitro; Example 2 discloses a segmental model of intestinal ischemia
in the rat; Example 3 describes the efficacy of HB-EGF in treating
intestinal ischemia in the rat model; and Example 4 details
treatment of human adults and infants with pathological conditions
associated with intestinal ischemia with HB-EGF.
EXAMPLE 1
[0019] The effects of treatment of rat intestinal epithelial cells
with recombinant human HB-EGF were examined. Specifically, HB-EGF
was tested for the ability to induce proliferation of intestinal
endothelial cells and also for the ability to protect intestinal
epithelial cells from hypoxia. Experiments were also performed to
examine the mechanism of HB-EGF cytoprotection. The rat intestinal
epithelial cells IEC-18 (ATCC CRL 1589) were used in the
experiments.
[0020] A. Production of Recombinant Human HB-EGF (rHB-EGF)
[0021] The maltose-binding protein (MBP) fusion system (New England
Biolabs, Beverly, Mass.) was used to produce recombinant human
HB-EGF. HB-EGF cDNA corresponding to nucleotides 220 to 444 of SEQ
ID NO: 1 (encoding amino acids 74-148 of the 208-amino acid HB-EGF
precursor molecule) was cloned into plasmid pMAL-c2 at the Xmnl and
HindIII sites. E. coli strain BL21 (F ompT r.sub.B.sup.-
m.sub.8.sup.-) (Novagen, Madison, Wis.) containing this construct
was grown at 37.degree. C. in LB broth (Gibco/BRL, Gaithersburg,
Md.) containing 2 g/L glucose (Gibco/BRL) and 100 .mu.g/ml
ampicillin (Sigma, St. Louis, Mo.) to an OD.sub.600 of 0.2-0.3. To
induce expression, IPTG (Promega, Madison, Wis.) was added to a
final concentration of 0.3 mM. After a 3 hour incubation at
37.degree. C., cells were harvested by centrifugation
(4,000.times.g, 20 minutes) and the cell pellet was resuspended in
MBP buffer (10 mM Tris-Ci, 200 mM NaCl, 1 mM EDTA) containing 1 mM
PMSF (Sigma) and frozen overnight at 20.degree. C. Thawed cell
sample was lysed with a french press (14,000 psi) and the insoluble
fraction was removed by centrifugation (9,000.times.g, 30 minutes).
The supernatant was passed over an amylose resin column (New
England Biolabs) and fusion protein was eluted in MBP buffer
containing 10 mM maltose (Sigma). rHB-EGF was cleaved from MBP with
Factor Xa (0.5%, w/w) (Boehringer Mannheim, Indianapolis, Ind.) at
23.degree. C. for 16 hours. Cleaved products were applied to a
TSK-heparin 5PW column (8.times.75 mm, TosoHaas, Philadelphia, Pa.)
that was equilibrated with buffer (10 mM Tris-HCI pH 7.4, 0.2M
NaCl). The column was washed with equilibration buffer and bound
proteins were eluted with a 40 ml linear gradient of 0.2-2.0M NaCl
in 10 mM Tris-HCI pH 7.4 at 1 ml/min using an FPLC system
(Pharmacia LKB Biotechnology, Piscataway, N.J.). One milliliter
fractions are collected and assayed in an EGF radioreceptor assay
essentially as described in Besner et al. (1992), supra. Fractions
showing peak displacement of .sup.125I-EGF binding were pooled,
adjusted to contain 5% acetonitrile and 0.1% trifluoroacetic acid
and subjected to reverse phase HPLC (RP-HPLC). RP-HPLC was
performed in a Hitachi (San Hose, Calif.) HPLC system using a Vydac
C.sub.4 column (0.46.times.25 cm, 5 .mu.m particle size; The
Separations Group, Hesperia, Calif.) that was equilibrated with
water containing 5% acetonitrile and 0.1% trifluoroacetic acid. The
HB-EGF sample was injected onto the column and the column was
eluted using a multilinear gradient of 5% acetonitrile (isocratic)
for 5 minutes, 5-15% acetonitrile over 5 minutes, 15-40% over 120
minutes, 40-90% over 1 minute, 90% isocratic for 10 minutes, 90-5%
over 1 minute, and 5% isocratic for 33 minutes. The flow rate was 1
ml/min throughout and 1 ml fractions were collected. After these
purification steps, the absorbance peak eluting at 19% acetonitrile
is shown by SDS-PAGE to be a single band migrating at approximately
13 kDA. NH.sub.2-terminal amino acid sequencing of the pure protein
produced in this fashion confirms the sequence for HB-EGF. The
rHB-EGF was biologically active in the EGF-radioreceptor assay and
a Balb/c 3T3 DNA synthesis assay [essentially as described in
Besner et al., Cell Regulation, 1: 811-819 (1990)].
[0022] B. Recombinant HB-EGF is Mitogenic for Intestinal Epithelial
Cells
[0023] IEC-18 cells were cultured in Dulbecco's Modified Eagle
Medium (DMEM) containing 5% fetal bovine serum (FBS), 50 .mu.g/ml
penicillin and 50 units/ml streptomycin in a humidified atmosphere
of 10% CO.sub.2 at 37.degree. C. Cells were then seeded at a
density of 1.times.10.sup.5 cells/well in a 24 well plate (2
ml/well). After a 24 hour incubation, medium was changed to DMEM/1%
FBS (2 ml/well) and cells were allowed to incubate for an
additional 24 hours. Cells in four of the wells were trypsinized
and counted at Day 0 using a Coulter Counter.RTM.. rHB-EGF was
added to duplicate wells on Day 0 at a concentration of 100 ng/ml.
This concentration of rHB-EGF was determined as optimal from
preliminary dose-response curves for rHB-EGF-stimulated
proliferation of IEC-18 cells. Cells in duplicate wells were
trypsinized and counted on days 1 through 5.
[0024] Under normoxic conditions, rHB-EGF-treated cells had 1.85
fold greater increase in number compared to non-treated cells by
day 4 (p<0.05). Further, the mitogenic response of IEC-18 cells
to rHB-EGF was dose-dependent, with maximal stimulation by 100
ng/ml.
[0025] C. Recombinant HB-EGF to Protects Intestinal Epithelial
Cells Against Hypoxia
[0026] Lactate dehyrogenase (LDH) efflux was used as a measure of
cell injury after hypoxia. IEC-18 cells were seeded at a density of
5.times.10.sup.4 cells/well in DMEM/5% FBS in 24 well plates (2
ml/well). After 24 hours, medium was changed to DMEM/1% FBS (2
ml/well). This plating density resulted in approximately 75%
confluency. After an additional 24 hour incubation period, the
medium was replaced with Kreb's buffer (116 mM NaCl, 1.0 mM
NaH.sub.2PO.sub.4, 25.0 mM NaHCO.sub.3, 5.4 mM KCl, 1.8 mM
CaCl.sub.2, and 0.8 mM MgSO.sub.4) and the cells were placed in an
anaerobic incubator with FiO.sub.2.ltoreq.1%. Aliquots (50 .mu.l)
of media were removed at specific time intervals and were assayed
for LDH efflux using a Cytox 96 assay. At the end of the
experiment, cells were lysed with PBS/0.1% Triton-100 and total LDH
content was determined. LDH efflux was expressed as the percentage
of total LDH activity. Based on preliminary experiments which
demonstrated approximately 20-25% cell death after 10 hours of
hypoxia and 100% cell death after 14 hours of hypoxia, a 10 hour
anaerobic period was used in the subsequent studies.
[0027] To test the growth factor effects, 100 ng/ml rHB/EGF was
added to some wells 12 hours prior to the initiation of hypoxia.
After 10 hours of hypoxia, plates were removed from the anaerobic
chamber, medium changed to DMEM/5% FBS, and cells were allowed to
recover for 48 hours. During recovery, some wells received
additional (post-hypoxia) rHB-EGF (100 ng/ml) treatment. Aliquots
(50 .mu.l) of media were removed from the wells at 0, 12, 24, 36,
and 48 hours of recovery and LDH efflux was measured.
[0028] Intestinal cells that received rHB-EGF either pre-hypoxia or
both pre- and post-hypoxia showed a significantly lower LDH release
during recovery from hypoxia compared to non-treated cells.
Although there was very little LDH efflux immediately after
hypoxia, by 48 hours, non-treated cells had an LDH release of 22.8%
compared to 7.48% for cells that had been pre-treated with HB-EGF
(p<0.009) or to 9.1% for cells that had been both pre-treated
and post-treated with HB-EGF (P<0.009). Cells that received
HB-EGF during only the post-hypoxic period did not have a
significantly lower LDH release compared to cells that were not
treated with HB-EGF.
[0029] D. Effects of rHB-EGF on Cytoskeletal Structure, ATP Stores,
and Post-Hypoxia Proliferation
[0030] To examine IEC-18 cytoskeletal structure, IEC-18 cells were
seeded at 5.times.10.sup.3 cells/well in DMEM/5% FBS (500
.mu.l/chamber) in 8-well chamber slides. Cells were incubated for
24 hours after which the medium was changed to DMEM/1% FBS. rHB-EGF
(100 ng/ml) was then added to specific wells. After an additional
12 hours, medium was changed to Kreb's buffer and cells were placed
in the anaerobic chamber for 10 hours. Following this, medium was
changed to DMEM/5% FBS and cells were incubated for an additional
24 or 48 hours. Medium was aspirated from the chambers, cells were
fixed for thirty minutes in 10% formalin (buffered with PBS), and
slides were washed twice in PBS. Cells were simultaneously stained
with rhodamine phalloidin to detect filamentous (F) actin and Dnase
I fluorescein to detect globular (G) actin. Confocal analysis of
the cells was performed with the Zeiss LSM inverted microscope
using a krypton/argon mixed gas laser and a Zeiss filter set,
utilizing emissions filters of 568 and 488. Images were digitally
recorded.
[0031] Under normal circumstances, monomeric G-actin is polymerized
to produce F-actin in an ATP-dependent manner. F-actin staining of
cells is present in the cortical region (peripheral) in cells
having an intact cytoskeleton, whereas G-actin accumulation is
indicative of cytoskeletal injury. Immediately after anaerobic
exposure, the respective proportions of F-actin and G-actin were
similar between rHB-EGF-treated and non-treated cells. However,
after 24 hours of recovery, IEC-18 cells that received rHB-EGF
prior to anaerobic exposure maintained the cortical F-actin
cytoskeletal structure compared to non-treated cells which had
increase levels of peri-nuclear G-actin staining. By 48 hours,
rHB-EGF-treated cells still maintained their F-actin structure,
whereas non-treated cells contained predominately G-actin with very
little F-actin.
[0032] To examine ATP stores in IEC-18 cells, the cells were seeded
at a density of 1.times.10.sup.5 cells/well in DMEM/5% FBS in 6
well palates (1 ml/well). Cells were incubated for 24 hours after
which the medium was changed to DMEM/1% FBS. rHB-EGF (100 ng/ml)
was then added to specific wells. After an additional 12 hours,
medium was changed to Kreb's buffer and cells were placed in the
anaerobic chamber for 10 hours. Cells were lysed with PBS/0.1%
Triton-100 at 0, 24 and 48 hours of recovery. ATP content was
measured using an ATP determination kit (Molecular Probes, Eugene,
Oreg.). This assay allows quantification of ATP through a
luciferin/luciferase-ATP reaction. Bioluminescence was measured
with a luminometer (Model LB9501). Initial experiments demonstrated
a linear relationship (r.sup.2=0.960) between ATP (pmoles) and
luminescence. A Bio-Rad D.sub.c protein assay was used to determine
total protein content in the samples. Optical density (OD) was
measured with a microplate reader (Model EL312). Preliminary
standard curves demonstrated a linear relationship (r.sup.2=0.988)
between protein (mg/ml) and OD.
[0033] Both non-treated and rHB-EGF treated cells had ATP levels in
the 11-12 nmole/mg range under normoxic conditions. rHB-EGF-treated
and non-treated cells had similar decreases in ATP levels in the
immediate post-hypoxic period (45% drop vs 50% drop, respectively).
However, during the later recovery periods (24 and 48 hours), the
HB/EGF-treated cells exhibited a rise in their ATP levels (6.1
nmole/mg), whereas the non-treated cells continued to have
decreased ATP content (4.5 nmole/mg).
[0034] Post-hypoxia IEC-18 cell proliferation was assessed with a
CytoQuant fluorescence assay (Molecular Probes, Eugene, Oreg.).
Preliminary experiments demonstrated a linear relationship
(r.sup.2=0.978) between cell number and fluorescence. IEC-18 cells
were seeded at 5.times.10.sup.3 cells/well in DMEM/5% FBS in
96-well plates (200 .mu.l/well). After 24 hours of incubation,
medium was changed to DMEM/1% FBS (200 .mu.l/well). Some wells
received HB-EGF (100 ng/ml) and the cells were incubated for an
additional 12 hours. Medium was then changed to Kreb's buffer and
the plate was placed in the anaerobic chamber for 10 hours. At 0,
12, 36, and 72 hours of recovery, media was removed and plates were
frozen at -70.degree. C. for thirty minutes. The Cytoquant
fluoroprobe was added after incubating plate at room temperature
for five minutes. Fluorescence was measured in a Cytofluor
fluorescent plate reader (Millipore, Bedford, Mass.).
[0035] Cytofluorometric measurement of post-hypoxia cellular
proliferation of IEC-18 cells showed that cells treated with HB-EGF
had a 1.23 fold increase in a cell number compared to non-treated
cells by 72 hours of recovery from hypoxia (p<0.05).
[0036] The experimental results described above relating to the
mitogenic and cytoprotective effects of HB-EGF were orally
disclosed at the Columbus Surgical Society Presidential Symposium
on Jan. 18, 1997 and at the Annual West Virginia University
Resident's Forum on Mar. 7, 1997. The experimental results
described above relating to LDH release were disclosed orally and
in abstract form at the Childrens' Hospital Research Foundation
Research Forum in Columbus, Ohio on Jun. 5, 1997.
[0037] HB-EGF was also demonstrated to preserve cytoskeletal
structure and increase cellular ATP levels in renal tubular
epithelial cells subject to hypoxia. The same cells also released a
lower level of LDH during recovery than untreated cells.
[0038] The results of the in vitro experiments indicate that in
addition to being a mitogen for intestinal epithelial cells, HB-EGF
is also a cytoprotective growth factor for these cells during
recovery from hypoxia. The in vitro cytoprotective effects of
rHB-EGF can be explained, at least in part, by increased cellular
ATP levels in rHB-EGF-treated cells with resultant preservation of
cytoskeletal structure. An additional beneficial effect of this
growth factor is that after the ischemic event, HB-EGF-treated
cells have a higher proliferative rate than non-treated cells. The
cytoprotective effects of HB-EGF may be enhanced by its ability to
bind to heparan sulfate proteoglycans expressed on the surface of
intestinal cells. See Carey et al., J. Cell Biology, 117(1):
191-201 (1992) for a discussion of heparan sulfate
proteoglycans.
EXAMPLE 2
[0039] While HB-EGF protected intestinal epithelial cells from
hypoxia in vitro, there was no model described in the literature
that was useful for examining the effect of HB-EGF on recovery from
intestinal ischemia in vivo. To determine whether HB-EGF was
efficacious in protecting the intestines from the deleterious
effects of ischemia in vivo, a novel animal model of segmental
intestinal ischemia was developed. The model provides the
opportunity to study ischemia-reperfusion injuries in localized
segments of bowel without the morbidity and mortality associated
with total SMA occlusion in prior animal models. By occluding a
first order branch of the superior mesenteric artery (SMA) and by
selectively ligating terminal collateral branches, reproducible
segmental intestinal ischemia was achieved. Bowel damage ranged
from alterations in the villus structure to frank hemorrhagic
necrosis of the intestinal wall.
[0040] The operative procedure was performed as follows. A total of
eighteen rats (age 7 to 9 weeks, 200-265 g) were induced with an
intraperitoneal injection of Ketamine-HCl (10 mg/kg) and Xylazine
(3 mg/kg). Intravenous access and/or fluid resuscitation were not
necessary during the procedure. The abdomen was shaved and painted
with betadine. The animal was placed supine on a warming pad set at
40.degree. C., and positioned under an operating microscope. A
midline skin incision was made, the linea alba was opened, and the
peritoneal cavity was entered. The small intestine, cecum and
proximal ascending colon were delivered into the operative field
and the superior mesenteric vein was identified. The SMA was
located postero-lateral to the vein and was exposed by carefully
dissecting apart the mesentery with 0.5 mm micro-surgical forceps.
Exposure of this artery and the subsequent steps were performed
using an operating microscope. Once the SMA was exposed, the first
order branches were evaluated for possible sites of occlusion.
Since the mesenteric arcades are longer in the ileal segments, the
middle and distal ileum were used as target segments for arterial
occlusion. Once a segment was identified, a micro-vascular
atraumatic clip (2.0 mm) was placed on the first order mesenteric
branch feeding this segment. After the clip was placed, the
terminal arterial and venous arcade branches, both proximal and
distal to the occluded arcade, were ligated with 5.0 silk sutures.
To create a 5 cm segment of ischemic bowel, six to seven terminal
branches need to be ligated. Arterial occlusion was maintained for
1 hour. A 4.times.4 gauze pad was placed over the bowel during this
time and was frequently moistened with warm saline. Prior to
removing the micro-clip, 0.1% Evan's blue solution (1.5 cc) was
injected into the renal vein with a 28 gauge needle to confirm
non-perfusion of the ischemic segment. The micro-clip was then
removed and Evan's blue, at the same concentration, was injected
into the contralateral renal vein to establish return of flow to
the ischemic segment. The silk ligatures were left on the terminal
branches. The abdomen was closed in a standard fashion. Animals
were then placed in a warm incubator (40.degree. C.) until awake
(approximately 20 minutes) and were then transferred to individual
cages. They received water, but not food, during the post-operative
period. Animals were euthanized with CO.sub.2 and segments of
intestine were removed for histologic analysis at 6 hours after
surgery in 6 animals and at 48 hours after surgery in 12
animals.
[0041] All eighteen animals survived the operation. Gross changes
of the bowel during the arterial occlusion were noted to occur in
stages. After 10-15 minutes of ischemia the serosa loses its sheen,
and after 15-20 minutes the bowel wall becomes edematous. After
25-35 minutes the bowel color changes from pink to white and later
develops a more dusky appearance. The proximal and distal ends of
the segment, where the terminal arteries and veins were ligated,
have a more bluish appearance, and the smaller veins become
dilated. Peristalsis of the affected segment ceases within the
first 25 minutes of ischemia. As ischemic time increases, the bowel
wall becomes increasingly edematous. Confirmation of ischemia was
shown with Evan's blue dye injection, with the dye taken up by the
normally perfused bowel, but not by the ischemic segment. Upon
termination of arterial occlusion, uptake of the blue dye
throughout the previously ischemic bowel confirmed resumption of
flow to this area.
[0042] After euthanization, the abdomen was re-explored and the
segment of ischemic bowel, as well as portions of intestine both
proximal and distal to the hypo-perfused segment, were excised and
fixed in Histochoice.TM. for 12 hours. The segments were then
cross-sectioned at 1 mm intervals, processed in a standard fashion,
and embedded in paraffin. Sections were H&E stained and
examined using a standard light microscope. All six animals that
were sacrificed at the 6 hour time point showed minor intestinal
villus structural change. Instead of the normal elongated distal
tip, the villi had more flattened tips with reduction of
cytoplasmic content and absence of the brush border. Of twelve
animals sacrificed after 48 hours, all showed evidence of villus
tip necrosis, and five also developed areas of hemorrhagic
transmural necrosis with extensive polymorphonuclear leukocyte
infiltration.
[0043] Animal care and experimentation described above conformed to
standards listed in the National Institute of Health's Guide for
the Care and Use of Laboratory Animals. The experimental protocol
was evaluated and approved by the Institutional Animal Care and Use
Committee of Children's Hospital (protocol #01496AR). Each
procedure was performed by a single operator without the need for
additional assistance. All procedures were performed using sterile
technique. The duration of the procedure was 11/4-11/2 hours,
depending on the difficulty of the dissection.
[0044] In comparison to ischemia-reperfusion injury models
involving total SMA occlusion the mortality rate of which
approaches 100%, the segmental model described herein had a 0%
postoperative mortality in the first 48 hours. The development of
the segmental model thus allowed the investigation of the effects
of potential therapeutic agents during the recovery period.
EXAMPLE 3
[0045] The effect of HB-EGF on intestinal ischemia in vivo was then
examined using the segmental model.
[0046] A total of twelve rHB-EGF-treated and twelve control male
Sprague-Dawley rats (220-310 g) were studied. Segmental intestinal
ischemia involving a 4.5-5.0 cm length of distal ileum was produced
as described in Example 2. Arterial occlusion was maintained for 1
hour. Fifteen minutes prior to removing the microvascular clip,
Evan's blue solution was injected intravenously to confirm
segmental intestinal ischemia. Experimental animals then received
HB-EGP (20 .mu.g/ml) which was injected intraluminally into the
duodenum in a volume of 5 ml of phosphate buffered saline (PBS),
which led to filing of the entire gastrointestinal tract from the
duodenum to the cecum. Control animals received an injection of 5
ml of PBS only. At the end of the 1 hour ischemic interval, the
microvascular clamp was removed and the Evan's blue solution was
reinjected intravenously to confirm segmental intestinal
reperfusion. Animals received water but no food postoperatively.
After 48 hours animals were sacrificed and necropsy performed. The
ischemic intestinal segment as well as the bowel just proximal and
distal to the ischemic segment were excised. Tissues were placed in
Histochoice fixative for 12 hours, cross-sectioned at random
intervals and embedded in paraffin. Sections were H & E stained
and examined using a standard light microscope. For each animal in
the experiment, 4-10 random sections of the ischemic bowel segment
were examined for histologic injury. Results are presented in Table
1 below.
1 TABLE 1 # sections Transmural Total # animals examined Tip
necrosis necrosis injury Non- 12 92 51/92 (55%) 21/92 (23%) 72/92
treated (78%) rHB- 12 94 12/94 (13%) 0/94 (0%) 12/94 EGF- (13%)
treated P < 0.05
[0047] Of the non-treated animals, 55% of the representative
sections studied had villous tip necrosis, and 23% of the sections
had transmural necrosis. In contrast, in the HB-EGF-treated
animals, only 13% of the sections studied had villous tip necrosis,
and none of the sections displayed transmural necrosis. Thus,
intraluminal HB-EGF administration, in this case delivered 45
minutes after the initiation of the ischemic event, protects the
intestine from ischemic injury.
EXAMPLE 4
[0048] The following section exemplifies administration of HB-EGF
to pediatric patients and adult patients. Administration of HB-EGF
is indicated in patients at risk for or suffering from any
pathological condition associated with ischemic injury.
[0049] A. Administration to Pediatric Patients
[0050] Intestinal injury related to an ischemic event is a major
risk factor for neonatal development of necrotizing enterocolitis
(NEC). NEC accounts for approximately 15% of all deaths occurring
after one week of life in small premature infants. Although most
babies who develop NEC are born prematurely, approximately 10% of
babies with NEC are full-term infants. Babies with NEC often suffer
severe consequences of the disease ranging from loss of a portion
of the intestinal tract to the entire intestinal tract. At present,
there are no known therapies to decrease the incidence of NEC in
neonates.
[0051] Babies considered to be at risk for NEC are those who are
premature (less than 36 weeks gestation) or those who are full-term
but exhibit, e.g., prenatal asphyxia, shock, sepsis, or congenital
heart disease. The presence and severity of NEC is graded using the
staging system of Bell et al., J. Ped. Surg., 15:569 (1980) as
follows:
2 Stage I Any one or more historical factors producing perinatal
stress (Suspected Systemic manifestations - temperature
instability, lethargy, NEC) apnea, bradycardia Gastrointestinal
manifestations - poor feeding, increased pregavage residuals,
emesis (may be bilious or test positive for occult blood), mild
abdominal distention, occult blood in stool (no fissure) Stage II
Any one or more historical factors (Definite Above signs and
symptoms plus persistant occult or gross NEC) gastrointestinal
bleeding, marked abdominal distention Abdominal radiographs showing
significant intestinal disten- tion with ileus, small-bowel
separation (edema in bowel wall or peritoneal fluid), unchanging or
persistent "rigid" bowel loops, pneumatosis intestinalls, portal
venous gas Stage III Any one or more historical factors (Advanced
Above sings and symptoms plus deterioration of vital signs, NEC)
evidence of septic shock, or marked gastrointestinal hemor- rhage
Abdominal radiographs showing pneumoperitoneum in addi- tion to
findings listed for Stage II
[0052] Babies at risk for or exhibiting HB-EGF are treated as
follows. Patients receive a daily liquid suspension of HB-EGF (1
mg/kg in saline). The medications are delivered via a nasogastric
tube if one is in place, or orally if there is no nasogastric tube
in place.
[0053] B. Administration to Adult Patients
[0054] Adults also receive a daily liquid suspension containing
HB-EGF (1 mg/kg) to drink or through a nasogastric tube if
necessary.
[0055] Numerous modifications and variations in the practice of the
invention are expected to occur to those skilled in the art upon
consideration of the foregoing description. Consequently, the only
limitations which should be placed on the invention are those which
appear in the following claims.
Sequence CWU 1
1
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