U.S. patent application number 13/526771 was filed with the patent office on 2012-12-06 for use of gcc ligands.
This patent application is currently assigned to Thomas Jefferson University. Invention is credited to Wilhelm Lubbe, Jason Park, Giovanni Mario Pitari, Stephanie Schulz, Scott A. Waldman, Henry Wolfe.
Application Number | 20120308583 13/526771 |
Document ID | / |
Family ID | 32869549 |
Filed Date | 2012-12-06 |
United States Patent
Application |
20120308583 |
Kind Code |
A1 |
Waldman; Scott A. ; et
al. |
December 6, 2012 |
USE OF GCC LIGANDS
Abstract
Proliferation of colorectal, gastric and esophageal cancer cells
is inhibited by administering ST receptor ligand. The number of ST
receptor molecules on the surface of a colorectal cell or
metastasized colorectal cancer cell are increased by administering
an ST receptor ligand such that ligand comes into contact with an
ST receptor on the surface of the colorectal cell. Pharmaceutical
compositions comprise sterile, pyrogen free ST receptor ligand and
a pharmaceutically acceptable carrier or diluent. Metastasized
colorectal cancer is treated or imaged by increasing the number of
ST receptor molecules on the surface of a metastasized colorectal
cancer cell and then administering a pharmaceutical composition
containing components that target the ST receptor for delivery of a
therapeutic agent or imaging agent. Methods of detecting
metastasized colorectal cancer are disclosed. Methods of delivering
active compounds to a colorectal cell in an individual are
disclosed.
Inventors: |
Waldman; Scott A.; (Ardmore,
PA) ; Pitari; Giovanni Mario; (Philadelphia, PA)
; Park; Jason; (Philadelphia, PA) ; Schulz;
Stephanie; (West Chester, PA) ; Wolfe; Henry;
(Glenmoore, PA) ; Lubbe; Wilhelm; (Philadelphia,
PA) |
Assignee: |
Thomas Jefferson University
Philadelphia
PA
|
Family ID: |
32869549 |
Appl. No.: |
13/526771 |
Filed: |
June 19, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10775481 |
Feb 10, 2004 |
8206704 |
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13526771 |
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60446730 |
Feb 10, 2003 |
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Current U.S.
Class: |
424/178.1 ;
514/19.3 |
Current CPC
Class: |
G01N 33/57446 20130101;
A61K 47/6415 20170801; A61P 35/00 20180101; A61P 35/04 20180101;
A61P 43/00 20180101; A61K 38/00 20130101; G01N 33/57419 20130101;
A61K 38/177 20130101; A61K 51/088 20130101 |
Class at
Publication: |
424/178.1 ;
514/19.3 |
International
Class: |
A61K 38/02 20060101
A61K038/02; A61P 35/00 20060101 A61P035/00; A61K 39/395 20060101
A61K039/395 |
Claims
1-131. (canceled)
132. A method of treating an individual who has metastasized
colorectal cancer or primary or metastasized gastric or esophageal
cancer in an individual who has been identified as having
metastasized colorectal cancer or primary or metastasized gastric
or esophageal cancer, said method comprising the steps in the
following order: a) administering to said individual an amount of
an guanylyl cyclase C ligand that activates guanylyl cyclase C on
cancer cells effective to increase the number of guanylyl cyclase C
molecules on the surface of cancer cells, and b) subsequently
administering a therapeutically effective amount of a guanylyl
cyclase C ligand that is conjugated to a cytotoxic moiety.
133. The method of claim 132 wherein the individual has been
identified as having metastatic colorectal, esophageal or stomach
cancer.
134. The method of claim 132 wherein said guanylyl cyclase C ligand
that is conjugated to a cytotoxic moiety is an anti-guanylyl
cyclase C antibody conjugated to a cytotoxic moiety or an
anti-guanylyl cyclase C binding fragment of an anti-guanylyl
cyclase C antibody conjugated to a cytotoxic moiety.
135. The method of claim 132 wherein said guanylyl cyclase C ligand
that is conjugated to a cytotoxic moiety is an anti-guanylyl
cyclase C antibody conjugated to a cytotoxic moiety.
136. The method of claim 132 wherein the guanylyl cyclase C ligand
that activates guanylyl cyclase C on cancer cells is administered
in an amount effective to increase the number of guanylyl cyclase C
molecules on the surface of cancer cells for at least 6 hours.
137. The method of claim 132 wherein the guanylyl cyclase C ligand
that activates guanylyl cyclase C on cancer cells is administered
in an amount effective to increase the number of guanylyl cyclase C
molecules on the surface of cancer cells for at least 8 hours.
138. The method of claim 132 wherein the guanylyl cyclase C ligand
that activates guanylyl cyclase C on cancer cells is administered
in an amount effective to increase the number of guanylyl cyclase C
molecules on the surface of cancer cells for at least 12 hours.
139. The method of claim 132 wherein the guanylyl cyclase C ligand
that activates guanylyl cyclase C on cancer cells is administered
in an amount effective to increase the number of guanylyl cyclase C
molecules on the surface of cancer cells for at least 16 hours.
140. The method of claim 132 wherein the guanylyl cyclase C ligand
that activates guanylyl cyclase C on cancer cells is administered
in an amount effective to increase the number of guanylyl cyclase C
molecules on the surface of cancer cells for at least 20 hours.
141. The method of claim 132 wherein the guanylyl cyclase C ligand
that activates guanylyl cyclase C on cancer cells is administered
in an amount effective to increase the number of guanylyl cyclase C
molecules on the surface of cancer cells for at least 24 hours.
142. The method of claim 132 wherein said guanylyl cyclase C ligand
that activates guanylyl cyclase C on cancer cells is administered
intravenously.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods of inhibiting
proliferation of human colorectal, gastric and esophageal cells
including: primary and metastasized human colorectal cancer cells,
primary and metastasized human gastric cancer cells, and primary
and metastasized human esophageal cancer cells. The present
invention relates to methods of treating individuals who have
diseases that effect colorectal, gastric and esophageal cells
including colorectal, gastric and esophageal cancer. The present
invention relates to improved methods of enhancing the therapeutic
efficacy of existing methods of treating gastric, esophageal and
colorectal cancers. The present invention relates to methods of
increasing the number of ST receptors on the surface of human
colorectal cells including metastasized human colorectal cancer
cells. The present invention relates to methods of inhibiting the
development of colorectal, gastric and esophageal cancer.
BACKGROUND OF THE INVENTION
[0002] The apical brush border membranes of normal mucosal cells
lining the small and large intestine express a specific receptor,
guanylyl cyclase C (gcC, hereinafter "ST receptor"). ST receptors
are unique in that they are localized in the apical brush border
membranes of normal cells lining the intestinal tract. Indeed, they
are not found in any other normal cell type in placental mammals.
It has been shown that normal gastric and esophageal cells do not
express the ST receptor. The discovery has been made that primary
and metastatic gastric and esophageal cancer cells express the ST
receptor. In addition, ST receptor transcription products, such as
mRNAs for the ST receptor protein and the ST receptor mRNA splice
variant CRCA-1 are markers for gastric and esophageal cancer
cells.
[0003] The ST receptors located in the lining of the small and
large intestine are almost exclusively localized to the apical
membranes, with little being found in the basolateral membranes on
the sides of intestinal cells. Of significance is the fact that
mucosal cells lining the intestine are joined together by tight
junctions which form a barrier against the passage of intestinal
contents into the blood stream and components of the blood stream
into the intestinal lumen. Therefore, the apical location of ST
receptors isolates these receptors from the circulatory system so
that they may be considered to exist "outside the body" and the
rest of the body is considered "outside the intestinal tract."
Compositions administered "outside the intestinal tract" are
maintained apart and segregated from the only cells which normally
express ST receptors.
[0004] E. coli produce a small heat-stable toxin (ST) that is
responsible for endemic diarrhea in developing countries and
travelers diarrhea. This toxin induces intestinal secretion by
binding to ST receptor in the apical brush border membranes of the
mucosal cells lining the small and large intestine. Binding of
toxin to these receptors triggers a cascade of biochemical
reactions in the apical membrane of these cells resulting in the
production of a signal which induces intestinal cells to secrete
fluids and electrolytes, resulting in diarrhea. Two homologous
peptides to ST are guanylin and uroguanylin, both of which are
produced locally in the intestine.
[0005] The discovery that a large proportion of metastasized
colorectal cells, and primary and metastatic gastric and esophageal
cancer cells, including those found in liver, lung, bone, brain,
nodes and peritoneum tissues, express ST receptors on their cell
surfaces, even at highly undifferentiated stages gave rise to
several inventions. Given that ST receptor expression is generally
tissue-specific to normal cells lining the inside of the colon, the
discovery of expression of ST receptors on the surfaces of
metastatic colorectal tumor cells, as well as primary and
metastatic gastric and esophageal cancer cells, provides a target
for delivering imaging and therapeutic agents to these cells and
provides a means for detecting and identifying cells of colorectal,
gastric and esophageal origin in samples.
[0006] U.S. Pat. No. 5,518,888, U.S. Pat. No. 5,879,656, U.S. Pat.
No. 6,060,037, and U.S. Pat. No. 6,268,159, which are each
incorporated herein by reference, describe technologies which
target metastasized colorectal tumor cells using compounds that
specifically bind to ST receptors. Compounds which bind to ST
receptors and which are either detectable or therapeutically active
are administered to an individual parenterally. The compounds
localize to the metastasized colorectal tumor cells through their
affinity to ST receptors expressed by the cells.
[0007] U.S. Pat. No. 5,601,990, U.S. Pat. No. 5,731,159, U.S. Pat.
No. 5,928,873, and U.S. Pat. No. 6,060,037, which are each
incorporated herein by reference, describe technologies which
detect ST receptor expression in extraintestinal samples as a means
of detecting the presence of metastatic colorectal tumor cells. ST
receptors and transcription products encoding ST receptors serve as
molecular markers whose presence in extraintestinal samples
indicates metastasized colorectal tumor cells and tumors of
colorectal origin. ST receptor protein and nucleic acid molecules
encoding ST receptor protein are detected in various ways such as,
for example, immunoassays and PCR assays.
[0008] In addition to its use for targeting in in vivo therapeutics
and diagnostic/imaging protocols as well as its use for targeting
in in vitro diagnostic protocols, the tissue specific expression of
ST receptor in cells of colorectal origin has also been exploited
in methods and compositions for drug delivery, for example gene
therapy. Cells that express ST receptor, i.e. cells of colorectal
origin, including normal as well as cancer cells or primary and
metastatic gastric and esophageal cells, can be specifically
targeted. U.S. Pat. No. 5,962,220 and U.S. Pat. No. 6,087,109,
which is incorporated herein by reference, describes the use of
compounds which bind to ST receptors such as native ST or other ST
receptor binding ligands, as ST receptor binding moieties used to
deliver nucleic acid molecules such as antisense agents to
colorectal cells by targeting ST receptors which are expressed by
such cells.
[0009] Moreover, vaccines have been designed which induce an immune
response against ST receptors and thus can be used to
prophylactically and/or therapeutically immunize an individual
against metastasized colorectal cancer. PCT application
PCT/US97/07565, which is incorporated herein by reference,
describes such vaccines and the uses therefor.
[0010] U.S. application Ser. No. 819,249 filed Mar. 27, 2000 and
published Oct. 11, 2001 as U.S. Application Publication Number
20010029019, which is incorporated herein by reference, describes
using ST receptor (GCC) as a marker for detection of Gastric and
Esophageal Cancer and as a target for delivery of imaging and
therapeutic agents against Gastric and Esophageal Cancer cells.
[0011] Moreover, vaccines have been designed which induce an immune
response against ST receptors and thus can be used to
prophylactically and/or therapeutically immunize an individual
against metastasized colorectal cancer. PCT application
PCT/US97/07565, which is incorporated herein by reference,
describes such vaccines and the uses therefor.
[0012] There remains a need for improved methods of inhibiting the
proliferation of colorectal, gastric and esophageal cancer cells.
There remains a need for improved methods of inhibiting primary
colorectal, gastric and esophageal cancer cells from metastasizing
and metastasized colorectal, gastric and esophageal cancer cells
from further metastasizing. There remains a need for improved
methods of treating primary and metastasized colorectal, gastric
and esophageal cancer in vivo. There remains a need for methods of
inhibiting the development of colorectal, gastric and esophageal
cancer from polyps and precancerous lesions in vivo. There remains
a need for improved in vitro methods of and reagents and kits for
treating metastasized colorectal and primary and metastatic gastric
and esophageal cancer. There remains an need for improved methods
of and compositions for treating, imaging and detecting
metastasized colorectal cancer in vivo. There remains an need for
improved in vitro methods of and reagents and kits for diagnosing
metastasized colorectal cancer and of identifying cancer as being
of colorectal origin. There remains an need for improved methods of
delivering compounds to colorectal cells. There remains a need for
improved vaccines for preventing and treating metastasized
colorectal cancer.
SUMMARY OF THE INVENTION
[0013] The present invention relates to in vivo and in vitro
methods of inhibiting the proliferation of human colorectal,
gastric and esophageal cancer cells.
[0014] The present invention further relates to methods of
administering to an individual an effective amount of ST receptor
binding agent for an effective amount of time to inhibit the of
proliferation of metastasized human colorectal cancer cells and
primary and metastatic human gastric and human esophageal cancer
cells.
[0015] The present invention relates to methods of inhibiting the
proliferation of metastasized colorectal, and primary and
metastasized gastric and esophageal cells in an individual. The
methods comprise the step of administering to the individual an
amount of an ST receptor ligand sufficient to result in an
inhibition of proliferation of human metastasized colorectal, and
primary and metastasized gastric and esophageal cancer cells. The
ST receptor ligand is administered to the individual in such a
manner as to provide a sufficient number of ST receptor ligand
molecules to come into contact with ST receptor proteins on the
surface of the colorectal, gastric and esophageal cells in the
individual for a sufficient time to result in an inhibition of the
proliferation of such cells.
[0016] The present invention relates to methods of treating an
individual identified as having metastatic colorectal, or primary
or metastatic gastric or esophageal cancer by inhibiting the
proliferation of metastasized colorectal cancer cells in an
individual who has metastasized colorectal cancer or primary or
metastasized gastric or esophageal cancer. The methods comprise the
step of administering to the individual an amount of an ST receptor
ligand sufficient to result in an inhibition of the proliferation
of metastatic colorectal cancer cells or primary or metastatic
gastric or esophageal cancer cells. The ST receptor ligand is
administered to the individual in such a manner as to provide a
sufficient number of ST receptor ligand molecules to come into
contact with ST receptor proteins on the surface of the
metastasized colorectal and primary and metastasized gastric and
esophageal cancer cells in the individual for a sufficient time to
result in an inhibition of the proliferation of the colorectal,
gastric and esophageal cancer cells or to induce a therapeutic
effect in the individual. This method may be followed by a further
step of administering an ST receptor ligand-therapeutic agent
conjugate.
[0017] The present invention relates to pharmaceutical compositions
that comprise sterile, pyrogen free ST receptor ligand and a
pharmaceutically acceptable carrier or diluent. The compositions
contain an amount of ST receptor ligand effective to have a
therapeutic effect in the treatment of cancer. The pharmaceutical
compositions may be formulated for delivery such that the patient
has a minimum titer of ST receptor ligand over a period of time
sufficient to have a therapeutic benefit.
[0018] The present invention relates to methods of treating an
individual who has metastasized colorectal cancer, as well as
primary and metastasized gastric or esophageal cancer. The methods
comprise the steps of administering an effective amount of ST
receptor ligand to an individual in a therapeutic pharmaceutical
composition for a period of time sufficient to result in an
inhibition of the proliferation of metastasized colorectal or
primary or metastasized gastric or esophageal cancer cells. The ST
receptor ligand is administered to the individual in such a manner
as to provide a sufficient number of ST receptor ligand molecules
to come into contact with ST receptor proteins on the surface of
the metastasized colorectal and primary and metastasized gastric
and esophageal cancer cells in the individual for a sufficient time
to result in an inhibition of proliferation or to cause a
therapeutic effect in the individual. This method may be followed
by a further step of administering a cell-division phase-specific
therapeutic agent. This method also may be followed by alternating
treatment steps using different phase-specific therapeutic
agents.
[0019] The present invention relates to methods of treating an
individual who has metastasized colorectal cancer, as well as
primary and metastasized gastric or esophageal cancer. A
pretreatment step of increasing the expression of ST receptor
proteins on the surface of such cells may be performed in order to
increase the number of ST receptor proteins expressed on the
surface of the cells. This step may be followed by the method of
the present invention directed to inhibiting the proliferation of
such cells. The methods comprise the steps of administering an
effective amount of ST receptor ligand to an individual in a
therapeutic pharmaceutical composition for a period of time
sufficient to result in an inhibition of the proliferation of
metastasized colorectal or primary or metastasized gastric or
esophageal cancer cells. The ST receptor ligand is administered to
the individual in such a manner as to provide a sufficient number
of ST receptor ligand molecules to come into contact with ST
receptor proteins on the surface of the metastasized colorectal and
primary and metastasized gastric and esophageal cancer cells in the
individual for a sufficient time to result in an inhibition of
proliferation or to cause a therapeutic effect in the
individual.
[0020] The present invention relates to in vivo and in vitro
methods of increasing the number of ST receptors on the surface of
human colorectal cells.
[0021] The present invention further relates to methods of
administering to an individual an effective amount of ST receptor
binding agent for an effective amount of time to induce an increase
in the number of ST receptors on the surface of metastasized human
colorectal cancer cells.
[0022] The present invention relates to methods of increasing the
number of ST receptor molecules on the surface of a colorectal cell
in an individual. The methods comprise the step of administering to
the individual an amount of an ST receptor ligand sufficient to
result in an increase in the presence of ST receptor protein on the
surface of the cell. The ST receptor ligand is administered to the
individual in such a manner to provide a sufficient number of ST
receptor ligand molecules to come into contact with ST receptor
proteins on the surface of the colorectal cell in the individual
for a sufficient time to result in an increase in the presence of
ST receptor protein on the surface of the cell.
[0023] The present invention relates to methods of increasing the
number of ST receptor molecules on the surface of a metastasized
colorectal cancer cell in an individual who has metastasized
colorectal cancer. The methods comprise the step of administering
to the individual an amount of an ST receptor ligand sufficient to
result in an increase in the presence of ST receptor protein on the
surface of the cell. The ST receptor ligand is administered to the
individual in such a manner to provide a sufficient number of ST
receptor ligand molecules to come into contact with ST receptor
proteins on the surface of the metastasized colorectal cancer cell
in the individual for a sufficient time to result in an increase in
the presence of ST receptor protein on the surface of the cell.
[0024] The present invention relates to pharmaceutical compositions
that comprise sterile, pyrogen free ST receptor ligand and a
pharmaceutically acceptable carrier or diluent. The compositions
contain at least 0.6 nM of ST receptor ligand.
[0025] The present invention relates to methods of treating an
individual who has metastasized colorectal cancer. The methods
comprise the steps of increasing the number of ST receptor
molecules on the surface of a metastasized colorectal cancer cell
in the individual and then administering a therapeutic
pharmaceutical composition that comprises components which target
ST receptors for delivery of a therapeutic agent to the individual.
The number of ST receptor molecules on the surface of a
metastasized colorectal cancer cell in the individual is increased
by administering to the individual an amount of an ST receptor
ligand sufficient to result in an increase in the number of ST
receptor protein molecules on the surface of the cell. The ST
receptor ligand is administered to the individual in such a manner
to provide a sufficient number of ST receptor ligand molecules to
come into contact with ST receptor proteins on the surface of the
metastasized colorectal cancer cell in the individual for a
sufficient time to result in an increase in the number of ST
receptor protein molecules on the surface of the cell.
[0026] The present invention relates to methods of imaging a
metastasized colorectal tumor in an individual who has metastasized
colorectal cancer. The methods comprise the steps of increasing the
number of ST receptor molecules on the surface of a metastasized
colorectal cancer cell in the individual and then administering an
pharmaceutical imaging composition that comprises components which
target ST receptor for delivery of an imaging agent. The number of
ST receptor molecules on the surface of a metastasized colorectal
cancer cell in the individual is increased by administering to the
individual an amount of an ST receptor ligand sufficient to result
in an increase in the number of ST receptor protein molecules on
the surface of the cell. The ST receptor ligand is administered to
the individual in such a manner to provide a sufficient number of
ST receptor ligand molecules to come into contact with ST receptor
proteins on the surface of the metastasized colorectal cancer cell
in the individual for a sufficient time to result in an increase in
the number of ST receptor protein molecules on the surface of the
cell.
[0027] The present invention relates to methods of determining
whether an individual has metastasized colorectal cancer. The
methods comprise the steps of increasing the number of ST receptor
molecules on the surface of a metastasized colorectal cancer cell
in the individual, obtaining a sample of extraintestinal body fluid
and/or tissue from the individual, and detecting the presence of
mRNA encoding ST receptor in the sample. The presence of the mRNA
indicates that the individual has metastatic colorectal cancer. The
number of ST receptor molecules on the surface of a metastasized
colorectal cancer cell in the individual is increased by
administering to the individual an amount of an ST receptor ligand
sufficient to result in an increase in the number of ST receptor
protein molecules on the surface of the cell. The ST receptor
ligand is administered to the individual in such a manner to
provide a sufficient number of ST receptor ligand molecules to come
into contact with ST receptor proteins on the surface of the
metastasized colorectal cancer cell in the individual for a
sufficient time to result in an increase in the number of ST
receptor protein molecules on the surface of the cell.
[0028] The present invention relates to methods of delivering an
active compound to a colorectal cell in an individual. The methods
comprise the steps of increasing the number of ST receptor
molecules on the surface of a colorectal cell in the individual.
The number of ST receptor molecules on the surface of a colorectal
cell in the individual is increased by administering to the
individual an amount of an ST receptor ligand sufficient to result
in an increase in the number of ST receptor protein molecules on
the surface of the cell. The ST receptor ligand is administered to
the individual in such a manner to provide a sufficient number of
ST receptor ligand molecules to come into contact with ST receptor
proteins on the surface of the colorectal cell in the individual
for a sufficient time to result in an increase in the number of ST
receptor protein molecules on the surface of the cell.
[0029] The present invention relates to methods of therapeutically
vaccinating an individual who has metastasized colorectal cancer,
i.e. inducing an immune response against metastatic colorectal
tumor cells. The methods comprise the steps of increasing the
number of ST receptor molecules on the surface of a metastasized
colorectal cancer cell in the individual. The number of ST receptor
molecules on the surface of a metastasized colorectal cancer cell
in the individual is increased by administering to the individual
an amount of an ST receptor ligand sufficient to result in an
increase in the number of ST receptor protein molecules on the
surface of the cell. The ST receptor ligand is administered to the
individual in such a manner to provide a sufficient number of ST
receptor ligand molecules to come into contact with ST receptor
proteins on the surface of the metastasized colorectal cancer cell
in the individual for a sufficient time to result in an increase in
the number of ST receptor protein molecules on the surface of the
cell.
[0030] The present invention relates to methods of treating an
individual who has primary colorectal, gastric or esophageal cancer
comprising the step of administering to the individual an effective
amount of ST receptor binding agent for an effective amount of time
to inhibit metastasis of human primary colorectal, gastric or
esophageal cancer cells. The ST receptor ligand is administered to
the individual in such a manner as to provide a sufficient number
of ST receptor ligand molecules to come into contact with ST
receptor proteins on the surface of primary colorectal, gastric or
esophageal cancer cells in the individual for a sufficient time to
result in an inhibition of metastasis.
[0031] The present invention relates to methods of treating an
individual who has metastatic colorectal, gastric or esophageal
cancer comprising the step of administering to the individual an
effective amount of ST receptor binding agent for an effective
amount of time to inhibit metastasis of human primary colorectal,
gastric or esophageal cancer cells. The ST receptor ligand is
administered to the individual in such a manner as to provide a
sufficient number of ST receptor ligand molecules to come into
contact with ST receptor proteins on the surface of metastatic
colorectal, gastric or esophageal cancer cells in the individual
for a sufficient time to result in an inhibition of metastasis.
[0032] The present invention further relates to pharmaceutical
compositions, including injectable pharmaceutical compositions,
that comprise an amount of an ST receptor ligand sufficient to
inhibit metastasis of human primary colorectal, gastric and
esophageal cancer in an individual who has primary colorectal,
gastric and esophageal cancer.
[0033] The present invention further relates to pharmaceutical
compositions, including injectable pharmaceutical compositions,
that comprise an amount of an ST receptor ligand sufficient to
inhibit further metastasis of human metastatic colorectal, gastric
and esophageal cancer in an individual who has metastatic
colorectal, gastric and esophageal cancer.
[0034] The present invention relates to methods of preventing
colorectal, gastric or esophageal cancer an individual who has
colorectal polyps or precancerous lesions in the stomach or
esophagus comprising the step of administering to the individual an
effective amount of ST receptor binding agent for an effective
amount of time to inhibit cells of the colorectal polyps or
precancerous lesions in the stomach or esophagus to transform into
cancer cells. The ST receptor ligand is administered to the
individual in such a manner as to provide a sufficient number of ST
receptor ligand molecules to come into contact with ST receptor
proteins on the surface of cells of the colorectal polyps or
precancerous lesions in the stomach or esophagus in the individual
for a sufficient time to result in an inhibit transformation of the
cells into cancer cells.
[0035] The present invention further relates to pharmaceutical
compositions, including injectable pharmaceutical compositions,
that comprise an amount of an ST receptor ligand sufficient to
inhibit transformation of cells of colorectal polyps or
precancerous lesions in the stomach or esophagus in colorectal,
gastric and esophageal cancer cells in an individual.
[0036] The present invention relates to methods of treating an
individual who has primary and/or metastatic human colorectal,
gastric or esophageal cancer. According to the present invention,
the methods comprise administering to such an individual, an
effective amount of ST receptor binding agent in combination with
an effective amount calcium to inhibit cell proliferation and
metastasis.
[0037] The present invention further relates to pharmaceutical
compositions, including injectable pharmaceutical compositions,
that comprise an ST receptor ligand in combination with Calcium in
an amount effective to inhibit cell proliferation and metastasis in
an individual with colorectal, gastric or esophageal in cancer.
[0038] The present invention relates to methods of treating an
individual who has polyps in the colon or precancerous lesions in
the stomach or esophagus. According to the present invention, the
methods comprise administering to such an individual, an ST
receptor binding agent in combination with Calcium in an amount
effective to inhibit transformation of the cells of colorectal
polyps or precancerous lesions in the stomach or esophagus into
cancer cells.
[0039] The present invention further relates to pharmaceutical
compositions, including injectable pharmaceutical compositions,
that comprise an ST receptor ligand, and Calcium in an amount
sufficient to inhibit transformation of cells of colorectal polyps
or precancerous lesions in the stomach or esophagus into
colorectal, gastric and esophageal cancer cells in an
individual.
BRIEF DESCRIPTION OF THE FIGURES
[0040] FIGS. 1a-1d illustrate the geographic imbalance between
colorectal cancer and infections with ETEC which produce
enterotoxins that suppress human colon carcinoma cell
proliferation. FIG. 1a shows worldwide geographic distribution of
ST-producing ETEC infections and the incidence of colorectal
cancer. The risk of infection with ST-producing ETEC was estimated
from the risk of travelers' diarrhea. The incidence of colorectal
cancer is represented as the age-adjusted rate using the World
Standard Population (ASR-W) and is expressed per 100,000. Linear
regression analysis was generated to fit the mean values for each
risk category. FIG. 1b shows data indicating ST (1 .mu.M) inhibits
DNA synthesis in colon cancer cells expressing GC-C [T84 cells;
P<0.01 for control vs. Student's t test (ST)] but not in those
cells that do not express GC-C (SW480 cells). Tumor cell
proliferation, % (y axis) is defined as the amount of DNA synthesis
in treated cells as a percentage of the amount of DNA synthesis in
parallel control cultures. FIG. 1c shows data indicating that in
T84 cells, the antiproliferative effects of ST correlate with
[cGMP]i but not intracellular cAMP accumulation. FIG. 1d shows that
inhibitors of downstream effectors of cGMP, including PKG (1 .mu.M
KT5823, 50 .mu.M RP8 pCPT-cGMP), cAMP-dependent protein kinase (0.5
.mu.M KT5720, 50 .mu.M RP-cAMPs), and cGMP-regulated
phosphodiesterase 3 [10 .mu.M milrinone (MRL)], did not alter the
inhibition of proliferation induced by ST. In contrast, an
inhibitor of CNG channels, L-DLT (200 .mu.M), blocked the effect of
ST on proliferation (P>0.1 for control vs. L-DLT, Student's t
test). The concentrations of inhibitors used are those that
selectively and completely inhibit their target enzymes: PKG-I and
(KT5823: Ki=234 nM; RP8 pCPT-cGMP: Ki=500 nM; refs. 20, 48, and
49), cAMP-dependent protein kinase I and II (KT5720: Ki=56 nM;
RP-cAMPs: Ki=10 .mu.M; refs. 50 and 51), and phosphodiesterase 3
(milrinone: Ki=300 nM; ref. 52). The concentration of L-DLT used
abolished ST-induced 45Ca2+ influx in the rat colon (40). Data are
the mean.+-.SEM of a representative experiment performed in
triplicate.
[0041] FIGS. 2a-2c show ST induces an L-DLT-sensitive current in
human colon carcinoma cells. FIG. 2a shows data from a time-course
of steady-state outward current recorded at the end of 200-ms-long
depolarizing rectangular pulses from a holding potential of -40 mV
to +30 mV. The period of drug application is indicated by
corresponding horizontal bars above the data plot. (Top) Original
current traces corresponding to specific points along the time
course under control conditions (1), following application of 500
nM ST (2), and in the combined presence of ST plus 200 .mu.M L-DLT
(3). FIG. 2b shows that average current at +30 mV under control
conditions, in the presence of ST, and in the presence of ST plus
L-DLT (n=6, meant SEM). FIG. 2c shows voltage-current relationships
obtained at the holding potential of -40 mV in response to 1-s-long
ramp pulses from -120 mV to +110 mV under control conditions, in
the presence of ST, and in the presence of ST plus L-DLT. ST
induced a significant shift of the reversal potential
(.DELTA.Em=-27.5.+-.2.6 mV, n=6, mean.+-.SEM) that was reversed by
L-DLT.
[0042] FIGS. 3a-3d show that 8-Br-cGMP induces an L-DLT-sensitive
current in, and inhibits proliferation of, human colon carcinoma
cells. FIG. 3a shows that [cGMP]i was increased by application of
the membrane-permeable analog, 8-br-cGMP (2.5. mM), which, in a
time-dependent manner, activated a membrane current sensitive to
200 .mu.M L-DLT. (Right) Original current traces recorded in
response to 200-ms depolarizing rectangular pulses from a holding
potential of -40 mV to +30 mV correspond to points along the time
course under control conditions (1), in the presence of 8-br-cGMP
(2), and in the combined presence of 8-br-cGMP plus L-DLT (3). FIG.
3b shows voltage-current relationships obtained at the holding
potential of -40 mV in response to 1-s-long ramp pulses from -100
mV to +110 mV under control conditions and in the presence of
8-br-cGMP. FIG. 3c shows average current at +30 mV in the presence
of 8-br-cGMP and in the presence of 8-br-cGMP plus L-DLT (n=4,
mean.+-.SEM). FIG. 3d shows inhibition of cell proliferation by
8-br-cGMP (5 mM) was blocked by 200 .mu.M L-DLT. Data are the
mean.+-.SEM of a representative experiment performed in
triplicate.
[0043] FIGS. 4a-4f show ST and 8-br-cGMP alter the membrane
conductance and proliferation of human colon carcinoma cells by
inducing CNG channel-mediated calcium influx. FIGS. 4a and 4b show
currents induced by ST (4a) and 8-br-cGMP (4b) are reversed in
calcium-free solution. Steady-state outward current recorded at the
end of 200-ms-long depolarizing rectangular pulses from a holding
potential of -40 mV to +10 mV. FIG. 4c shows average effect of ST
(white bars) and 8-br-cGMP (gray bars) in the presence (control;
1.8 mM CaCl2) and absence (Ca-free) of [Ca2+]ext, as well as in the
presence of 100 nM charybdotoxin, a specific inhibitor of Kca
channels (CTX in the presence of 1.8 mM CaCl2). Data are the
mean.+-.SEM of five separate experiments. (Inset) A representative
current-voltage relationship defining the effect of charybdotoxin
on ST-induced current obtained in response to 0.25 V/s ramped
membrane depolarization. FIG. 4d shows ST (1 .mu.M, 20 min) or
8-br-cGMP (5 mM, 40 min) induced influx of 45Ca2+ into colon cancer
cells, which was abolished by pretreatment (30 min) with 250 .mu.M
L-DLT. Results are expressed as percent increase over respective
controls and are the mean.+-.SEM of six (ST) or five (8-br-cGMP)
experiments performed in duplicate. FIG. 4e shows ST inhibits cell
proliferation, an effect abolished by the cytosolic calcium
chelator BAPTA-AM (BAPTA, 20 .mu.M). Dantrolene (DTR, 50 .mu.M),
which blocks Ca2+ mobilization from the endoplasmic reticulum, and
phenamil (PHE, 1 .mu.M), which blocks Na+/Ca2+ exchange as well as
CTX (100 nM), did not reverse ST-mediated inhibition of
proliferation. Ionomycin (INM, 1 .mu.M), a calcium ionophore,
mimicked on its own the effects of ST (1 .mu.M). Results are
expressed as percentage of respective controls and are the
mean.+-.SEM of a representative experiment performed in triplicate.
FIG. 4f shows ST inhibition of cell proliferation (left scale)
depended on [Ca2+]ext, with an EC50 estimated at 127 .mu.M. The
inability of ST to inhibit proliferation in the absence of
[Ca2+]ext did not reflect failure of ST to bind to GC-C, because
induction of [cGMP]i accumulation (right scale) by ST was
independent of [Ca2+]ext. Data are the mean.+-.SEM of a
representative experiment performed in triplicate.
DESCRIPTIONS OF PREFERRED EMBODIMENTS OF THE INVENTION
[0044] As used herein, the terms "ST" and "native ST" are used
interchangeably and are meant to refer to heat-stable toxin (ST)
which is a peptide produced by E. coli, as well as other organisms.
STs are naturally occurring peptides which 1) are naturally
produced by organisms, 2) which bind to the ST receptor and 3)
which activate the signal cascade that mediates ST-induced
diarrhea.
[0045] As used herein, the terms "ST receptor", "guanylyl cyclase
C" and "gcC" are meant to refer to the receptors found on
colorectal cells, including local and metastasized colorectal
cancer cells, as well as primary and metastatic gastric and
esophageal cancer cells, which bind to ST. In normal individuals,
ST receptors are found exclusively in cells of intestine, in
particular in cells in the duodenum, small intestine (jejunum and
ileum), the large intestine, colon (cecum, ascending colon,
transverse colon, descending colon and sigmoid colon) and rectum.
The nucleotide sequence of cDNA that encodes the human ST receptor
protein and the protein sequence are reported in de Sauvage F. J.
et al. (Sep. 25, 1991) Journal of Biological Chemistry
266(27):17912-8, which is incorporated herein by reference.
[0046] As used herein, the term "ST receptor ligand" is meant to
refer to compounds which specifically bind to the ST receptor. STs
are ST receptor ligands. Other ST receptor ligands include, but are
not limited to, guanylin and uroguanylin. An ST receptor ligand may
be a peptide or a non-peptide. ST receptor ligands may be
conjugated or unconjugated.
[0047] As used herein, the term "ST receptor binding peptide" is
meant to refer to ST receptor ligands that are peptides. STs are ST
receptor binding peptides. Guanylin and uroguanylin are homologous
ST receptor binding peptides.
[0048] As used herein, the term "ST peptides" is meant to refer to
ST receptor binding peptides selected from the group consisting of
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NOS:5-56 and fragments and
derivatives thereof.
[0049] As used herein, the term "fragment" is meant to refer to
peptide a) which has an amino acid sequence identical to a portion
of an ST receptor binding peptide and b) which is capable of
binding to the ST receptor.
[0050] As used herein, the term "derivative" is meant to refer to a
peptide a) which has an amino acid sequence substantially identical
to at least a portion of an ST receptor binding peptide and b)
which is capable of binding to the ST receptor.
[0051] As used herein, the term "substantially identical" is meant
to refer to an amino acid sequence that is the same as the amino
acid sequence of an ST peptide except some of the residues are
deleted or substituted with conservative amino acids or additional
amino acids are inserted.
[0052] As used herein, the term "homologous ST receptor peptide" is
meant to refer to peptides, other than ST, that bind to the ST
receptor.
[0053] As used herein, the term "active agent" is meant to refer to
compounds that are therapeutic agents or imaging agents.
[0054] As used herein, the term "radiostable" is meant to refer to
compounds which do not undergo radioactive decay; i.e. compounds
which are not radioactive.
[0055] As used herein, the term "therapeutic agent" is meant to
refer to chemotherapeutics, toxins, radiotherapeutics, targeting
agents or radiosensitizing agents.
[0056] As used herein, the term "chemotherapeutic" is meant to
refer to compounds that, when contacted with and/or incorporated
into a cell, produce an effect on the cell including causing the
death of the cell, inhibiting cell division or inducing
differentiation.
[0057] As used herein, the term "toxin" is meant to refer to
compounds that, when contacted with and/or incorporated into a
cell, produce the death of the cell.
[0058] As used herein, the term "radiotherapeutic" is meant to
refer to radionuclides which when contacted with and/or
incorporated into a cell, produce the death of the cell.
[0059] As used herein, the term "targeting agent" is meant to refer
compounds which can be bound by and or react with other compounds.
Targeting agents may be used to deliver chemotherapeutics, toxins,
enzymes, radiotherapeutics, antibodies or imaging agents to cells
that have targeting agents associated with them and/or to convert
or otherwise transform or enhance co-administered active agents. A
targeting agent may include a moiety that constitutes a first agent
that is localized to the cell which when contacted with a second
agent either is converted to a third agent which has a desired
activity or causes the conversion of the second agent into an agent
with a desired activity. The result is the localized agent
facilitates exposure of an agent with a desired activity to the
metastasized cell.
[0060] As used herein, the term "radiosensitizing agent" is meant
to refer to agents which increase the susceptibility of cells to
the damaging effects of ionizing radiation. A radiosensitizing
agent permits lower doses of radiation to be administered and still
provide a therapeutically effective dose.
[0061] As used herein, the term "imaging agent" is meant to refer
to compounds whose localization can be detected in vivo.
[0062] As used herein, the term "ST receptor binding moiety" is
meant to refer to the portion of a compound that constitutes an ST
receptor ligand.
[0063] As used herein, the term "active moiety" is meant to refer
to the portion of a compound that constitutes an active agent.
[0064] As used herein, the terms "conjugated compound" and
"conjugated composition" are used interchangeably and meant to
refer to a compound which comprises an ST receptor binding moiety
and an active moiety and which is capable of binding to the ST
receptor. Conjugated compounds according to the present invention
comprise a portion which constitutes an ST receptor ligand and a
portion which constitutes an active agent. Thus, conjugated
compounds according to the present invention are capable of
specifically binding to the ST receptor and include a portion which
is a therapeutic agent or imaging agent. Conjugated compositions
may comprise crosslinkers and/or molecules that serve as spacers
between the moieties.
[0065] As used herein, the term "non-colorectal sample" and
"extra-intestinal sample" are used interchangeably and meant to
refer to a sample of tissue or body fluid from a source other than
colorectal tissue. In some preferred embodiments, the
non-colorectal sample is a sample of tissue such as lymph nodes. In
some preferred embodiments, the non-colorectal sample is a sample
of extra-intestinal tissue which is an adenocarcinoma of
unconfirmed origin. In some preferred embodiments, the
non-colorectal sample is a blood sample.
[0066] As used herein, the term "colorectal cancer" is meant to
include the well-accepted medical definition that defines
colorectal cancer as a medical condition characterized by cancer of
cells of the intestinal tract below the small intestine (i.e. the
large intestine (colon), including the cecum, ascending colon,
transverse colon, descending colon, and sigmoid colon, and rectum).
Additionally, as used herein, the term "colorectal cancer" is meant
to further include medical conditions which are characterized by
cancer of cells of the duodenum and small intestine (jejunum and
ileum). The definition of colorectal cancer used herein is more
expansive than the common medical definition but is provided as
such since the cells of the duodenum and small intestine also
contain ST receptors and are therefore amenable to the methods of
the present invention using the compounds of the present
invention.
[0067] As used herein, the term "gastric cancer" is meant to
include the well-accepted medical definition that defines "gastric
cancer" as a medical condition characterized by cancer of cells of
the gastric cavity as defined by the esophogastric junction to the
pyloric sphincter. The term as used herein is also meant to refer
to the various forms of cancer of the stomach and esophagus and may
include some poorly defined precancerous conditions, such as, for
example, Barrett's esophagus and GERD.
[0068] As used herein, the term "esophageal cancer" is meant to
include the well-accepted medical definition that defines
"esophageal cancer" as a medical condition characterized by cancer
of cells of the esophagus as defined by the oral cavity, esophagus
and including the esophogastric junction. The term as used herein
is also meant to refer to the various forms of cancer of the
stomach and esophagus and may include some poorly defined
precancerous conditions, such as, for example, Barrett's esophagus
and GERD.
[0069] As used herein, the term "primary" tumor cell is meant to
refer to cancer cells which are not metastatic in character and are
located in the specific tissue or organ in which the cancer
originated. The present invention relates to methods of delivering
active agents to primary gastric and esophageal cancer cells.
[0070] As used herein, the term "metastasis" or "metastatic" is
meant to refer to the process in which cancer cells originating in
one organ or part of the body relocate to another part of the body
and continue to replicate. Metastasized cells subsequently form
tumors which may further metastasize. Metastasis thus refers to the
spread of cancer from the part of the body where it originally
occurs to other parts of the body. The present invention relates to
methods of delivering active agents to metastasized colorectal,
gastric and esophageal cancer cells.
[0071] As used herein, the terms "the presence of mRNA encoding ST
receptor in a non-colorectal or an extra-intestinal sample" and
"the presence of mRNA encoding ST receptor in said sample" are
meant to refer to mRNA levels above those observed due to
illegitimate transcription. Illegitimate transcription is
responsible for background levels of ST receptor expression in
non-colorectal cells.
[0072] It has been discovered that the proliferation of colorectal,
gastric and esophageal cancer cells can be inhibited by contacting
the cell with an ST receptor ligand that binds to ST receptors on
the cell surface, i.e. an ST receptor ligand. It is known that the
cell cycle effect of ST on colorectal, esophageal and gastric
cancer cells is instantaneous and % that the cells take a complete
cell cycle to recover. It also has been shown that the binding of
an ST receptor ligand to an ST receptor protein stimulates the
accumulation of intracellular concentrations of cGMP. The
cytostatic effect of cGMP induced by ST receptor ligands, inhibits
the proliferation of human colorectal, gastric and esophageal
cancer cells. It has also been discovered that the cytostatic
effect of the cGMP accumulation is not mediated by specific cyclic
nucleotide-dependent kinases, but, rather a novel molecular
mechanism, not previously defined.
[0073] There are several applications for methods of inhibiting the
proliferation of cancer cells of colorectal, gastric or esophageal
origin. For example, cells outside the intestinal tract that
express ST receptors are metastasized colorectal cancer cells or
primary or metastatic gastric or esophageal cancer cells. The ST
receptors are targets for in vivo delivery of ST receptor binding
ligand to inhibit the proliferation of colorectal, gastric and
esophageal cancer cells. The discovery of a means to inhibit the
proliferation of such cancer cells by contacting the ST receptor
with an effective amount of ST receptor binding protein is useful
in conjunction with known therapeutic methods to enhance the
effectiveness of such methods. Accordingly, the present invention
provides for improved in vivo therapeutic methods comprising the
steps of first inhibiting the proliferation of metastatic
colorectal and primary and metastatic gastric and esophageal cancer
cells and then treating with a therapeutic agent. The present
invention also provides therapeutic methods for treating primary
colorectal cancer.
[0074] In addition, it has been discovered that metastasis of
primary colorectal, gastric and esophageal cancer cells as well as
further metastasis of metastasized colorectal, gastric and
esophageal cancer cells can be inhibited by contacting the cell
with an ST receptor ligand that binds to ST receptors on the cell
surface, i.e. an ST receptor ligand. There are several applications
for methods of inhibiting metastasis of colorectal, gastric or
esophageal cancer cells. For example, administration of an amount
of ST receptor ligand effective to inhibit metastasis can be
undertaken upon initial diagnosis of primary cancer. The
administration can be directed at the site of the primary cancer as
well as administered systemically. In some embodiments, the
administration of an amount of ST receptor ligand effective to
inhibit metastasis is undertaken before during or after surgical
removal of the cancer and surrounding tissue. The site of cancer as
well as other tissue of the effected organ is contacted directly
with ST receptor ligand. If performed during surgery, the organ may
be washed with a solution containing the ST receptor ligand. If
performed prior to or post-surgery, the ST receptor ligand is
delivered to the organ. Systemic administration also may be
performed in newly diganosed patients in order to prevent
metastasis arising from any undetected metastasized cancer
including micrometastses. The administration of an amount of ST
receptor ligand effective to inhibit metastasis may be undertaken
upon discovery of metastatic disease including directly at the site
of any metastic cancer as well as systemically. The ST receptors
are targets for in vivo delivery of ST receptor binding ligand to
inhibit the metastasis of colorectal, gastric and esophageal cancer
cells. The effective amount of ST receptor binding protein to
inhibit metastas may be delivered in conjunction with known
therapeutic methods to enhance the effectiveness of such methods.
Accordingly, the present invention provides for improved in vivo
therapeutic methods comprising the steps of first inhibiting
metastasis of primary and metastatic colorectal, gastric and
esophageal cancer cells and then treating with a therapeutic agent.
The present invention also provides therapeutic methods for
treating primary colorectal cancer.
[0075] Another embodiment of the invention arises from the
discovery that treatment of precancerous polyps and lesions in the
colon, stomach or esophagus can prevent such precancerous cells and
tissue from becoming transformed into cancer. Accordingly, the
present invention provides methods of preventing precancerous
polyps and lesions in the colon, stomach or esophagus in an
individual from becoming transformed into cancer by administering
an amount of ST receptor ligand sufficient to inhibit such
transformation. The ST receptor ligand may be administered directly
to the polyps or lesions or in some cases administered
systemically. In some embodiments, upon detection of precancerous
polyps or lesions in an individual, an amount of ST receptor ligand
sufficient to inhibit transformation of such polyps or lesions is
administered to the individual. In some embodiments, individuals
may undergo a preventive course of ST receptor ligand without prior
detection of precancerous conditions. In such embodiments, the
individual is administered an amount of ST receptor ligand
sufficient to inhibit transformation of such polyps or lesions
either by administration of an amount of ST receptor ligand
effective to inhibit transformation directly to the esophagus,
stomach or colorectal tract or systemically.
[0076] The method of inhibiting the proliferation of colorectal,
gastric and esophageal cancer cells followed by treatment with
known therapeutic agents described above can be followed by another
step of imaging in order to determine the extent of the decrease in
proliferation of the colorectal, gastric and esophageal cancer
cells.
[0077] It has been discovered that the number of ST receptors on
the cell surface of a cell that expresses ST receptors, i.e. a cell
of colorectal origin, can be increased by contacting the cell with
a ligand which binds to ST receptors on the cell surface, i.e. an
ST receptor ligand. ST receptor protein/ST receptor ligand binding
upregulates ST receptor gene expression or otherwise affects the
cells to result in an increase in the number of ST receptors on the
cell surface of a cell. There are several applications for methods
of increasing the number of ST receptors on cells of colorectal
origin. For example, cells outside the intestinal tract that
express ST receptors are metastasized colorectal cancers and the ST
receptors are targets for in vivo delivery of therapeutic and
imaging agents to treat and image, respectively, metastatic
colorectal cancer. The discovery of a means to upregulate ST
receptor expression is useful to enhance the effectiveness of such
methods. Accordingly, the present invention provides for improved
in vivo therapeutic and imaging/diagnostic methods by increasing
the number of targets for therapeutic and imaging agents on
metastatic colorectal cancer cells. Similarly, in vitro
screening/diagnostic methods which employ the detection of ST
receptors or evidence of the expression thereof in extraintestinal
samples as a marker for metastatic colorectal cancer. Upregulation
of ST receptor expression is useful to improve in vitro diagnostic
assays by increasing the amount of marker, protein or nucleic acid,
to be detected. In addition, ST receptors are used to target and
facilitate delivery of material to cells of colorectal origin
including normal cells and cells from primary tumors as well as
metastatic disease. Compounds such as gene therapeutics, antisense
compounds, therapeutic proteins and pharmacologically active
compounds conjugated to ST receptor ligands are targeted to and
taken up by cells expressing ST receptors. In applications where ST
receptors are targeted for delivery of compounds to cells, such as,
for example, the delivery of therapeutic or imaging agents to
metastasized colorectal cancer cells or the delivery of therapeutic
or prophylactic compounds such as, for example, genetic material to
normal colon cells or primary colon cancer tumors, increasing the
number of ST receptors on the surface of the cell increases the
delivery of the compounds to the cell.
[0078] High specificity of binding of ST receptor ligand to ST
receptors is desired for better delivery of compounds. Specificity
has been found to be directly correlated to the number of
binding-site receptors on the subject cell. The only theoretical
limitation to the utilization of targeting imaging and therapeutic
agents to ST receptors is the number of ST receptors on the
colorectal cells. The present invention has the potential of
increasing the number of guanylyl cyclase C molecules on the
surface of colorectal cancer cells 5- to 10-fold. The occurrence of
ST receptors on individual colorectal cells occur on the order of
10.sup.4 to 10.sup.6 receptors per cell and have an affinity of
10.sup.-7 or better. Metastasized colorectal tumors exhibit similar
features.
[0079] According to the invention, proliferation in cells that
express ST receptors is inhibited using ST receptor ligands, or
homologues of ST receptor ligands. In some preferred embodiments,
the ST receptor ligand is a homologue of ST receptor binding
ligand, such as uroguanylin or guanylin. In other preferred
embodiments, the ST receptor ligand is an ST receptor binding
peptide. In some preferred embodiments, the ST receptor ligand is
an ST receptor peptide selected from the group consisting of: SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NOS:5-56 and fragments and derivatives
thereof. In some preferred embodiments, the ST receptor ligand is a
modified ST receptor binding peptide which is chemically altered to
prevent or inhibit degradation in vivo. For example, N terminal
capping to prevent or inhibit N terminal peptidases is well known
and can be routinely undertaken. In some preferred embodiments, the
ST receptor ligand is an anti-ST receptor antibody. In some
preferred embodiments, the ST receptor ligand is an anti-ST
receptor monoclonal antibody.
[0080] According to the invention, ST receptor expression in cells
which express ST receptors is increased using ST receptor ligands.
In some preferred embodiments, the ST receptor ligand is an ST
receptor binding peptide. In some preferred embodiments, the ST
receptor ligand is an ST receptor peptide selected from the group
consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NOS:5-56 and
fragments and derivatives thereof. In some preferred embodiments,
the ST receptor ligand is a modified ST receptor binding peptide
which is chemically altered to prevent or inhibit degradation in
vivo. For example, N terminal capping to prevent or inhibit N
terminal peptidases is well known and can be routinely undertaken.
In some preferred embodiments, the ST receptor ligand is an anti-ST
receptor antibody. In some preferred embodiments, the ST receptor
ligand is an anti-ST receptor monoclonal antibody.
[0081] SEQ ID NO:1 discloses a nucleotide sequence which encodes 19
amino acid ST, designated ST Ia, reported by So and McCarthy (1980)
Proc. Natl. Acad. Sci. USA 77:4011, which is incorporated herein by
reference.
[0082] The amino acid sequence of ST Ia is disclosed in SEQ ID
NO:2.
[0083] SEQ ID NO:3 discloses the amino acid sequence of an 18 amino
acid peptide which exhibits ST activity, designated ST I*, reported
by Chan and Giannella (1981) J. Biol. Chem. 256:7744, which is
incorporated herein by reference.
[0084] SEQ ID NO:4 discloses a nucleotide sequence which encodes 19
amino acid ST, designated ST Ib, reported by Mosely et al. (1983)
Infect. Immun. 39:1167, which is incorporated herein by
reference.
[0085] The amino acid sequence of ST Ib is disclosed in SEQ ID
NO:5.
[0086] A 15 amino acid peptide called guanylin which has about 50%
sequence homology to ST has been identified in mammalian intestine
(Currie, M. G. et al. (1992) Proc. Natl. Acad. Sci. USA 89:947-951,
which is incorporated herein by reference). Guanylin binds to ST
receptors and activates guanylate cyclase at a level of about 10-
to 100-fold less than native ST. Guanylin may not exist as a 15
amino acid peptide in the intestine, gastric or esophagus but
rather as part of a larger protein in that organ. The amino acid
sequence of guanylin from rodent is disclosed as SEQ ID NO:6.
[0087] SEQ ID NO:7 is an 18 amino acid fragment of SEQ ID NO:2. SEQ
ID NO:8 is a 17 amino acid fragment of SEQ ID NO:2. SEQ ID NO:9 is
a 16 amino acid fragment of SEQ ID NO:2. SEQ ID NO:10 is a 15 amino
acid fragment of SEQ ID NO:2. SEQ ID NO:11 is a 14 amino acid
fragment of SEQ ID NO:2. SEQ ID NO:12 is a 13 amino acid fragment
of SEQ ID NO:2. SEQ ID NO:13 is an 18 amino acid fragment of SEQ ID
NO:2. SEQ ID NO:14 is a 17 amino acid fragment of SEQ ID NO:2. SEQ
ID NO:15 is a 16 amino acid fragment of SEQ ID NO:2. SEQ ID NO:16
is a 15 amino acid fragment of SEQ ID NO:2. SEQ ID NO:17 is a 14
amino acid fragment of SEQ ID NO:2.
[0088] SEQ ID NO:18 is a 17 amino acid fragment of SEQ ID NO:3. SEQ
ID NO:19 is a 16 amino acid fragment of SEQ ID NO:3. SEQ ID NO:20
is a 15 amino acid fragment of SEQ ID NO:3. SEQ ID NO:21 is a 14
amino acid fragment of SEQ ID NO:3. SEQ ID NO:22 is a 13 amino acid
fragment of SEQ ID NO:3. SEQ ID NO:23 is a 17 amino acid fragment
of SEQ ID NO:3. SEQ ID NO:24 is a 16 amino acid fragment of SEQ ID
NO:3. SEQ ID NO:25 is a 15 amino acid fragment of SEQ ID NO:3. SEQ
ID NO:26 is a 14 amino acid fragment of SEQ ID NO:3.
[0089] SEQ ID NO:27 is an 18 amino acid fragment of SEQ ID NO:5.
SEQ ID NO:28 is a 17 amino acid fragment of SEQ ID NO:5. SEQ ID
NO:29 is a 16 amino acid fragment of SEQ ID NO:5. SEQ ID NO:30 is a
15 amino acid fragment of SEQ ID NO:5. SEQ ID NO:31 is a 14 amino
acid fragment of SEQ ID NO:5. SEQ ID NO:32 is a 13 amino acid
fragment of SEQ ID NO:5. SEQ ID NO:33 is an 18 amino acid fragment
of SEQ ID NO:5. SEQ ID NO:34 is a 17 amino acid fragment of SEQ ID
NO:5. SEQ ID NO:35 is a 16 amino acid fragment of SEQ ID NO:5. SEQ
ID NO:36 is a 15 amino acid fragment of SEQ ID NO:5. SEQ ID NO:37
is a 14 amino acid fragment of SEQ ID NO:5.
[0090] SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:36 AND SEQ ID NO:37
are disclosed in Yoshimura, S., et al. (1985) FEBS Lett. 181:138,
which is incorporated herein by reference.
[0091] SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, which are
derivatives of SEQ ID NO:3, are disclosed in Waldman, S. A. and
O'Hanley, P. (1989) Infect. Immun. 57:2420, which is incorporated
herein by reference.
[0092] SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and
SEQ ID NO:45, which are a derivatives of SEQ ID NO:3, are disclosed
in Yoshimura, S., et al. (1985) FEBS Lett. 181:138, which is
incorporated herein by reference.
[0093] SEQ ID NO:46 is a 25 amino acid peptide derived from Y.
enterocolitica which binds to the ST receptor.
[0094] SEQ ID NO:47 is a 16 amino acid peptide derived from V.
cholerae which binds to the ST receptor. SEQ ID NO:47 is reported
in Shimonishi, Y., et al. FEBS Lett. 215:165, which is incorporated
herein by reference.
[0095] SEQ ID NO:48 is an 18 amino acid peptide derived from Y.
enterocolitica which binds to the ST receptor. SEQ ID NO:48 is
reported in Okamoto, K., et al. Infec. Immun. 55:2121, which is
incorporated herein by reference.
[0096] SEQ ID NO:49, is a derivative of SEQ ID NO:5.
[0097] SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53
are derivatives.
[0098] SEQ ID NO:54 is the amino acid sequence of guanylin from
human.
[0099] SEQ ID NO:55 is the amino acid sequence of uroguanylin from
rat. Miyazato, M., et al, FEBS Lett. 398 (2-3), 170-174 (1996); and
Blanchard, R. K. and Cousins, R. J., Am. J. Physiol. 272 (5 Pt 1),
G972-G978 (1997) which are incorporated herein by reference.
[0100] SEQ ID NO:56 is the amino acid sequence of uroguanylin from
human. Hill, O., et al, Biochim. Biophys. Acta 1253 (2), 146-149
(1995); Miyazato,M., et al., Biochem. Biophys. Res. Commun. 219
(2), 644-648 (1996); and Miyazato, M., et al., Genomics 43 (3),
359-365 (1997) which are incorporated herein by reference.
[0101] In some preferred embodiments, conjugated compounds comprise
ST receptor binding moieties that comprise amino acid sequences
selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NOS:5-56 and fragments and derivatives thereof.
[0102] Those having ordinary skill in the art can readily design
and produce derivatives having substantially identical amino acid
sequences of ST peptides with deletions and/or insertions and/or
conservative substitutions of amino acids. For example, following
what are referred to as Dayhofs rules for amino acid substitution
(Dayhof, M.D. (1978) Nat. Biomed. Res. Found., Washington, D.C.
Vol. 5, supp. 3), amino acid residues in a peptide sequence may be
substituted with comparable amino acid residues. Such substitutions
are well known and are based the upon charge and structural
characteristics of each amino acid. Derivatives include fragments
of ST receptor binding peptides with deletions and/or insertions
and/or conservative substitutions.
[0103] In some embodiments, ST receptor binding peptides comprise D
amino acids. As used herein, the term "D amino acid peptides" is
meant to refer to ST receptor binding peptides, fragments or
derivatives which comprise at least one and preferably a plurality
of D amino acids which are capable of binding to the ST receptor.
The use of D amino acid peptides is desirable as they are less
vulnerable to degradation and therefore have a longer
half-life.
[0104] In some embodiments, ST receptor binding peptides, including
D amino acid peptides, are conformationally restricted to present
and maintain the proper structural conformation for binding to the
ST receptor. The compositions may comprise additional amino acid
residues required to achieve proper three dimensional conformation
including residues which facilitate circularization or desired
folding.
[0105] It is preferred that the ST receptor ligand be as small as
possible. Thus it is preferred that the ST receptor ligand be a
non-peptide small molecule or small peptide, preferably less than
25 amino acids, more preferably less than 20 amino acids. In some
embodiments, the ST receptor ligand is less than 15 amino acids. ST
receptor binding peptides comprising less than 10 amino acids and
ST receptor binding peptides less than 5 amino acids may also be
used. It is within the scope of the present invention to include
larger molecules which serve as ST receptor binding moieties
including, but not limited to molecules such as antibodies,
fragments of antibodies, FAbs and F(Ab).sub.2s which specifically
bind to ST receptor.
[0106] ST may be isolated from natural sources using standard
techniques. Additionally, ST receptor binding peptides and
conjugated compositions or portions thereof which are peptides may
be prepared routinely by any of the following known techniques.
[0107] Antibodies include polyclonal and monoclonal antibodies as
well as Fab fragments, F(ab).sub.2 fragments and other
modifications and products of antibody engineering. Humanized,
primatized and modified forms of antibodies to render them less
immunogenic are antibodies according to the invention and may be
used in the methods of the invention. Antibodies which specifically
recognize ST receptor protein have been generated previously and
are reported in Vaandrager et al., 1993 J. Biolog. Chem. 268:2174,
which is incorporated herein by reference. Those having ordinary
skill in the art can produce monoclonal antibodies which
specifically bind to ST receptor using standard techniques and
readily available starting materials. The techniques for producing
monoclonal antibodies are outlined in Harlow, E. and D. Lane,
(1988) ANTIBODIES: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor N.Y., which is incorporated herein
by reference, provide detailed guidance for the production of
hybridomas and monoclonal antibodies which specifically bind to
target proteins. Briefly, the ST receptor molecule or a molecule
which includes an epitope that is immunogenically cross reactive to
ST receptor is injected into mice. The spleen of the mouse is
removed, the spleen cells are isolated and fused with immortalized
mouse cells. The hybrid cells, or hybridomas, are cultured and
those cells which secrete antibodies are selected. The antibodies
are analyzed and, if found to specifically bind to the protein of
interest, the hybridoma which produces them is cultured to produce
a continuous supply of antigen specific antibodies. Techniques for
engineering antibodies are described in U.S. Pat. No. 5,530,101,
U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,225,539, Winter and
Millstein (1991) Nature 349:293, and Larrich and Fry (1991) Hum.
Antibod. and Hybridomas 2:17, which are each incorporated herein by
reference.
[0108] ST receptor binding ligands may be identified by routine
screening technology to identify compounds that bind to ST
receptor. It is preferred that such compounds bind to ST receptor
with an affinity of greater than about that of lymphoguanylin (SEQ
ID NO:54) and more preferably that of E. coli heat stable
enterotoxin (SEQ ID NO:2).
[0109] Technology is widely available for screening libraries of
compounds including peptides and non-peptides to identify those
that bind to proteins such as Brenner, S, and R. A. Lerner, Proc.
Natl. Acad. Sci. USA 89:5381-5383 (June 1992); Cull, M. G. et al.,
Proc. Natl. Acad. Sci. USA 89:1865-1869 (March 1992); and Fodor, S.
P. A. et al. Science 251:767-773 (Feb. 15, 1991), which are each
incorporated herein by reference. The following patents, which are
each incorporated herein by reference, describe methods of making
random peptide or non-peptide libraries and screening such
libraries to identify compounds that bind to target proteins. As
used in the present invention, ST receptors can be the targets used
to identify the peptide and non-peptide ST receptor ligands
generated and screened as disclosed in the patents. U.S. Pat. No.
5,270,170 issued to Schatz et al. on Dec. 14, 1993, and U.S. Pat.
No. 5,338,665 issued to Schatz et al. on Aug. 16, 1994, which are
both incorporated herein by reference, refer to peptide libraries
and screening methods which can be used to identify ST receptor
ligands according to the invention. U.S. Pat. No. 5,395,750 issued
to Dillon et al. on Mar. 7, 1995, which is incorporated herein by
reference, refers to methods of producing proteins which bind to
predetermined antigens. Such methods can be used to produce ST
receptor ligands according to the invention. U.S. Pat. No.
5,223,409 issued to Ladner et al. on Jun. 29, 1993, which is
incorporated herein by reference, refers to the directed evolution
to novel binding proteins. Such proteins may be produced and
screened as disclosed therein to identify ST receptor ligands
according to the invention. U.S. Pat. No. 5,366,862 issued to
Venton et al. on Nov. 22, 1994, which is incorporated herein by
reference, refers to methods for generating and screening useful
peptides. The methods herein described can be used to identify ST
receptor ligands according to the invention. U.S. Pat. No.
5,340,474 issued to Kauvar on Aug. 23, 1994 as well as U.S. Pat.
No. 5,133,866, U.S. Pat. No. 4,963,263 and U.S. Pat. No. 5,217,869,
which are each incorporated herein by reference, can be used to
identify ST receptor ligands according to the invention. U.S. Pat.
No. 5,405,783 issued to Pirrung et al. on Apr. 11, 1995, which is
incorporated herein by reference, refers to large scale
photolithographic solid phase synthesis of an array of polymers.
The teachings therein can be used to identify ST receptor ligands
according to the invention. U.S. Pat. No. 5,143,854 issued to
Pirrung et al. on Sep. 1, 1992, which is incorporated herein by
reference, refers to a large scale photolithographic solid phase
synthesis of polypeptides and receptor binding screening thereof.
U.S. Pat. No. 5,384,261 issued to Winkler et al. on Jan. 24, 1995,
which is incorporated herein by reference, refers to very large
scale immobilized polymer synthesis using mechanically directed
flow patterns. Such methods are useful to identify ST receptor
ligands according to the invention. U.S. Pat. No. 5,221,736 issued
to Coolidge et al. on Jun. 22, 1993, which is incorporated herein
by reference, refers to sequential peptide and oligonucleotide
synthesis using immunoaffinity techniques. Such techniques may be
used to identify ST receptor ligands according to the invention.
U.S. Pat. No. 5,412,087 issued to McGall et al. on May 2, 1995,
which is incorporated herein by reference, refers to spatially
addressable immobilization of oligonucleotides and other biological
polymers on surfaces. Such methods may be used to identify ST
receptor ligands according to the invention. U.S. Pat. No.
5,324,483 issued to Cody et al. on Jun. 28, 1994, which is
incorporated herein by reference, refers to apparatus for multiple
simultaneous synthesis. The apparatus and method disclosed therein
may be used to produce multiple compounds which can be screened to
identify ST receptor ligands according to the invention. U.S. Pat.
No. 5,252,743 issued to Barrett et al. on Oct. 12, 1993, which is
incorporated herein by reference, refers to spatially addressable
immobilization of anti-ligands on surfaces. The methods and
compositions described therein may be used to identify ST receptor
ligands according to the invention. U.S. Pat. No. 5,424,186 issued
to Foder et al. on Jun. 13, 1995, which is incorporated herein by
reference, refers to a very large scale immobilized polymer
synthesis. The method of synthesizing oligonucleotides described
therein may be used to identify ST receptor ligands according to
the invention. U.S. Pat. No. 5,420,328 issued to Campbell on May
30, 1995, which is incorporated herein by reference, refers to
methods of synthesis of phosphonate esters. The phosphonate esters
so produced may be screened to identify compounds which are ST
receptor ligands. U.S. Pat. No. 5,288,514 issued to Ellman on Feb.
22, 1994, which is incorporated herein by reference, refers to
solid phase and combinatorial synthesis of benzodiazepine compounds
on a solid support. Such methods and compounds may be used to
identify ST receptor ligands according to the invention.
[0110] An assay may be used to test both peptide and non-peptide
compositions to determine whether or not they are ST receptor
ligands or, to test conjugated compositions to determine if they
possess ST receptor binding activity. Such compositions that
specifically bind to ST receptors can be identified by a
competitive binding assay. The competitive binding assay is a
standard technique in pharmacology which can be readily performed
by those having ordinary skill in the art using readily available
starting materials. Competitive binding assays have been shown to
be effective for identifying compositions that specifically bind to
ST receptors. Briefly, the assay consists of incubating a
preparation of ST receptors (e.g. intestinal membranes from rat
intestine, human intestine, T84 cells) with a constant
concentration (1.times.10.sup.-10 M to 5.times.10.sup.-10 M) of
.sup.125I-ST (any ST receptor ligand such as native STs SEQ ID
NO:2, SEQ ID NO:3 or SEQ ID NO:5 may be used) and a known
concentration of a test compound. As a control, a duplicate
preparation of ST receptors are incubated with a duplicate
concentration of .sup.125I-ST in the absence of test compound.
Assays are incubated to equilibrium (2 hours) and the amount of
.sup.125I-ST bound to receptors is quantified by standard
techniques. The ability of the test compound to bind to receptors
is measured as its ability to prevent (compete with) the
.sup.125I-ST from binding. Thus, in assays containing the test
compound which bind to the receptor, there will be less
radioactivity associated with the receptors. This assay, which is
appropriate for determining the ability of any molecule to bind to
ST receptors, is a standard competitive binding assay which can be
readily employed by those having ordinary skill in the art using
readily available starting materials.
[0111] ST receptor binding peptides and conjugated compositions or
portions thereof which are peptides may be prepared using the
solid-phase synthetic technique initially described by Merrifield,
in J. Am. Chem. Soc., 15:2149-2154 (1963). Other peptide synthesis
techniques may be found, for example, in M. Bodanszky et al.,
(1976) Peptide Synthesis, John Wiley & Sons, 2d Ed.; Kent and
Clark-Lewis in Synthetic Peptides in Biology and Medicine, p.
295-358, eds. Alitalo, K., et al. Science Publishers, (Amsterdam,
1985); as well as other reference works known to those skilled in
the art. A summary of peptide synthesis techniques may be found in
J. Stuart and J. D. Young, Solid Phase Peptide Synthelia, Pierce
Chemical Company, Rockford, Ill. (1984), which is incorporated
herein by reference. The synthesis of peptides by solution methods
may also be used, as described in The Proteins, Vol. II, 3d Ed., p.
105-237, Neurath, H. et al., Eds., Academic Press, New York, N.Y.
(1976). Appropriate protective groups for use in such syntheses
will be found in the above texts, as well as in J. F. W. McOmie,
Protective Groups in Organic Chemistry, Plenum Press, New York,
N.Y. (1973), which is incorporated herein by reference. In general,
these synthetic methods involve the sequential addition of one or
more amino acid residues or suitable protected amino acid residues
to a growing peptide chain. Normally, either the amino or carboxyl
group of the first amino acid residue is protected by a suitable,
selectively removable protecting group. A different, selectively
removable protecting group is utilized for amino acids containing a
reactive side group, such as lysine.
[0112] Using a solid phase synthesis as an example, the protected
or derivatized amino acid is attached to an inert solid support
through its unprotected carboxyl or amino group. The protecting
group of the amino or carboxyl group is then selectively removed
and the next amino acid in the sequence having the complementary
(amino or carboxyl) group suitably protected is admixed and reacted
with the residue already attached to the solid support. The
protecting group of the amino or carboxyl group is then removed
from this newly added amino acid residue, and the next amino acid
(suitably protected) is then added, and so forth. After all the
desired amino acids have been linked in the proper sequence, any
remaining terminal and side group protecting groups (and solid
support) are removed sequentially or concurrently, to provide the
final peptide. The peptide of the invention are preferably devoid
of benzylated or methylbenzylated amino acids. Such protecting
group moieties may be used in the course of synthesis, but they are
removed before the peptides are used. Additional reactions may be
necessary, as described elsewhere, to form intramolecular linkages
to restrain conformation.
[0113] ST receptor binding peptides and conjugated compositions or
portions thereof which are peptides may also be prepared by
recombinant DNA techniques. Provision of a suitable DNA sequence
encoding the desired peptide permits the production of the peptide
using recombinant techniques now known in the art. The coding
sequence can be obtained from natural sources or synthesized or
otherwise constructed using widely available starting materials by
routine methods. When the coding DNA is prepared synthetically,
advantage can be taken of known codon preferences of the intended
host where the DNA is to be expressed.
[0114] To produce an ST receptor binding peptide which occurs in
nature, one having ordinary skill in the art can, using well-known
techniques, obtain a DNA molecule encoding the ST receptor binding
peptides from the genome of the organism that produces the ST
receptor binding peptide and insert that DNA molecule into a
commercially available expression vector for use in well-known
expression systems.
[0115] Likewise, one having ordinary skill in the art can, using
well known techniques, combine a DNA molecule that encodes an ST
receptor binding peptide, such as SEQ ID NO:1 and SEQ ID NO:4,
which can be obtained from the genome of the organism that produces
the ST, with DNA that encodes a toxin, another active agent that is
a peptide or additionally, any other amino acid sequences desired
to be adjacent to the ST receptor binding peptide amino acid
sequence in a single peptide and insert that DNA molecule into a
commercially available expression vector for use in well-known
expression systems.
[0116] For example, the commercially available plasmid pSE420
(Invitrogen, San Diego, Calif.) may be used for recombinant
production in E. coli. The commercially available plasmid pYES2
(Invitrogen, San Diego, Calif.) may be used for production in S.
cerevisiae strains of yeast. The commercially available MaxBac.TM.
(Invitrogen, San Diego, Calif.) complete baculovirus expression
system may be used for production in insect cells. The commercially
available plasmid pcDNA I (Invitrogen, San Diego, Calif.) may be
used for production in mammalian cells such as Chinese Hamster
Ovary cells.
[0117] One having ordinary skill in the art may use these or other
commercially available expression vectors and systems or produce
vectors using well-known methods and readily available starting
materials. Expression systems containing the requisite control
sequences, such as promoters and polyadenylation signals, and
preferably enhancers, are readily available and known in the art
for a variety of hosts. See e.g., Sambrook et al., Molecular
Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press
(1989). Thus, the desired proteins can be prepared in both
prokaryotic and eukaryotic systems, resulting in a spectrum of
processed forms of the protein.
[0118] The most commonly used prokaryotic system remains E. coli,
although other systems such as B. subtilis and Pseudomonas are also
useful. Suitable control sequences for prokaryotic systems include
both constitutive and inducible promoters including the lac
promoter, the trp promoter, hybrid promoters such as tac promoter,
the lambda phage P1 promoter. In general, foreign proteins may be
produced in these hosts either as fusion or mature proteins. When
the desired sequences are produced as mature proteins, the sequence
produced may be preceded by a methionine which is not necessarily
efficiently removed. Accordingly, the peptides and proteins claimed
herein may be preceded by an N-terminal Met when produced in
bacteria. Moreover, constructs may be made wherein the coding
sequence for the peptide is preceded by an operable signal peptide
which results in the secretion of the protein. When produced in
prokaryotic hosts in this matter, the signal sequence is removed
upon secretion.
[0119] A wide variety of eukaryotic hosts are also now available
for production of recombinant foreign proteins. As in bacteria,
eukaryotic hosts may be transformed with expression systems which
produce the desired protein directly, but more commonly signal
sequences are provided to effect the secretion of the protein.
Eukaryotic systems have the additional advantage that they are able
to process introns which may occur in the genomic sequences
encoding proteins of higher organisms. Eukaryotic systems also
provide a variety of processing mechanisms which result in, for
example, glycosylation, carboxy-terminal amidation, oxidation or
derivatization of certain amino acid residues, conformational
control, and so forth.
[0120] Commonly used eukaryotic systems include, but are not
limited to, yeast, fungal cells, insect cells, mammalian cells,
avian cells, and cells of higher plants. Suitable promoters are
available which are compatible and operable for use in each of
these host types as well as are termination sequences and
enhancers, as e.g. the baculovirus polyhedron promoter. As above,
promoters can be either constitutive or inducible. For example, in
mammalian systems, the mouse metallothionene promoter can be
induced by the addition of heavy metal ions.
[0121] The particulars for the construction of expression systems
suitable for desired hosts are known to those in the art. For
recombinant production of the protein, the DNA encoding it is
suitably ligated into the expression vector of choice and then used
to transform the compatible host which is then cultured and
maintained under conditions wherein expression of the foreign gene
takes place. The protein of the present invention thus produced is
recovered from the culture, either by lysing the cells or from the
culture medium as appropriate and known to those in the art.
[0122] One having ordinary skill in the art can, using well-known
techniques, isolate the protein that is produced.
[0123] In some embodiments the ST receptor ligand is formulated as
a injectable pharmaceutical composition suitable for parenteral
administration. Accordingly, the ST receptor ligand is a sterile,
pyrogen-free preparation that has the structural/physical
characteristics required for injectable products; i.e. it meets
well known standards recognized by those skilled in the art for
purity, pH, isotonicity, sterility, and particulate matter.
[0124] In some preferred embodiments, the ST receptor ligand
administered orally or rectally and the ST receptor ligand is
formulated as pharmaceutical composition suitable for oral or
rectal administration. Some embodiments providing ST receptor
ligand suitable for oral administration provide ST receptor ligand
formulated for sustained release. Some embodiments providing ST
receptor ligand suitable for oral administration provide ST
receptor ligand formulated by enteric coating to release the active
agent in the intestine. Enteric formulations are described in U.S.
Pat. No. 4,601,896, U.S. Pat. No. 4,729,893, U.S. Pat. No.
4,849,227, U.S. Pat. No. 5,271,961, U.S. Pat. No. 5,350,741, and
U.S. Pat. No. 5,399,347, which are each hereby incorporated herein
by reference. Oral and rectal formulation are taught in Remington's
Pharmaceutical Sciences, 18th Edition, 1990, Mack Publishing Co.,
Easton Pa. which is incorporated herein by reference.
[0125] Alternative embodiments include sustained release
formulations and implant devices which provide continuous delivery
of ST receptor ligand. In another embodiment, the ST receptor
ligand is administered topically, or for application
intratumorally, intrathecally, intraventricularly, intrapleurally,
intrabronchially, intracranially, or subcutaneously.
[0126] The ST receptor ligand is administered to the individual in
an amount effective to inhibit the proliferation of cells that
express the ST receptor, such as metastatic colorectal cancer cells
and primary and metastatic gastric and esophageal cancer cells. In
some preferred embodiments, the cells are metastasized human
colorectal cancer cells. In other preferred embodiments, the cells
are primary or metastasized gastric cancer cells. In other
preferred embodiments, the cells are primary or metastasized
esophageal cancer cells.
[0127] The ST receptor ligand is administered to the individual by
any route that will allow for the delivery of the ligand to cells
that express ST receptors. In some preferred embodiments, the ST
receptor ligand is administered parenterally. In some preferred
embodiments, the ST receptor ligand is administered into the
circulatory system of the individual. In some embodiments, the ST
receptor ligand is administered intravenously. In some embodiments,
the ST receptor ligand is administered into the cerebral spinal
fluid. In some embodiments, the ST receptor ligand is administered
into the lymph system. In some embodiments, the ST receptor ligand
is administered intratumorally. In some preferred embodiments, the
ST receptor ligand administered orally, rectally, topically,
intratumorally, intrathecally, intraventricularly, intrapleurally,
intrabronchially, intracranially, or subcutaneously.
[0128] When the ST receptor ligand binds to the ST receptor protein
on cells expressing the ST receptor, proliferation of the cells is
inhibited. An effective amount of ST receptor ligand must be
administered to achieve inhibition of proliferation. Generally, ST
receptor ligand must be present at a sufficient level for a
sustained amount of time to expose cells that express ST receptors
to the ST receptor ligand. Generally, enough ST receptor ligand
must be administered initially and/or by continuous administration
to maintain the concentration of ST ligand to be greater than about
10.sup.-10 M, and preferably about 10.sup.-9M or more. I is
preferred that such a concentration be maintained for at least
about 6 hours, preferably about for at least about 8 hours, more
preferably about for at least about 12 hours, in some embodiments
at least 16 hours, in some embodiments at least 20 hours and up to
about 24 hours or more. Regardless of whether the compound is an ST
receptor ligand or ST receptor ligand conjugate which has
additional functions such as cytotoxic activity or detectability,
it is important that the dosage and administration be sufficient
for the ST receptor binding to occur at a sufficient level for
sufficient time to inhibit proliferation or to induce a therapeutic
effect. Generally, the plasma concentration of ST receptor greater
than about 10.sup.-10 M must be maintained for at least about 6
hours. Pharmaceutical compositions according to the invention are
therefore provided in dosages sufficient to maintain such plasma
concentration. Dosage varies depending upon known factors such as
the pharmacodynamic characteristics of the particular agent, and
its mode and route of administration; age, health, and weight of
the recipient; nature and extent of symptoms, kind of concurrent
treatment, frequency of treatment, and the effect desired.
[0129] In some embodiments, the ST receptor ligand is initially
administered to the individual in a loading dose of at least 0.1 nM
per 10 kg. bodyweight of the individual. In some embodiments, the
ST receptor ligand is initially administered to the individual in a
loading dose of at least 0.25 nM per 10 kg. bodyweight of the
individual. In some embodiments, the ST receptor ligand is
initially administered to the individual in a loading dose of at
least 0.5 nM per 10 kg. bodyweight of the individual. In some
embodiments, the ST receptor ligand is initially administered to
the individual in a loading dose of at least 0.75 nM per 10 kg.
bodyweight of the individual. In some embodiments, the ST receptor
ligand is initially administered to the individual in a loading
dose of at least 1 nM per 10 kg. bodyweight of the individual. In
some embodiments, the ST receptor ligand is initially administered
to the individual in a loading dose of at least 1.5 nM per 10 kg.
bodyweight of the individual. In some embodiments, the ST receptor
ligand is initially administered to the individual in a loading
dose of at least 2 nM per 10 kg. bodyweight of the individual. In
some embodiments, the ST receptor ligand is initially administered
to the individual in a loading dose of at least 2.5 nM per 10 kg.
bodyweight of the individual. In some embodiments, the ST receptor
ligand is initially administered to the individual in a loading
dose of at least 3 nM per 10 kg. bodyweight of the individual. In
some embodiments, the ST receptor ligand is initially administered
to the individual in a loading dose of at least 4 nM per 10 kg.
bodyweight of the individual. In some embodiments, the ST receptor
ligand is initially administered to the individual in a loading
dose of at least 5 nM per 10 kg. bodyweight of the individual. In
some embodiments, the ST receptor ligand is initially administered
to the individual in a loading dose of at least 7.5 nM per 10 kg.
bodyweight of the individual. In some embodiments, the ST receptor
ligand is initially administered to the individual in a loading
dose of at least 10 nM per 10 kg. bodyweight of the individual. In
some embodiments, the loading dose is 0.1-10 nM of ST receptor
ligand per 10 kg. bodyweight of said individual. In some
embodiments, the loading dose is 0.1-7.5 nM of ST receptor ligand
per 10 kg. bodyweight of said individual. In some embodiments, the
loading dose is 0.1-5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual. In some embodiments, the loading
dose is 0.1-2.5 nM of ST receptor ligand per 10 kg. bodyweight of
said individual. In some embodiments, the loading dose is 0.1-1.5
nM of ST receptor ligand per 10 kg. bodyweight of said individual.
In some embodiments, the loading dose is 0.1-1.0 nM of ST receptor
ligand per 10 kg. bodyweight of said individual. In some
embodiments, the loading dose is 0.1-0.5 nM of ST receptor ligand
per 10 kg. bodyweight of said individual. In some embodiments, the
loading dose is 0.1-0.25 nM of ST receptor ligand per 10 kg.
bodyweight of said individual. In some embodiments, the loading
dose is 0.25-10 nM of ST receptor ligand per 10 kg. bodyweight of
said individual. In some embodiments, the loading dose is 0.25-7.5
nM of ST receptor ligand per 10 kg. bodyweight of said individual.
In some embodiments, the loading dose is 0.25-5 nM of ST receptor
ligand per 10 kg. bodyweight of said individual. In some
embodiments, the loading dose is 0.25-2.5 nM of ST receptor ligand
per 10 kg. bodyweight of said individual. In some embodiments, the
loading dose is 0.25-1.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual. In some embodiments, the loading
dose is 0.25-1.0 nM of ST receptor ligand per 10 kg. bodyweight of
said individual. In some embodiments, the loading dose is 0.5-0.75
nM of ST receptor ligand per 10 kg. bodyweight of said individual.
In some embodiments, the loading dose is 0.25-0.5 nM of ST receptor
ligand per 10 kg. bodyweight of said individual.
[0130] In some embodiments, the loading dose is 0.5-10 nM of ST
receptor ligand per 10 kg. bodyweight of said individual. In some
embodiments, the loading dose is 0.5-7.5 nM of ST receptor ligand
per 10 kg. bodyweight of said individual. In some embodiments, the
loading dose is 0.5-5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual. In some embodiments, the loading
dose is 0.5-2.5 nM of ST receptor ligand per 10 kg. bodyweight of
said individual. In some embodiments, the loading dose is 0.5-1.5
nM of ST receptor ligand per 10 kg. bodyweight of said individual.
In some embodiments, the loading dose is 0.5-1.0 nM of ST receptor
ligand per 10 kg. bodyweight of said individual. In some
embodiments, the loading dose is 0.5-0.75 nM of ST receptor ligand
per 10 kg. bodyweight of said individual.
[0131] In some embodiments, the loading dose is 0.75-10 nM of ST
receptor ligand per 10 kg. bodyweight of said individual. In some
embodiments, the loading dose is 0.75-7.5 nM of ST receptor ligand
per 10 kg. bodyweight of said individual. In some embodiments, the
loading dose is 0.75-5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual. In some embodiments, the loading
dose is 0.75-2.5 nM of ST receptor ligand per 10 kg. bodyweight of
said individual. In some embodiments, the loading dose is 0.75-1.5
nM of ST receptor ligand per 10 kg. bodyweight of said individual.
In some embodiments, the loading dose is 0.75-1.0 nM of ST receptor
ligand per 10 kg. bodyweight of said individual. In some
embodiments, the loading dose is 1.-10 nM of ST receptor ligand per
10 kg. bodyweight of said individual. In some embodiments, the
loading dose is 1.-7.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual. In some embodiments, the loading
dose is 1.-5 nM of ST receptor ligand per 10 kg. bodyweight of said
individual. In some embodiments, the loading dose is 1.-2.5 nM of
ST receptor ligand per 10 kg. bodyweight of said individual. In
some embodiments, the loading dose is 1.-1.5 nM of ST receptor
ligand per 10 kg. bodyweight of said individual. In some
embodiments, the loading dose is 1.5-10 nM of ST receptor ligand
per 10 kg. bodyweight of said individual. In some embodiments, the
loading dose is 1.5-7.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual. In some embodiments, the loading
dose is 1.5-5 nM of ST receptor ligand per 10 kg. bodyweight of
said individual. In some embodiments, the loading dose is 1.5-2.5
nM of ST receptor ligand per 10 kg. bodyweight of said individual.
In some embodiments, the loading dose is 2.5-10 nM of ST receptor
ligand per 10 kg. bodyweight of said individual. In some
embodiments, the loading dose is 2.5-7.5 nM of ST receptor ligand
per 10 kg. bodyweight of said individual. In some embodiments, the
loading dose is 2.5-5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual. In some embodiments, the loading
dose is 5-10 nM of ST receptor ligand per 10 kg. bodyweight of said
individual. In some embodiments, the loading dose is 5-7.5 nM of ST
receptor ligand per 10 kg. bodyweight of said individual.
[0132] In some embodiments, the ST receptor ligand is administered
to the individual by continuous infusion of at least 0.1 nM per 10
kg. bodyweight of the individual per hour. In some embodiments, the
ST receptor ligand is administered to the individual by continuous
infusion of at least 0.25 nM per 10 kg. bodyweight of the
individual per hour. In some embodiments, the ST receptor ligand is
administered to the individual by continuous infusion of at least
0.5 nM per 10 kg. bodyweight of the individual per hour. In some
embodiments, the ST receptor ligand is administered to the
individual by continuous infusion of at least 0.75 nM per 10 kg.
bodyweight of the individual per hour. In some embodiments, the ST
receptor ligand is administered to the individual by continuous
infusion of at least 1 nM per 10 kg. bodyweight of the individual
per hour. In some embodiments, the ST receptor ligand is
administered to the individual by continuous infusion of at least
1.5 nM per 10 kg. bodyweight of the individual per hour. In some
embodiments, the ST receptor ligand is administered to the
individual by continuous infusion of at least 2 nM per 10 kg.
bodyweight of the individual per hour. In some embodiments, the ST
receptor ligand is administered to the individual by continuous
infusion of at least 2.5 nM per 10 kg. bodyweight of the individual
per hour. In some embodiments, the ST receptor ligand is
administered to the individual by continuous infusion of at least 3
nM per 10 kg. bodyweight of the individual per hour. In some
embodiments, the ST receptor ligand is administered to the
individual by continuous infusion of at least 4 nM per 10 kg.
bodyweight of the individual per hour. In some embodiments, the ST
receptor ligand is administered to the individual by continuous
infusion of at least 5 nM per 10 kg. bodyweight of the individual
per hour. In some embodiments, the ST receptor ligand is
administered to the individual by continuous infusion of at least
7.5 nM per 10 kg. bodyweight of the individual per hour. In some
embodiments, the ST receptor ligand is administered to the
individual by continuous infusion of at least 10 nM per 10 kg.
bodyweight of the individual per hour. In some embodiments, the
dose infused is 0.1-10 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.1-7.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.1-5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.1-2.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.1-1.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.1-1.0 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.1-0.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.1-0.25 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.25-10 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.25-7.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.25-5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.25-2.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.25-1.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.25-1.0 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.5-0.75 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.25-0.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.5-10 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.5-7.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.5-5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.5-2.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.5-1.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.5-1.0 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.5-0.75 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.75-10 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.75-7.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.75-5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.75-2.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.75-1.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 0.75-1.0 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 1.-10 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 1.-7.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 1.-5 nM of ST receptor ligand per 10 kg. bodyweight
of said individual per hour. In some embodiments, the dose infused
is 1.-2.5 nM of ST receptor ligand per 10 kg. bodyweight of said
individual per hour. In some embodiments, the dose infused is
1.-1.5 nM of ST receptor ligand per 10 kg. bodyweight of said
individual per hour. In some embodiments, the dose infused is
1.5-10 nM of ST receptor ligand per 10 kg. bodyweight of said
individual per hour. In some embodiments, the dose infused is
1.5-7.5 nM of ST receptor ligand per 10 kg. bodyweight of said
individual per hour. In some embodiments, the dose infused is 1.5-5
nM of ST receptor ligand per 10 kg. bodyweight of said individual
per hour. In some embodiments, the dose infused is 1.5-2.5 nM of ST
receptor ligand per 10 kg. bodyweight of said individual per hour.
In some embodiments, the dose infused is 2.5-10 nM of ST receptor
ligand per 10 kg. bodyweight of said individual per hour. In some
embodiments, the dose infused is 2.5-7.5 nM of ST receptor ligand
per 10 kg. bodyweight of said individual per hour. In some
embodiments, the dose infused is 2.5-5 nM of ST receptor ligand per
10 kg. bodyweight of said individual per hour. In some embodiments,
the dose infused is 5-10 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour. In some embodiments, the
dose infused is 5-7.5 nM of ST receptor ligand per 10 kg.
bodyweight of said individual per hour.
[0133] In some embodiments, the ST receptor ligand is infused into
the individual for at least 8 hours. In some embodiments, the ST
receptor ligand is infused into the individual for at least 12
hours. In some embodiments, the ST receptor ligand is infused into
the individual for at least 16 hours. In some embodiments, the ST
receptor ligand is infused into the individual for at least 20
hours. In some embodiments, the ST receptor ligand is infused into
the individual for at least 24 hours.
[0134] In some embodiments, a pharmaceutical composition is
provided which comprises sterile, pyrogen free ST receptor ligand
in an amount sufficient for continuous infusion of at least 0.1-10
nM of ST receptor ligand per hour for at least 6 hours, in some
embodiments at least 12 hours, in some embodiments at least 16
hours, in some embodiments at least 20 hours and in some
embodiments at least 24 hours. In some embodiments, a
pharmaceutical composition is provided which comprises sterile,
pyrogen free ST receptor ligand in an amount sufficient for
continuous infusion of at least 0.1-10 nM of ST receptor ligand per
hour for at least 6 hours, in some embodiments at least 12 hours,
in some embodiments at least 16 hours, in some embodiments at least
20 hours and in some embodiments at least 24 hours. In some
embodiments, a pharmaceutical composition is provided which
comprises sterile, pyrogen free ST receptor ligand in an amount
sufficient for continuous infusion of at least 0.1-10 nM of ST
receptor ligand per hour for at least 6 hours, in some embodiments
at least 12 hours, in some embodiments at least 16 hours, in some
embodiments at least 20 hours and in some embodiments at least 24
hours.
[0135] In a preferred embodiment, the ST receptor ligand is the E.
coli heat stable enterotoxin (ST), in its native form or modified
to inhibit degradation, and the proper concentration of ST in the
circulation is achieved by administering a continuous infusion of
at least 0.5 micrograms per 10 kg. bodyweight of the individual per
hour for at least 6 hours. In some embodiments, the ST is initially
administered to an individual in a loading dose of at least 1
microgram of ST per 10 kg. bodyweight of the individual. In some
embodiments, the loading dose is 1-10 micrograms of ST per 10 kg.
bodyweight of the individual. In some embodiments, the loading dose
is 3-5 micrograms of ST per 10 kg. bodyweight of the individual. In
some embodiments, the loading dose is about 4 micrograms of ST per
10 kg. bodyweight of the individual. In some embodiments, the ST is
infused into the individual in a dose of 0.5-8 micrograms of ST per
10 kg. bodyweight of the individual. In some embodiments, the ST is
infused into the individual in a dose of 1-5 micrograms of ST per
10 kg. bodyweight of the individual. In some embodiments, the ST is
infused into the individual in a dose of about 3 micrograms of ST
per 10 kg. bodyweight of the individual. In some embodiments, the
ST is infused into the individual for at least 8 hours. In some
embodiments, the ST is infused into the individual for at least 12
hours. In some embodiments, the ST is infused into the individual
for at least 16 hours. In some embodiments, the ST is infused into
the individual for at least 20 hours. In some embodiments, the ST
is infused into the individual for at least 24 hours. In some
embodiments, the ST is infused into the individual for 12-24
hours.
[0136] In a preferred embodiment, the ST ligand is the E. coli heat
stable enterotoxin (ST) and the proper concentration of ST in the
circulation is achieved by administering a loading dose of about 30
micrograms of ST, and a continuous infusion of 20 micrograms per
hour for a 70 kg individual. A continuous infusion is administered
to maintain the plasma concentration of ST greater than 10.sup.-9
M. The infusion is continued for at least 12 hours and up to 24
hours.
[0137] After the infusion has been completed, the number of the
patient's colorectal, gastric and esophageal cancer cells
decreases, and imaging or therapy with a compound targeted by ST or
another agent which binds to ST receptors is initiated.
[0138] The ability to inhibit the proliferation of cancer cells has
been demonstrated with T84 and Caco 2 human colon carcinoma cells
after exposure to STa and uroguanylin In embodiments of the present
invention in which patients with metastatic colorectal and primary
and metastatic gastric and esophageal cancer are treated or their
cancer tumors are imaged, the ST receptor targeted therapeutic or
imaging compounds are administered following initiation of the
method of inhibiting proliferation of the cancer cells. Thus,
generally before imaging or therapeutic agents targeted with ST
receptor-binding compounds are employed in a patient with
colorectal or primary or metastatic gastric or esophageal cancer,
the patient is pretreated to inhibit the proliferation of the
cancer cells. The actual timing of administration of the ST
receptor ligand and the active ST receptor binding compounds
relative to each other is not critical. Rather, the methods require
that the sustained presence of an ST receptor ligand occur for a
period sufficient to inhibit the proliferation of cancer cells and
the active compound which binds to ST receptors is administered at
such time to be present in the body after the number of cells has
begun to decrease.
[0139] In some embodiments, therapeutic compounds which comprise a
ST receptor binding compound such as an ST receptor binding peptide
and a cytotoxic or cytostatic agent, including radioactive and
radiostable agents, such as those disclosed in U.S. Pat. No.
5,518,888, U.S. Pat. No. 5,879,656, U.S. Pat. No. 6,060,037, and
U.S. Pat. No. 6,268,159, ad PCT application number PCT/US94/12232
are administered following or contemporaneous with inhibition of
proliferation of the cancer cells. In some embodiments, the method
of treating an individual who has metastasized colorectal or
primary or metastatic gastric or esophageal cancer comprises the
steps of first inhibiting proliferation of cancer cells in the
individual by administering to said individual by substantially
continuous infusion, at least 0.1-10 nM of an ST receptor ligand
per 10 kg. bodyweight of the individual per hour for at least 6
hours. A therapeutic pharmaceutical composition that comprises
components which target ST receptor for delivery of a therapeutic
agent is then administered to the individual. In some embodiments,
the therapeutic pharmaceutical composition comprises a conjugated
composition that comprises an ST receptor binding moiety and an
active moiety which is a therapeutic agent. In some embodiments,
the ST receptor binding moiety is a peptide. In some embodiments,
the ST receptor binding moiety is selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NOS:5-56 and
fragments and derivatives thereof. In some embodiments, the
therapeutic agent is radioactive. In some embodiments, the
therapeutic agent is selected from the group consisting of:
.sup.43K, .sup.52Fe, .sup.57Co, .sup.67Cu, .sup.67Ga, .sup.68Ga,
.sup.77Br, .sup.81Rb/.sup.81MKr, .sup.87MSr, .sup.166Ho,
.sup.211Bi, .sup.153Sm, .sup.113MIn, .sup.123I, .sup.125I,
.sup.127Cs, .sup.129Cs, .sup.131I, .sup.132I, .sup.197Hg,
.sup.203Pb and .sup.206Bi. In some embodiments, the therapeutic
agent is selected from the group consisting of: .sup.47Sc,
.sup.67Cu, .sup.90Y, .sup.109Pd, .sup.123I, .sup.125I, .sup.131I,
.sup.186Re, .sup.188Re, .sup.199Au, .sup.212At, .sup.212Pb,
.sup.212B, .sup.32P and .sup.33P, .sup.71Ge, .sup.77As, .sup.103Pb,
.sup.105Rh, .sup.111Ag, .sup.119Sb, .sup.121Sn, .sup.131Cs,
.sup.143Pr, .sup.161Tb, .sup.177Lu, .sup.191Os, .sup.193MPt and
.sup.197Hg. In some embodiments, the therapeutic agent is
radiostable. In some embodiments, the therapeutic agent is selected
from the group consisting of: compounds that cause cell death,
compounds that inhibit cell division, and compounds that induce
cell differentiation. In some embodiments, the therapeutic agent is
selected from the group consisting of: chemotherapeutics, toxins
and radiosensitizing agents. In some embodiments, the therapeutic
agent is selected from the group consisting of: methotrexate,
doxorubicin, daunorubicin, cytosinarabinoside, etoposide, 5-4
fluorouracil, melphalan, chlorambucil, cis-platin, vindesine,
mitomycin, bleomycin, purothionin, macromomycin, 1,4-benzoquinone
derivatives, trenimon, ricin, ricin A chain, Pseudomonas exotoxin,
diphtheria toxin, Clostridium perfringens phospholipase C, bovine
pancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin A
chain, cobra venom factor, gelonin, saporin, modeccin, viscumin,
volkensin, nitroimidazole, metronidazole, misonidazole, porfimer
and compounds that enchance the accumulation of intracellular cGMP
such as phosphodiesterase inhibitors, for example exisulind,
zaprinast, and sildenafil.
[0140] In some embodiments, detectable compounds which comprise a
ST receptor binding compound such as an ST receptor binding peptide
and a detectable agent such as those disclosed in U.S. Pat. No.
5,518,888, U.S. Pat. No. 5,879,656, U.S. Pat. No. 6,060,037, and
U.S. Pat. No. 6,268,159, and PCT application number PCT/US94/12232
are administered following or contemporaneous with the inhibition
of proliferation of cells expressing the ST receptor protein. In
some embodiments, the method of imaging a metastasized colorectal
tumor or primary or metastatic gastric or esophageal tumor in an
individual who has metastasized colorectal or primary or
metastasized gastric or esophageal cancer comprises the step of
first inhibiting the proliferation of cancer cells in the
individual by administering to the individual by substantially
continuous infusion, at least 0.1-10 nM of an ST receptor ligand
per 10 kg. bodyweight of the individual per hour for at least 6
hours. A pharmaceutical imaging composition that comprises
components which target ST receptor for delivery of an imaging
agent is then administered to the individual. In some embodiments,
the imaging is performed by radioscintigraphy, nuclear magnetic
resonance imaging (MRI) or computed tomography (CT scan). In some
embodiments, the pharmaceutical imaging composition comprises a
conjugated composition that comprises an ST receptor binding moiety
and an active moiety that is an imaging agent. In some embodiments,
the ST receptor binding moiety is a peptide. In some embodiments,
the ST receptor binding moiety is selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NOS:5-56 and
fragments and derivatives thereof. In some embodiments, the imaging
agent is radioactive. In some embodiments, the therapeutic agent is
selected from the group consisting of: .sup.43K, .sup.52Fe,
.sup.57Co, .sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.77Br,
.sup.81Rb/.sup.81MKr, .sup.87MSr, .sup.99MTc, .sup.111In,
.sup.113MIn, .sup.123I, .sup.125I, .sup.127Cs, .sup.129Cs,
.sup.131I, .sup.132I, .sup.197Hg, .sup.203Pb and .sup.206Bi. In
some embodiments, the therapeutic agent is selected from the group
consisting of: .sup.47Sc, .sup.67Cu, .sup.90Y, .sup.109Pd,
.sup.123I, .sup.125I, .sup.131I, .sup.186Re, .sup.188Re,
.sup.199Au, .sup.211At, .sup.212Pb, .sup.212B, .sup.32P and
.sup.33P, .sup.71Ge, .sup.77As, .sup.103Pb, .sup.105Rh, .sup.111Ag,
.sup.119Sb, .sup.121Sn, .sup.131Cs, .sup.143Pr, .sup.161Tb,
.sup.177Lu, .sup.181Os, .sup.193MPt and .sup.197Hg. In some
embodiments, the imaging agent is radiostable. In some embodiments,
the imaging agent is a heavy metal such as iron chelates, or
chelates of gadolinium or manganese. In some embodiments, the
imaging is performed by positron emission tomography (PET) using
positron emitters of oxygen, nitrogen, iron, carbon, or gallium. In
other embodiments, the imaging is performed by light imaging dyes
such as pthallocyanine derivatives and indocyanine derivatives.
[0141] In some embodiments, compounds which comprise an ST receptor
binding compound such as an ST receptor binding peptide and an
active agent such as those disclosed in U.S. Pat. No. 5,518,888,
U.S. Pat. No. 5,879,656, U.S. Pat. No. 5,962,220, U.S. Pat. No.
6,060,037, U.S. Pat. No. 6,087,109 and U.S. Pat. No. 6,268,159, PCT
application number PCT/US94/12232 and Ser. No. 08/467,920 filed
Jun. 6, 1995 are administered following or contemporaneous with
inhibition of proliferation of cells expressing the ST receptor
protein. In some embodiments, the method of treating an individual
who has a genetic disease, a prediposition for a genetic disease,
metastasized colorectal or primary or metastasized gastric or
esophageal cancer comprises the steps of first inhibiting the
proliferation of cancer cells in the individual by administering to
said individual by oral or rectal administration, at least 0.1-10
nM of an ST receptor ligand per 10 kg. bodyweight of the individual
per hour for at least 6 hours. A pharmaceutical composition that
comprises components which target ST receptor for delivery of an
active agent, such as a gene therapeutic, antisense compound, or
ribozyme is then administered to the individual. In some
embodiments, the pharmaceutical composition comprises a conjugated
composition that comprises an ST receptor binding moiety and an
active moiety which is a therapeutic agent.
[0142] In embodiments of the present invention, individuals are
being screened for the detection of evidence of ST receptor gene
expression in extraintestinal samples, such as by those methods
described U.S. Pat. No. 5,601,990, U.S. Pat. No. 5,731,159, U.S.
Pat. No. 5,928,873, and U.S. Pat. No. 6,060,037, PCT application
number PCT/US94/12232, and PCT application No. PCT/US97/07467, the
detection of which in such samples indicates metastasized
colorectal cancer or primary or metastasized gastric or esophageal
cancer. In such embodiments, the individuals are administered
parenterally ST receptor ligand in sufficient quantity for
sufficient time to inhibit proliferation of cancer cells of
colorectal, gastric or esophageal origin.
[0143] Following initiation of the method of inhibiting
proliferation, the extraintestinal samples are obtained and
screened for evidence of ST receptor expression. Generally, assays
to detect ST receptor protein, mRNA encoding ST receptor protein or
cDNA generated from such mRNA are typically the evidence whose
presence is detected. Before samples are obtained, the patient is
typically pretreated to inhibit proliferation of colorectal,
gastric and esophageal cancer cells. The actual timing of
administration of the ST receptor ligand and the active ST receptor
binding compounds relative to each other is not critical. Rather,
the methods require that the sustained presence of an ST receptor
ligand occur for a period sufficient to inhibit the proliferation
of a cell and that the extraintestinal sample to be screened is
obtained after the number of cells has decreased.
[0144] In some embodiments, the method of determining whether an
individual has metastasized colorectal cancer or primary or
metastasized gastric or esophageal cancer comprises the step of
first increasing the number of ST receptor molecules on the surface
of a cancer cell in the individual by administering to the
individual by substantially continuous infusion, at least 0.1-10 nM
of an ST receptor ligand per 10 kg. bodyweight of the individual
per hour for at least 6 hours. A sample of extraintestinal body
fluid and/or tissue is then obtained from the individual and an
assay is run to detect the presence of protein or mRNA encoding ST
receptor in the sample. The presence of the protein or mRNA
indicates that the individual has metastatic colorectal cancer or
primary or metastatic gastric or esophageal cancer. In some
embodiments, the presence of the mRNA is detected using a
polymerase chain reaction. In some embodiments, the extraintestinal
sample is tissue or body fluid. In some embodiments, the
extraintestinal sample is blood. In some embodiments, the
extraintestinal sample is screened to detect the presence of ST
receptor protein in the sample using an immunoassay.
EXAMPLES
Example 1
ST as an Anticancer Agent
[0145] ST is a cytostatic agent, but not an apoptotic or cytotoxic
drug. It can be used alone or in combination therapies to treat: 1)
adenomas and polyps of the small and large intestine; 2) intestinal
and colorectal carcinomas and their metastases; 3) cancer
recurrence of the gastrointestinal (GI tract; 4) at-risk population
for polyp and cancer of the GI tract.
[0146] The combination therapy is an alternative approach to the
mono-therapy with ST and it has been conceived to maximize the
tumor to normal cells toxicity ratio of the anti-neoplastic
intervention. The combination therapy should include:
[0147] 1. ST receptor ligand such as ST plus surgery;
[0148] 2. ST receptor ligand such as ST plus one or more of the
classic chemotherapeutic drugs (e.g. fluorouracil, leucovorin,
cisplatin, levamisole, H.sub.2 antagonists, folate);
[0149] 3. ST receptor ligand such as ST plus one or more of the new
anti-neoplastic drugs (e.g. genasense (Genta); SAHA (Aton Pharma),
vitaxin (applied Molecular Evolution), EMD121974 (E-Merck));
[0150] 4. ST receptor ligand such as ST plus one or more vitamins
(e.g. vitamin D; vitamin A; vitamin C, vitamin E,
.beta.-carotene);
[0151] 5. ST receptor ligand such as ST plus radiation therapy;
[0152] 6. ST receptor ligand such as ST plus calcium (e.g.
CaCl.sub.2 1 mM) and/or calcium mimics (e.g. cations as Barium,
Nichel);
[0153] 7. ST receptor ligand such as ST plus compounds that
enchance the accumulation of intracellular cGMP such as
phosphodiesterase inhibitors, for example exisulind, zaprinast, and
sildenafil. (e.g. zaprinast 10 .mu.M; dipyridamole 10 .mu.M)
[0154] 8. ST receptor ligand such as ST plus cyclic GMP analogs
(e.g. 8-br-cGMP 5 mM; dibutyryl-cGMP 5 mM)
[0155] 9. ST receptor ligand such as ST plus agonists of cyclic
nucleotide gated (CNG) channels (e.g. 8-(4-chlorophenylthio)-cGMP
500 .mu.M; 1000-2000PEG-(cGMP).sub.2 1 .mu.M);
[0156] 10. ST receptor ligand such as ST plus cyclic AMP analogs
(e.g. dibutyryl-cAMP 0.1 mM);
[0157] 11. ST receptor ligand such as ST plus agents that causes
intracellular mobilization of cyclic nucleotydes (e.g. VIP 10 nM,
forskolin 1 .mu.M, NO donors);
[0158] 12. ST receptor ligand such as ST plus calcium influx
inhibitors (e.g. carboxymido-triazole (CAI) 2.3 .mu.M);
[0159] 13. ST receptor ligand such as ST plus inhibitors of
store-operated calcium entry (e.g. La.sup.3+100 .mu.M,
N-1-n-octyl-3,5-bis-(4-pyridyl)triazole (DPT) 10 .mu.M, SFK
96364);
[0160] 14. ST receptor ligand such as ST plus non-steroidal
anti-inflammatory drugs (NSAIDs) (e.g. acetyl-salicylic-acid 2 mM,
mefenamic acid 200 .mu.M, sulindac sulfide 60 .mu.M);
[0161] 15. ST receptor ligand such as ST plus sulindac derivatives
(e.g. sulindac sulfone (exisulind) 0.6 mM, CP248 1 .mu.M, CP461 10
.mu.M).
Routes of Administration:
[0162] ST receptor ligand such as ST should be administered
parentally as anti-neoplastic drug. Oral administration should be
avoided for the hyper-secretory and diarrheic effect of ST receptor
ligand in the intestine. Depending on disease staging and
metastasis spreading, ST receptor ligand could be administered
intravenously (I.V.), subcutaneously, intramuscularly,
intraperitoneally or intrathecally. In addition, ST receptor ligand
could be administered regionally via direct injection into the main
artery supplying the diseased organ.
[0163] ST Dosages for Mono- and Combination Therapy
[0164] The IC.sub.50 for ST as an anticancer agent in vitro is 14
nM
[0165] The predicated target concentration for ST in vivo is 140
nM
[0166] I.V. Loading Dose (bolus dose) for a 70 Kg patient is 3.3
mg
[0167] I.V. Maintenance Dose (infusion rate) is 1 .mu.g/min/Kg
(equivalent to 4.3 mg/hour for a 70 Kg patient)
Dosage Regimens
[0168] 1. Continuous infusion (infusion rate: 1 .mu.g/min/Kg) for a
minimum of 7 days up to a maximum of 15 day, with a 7 day interval
periods during which the patients undergo to a chemotherapy
treatment with a cytotoxic drug (e.g. fluorouracil, cisplatin,
etc.) A mini-pump system may be employed for I.V. infusions.
[0169] 2. Intermittent dosage regimens with morning (7-9 a.m.)
single dose of 3.3 mg every 24 h for 30 consecutive days. This
30-day treatment can be repeated after 15-day intervals during
which patients can be dosed with cytotoxic drugs (e.g.
fluorouracil, cisplatin, etc.)
Example 2
[0170] The heat stable enterotoxin, ST, has been demonstrated to
induce a prolongation of the cell cycle (a cytostatic effect). This
cytostatic effect is specific to cells expressing guanylyl cyclase
C, such as cancer cells from colorectal, gastric and esophageal
adenocarcinomas. When used in conjunction with standard chemo-
(5FU, irenotecan, leukovorin, etc) or radiation (external bean,
targeted radiotherapy, etc.) therapies, the therapeutic index of
these standard therapies will be enhanced due to the ability to
achieve a similar clinical response at a lower cytotoxin dose. This
concept applies equally to the use of ST and all GCC ligands,
irrespective of their structure or origin
[0171] Pre-clinical data has shown that the overall cell cycle time
is slowed, yet no particular phase was favored over another after
ST treatment of GCC-bearing cells. Data from the effect of
treatment with ST alone on cell cycle indicates that the time spent
in various phases of the cell cycle increases equally for all four
phases of the cell cycle as a result of ST treatment. Data from the
effect of treatment with ST plus doxorubicin on cell cycle
indicates a profound shift whereby the cell does not progress
through the cell cycle. The implications of this finding is that ST
can be used as a cytostatic drug in combination with current
chemotherapies, decreasing their adverse side effects while
maintaining the same therapeutic effect (an improved therapeutic
index). A combination of ST with phase-specific therapeutics, such
as 5-Fluorouracil, enhances killing of cells as they spend more
time in the S phase. As a result, the proportion of cells existing
in other phases would be modified and they could then be attacked
either with prolonged 5-Fluorouracil therapy or by adjunct therapy
using a different cytotoxin, such as bleomycin, which selectively
kills those cells in G1 phase. This latter approach, wherein,
different cytotoxins are used in conjunction with ST in an
alternating regimen may be preferable to avoid the selective
survival of cytotoxin-resistant cells.
[0172] Information in Table 1 reflects dosage calculations. A
sample dosage regimen in humans is described below. ST is used to
attain an acute cytostatic effect by bolus injection or to maintain
this effect indefinitely by continuous infusion following the bolus
injection.
TABLE-US-00001 TABLE 1 Item Measure Reference Molecular weight of
ST 1972 g/mole Primary technical literature EC.sub.50 for ST 10 nM
In vitro TJU experiments Colorectal cancer patient 70 kg; blood
volume = 5.5 liters; Goodman & Gilman 12 liters ECF volume
Blood half-life for ST (rat) 6 min (.alpha.); 2.68 hrs (.beta.)
data Blood half-life for ST (man) 24 min (.alpha.); 11.12 hrs
(.beta.) Allometric scaling theory Clearance 14.9 hr.sup.-1 in rats
(Primarily renal) data Volume of distribution, V.sub.d 49 mls in
rats (An ECF agent) data
Calculations of Dosages:
[0173] To achieve therapeutic efficacy, the steady state
concentration (C.sub.SS) should be .gtoreq.10 EC.sub.50. This is
readily attained by multiple dosage regimens, three of which are
illustrated below. Additionally, combinations of methods A-C, or
other regimens will serve to produce a therapeutic effect using
ST.
Minimal Bolus Dose
[0174] From the above information, a bolus I.V. administration of
2.4 milligrams of ST (1.2 .mu.moles) as a loading dose will result
in a target concentration of approximately 100 nM (10-fold higher
than the EC.sub.50), and the concentration in extracellular fluid
should stay at or above the EC.sub.50 for approximately 1.5
hours.
Loading Dose = ( Target Concentration ) ( Volume of distribution )
= ( 100 nM ) ( 12 liters ) = 1.2 .mu.moles ( 2.4 milligrams )
##EQU00001##
Maximal Bolus Dose
[0175] An amount of ST is injected intravenously to maintain the
concentration at .gtoreq.10 EC.sub.50 for a prolonged period of
time to induce a therapeutic effect. It is known that the cell
cycle effect of ST is instantaneous and cells take a complete cell
cycle to full recover. Given this, it is reasonable to postulate
that the therapeutic dose must be maintained for at least 6 hours
to achieve a clinical effect: (24 hrs/cycle)/4 phases/cycle).
Dose=[(C.sub.t)/e.sup.-kt](V.sub.d)
[0176] Where, [0177] C.sub.t is the concentration of ST at time
"t"; equal to .gtoreq.10 EC.sub.50 (.gtoreq.100 nM). [0178] V.sub.d
is the volume of distribution of ST (12 liters) [0179] k is the
rate constant for ST elimination (k=0.693/half-life in minutes)
[0180] The t.sub.1/2 (.alpha.) in man was calculated to be 24
minutes for ST. [0181] The time to maintain this minimal
concentration is 6 hours
[0182] Therefore, the bolus dose administered would need to be:
ST=[(100 nM)(12 liters)]/e.sup.-(6.93 hr-1)(6 hrs)=39.5 millimoles
(78 grams)
Continuous Intravenous Infusion
[0183] An alternative approach is to provide the patient with
continual intravenous dosing of ST, thereby maintaining the
cytostatic effect through adjuvant chemotherapy, while minimizing
any adverse effects associated with ST. To achieve therapeutic
efficacy, the steady state concentration (C.sub.ss) should be
.gtoreq.10 EC.sub.50. To maintain a value of 10*EC.sub.50
indefinitely (C.sub.ss) would require an infusion rate of:
Infusion Rate=(Target concentration)(Clearance)
t.sub.1/2=0.53 hours (.alpha.+.beta.)
Cl=(0.693)(V.sub.d/70 kg man)/(t.sub.1/2)=3.7 ml/min/kg
From this,
Infusion rate=(100 nM)(3.7 ml/min/kg)=370 pmoles/min/kg
[0184] Or
[0185] 25.9 nanomoles/min (51 micrograms/min) for a 70 kg man over
a six hour period for a total of 184 milligrams administered.
Example 3
Bacterial Enterotoxins are Associated with Resistance to Colon
Cancer
[0186] One half million patients suffer from colorectal cancer in
industrialized nations, yet this disease exhibits a low incidence
in under-developed countries. This geographic imbalance suggests an
environmental contribution to the resistance of endemic populations
to intestinal neoplasia. A common epidemiological characteristic of
these colon cancer-spared regions is the prevalence of
enterotoxigenic bacteria associated with diarrheal disease. Here, a
bacterial heat-stable enterotoxin was demonstrated to suppress
colon cancer cell proliferation by a guanylyl cyclase C-mediated
signaling cascade. The heat-stable enterotoxin suppressed
proliferation by increasing intracellular cGMP, an effect mimicked
by the cell-permeant analog 8-br-cGMP. The antiproliferative
effects of the enterotoxin and 8-br-cGMP were reversed by
L-cis-diltiazem, a cyclic nucleotide-gated channel inhibitor, as
well as by removal of extracellular Ca2+, or chelation of
intracellular Ca2+. In fact, both the enterotoxin and 8-br-cGMP
induced an L-cis-diltiazem-sensitive conductance, promoting Ca2+
influx and inhibition of DNA synthesis in colon cancer cells.
Induction of this previously unrecognized antiproliferative
signaling pathway by bacterial enterotoxin could contribute to the
resistance of endemic populations to intestinal neoplasia, and
offers a paradigm for targeted prevention and therapy of primary
and metastatic colorectal cancer.
[0187] Colorectal cancer is the fourth leading cause of cancer and
cancer-related mortality in the world, with a geographic
distribution primarily affecting patients in developed countries
(FIG. 1a; ref. 1). Although the epidemiology of this disease
remains poorly understood, there is an unexplained inverse
relationship between the incidence of colorectal cancer and
enterotoxigenic Escherichia coli (ETEC) infections (FIG. 1a).
Indeed, the age-adjusted incidence of colorectal cancer is lowest
in under-developed countries (1), where ETEC infections are the
highest (FIG. 1a; refs. 2 and 3).
[0188] ETEC produce heat-stable enterotoxins (STs; refs. 4 and 5),
a principle cause of secretory diarrhea in endemic populations,
travelers, and agriculturally important animal herds (6, 7). These
enterotoxins are plasmid-encoded small (9 amino acids) peptides
that bind to guanylyl cyclase C (GC-C), specifically expressed in
intestinal epithelial cells (4, 8). Association of STs with the
extracellular domain of GC-C activates the intracellular catalytic
domain that converts GTP into cGMP (9, 10). The second messenger
cGMP, in turn, activates cGMP-dependent protein kinase (PKG) II,
the conventional downstream effector for this cyclic nucleotide,
resulting in secretory diarrhea (9-11). In this way, STs represent
molecular mimicry wherein enterotoxigenic bacteria have evolved a
strategy for dissemination and propagation that exploits normal
intestinal physiology. Indeed, STs are structurally and
functionally homologous to the endogenous peptides guanylin and
uroguanylin (12, 13), which mediate autocrine/paracrine control of
intestinal fluid and electrolyte homeostasis (14).
[0189] Beyond volume homeostasis, GC-C and its ligands have been
implicated in the regulation of the balance of proliferation and
differentiation along the crypt-to-villus axis in the intestine
(15). Expression of endogenous GC-C ligands is frequently lost
during tumorigenesis, and subsequent loss of signaling may
represent one key mutational event underlying neoplastic
transformation in the colon (16-18). In principle, this putative
role for GC-C as a tumor suppressor may contribute to the inverse
epidemiological association between colorectal cancer (1) and ETEC
infections (2, 3) reflecting, in part, longitudinal exposure in
under-developed countries to ST-producing bacteria. However, the
mechanisms by which STs repress colorectal tumorigenesis are
unknown. The present study revealed a previously unrecognized
ST-induced cGMP-dependent signaling pathway, through cyclic
nucleotide-gated (CNG) channels and calcium, responsible for the
antiproliferative action of bacterial enterotoxins on human colon
carcinoma cells.
Materials and Methods
[0190] Cell Culture.
[0191] T84 (passages 50-70) and SW480 (passage 100-120) human colon
carcinoma cells, obtained from the American Type Culture
Collection, were maintained at 37.degree. C. (5% CO2) in DMEM/F12
containing 2.5 mM L-glutamine, 100 units/ml penicillin, 100
.mu.g/ml streptomycin, and 10% (vol/vol) FBS.
[0192] DNA Synthesis.
[0193] Exponentially growing cancer cells (.apprxeq.60% confluent
in 96-well plates) were synchronized by serum starvation in Eagle's
minimal essential medium (EMEM) for 48 h, followed by proliferative
induction with 10 mM L-glutamine (in EMEM) for 24 h. ST and other
agents were added to cells 15 min before 0.2 .mu.Ci per well (1
Ci=37 GBq) of [methyl-3H]thymidine, which was added for 3 h,
followed by quantification of [3H]thymidine incorporation into DNA
(15).
[0194] Current Measurements.
[0195] The perforated mode of the whole-cell patch-clamp recording
was applied to human T84 colon cancer cells. Membrane potential was
controlled through the electrical access obtained by membrane
perforation induced by amphotericin B (9; 10). The pipette solution
supplemented by amphotericin B (200-240 mg/ml) contained: 140 mM
KCl, 1 mM MgCl2, 5 mM EGTA, and 20 mM Hepes-KOH (pH 7.3). The bath
solution contained (in mM): 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl.sub.2, 0.5 mM MgCl2, 5.5 mM glucose, and 5 mM Hepes-NaOH (pH
7.4). Voltage-clamp recordings were performed with an Axopatch 1-C
patch-clamp amplifier (Axon Instruments, Foster City, Calif.) at
31.+-.1.degree. C. using an HCC-100 temperature controller (Dagan
Instruments, Minneapolis; ref. 19).
[0196] Cyclic Nucleotides.
[0197] cGMP and cAMP were quantified in exponentially growing T84
cells (.apprxeq.60% confluent in 96-well plates) employing ELISA
(Amersham Pharmacia) or RIA. In experiments employing ELISA, media
was aspirated, reactions were terminated by the addition of a lysis
solution (200 .mu.l per well) containing 0.5%
dodecyltrimethylammonium bromide, and aliquots (100 .mu.l) from
each well were processed for cyclic nucleotide determinations. In
experiments employing RIA, reactions were terminated with ice-cold
100% ethanol, and supernatants were separated from pellets by
centrifugation and processed for cGMP determinations (8).
[0198] Calcium Transport.
[0199] Exponentially growing T84 cells (40-80% confluent in 24-well
plates) were incubated in media (S-MEM, Life Technologies,
Rockville, Md.) containing low (300 .mu.M) CaCl2 to maximize the
signal-to-noise ratio for L-cis-diltiazem (L-DLT)-sensitive 45Ca2+
transport determinations. Cells were treated with ST, 8-br-cGMP,
and/or L-DLT for the indicated times, followed by the addition of 1
.mu.Ci per well 45Ca2+ for the last 15 min. Incubations were
terminated by washing four times in cold PBS buffer (145 mM NaCl/5
mM KCl/1 mM MgCl2/10 mM glucose/5 mM Hepes, pH 7.4) containing 0.1
mM EGTA, cells were solubilized with cold NaOH (0.1 M), and
radioactivity was quantified in 100-.mu.l aliquots.
Results and Discussion
[0200] ST inhibited DNA synthesis in human colon cancer cells that
express GC-C, but not in tumor cells deficient in GC-C (FIG. 1b).
The concentration-dependence of ST inhibition of proliferation
corresponded to the accumulation of intracellular cGMP [cGMP]i, but
not other cyclic nucleotides (FIG. 1c). Yet, selective inhibitors
of PKG, which disrupt ST induction of intestinal secretion (20),
did not prevent the antiproliferative action of the enterotoxin
(FIG. 1d). Moreover, inhibitors of cAMP-dependent protein kinase or
cGMP-regulated phosphodiesterase 3 did not influence inhibition of
proliferation by ST (FIG. 1d). Thus, the antiproliferative actions
of ST on human colon carcinoma cells were not mediated by
conventional downstream effectors of cGMP.
[0201] Rather, a specific reversible inhibitor of CNG channels,
L-DLT (21), prevented ST inhibition of proliferation (FIG. 1d). In
fact, ST induced membrane current in voltage-clamped human colon
cancer cells that was reversibly blocked by L-DLT, indicating that
the enterotoxin activates CNG channels (FIGS. 2a and 2b).
Throughout the range of the imposed membrane potential ramp, ST
significantly increased current, an effect largely reversed by
L-DLT (FIG. 2c). Although the reversal potential (Em) of colon
cancer cells was -39.3.+-.3.9 mV (n=6), ST shifted Em to
-66.3.+-.3.6 mV (n=6; FIG. 2c), an effect abolished by L-DLT that
restored Em to -39.5.+-.5.2 mV (n=4; FIG. 2c). The action of ST on
membrane current in colon cancer cells was mimicked by the
membrane-permeant cGMP analog, 8-br-cGMP, which also induced an
L-DLT-sensitive current and produced a significant shift in Em
(.DELTA.m=-25.8.+-.4.6 mV, n=3; FIG. 3 a-c). Moreover, 8-br-cGMP
inhibited tumor cell DNA synthesis in an L-DLT sensitive manner
(FIG. 3d). Identical actions of ST and 8-br-cGMP in colon cancer
cells demonstrate that a [cGMP]i-signaling pathway mediates the
action of the enterotoxin on activation of CNG channels and
suppression of tumor cell proliferation.
[0202] The nonselective CNG channel preferentially conducts Ca2+
ions (22). Accordingly, removal of extracellular Ca2+ ([Ca2+]ext)
reduced both ST and 8-br-cGMP currents, from 0.23.+-.0.07 to
0.02.+-.0.01 nA and from 0.21.+-.0.04 to 0.07.+-.0.04 nA,
respectively (n=5; FIG. 4 a-c). As a further indicator of Ca2+
entry, Ca2+-dependent K+ current (KCa), previously reported in this
cancer cell line (23) and identified here by sensitivity to the
specific blocker charybdotoxin, was found to be induced by both ST
and 8-br-cGMP (FIG. 4c). In accordance, reversal potentials derived
from the ST- and 8-br-cGMP-induced current-voltage relationships
approached -70 mV (FIGS. 2c, 3b, and 4c Inset), which closely
approximates the theoretical equilibrium potential for K+ ions
(EK.apprxeq.-75 mV) calculated from the Nernst equation (24). Such
shift in Em away from Ca2+ equilibrium would drive Ca2+ influx.
However, failure to exactly match the theoretical K+ equilibrium
reflects current contribution by non-K.sup.+ conductances, such as
the chloride-carrying CFTR and/or nonselective current through CNG
channels. Finally, ST and 8-br-cGMP-mediated Ca2+ entry through CNG
channels was demonstrated by direct measurement of radioactive
calcium flux into colon cancer cells that was inhibited by L-DLT
(FIG. 4d).
[0203] The essential role of Ca2+ influx in the ST-mediated
regulation of cancer cell proliferation is underscored by reversal
of ST antiproliferative action through cytosolic Ca2+ chelation
with BAPTA-AM (FIG. 4e). Furthermore, without influencing [cGMP]i,
depletion of [Ca2+]ext abolished the ability of ST to inhibit
cancer cell proliferation, whereas increases in [Ca2+]ext restored
in a concentration-dependent manner the antiproliferative effect of
ST (FIG. 40. In fact, ionomycin, a Ca2+ ionophore, mimicked,
whereas dantrolene, which blocks Ca2+ release from intracellular
pools, or phenamil, an inhibitor of electrogenic Na+ influx, did
not affect the ability of ST to inhibit cancer cell proliferation
(FIG. 4e). The absence of charybdotoxin effect on ST inhibition of
cancer cell proliferation further indicates that Ca2+ entry rather
than the consequent Ca2+-sensitive K+ current is required for
enterotoxin-mediated antiproliferation (FIG. 4e). Thus, ST inhibits
DNA synthesis in colon carcinoma cells by a signaling mechanism
initiated by activation of GC-C, accumulation of [cGMP]i, and Ca2+
influx through CNG channels.
[0204] cGMP is emerging as an important regulator of cell
proliferation, although the molecular mechanisms mediating that
activity appear to be varied and cell-specific. Thus, in human
vascular smooth muscle cells, cGMP delays the G1/S transition by
down-regulation of cyclin D1 and cyclin-dependent kinase 4
activities after platelet-derived growth factor stimulation (25).
Also, cGMP suppresses human T cell proliferation by inhibiting IL-2
release (26). In addition, proliferation of glomerular mesangial
cells by phorbol 12,13-dibutyrate-mediated activation of
mitogen-activated protein kinase is antagonized by cGMP-induced
expression of the specific phosphatase MKP-1 (27). Further, in
colorectal cancer cells, the antiproliferative effects of cGMP may
reflect activation of apoptotic pathways (28, 29) or regulation of
cell cycle (15). Conversely, cGMP-dependent activation of PKG
promotes human umbilical vein endothelial cell proliferation by
stimulating Raf-1 kinase activity (30).
[0205] In the present study, proliferation of intestinal epithelial
cells was suppressed by a signaling mechanism initiated by ST
interaction with GC-C. Indeed, cells that lack this receptoenzyme
molecule and do not exhibit ST-induced accumulation of [cGMP]i (31)
were unresponsive to the antiproliferative effects of ST. ST/GC-C
interaction induced the accumulation of [cGMP]i, which mediated
suppression of proliferation by activating CNG channels. It is
notable that cGMP inhibition of proliferation in intestinal cells
is mediated through the L-DLT-sensitive CNG pathway, because
specific inhibitors of cAMP-dependent protein kinase (32),
phosphodiesterase 3 (11), and PKG (20), which mediate
GC-C-dependent secretion, did not alter the effect of ST on DNA
synthesis. Thus, ST-mediated [cGMP]i accumulation does not cause
functional transactivation of cAMP-dependent pathways because ST
did not induce [cAMP]i accumulation (FIG. 1c), activation of
cAMP-dependent protein kinase, or inhibition of phosphodiesterase 3
(FIG. 1d). This report details the regulation of cell proliferation
by a cGMP-dependent mechanism mediated by CNG channels.
[0206] Ca2+ serves as the third messenger in the signaling cascade
linking GC-C at the cell surface to regulation of proliferation in
the nucleus. Indeed, the ability of the second messenger cGMP to
inhibit DNA synthesis was mediated by [Ca2+]ext influx through CNG
channels (see FIG. 4 d-f). cGMP-dependent CNG channel-mediated Ca2+
influx has been described in excitable cells, although it mediates
different functions. In this way, in cone and rod photoreceptors,
high [cGMP]i sustains dark-state functions, in part, by inducing
Ca2+ influx through CNG channels (33). Similarly, in the brain,
cGMP-dependent activation of CNG channels regulates
neurotransmitter release and potentiates synaptic transmission
through Ca2+ influx (34). Additionally, regulation of intracellular
Ca2+ by cGMP has been described in several cell systems, including
vascular smooth muscle (35), platelets (36), and neurons (37). In
intestinal cells, ST activation of GC-C mobilizes intracellular
Ca2+ by a cGMP-dependent mechanism (38, 39). Further, ST and
8-br-cGMP induce Ca2+ influx through CNG channels in colon (40). In
close agreement, the present study demonstrates that ST and
8-br-cGMP induce a structurally and functionally compartmentalized
influx of Ca2+ through CNG channels in human colon carcinoma cells.
Indeed, cGMP does not evoke electrophysiologically detectable Ca2+
currents in these cells (41), although its influx could be detected
with radioactive Ca2+. The precise mode of Ca2+ delivery to its
intracellular site of action to induce intestinal cell cytostasis
remains undefined. Yet, this antiproliferative process must be
tightly regulated because Ca2+ can, upon intracellular
accumulation, promote apoptosis (42), which is not induced by ST
(15).
[0207] In summary, these data demonstrate that bacterial
enterotoxins suppress colon carcinoma cells through a GC-C-based
calcium-dependent signaling pathway with a newly identified role in
regulating cell proliferation. Endogenous GC-C ligands, guanylin
and uroguanylin, may activate this pathway and promote the
transition from proliferation to differentiation of enterocytes
along the crypt-villus axis in normal intestine (15). In addition,
these observations offer a possible mechanistic insight into the
resistance to colorectal cancer observed in geographic areas in
which ETEC is endemic. Although additional factors contribute to
the epidemiology of colorectal cancer (43, 44), the significance of
this antiproliferative pathway is highlighted by the neoplastic
transformation of epithelial cells that follows loss of expression
of endogenous GC-C ligands in the intestine (16-18). In turn, the
conservation of GC-C itself and its downstream effectors by
colorectal tumors provides a therapeutic target for restoration of
this signaling cascade and maintenance of the tumor-suppressor
phenotype. Indeed, oral administration of GC-C ligands or
downstream effectors of that pathway, such as calcium, offer a
hitherto unknown approach to the primary prevention of intestinal
neoplasia and/or therapy of colorectal cancer metastases (29,
45-47).
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Bhattacharya, J. & Chakrabarti, M. K. (1998). Biochim. Biophys.
Acta 1403, 14 [0247] 40. Qiu, W., Lee, B., Lancaster, M., Xu, W.,
Leung, S., & Guggino, S. E. (2000). Am. J. Physiol. 278,
C336-C343 [0248] 41. Biel, M., Sautter, A., Ludwig, A., Hofmann,
F., & Zong, X. (1998). Naunyn-Schmiedeberg's Arch. Pharmacol.
358, 140-144 [0249] 42. Berridge, M. J., Bootman, M. D., &
Lipp, P. (1998). Nature 395, 645-648 [0250] 43. Briskey, E. N.
& Pamies, R. J. (2000). J. Natl. Med. Assoc. 92, 222-230 [0251]
44. Wilmink, A. B. (1997). Dis. Colon Rectum 40, 483-493 [0252] 45.
Buset, M., Lipkin, M., Winawer, S., Swaroop, S., & Friedman, E.
(1986). Cancer Res. 46, 5426-5430 [0253] 46. Penman, I. D., Liang,
Q. L., Bode, J., Eastwood, M. A., & Arends, M. J. (2000). J.
Clin. Pathol. 53, 302-307 [0254] 47. Sesink, A. L., Termont, D. S.,
Kleibeuker, J. H., & Van der Meer, R. (2001). Carcinogenesis
22, 1653-1659 [0255] 48. Grider, J. R. (1993). Am. J. Physiol. 264,
G334-G340 [0256] 49. Butt, E., Eigenthaler, M., & Genieser, H.
G. (1994). Eur. J. Pharmacol. 269, 265-268 [0257] 50. Gadbois, D.
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(1995). FEBS Lett. 375, 231-234 [0259] 52. Harrison, S. A.,
Reifsnyder, D. H., Gallis, B., Cadd, G. G., & Beavo, J. A.
(1986). Mol. Phar.
Example 3
[0260] The effect of GCC ligands on the component processes
underlying metastasis cancer which has cells that expresses GCC has
been studied. The metastatic process nominally involves three
steps: digesting the underlying connective tissue, moving through
the hole created by that digestion, and evading the trap of
connective tissue underneath the hole that traps cells trying to
escape from their normal compartment. Experiments used ST as the
GCC ligand and colorectal cancer cells as the cancer which has
cells that expresses GCC. Results indicate the following.
[0261] ST inhibited the release of matrix metalloproteinase 9, an
enzyme that digests collagen upon which intestinal epithelial cells
sit. Preventing the secretion of this digestive enzyme will inhibit
the ability of these tumor cells to invade the wall of the
intestine. A decrease in secretion was demonstrated by employing
enzymology (MMP 9 activity) and protein analysis (western blot) in
human colorectal cancer cells in vitro. ST did this at a
concentration of 1 micromolar. The effects of ST are precisely
mimicked by cylic GMP.
[0262] ST prevented human colorectal cancer cells from organizing
their actin cytoskeleton. ST (1 micromolar) and cyclic GMP
prevented this organization as observed by confocal microscopy,
specifically staining actin filaments, and by cell spreading. The
latter is a functional readout of the ability of cells to organize
actin. Human colorectal cancer cells treated with ST cannot spread
their bodies out in vitro. An inability to organize their actin
cytoskeletons will prevent these cells from being able to migrate
through tissues to metastasize.
[0263] ST selectively increased the affinity of human colorectal
cancer cells for type IV collagen in vitro (ST at 1 micromolar).
Type IV collagen forms the connective tissue meshwork supporting
the lamina propria and specifically serves to trap cells through
specific interactions with beta integrins. Cancer cells evade this
trap by diminishing their ability to bind to type IV collagen. ST
treatment increased by 2-fold, a significant change, the
association of human colorectal cancer cells with type IV collagen,
but not with laminin or fibronectin, making this a specific
interaction. Enhancing this interaction will prevent metastases by
trapping cells in the laminapropria underlying epithelial cells in
the intestine. In addition to demonstrating selective increase of
colorectal cancer cell affinity for type IV collagen, a component
of the laminopropria, these data demonstrate the effects of ST on
functions important to metastasis of gastric and esophageal cancer.
Specifically the data demonstrate that ST inhibited the release of
an enzyme involved in metastasis and prevented actin organization
necessary for cell migration. These data support the conclusion
that GCC ligands will inhibit metastasis of cancer cells that
express GCC such as primary and metastatic colorectal, gastric and
esophageal.
Sequence CWU 1
1
56157DNAArtificial Sequenceencodes heat stable toxin peptide of SEQ
ID NO 2 1aac aac aca ttt tac tgc tgt gaa ctt tgt tgt aat cct gcc
tgt gct 48Asn Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala
Cys Ala1 5 10 15gga tgt tat 57Gly Cys Tyr219PRTArtificial
Sequenceheat stable toxin peptide Ia 2Asn Asn Thr Phe Tyr Cys Cys
Glu Leu Cys Cys Asn Pro Ala Cys Ala1 5 10 15Gly Cys
Tyr318PRTArtificial Sequenceheat stable toxin peptide I* 3Asn Thr
Phe Tyr Cys Cys Glu Leu Cys Cys Tyr Pro Ala Cys Ala Gly1 5 10 15Cys
Asn457DNAArtificial Sequenceencodes heat stable toxin peptide of
SEQ ID NO 5 4aat agt agc aat tac tgc tgt gaa ttg tgt tgt aat cct
gct tgt aac 48Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys Cys Asn Pro
Ala Cys Asn1 5 10 15ggg tgc tat 57Gly Cys Tyr519PRTArtificial
Sequenceheat stable toxin peptide Ib 5Asn Ser Ser Asn Tyr Cys Cys
Glu Leu Cys Cys Asn Pro Ala Cys Asn1 5 10 15Gly Cys
Tyr615PRTArtificial Sequenceguanylin 6Pro Asn Thr Cys Glu Ile Cys
Ala Tyr Ala Ala Cys Thr Gly Cys1 5 10 15718PRTArtificial
Sequencefragment of SEQ ID NO 2 7Asn Asn Thr Phe Tyr Cys Cys Glu
Leu Cys Cys Asn Pro Ala Cys Ala1 5 10 15Gly Cys817PRTArtificial
Sequencefragment of SEQ ID NO 2 8Asn Thr Phe Tyr Cys Cys Glu Leu
Cys Cys Asn Pro Ala Cys Ala Gly1 5 10 15Cys916PRTArtificial
Sequencefragment of SEQ ID NO 2 9Thr Phe Tyr Cys Cys Glu Leu Cys
Cys Asn Pro Ala Cys Ala Gly Cys1 5 10 151015PRTArtificial
Sequencefragment of SEQ ID NO 2 10Phe Tyr Cys Cys Glu Leu Cys Cys
Asn Pro Ala Cys Ala Gly Cys1 5 10 151114PRTArtificial
Sequencefragment of SEQ ID NO 2 11Tyr Cys Cys Glu Leu Cys Cys Asn
Pro Ala Cys Ala Gly Cys1 5 101213PRTArtificial Sequencefragment of
SEQ ID NO 2 12Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Ala Gly Cys1
5 101318PRTArtificial Sequencefragment of SEQ ID NO 2 13Asn Thr Phe
Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Ala Gly1 5 10 15Cys
Tyr1417PRTArtificial Sequencefragment of SEQ ID NO 2 14Thr Phe Tyr
Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Ala Gly Cys1 5 10
15Tyr1516PRTArtificial Sequencefragment of SEQ ID NO 2 15Phe Tyr
Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Ala Gly Cys Tyr1 5 10
151615PRTArtificial Sequencefragment of SEQ ID NO 2 16Tyr Cys Cys
Glu Leu Cys Cys Asn Pro Ala Cys Ala Gly Cys Tyr1 5 10
151714PRTArtificial Sequencefragment of SEQ ID NO 2 17Cys Cys Glu
Leu Cys Cys Asn Pro Ala Cys Ala Gly Cys Tyr1 5 101817PRTArtificial
Sequencefragment of SEQ ID NO 3 18Asn Thr Phe Tyr Cys Cys Gly Leu
Cys Cys Tyr Pro Ala Cys Ala Gly1 5 10 15Cys1916PRTArtificial
Sequencefragment of SEQ ID NO 3 19Thr Phe Tyr Cys Cys Glu Leu Cys
Cys Tyr Pro Ala Cys Ala Gly Cys1 5 10 152015PRTArtificial
Sequencefragment of SEQ ID NO 3 20Phe Tyr Cys Cys Glu Leu Cys Cys
Tyr Pro Ala Cys Ala Gly Cys1 5 10 152114PRTArtificial
Sequencefragment of SEQ ID NO 3 21Tyr Cys Cys Glu Leu Cys Cys Tyr
Pro Ala Cys Ala Gly Cys1 5 102213PRTArtificial Sequencefragment of
SEQ ID NO 3 22Cys Cys Glu Leu Cys Cys Tyr Pro Ala Cys Ala Gly Cys1
5 102317PRTArtificial Sequencefragment of SEQ ID NO 3 23Thr Phe Tyr
Cys Cys Glu Leu Cys Cys Tyr Pro Ala Cys Ala Gly Cys1 5 10
15Asn2416PRTArtificial Sequencefragment of SEQ ID NO 3 24Phe Tyr
Cys Cys Glu Leu Cys Cys Tyr Pro Ala Cys Ala Gly Cys Asn1 5 10
152515PRTArtificial Sequencefragment of SEQ ID NO 3 25Tyr Cys Cys
Glu Leu Cys Cys Tyr Pro Ala Cys Ala Gly Cys Asn1 5 10
152614PRTArtificial Sequencefragment of SEQ ID NO 3 26Cys Cys Glu
Leu Cys Cys Tyr Pro Ala Cys Ala Gly Cys Asn1 5 102718PRTArtificial
Sequencefragment of SEQ ID NO 5 27Asn Ser Ser Asn Tyr Cys Cys Glu
Leu Cys Cys Asn Pro Ala Cys Thr1 5 10 15Gly Cys2817PRTArtificial
Sequencefragment of SEQ ID NO 5 28Ser Ser Asn Tyr Cys Cys Glu Leu
Cys Cys Asn Pro Ala Cys Thr Gly1 5 10 15Cys2916PRTArtificial
Sequencefragment of SEQ ID NO 5 29Ser Asn Tyr Cys Cys Glu Leu Cys
Cys Asn Pro Ala Cys Thr Gly Cys1 5 10 153015PRTArtificial
Sequencefragment of SEQ ID NO 5 30Asn Tyr Cys Cys Glu Leu Cys Cys
Asn Pro Ala Cys Thr Gly Cys1 5 10 153114PRTArtificial
Sequencefragment of SEQ ID NO 5 31Tyr Cys Cys Glu Leu Cys Cys Asn
Pro Ala Cys Thr Gly Cys1 5 103213PRTArtificial Sequencefragment of
SEQ ID NO 5 32Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys1
5 103318PRTArtificial Sequencefragment of SEQ ID NO 5 33Ser Ser Asn
Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly1 5 10 15Cys
Tyr3417PRTArtificial Sequencefragment of SEQ ID NO 5 34Ser Asn Tyr
Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys1 5 10
15Tyr3516PRTArtificial Sequencefragment of SEQ ID NO 5 35Asn Tyr
Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
153615PRTArtificial Sequencefragment of SEQ ID NO 5 36Tyr Cys Cys
Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
153714PRTArtificial Sequencefragment of SEQ ID NO 5 37Cys Cys Glu
Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 103818PRTArtificial
Sequencederivative 38Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Asn
Pro Ala Cys Ala Gly1 5 10 15Cys Tyr3918PRTArtificial
Sequencederivative 39Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Ala
Pro Ala Cys Ala Gly1 5 10 15Cys Tyr4018PRTArtificial
Sequencederivative 40Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Asn
Ala Ala Cys Ala Gly1 5 10 15Cys Tyr4117PRTArtificial
Sequencederivative 41Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Asn
Pro Ala Cys Ala Gly1 5 10 15Cys4215PRTArtificial Sequencederivative
42Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Ala Gly Cys Tyr1 5 10
154314PRTArtificial Sequencederivative 43Tyr Cys Cys Glu Leu Cys
Cys Asn Pro Ala Cys Ala Gly Cys1 5 104414PRTArtificial
Sequencederivative 44Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Ala
Gly Cys Tyr1 5 104513PRTArtificial Sequencederivative 45Cys Cys Glu
Leu Cys Cys Asn Pro Ala Cys Ala Gly Cys1 5 104625PRTArtificial
Sequencederivative 46Gln Ala Cys Asp Pro Pro Ser Pro Pro Ala Glu
Val Cys Cys Asp Val1 5 10 15Cys Cys Asn Pro Ala Cys Ala Gly Cys 20
254716PRTArtificial Sequencederivative 47Ile Asp Cys Cys Ile Cys
Cys Asn Pro Ala Cys Phe Gly Cys Leu Asn1 5 10 154818PRTArtificial
Sequencederivative 48Ser Ser Asp Trp Asp Cys Cys Asp Val Cys Cys
Asn Pro Ala Cys Ala1 5 10 15Gly Cys4919PRTArtificial
Sequencederivative 49Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys Cys
Tyr Pro Ala Cys Thr1 5 10 15Gly Cys Tyr5013PRTArtificial
Sequencederivative 50Cys Cys Asp Val Cys Cys Asn Pro Ala Cys Thr
Gly Cys1 5 105114PRTArtificial Sequencederivative 51Cys Cys Asp Val
Cys Cys Tyr Pro Ala Cys Thr Gly Cys Tyr1 5 105214PRTArtificial
SequenceOTHER INFORMATION derivative 52Cys Cys Asp Leu Cys Cys Asn
Pro Ala Cys Ala Gly Cys Tyr1 5 105314PRTArtificial
Sequencederivative 53Cys Cys Gln Leu Cys Cys Asn Pro Ala Cys Thr
Gly Cys Tyr1 5 105415PRTHomo sapiens 54Pro Gly Thr Cys Glu Ile Cys
Ala Tyr Ala Ala Cys Thr Gly Cys1 5 10 1555106PRTRattus norvegicus
55Met Ser Gly Ser Gln Leu Trp Ala Ala Val Leu Leu Leu Leu Val Leu1
5 10 15Gln Ser Ala Gln Gly Val Tyr Ile Lys Tyr His Gly Phe Gln Val
Gln 20 25 30Leu Glu Ser Val Lys Lys Leu Asn Glu Leu Glu Glu Lys Gln
Met Ser 35 40 45Asp Pro Gln Gln Gln Lys Ser Gly Leu Leu Pro Asp Val
Cys Tyr Asn 50 55 60Pro Ala Leu Pro Leu Asp Leu Gln Pro Val Cys Ala
Ser Gln Glu Ala65 70 75 80Ala Ser Thr Phe Lys Ala Leu Arg Thr Ile
Ala Thr Asp Glu Cys Glu 85 90 95Leu Cys Ile Asn Val Ala Cys Thr Gly
Cys 100 10556112PRTHomo sapiens 56Met Gly Cys Arg Ala Ala Ser Gly
Leu Leu Pro Gly Val Ala Val Val1 5 10 15Leu Leu Leu Leu Leu Gln Ser
Thr Gln Ser Val Tyr Ile Gln Tyr Gln 20 25 30Gly Phe Arg Val Gln Leu
Glu Ser Met Lys Lys Leu Ser Asp Leu Glu 35 40 45Ala Gln Trp Ala Pro
Ser Pro Arg Leu Gln Ala Gln Ser Leu Leu Pro 50 55 60Ala Val Cys His
His Pro Ala Leu Pro Gln Asp Leu Gln Pro Val Cys65 70 75 80Ala Ser
Gln Glu Ala Ser Ser Ile Phe Lys Thr Leu Arg Thr Ile Ala 85 90 95Asn
Asp Asp Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu 100 105
110
* * * * *