U.S. patent application number 16/546707 was filed with the patent office on 2020-02-06 for process of preparing guanylate cyclase c agonists.
The applicant listed for this patent is Bausch Health Ireland Limited. Invention is credited to Juncai Bai, Stephen Comiskey, Rong Feng, Jun Jian, Kunwar Shailubhai, Guoquing Zhang, Ruoping Zhang, Qiao Zhao, Junfeng Zhou.
Application Number | 20200040038 16/546707 |
Document ID | / |
Family ID | 46758495 |
Filed Date | 2020-02-06 |
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United States Patent
Application |
20200040038 |
Kind Code |
A1 |
Bai; Juncai ; et
al. |
February 6, 2020 |
Process of Preparing Guanylate Cyclase C Agonists
Abstract
The invention provides processes of preparing a peptide
including a GCC agonist sequence selected from the group consisting
of SEQ ID NOs: 1-249 described herein.
Inventors: |
Bai; Juncai; (North Augusta,
SC) ; Zhang; Ruoping; (North Augusta, SC) ;
Jian; Jun; (Shanghai, CN) ; Zhou; Junfeng;
(Shanghai, CN) ; Zhao; Qiao; (Shanghai, CN)
; Zhang; Guoquing; (Shanghai, CN) ; Shailubhai;
Kunwar; (Audubon, PA) ; Comiskey; Stephen;
(Doylestown, PA) ; Feng; Rong; (Langhorne,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bausch Health Ireland Limited |
Dublin |
|
IE |
|
|
Family ID: |
46758495 |
Appl. No.: |
16/546707 |
Filed: |
August 21, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15405787 |
Jan 13, 2017 |
10421787 |
|
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16546707 |
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14001638 |
Feb 11, 2014 |
9580471 |
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PCT/US2012/027287 |
Mar 1, 2012 |
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15405787 |
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61447891 |
Mar 1, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/195 20130101;
C07K 7/64 20130101; C07K 7/08 20130101 |
International
Class: |
C07K 7/64 20060101
C07K007/64; C07K 7/08 20060101 C07K007/08; C07K 14/195 20060101
C07K014/195 |
Claims
1. A process of preparing a peptide comprising a GCC agonist
sequence selected from the group consisting of SEQ ID NOs: 1-249,
wherein the GCC agonist sequence is n amino acid units in length,
with the N-terminal unit at position 1 and the C-terminal unit at
position n, the process comprising: providing a first fragment
having a first sequence of amino acid units from position j through
position k of the GCC agonist sequence, wherein j is an integer
between 1 and n-1, k is an integer between 2 and n and is greater
than j, and the first fragment is protected except for an amino
group of the amino acid unit at position j, or alternatively, a
carboxyl group of the amino acid unit at position k, providing a
second fragment having a second sequence of amino acid units from
position h through position j-1 of the GCC agonist sequence or
having a third sequence of amino acid units from position k+1
through position m of the GCC agonist sequence, wherein h is an
integer between 1 and n-2 and is smaller than j, m is an integer
between k+2 and n, and the second fragment is protected except for
a carboxyl group of the amino acid unit at position j-1 or an amino
group of the amino acid unit at position k+1, and coupling the
first and the second fragments via a solution-phase synthesis to
yield a protected peptide having a sequence of amino acid units
from position h through position k of the GCC agonist sequence or a
protected peptide having a sequence of amino acid units from
position j through position m of the GCC agonist sequence.
2. The process of claim 1, wherein the GCC agonist sequence is
selected from the group consisting of SEQ ID NOs: 1, 8, 9, 55, 56,
58, and 59.
3. The process of claim 1, wherein the GCC agonist sequence is
selected from the group consisting of SEQ ID NOs: 1 and 9.
4. The process of claim 3, wherein at least one of the first and
second fragments is provided via a solid-phase peptide
synthesis.
5. The process of claim 4, wherein the solid-phase peptide
synthesis is a Fmoc solid-phase synthesis.
6. The process of claim 5, wherein the Fmoc solid-phase synthesis
is performed on 2-chlorotrityl resin.
7. The process of claim 3, wherein h is 1, j is 7, and k is 16.
8. The process of claim 3, wherein h is 7, j is 15, and k is
16.
9. The process of claim 8, further comprising deprotecting an amino
group of the amino acid unit at position 7 of the protected peptide
having the sequence of amino acid units from position 7 through
position 16 of the GCC agonist sequence to yield a position-7
reactive peptide.
10. The process of claim 9, further comprising providing a third
fragment having a sequence of amino acid units from position 1
through position 6 of the GCC agonist sequence, wherein the third
fragment is protected except for a carboxyl group of the amino acid
at position 6.
11. The process of claim 10, wherein the third fragment is provided
via a solid-phase peptide synthesis.
12. The process of claim 11, wherein the solid-phase peptide
synthesis is Fmoc solid-phase synthesis.
13. The process of claim 10, further comprising coupling the third
fragment and the position-7 reactive peptide via a solution-phase
synthesis to yield a protected linear peptide having a sequence of
amino acid units from position 1 through position 16 of the GCC
agonist sequence.
14. The process of claim 13, further comprising deprotecting the
protected linear peptide to yield a deprotected linear peptide.
15. The process of claim 14, further comprising oxidizing the
deprotected linear peptide to yield the peptide comprising the GCC
agonist sequence selected from the group consisting of SEQ ID NOs:
1 and 9.
16. The process of claim 1, wherein at least one of the first and
second fragments is provided via a solid-phase peptide
synthesis.
17. The process of claim 16, wherein the solid-phase peptide
synthesis is Fmoc solid-phase synthesis.
18. The process of claim 1, wherein each of the first and second
fragments is not more than 10 amino acid units in length.
19. The process of claim 18, wherein the second fragment has the
second sequence of amino acid units from position h through
position j-1 of the GCC agonist sequence and either one or both of
the amino acid unit of the second fragment at position j-1 and the
amino acid unit of the first fragment at position k is selected
from the group consisting of glycine, proline, leucine, alanine,
and arginine.
20. The process of claim 18, wherein the second fragment has the
second sequence of amino acid units from position h through
position j-1 of the GCC agonist sequence and either one or both of
the amino acid unit of the second fragment at position j-1 and the
amino acid unit of the first fragment at position k is selected
from the group consisting of glycine and proline.
21. The process of claim 18, wherein the second fragment has the
third sequence of amino acid units from position k+1 through
position m of the GCC agonist sequence and either one or both of
the amino acid unit of the second fragment at position m and the
amino acid unit of the first fragment at position k is selected
from the group consisting of glycine, proline, leucine, alanine,
and arginine.
22. The process of claim 18, wherein the second fragment has the
third sequence of amino acid units from position k+1 through
position m of the GCC agonist sequence and either one or both of
the amino acid unit of the second fragment at position m and the
amino acid unit of the first fragment at position k is selected
from the group consisting of glycine and proline.
23. The process of claim 1, wherein k is n, the second fragment has
the second sequence of amino acid units from position h through
position j-1 of the GCC agonist sequence, and the amino acid unit
at positions j-1 is selected from the group consisting of glycine,
proline, leucine, alanine, and arginine.
24. The process of claim 23, further comprising deprotecting an
amino group of the amino acid unit at position h of the protected
peptide having a sequence of amino acid units from position h
through position k of the GCC agonist sequence to yield a
position-h reactive peptide.
25. The process of claim 24, further comprising providing a fourth
fragment having a sequence of amino acid units from position 1
through position h-1 of the GCC agonist sequence, wherein the
fourth fragment is protected except for a carboxyl group of the
amino acid unit at position h-1.
26. The process of claim 25, wherein the fourth fragment is
provided via a solid-phase peptide synthesis.
27. The process of claim 26, wherein the solid-phase peptide
synthesis is Fmoc solid-phase synthesis.
28. The process of claim 25, wherein the fourth fragment is not
more than 10 amino acid units in length.
29. The process of claim 25, further comprising coupling the fourth
fragment and the position-h reactive peptide via a solution-phase
synthesis to yield a protected linear peptide having a sequence of
amino acid units from position 1 through position n of the GCC
agonist sequence.
30. The process of claim 29, further comprising deprotecting the
protected linear peptide to yield a deprotected linear peptide
having a sequence of amino acid units from position 1 through
position n of the GCC agonist sequence.
31. The process of claim 30, further comprising oxidizing the
deprotected linear peptide to yield the peptide comprising the GCC
agonist sequence selected from the group consisting of SEQ ID NOs:
1-249.
32. The process of claim 31, further comprising purifying the
peptide comprising the GCC agonist sequence selected from the group
consisting of SEQ ID NOs: 1-249, wherein the purification comprises
adsorbing the peptide onto a polymeric adsorbent column, optionally
rinsing the peptide with deionized water, eluting the optionally
rinsed peptide off the polymeric adsorbent column with an alcohol
aqueous solution to form a peptide solution, removing water and the
alcohol from the peptide solution to precipitate the peptide, and
optionally adding an ether to the dewatered peptide to facilitate
precipitation of the peptide.
33. The process of claim 32, wherein the alcohol aqueous solution
comprises isopropanol.
34. The process of claim 32, wherein the ether comprises diethyl
ether.
35. The process of claim 32, further comprising salt exchanging the
peptide by washing the peptide with an aqueous solution comprising
an ammonium salt before the purification.
36. The process of claim 35, further comprising, lyophilizing the
peptide after the salt exchanging step before the purification.
37. A process of purifying the peptide comprising the GCC agonist
sequence selected from the group consisting of SEQ ID NOs: 1-249,
the process comprising adsorbing the peptide onto a polymeric
adsorbent column, desalting the peptide, eluting the desalted
peptide off the polymeric adsorbent column with an alcohol aqueous
solution to form a peptide solution, removing water from the
peptide solution, and adding an ether to the dewatered peptide to
precipitate the peptide.
38. The process of claim 37, wherein the alcohol aqueous solution
comprises isopropanol.
39. The process of claim 37, wherein the ether comprises diethyl
ether.
40. The process of claim 37, further comprising salt exchanging the
peptide by washing the peptide with an aqueous solution comprising
an ammonium salt before the purification.
41. The process of claim 40, further comprising, lyophilizing the
peptide after the salt exchanging step before the purification.
42. A purified peptide comprising the GCC agonist sequence selected
from the group consisting of SEQ ID NOs: 1-249, wherein the peptide
has a bulk density of no less than 0.1 g/mL, no less than 0.2 g/mL,
no less than 0.3 g/mL, no less than 0.4 g/mL, or no less than 0.5
g/mL.
43. A purified peptide, wherein the peptide is purified by the
process of any of claims 37-41.
44. The purified peptide of claim 42, wherein the peptide is
purified by the process of any of claims 37-41.
45. The purified peptide of any of claims 42-44, wherein the
peptide has a tap density of no less than 0.1 g/mL, no less than
0.2 g/mL, no less than 0.3 g/mL, no less than 0.4 g/mL, no less
than 0.5 g/mL, or no less than 0.6 g/mL.
46. The purified peptide of any of claims 42-45, wherein the
peptide has a chromatographic purity of no less than 95%, no less
than 97%, or no less than 98%.
47. The purified peptide of any of claims 42-46, wherein peptide is
substantially free of water.
48. The purified peptide of any of claims 42-47, wherein peptide is
substantially free of impurities selected from acetonitrile,
acetamide, alcohols, ammonium, and TFA.
49. The purified peptide of any of claims 42-48, wherein the
peptide is substantially free of topoisomers.
50. The purified peptide of any of claims 42-49, wherein the
peptide comprises the GCC agonist sequence of SEQ ID NO: 1.
51. The purified peptide of any of claims 42-49, wherein the
peptide comprises the GCC agonist sequence of SEQ ID NO: 9.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of, and claims priority
to, U.S. application Ser. No. 15/405,787 filed Jan. 13, 2017, which
is a continuation of, and claims priority to, U.S. application Ser.
No. 14/001,638 filed Feb. 11, 2014, which is a 35 U.S.C. .sctn. 371
National Phase Application of and claims priority to
PCT/US2012/027287, filed Mar. 1, 2012 which claims priority to U.S.
Provisional Application No. 61/447,891 filed Mar. 1, 2011, the
contents of each of which are incorporated by reference in their
entireties.
INCORPORATION OF SEQUENCE LISTING
[0002] The contents of the text file named "376464-2003US3 Sequence
Listing.txt", which was created on Aug. 21, 2019 and is 112 KB in
size, are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to processes of preparing
guanylate cyclase C peptide agonists useful for the treatment and
prevention of various diseases and disorders.
BACKGROUND OF THE INVENTION
[0004] Guanylate cyclase C is a transmembrane form of guanylate
cyclase that is expressed on various cells, including
gastrointestinal epithelial cells (reviewed in Vaandrager 2002 Mol.
Cell. Biochem. 230:73-83). It was originally discovered as the
intestinal receptor for the heat-stable toxin (ST) peptides
secreted by enteric bacteria and which cause diarrhea. The ST
peptides share a similar primary amino acid structure with two
peptides isolated from intestinal mucosa and urine, guanylin and
uroguanylin (Currie, et al., Proc. Nat'l Acad. Sci. USA 89:947-951
(1992); Hamra, et al., Proc. Nat'l Acad. Sci. USA 90:10464-10468
(1993); Forte, L., Reg. Pept. 81:25-39 (1999); Schulz, et al., Cell
63:941-948 (1990); Guba, et al., Gastroenterology 111:1558-1568
(1996); Joo, et al., Am. J. Physiol. 274:G633-G644 (1998)).
[0005] In the intestines, guanylin and uroguanylin act as
regulators of fluid and electrolyte balance. In response to high
oral salt intake, these peptides are released into the intestinal
lumen where they bind to guanylate cyclase C localized on the
luminal membrane of enterocytes (simple columnar epithelial cells
of the small intestines and colon). The binding of the guanylin
peptides to guanylate cyclase C induces electrolyte and water
excretion into the intestinal lumen via a complex intracellular
signaling cascade that is initiated by an increase in cyclic
guanosine monophosphate (cGMP).
[0006] The cGMP-mediated signaling that is initiated by the
guanylin peptides is critical for the normal functioning of the
gut. Any abnormality in this process could lead to gastrointestinal
disorders such as irritable bowel syndrome (IBS) and inflammatory
bowel diseases. Inflammatory bowel disease is a general name given
to a group of disorders that cause the intestines to become
inflamed, characterized by red and swollen tissue. Examples include
ulcerative colitis and Crohn's disease. Crohn's disease is a
serious inflammatory disease that predominantly affects the ileum
and colon, but can also occur in other sections of the
gastrointestinal tract. Ulcerative colitis is exclusively an
inflammatory disease of the colon, the large intestine. Unlike
Crohn's disease, in which all layers of the intestine are involved,
and in which there can be normal healthy bowel in between patches
of diseased bowel, ulcerative colitis affects only the innermost
lining (mucosa) of the colon in a continuous manner. Depending on
which portion of the gastrointestinal tract is involved, Crohn's
disease may be referred to as ileitis, regional enteritis, colitis,
etc. Crohn's disease and ulcerative colitis differ from spastic
colon or irritable bowel syndrome, which are motility disorders of
the gastrointestinal tract. Gastrointestinal inflammation can be a
chronic condition. It is estimated that as many as 1,000,000
Americans are afflicted with inflammatory bowel disease, with male
and female patients appearing to be equally affected. Most cases
are diagnosed before age 30, but the disease can occur in the
sixth, seventh, and later decades of life.
[0007] IBS and chronic idiopathic constipation are pathological
conditions that can cause a great deal of intestinal discomfort and
distress but unlike the inflammatory bowel diseases, IBS does not
cause the serious inflammation or changes in bowel tissue and it is
not thought to increase the risk of colorectal cancer. In the past,
inflammatory bowel disease, celiac disease, and IBS were regarded
as completely separate disorders. Now, with the description of
inflammation, albeit low-grade, in IBS, and of symptom overlap
between IBS and celiac disease, this contention has come under
question. Acute bacterial gastroenteritis is the strongest risk
factor identified to date for the subsequent development of
postinfective irritable bowel syndrome. Clinical risk factors
include prolonged acute illness and the absence of vomiting. A
genetically determined susceptibility to inflammatory stimuli may
also be a risk factor for irritable bowel syndrome. The underlying
pathophysiology indicates increased intestinal permeability and
low-grade inflammation, as well as altered motility and visceral
sensitivity. Serotonin (5-hydroxytryptamine [5-HT]) is a key
modulator of gut function and is known to play a major role in
pathophysiology of IBS. The activity of 5-HT is regulated by
cGMP.
[0008] While the precise causes of IBS and inflammatory bowel
diseases (IBD) are not known, a disruption in the process of
continual renewal of the gastrointestinal mucosa may contribute to
disease pathology in IBD and aggravate IBS. The renewal process of
the gastrointestinal lining is an efficient and dynamic process
involving the continual proliferation and replenishment of unwanted
damaged cells. Proliferation rates of cells lining the
gastrointestinal mucosa are very high, second only to the
hematopoietic system. Gastrointestinal homeostasis depends on both
the proliferation and programmed cellular death (apoptosis) of
epithelial cells lining the gut mucosa. Cells are continually lost
from the villus into the lumen of the gut and are replenished at a
substantially equal rate by the proliferation of cells in the
crypts, followed by their upward movement to the villus. The rates
of cell proliferation and apoptosis in the gut epithelium can be
increased or decreased in a variety of circumstances, e.g., in
response to physiological stimuli such as aging, inflammatory
signals, hormones, peptides, growth factors, chemicals and dietary
habits. In addition, an enhanced proliferation rate is frequently
associated with a reduction in turnover time and an expansion of
the proliferative zone. The proliferation index is much higher in
pathological states such as ulcerative colitis and other
gastrointestinal disorders. Intestinal hyperplasia is a major
promoter of gastrointestinal inflammation. Apoptosis and cell
proliferation together regulate cell number and determine the
proliferation index. Reduced rates of apoptosis are often
associated with abnormal growth, inflammation, and neoplastic
transformation. Thus, both increased proliferation and/or reduced
cell death may increase the proliferation index of intestinal
tissue, which may in turn lead to gastrointestinal inflammatory
diseases.
[0009] In addition to a role for uroguanylin and guanylin as
modulators of intestinal fluid and ion secretion, these peptides
may also be involved in the continual renewal of gastrointestinal
mucosa by maintaining the balance between proliferation and
apoptosis. For example, uroguanylin and guanylin peptides appear to
promote apoptosis by controlling cellular ion flux. Given the
prevalence of inflammatory conditions in Western societies a need
exists to improve the treatment options for inflammatory
conditions, particularly of the gastrointestinal tract.
[0010] Peptide agonists of guanylate cyclase C agonists ("GCC
agonists") are described in U.S. Pat. Nos. 7,041,786, 7,799,897,
and U.S. Patent Application Publication Nos. US2009/0048175, US
2010/0069306, US 2010/0120694, US 2010/0093635, and US
2010/0221329. However, the solid phase synthesis of peptides for
pharmaceutical application presents a number of special problems
such as an overall low yield (e.g., less than 10%).
SUMMARY OF THE INVENTION
[0011] The present invention provides a process of preparing a
peptide, particularly a peptide comprising the sequence of a
peptide agonist of guanylate cyclase C ("GCC"). The GCC agonist
sequence is selected from the group consisting of SEQ ID NOs:
1-249. The GCC agonist sequence is n amino acid units in length,
with the N-terminal unit at position 1 and the C-terminal unit at
position n.
[0012] The process of the invention includes solid phase and/or
solution phase syntheses of suitable peptide fragments, subsequent
fragment condensation in a solution to form a linear crude peptide,
and optional oxidative cyclization of cysteine amino acid residues
of the linear crude peptide to form the cyclized final product.
Particularly, the process includes the following steps: [0013]
providing a first fragment having a first sequence of amino acid
units from position j through position k of the GCC agonist
sequence, wherein j is an integer between 1 and n-1, k is an
integer between 2 and n and is greater than j, and the first
fragment is protected except for an amino group of the amino acid
unit at position j, or alternatively, a carboxyl group of the amino
acid unit at position k, [0014] providing a second fragment having
a second sequence of amino acid units from position h through
position j-1 of the GCC agonist sequence or having a third sequence
of amino acid units from position k+1 through position m of the GCC
agonist sequence, wherein h is an integer between 1 and n-2 and is
smaller than j, m is an integer between k+2 and n, and the second
fragment is protected except for a carboxyl group of the amino acid
unit at position j-1 or an amino group of the amino acid unit at
position k+1, and [0015] coupling the first and the second
fragments via a solution-phase synthesis to yield a protected
peptide having a sequence of amino acid units from position h
through position k of the GCC agonist sequence or a protected
peptide having a sequence of amino acid units from position j
through position m of the GCC agonist sequence.
[0016] The process of the invention may include one or more of the
features described in the following embodiments.
[0017] In one embodiment, the GCC agonist sequence is selected from
the group consisting of SEQ ID NOs: 1, 8, 9, 55, 56, 58, and
59.
[0018] In one embodiment, the GCC agonist sequence is SEQ ID NO: 1
or 9. In one embodiment, h is 1, j is 7, and k is 16. More
specifically, the first fragment has a sequence of amino acid units
from position 7 through position 16 of SEQ ID NO: 1 or 9 and the
second fragment has a sequence of amino acid units from position 1
through position 6 of SEQ ID NO: 1 or 9. In another embodiment, h
is 7, j is 15, and k is 16. More specifically, the first fragment
has a sequence of amino acid units from position 15 through
position 16 of SEQ ID NO: 1 or 9, the second fragment has a
sequence of amino acid units from position 7 through position 14 of
SEQ ID NO: 1 or 9, and the protected peptide produced via coupling
the first and second fragments has a sequence of amino acid units
from position 7 through position 16 of SEQ ID NO: 1 or 9. In one
embodiment, the process of the invention further includes
deprotecting an amino group of the amino acid unit at position 7 of
the protected peptide having the sequence of amino acid units from
position 7 through position 16 of SEQ ID NO: 1 or 9 to yield a
position-7 reactive peptide. In one embodiment, the process of the
invention further includes providing a third fragment having a
sequence of amino acid units from position 1 through position 6 of
SEQ ID NO: 1 or 9, wherein the third fragment is protected except
for a carboxyl group of the amino acid at position 6. In one
embodiment, the process further includes coupling the third
fragment and the position-7 reactive peptide via a solution-phase
synthesis to yield a protected linear peptide having a sequence of
amino acid units from position 1 through position 16 of SEQ ID NO:
1 or 9. In one embodiment, the process further includes
deprotecting the protected linear peptide to yield a deprotected
linear peptide. In one embodiment, the process further comprises
oxidizing the deprotected linear peptide to yield the peptide
having the GCC agonist sequence of SEQ ID NOs: 1 or 9.
[0019] In one embodiment, each of the first and second fragments is
not more than 10 amino acid units in length (e.g., 2-10 amino acid
units in length, 3-9 amino acid units in length, or 4-8 amino acid
units in length).
[0020] In one embodiment, the second fragment has the second
sequence of amino acid units from position h through position j-1
of the GCC agonist sequence and either one or both of the amino
acid unit of the second fragment at position j-1 and the amino acid
unit of the first fragment at position k is selected from the group
consisting of glycine, proline, leucine, alanine, and arginine. In
one embodiment, at least one of the amino acid units at positions
j-1 and k is either glycine or proline.
[0021] In one embodiment, the second fragment has the third
sequence of amino acid units from position k+1 through position m
of the GCC agonist sequence and either one or both of the amino
acid unit of the third fragment at position m and the amino acid
unit of the first fragment at position k is selected from the group
consisting of glycine, proline, leucine, alanine, and arginine. In
one embodiment, at least one of the amino acid units at positions m
and k is either glycine or proline.
[0022] In one embodiment, k is n. In one embodiment, the second
fragment has the second sequence of amino acid units from position
h through position j-1 of the GCC agonist sequence, and the amino
acid unit at positions j-1 is selected from the group consisting of
glycine, proline, leucine, alanine, and arginine. In one
embodiment, the amino acid unit at position j-1 is either glycine
or proline. In one embodiment, the process of the invention further
comprises deprotecting an amino group of the amino acid unit at
position h of the protected peptide having a sequence of amino acid
units from position h through position k of the GCC agonist
sequence to yield a position-h reactive peptide. In one embodiment,
the process further comprises providing a fourth fragment having a
sequence of amino acid units from position 1 through position h-1
of the GCC agonist sequence, wherein the fourth fragment is
protected except for a carboxyl group of the amino acid unit at
position h-1. In one embodiment, the fourth fragment is not more
than 10 amino acid units in length (e.g., 2-10 amino acid units in
length, 3-9 amino acid units in length, or 4-8 amino acid units in
length). In one embodiment, the process further includes coupling
the fourth fragment and the position-h reactive peptide via a
solution-phase synthesis to yield a protected linear peptide having
a sequence of amino acid units from position 1 through position n
of the GCC agonist sequence. In one embodiment, the process further
comprises deprotecting the protected linear peptide to yield a
deprotected linear peptide having a sequence of amino acid units
from position 1 through position n of the GCC agonist sequence. In
one embodiment, the process of the invention further comprises
oxidizing the deprotected linear peptide to yield the peptide
comprising the GCC agonist sequence selected from the group
consisting of SEQ ID NOs: 1-249.
[0023] In one embodiment, at least one of the fragments of the
peptide of interest (e.g., the first, second, third, and/or fourth
fragments) is provided via a solid-phase peptide synthesis. In one
embodiment, the solid-phase peptide synthesis is a Fmoc solid-phase
synthesis. In one embodiment, the Fmoc solid-phase synthesis is
performed on 2-chlorotrityl resin, such as those having 1% DVB and
a substitution rate ranging from 0.3 mmol/g to 1.2 mmol/g (e.g.,
0.9-1.1 mmol/g).
[0024] The methods of this invention unexpectedly produce GCC
agonist peptides of high purities (e.g., >96%) at high yields
(e.g., >14%) compared to conventional step-by-step solid-phase
peptide synthesis (SPPS), where the overall yield of the peptides
having a 96% purity level is approximately 5%. The fragment
condensation process (i.e., hybrid solution- and solid-phase
process) of this invention also requires much less time for
synthesizing GCC peptides than conventional SPPS. In addition to
the cost effectiveness afforded by the increased yields and reduced
time, the process of this invention is also readily scalable for
commercial production.
[0025] In another aspect, the invention features the peptides,
e.g., GCC agonist peptides, prepared by the methods described
herein.
[0026] In still another aspect, the invention also provides a
process of purifying the peptide comprising the GCC agonist
sequence selected from the group consisting of SEQ ID NOs: 1-249.
The process comprises adsorbing the peptide onto a polymeric
adsorbent column, optionally rinsing the peptide with deionized
water, eluting the optionally rinsed peptide off the polymeric
adsorbent column with a solvent mixture (e.g., an alcohol aqueous
solution) to form a peptide solution, removing part or all of the
solvent mixture (e.g., water and the alcohol) from the peptide
solution to precipitate the peptide, and optionally adding an ether
to the dewatered peptide to facilitate precipitation of the
peptide.
[0027] In one embodiment, the peptide is precipitated by
concentrating the peptide solution by removing water and the
alcohol under vacuum without the need of adding an ether. In
another embodiment, an ether is added to facilitate the
precipitation, e.g., by hastening the precipitation process.
[0028] In one embodiment, rinsing the peptide after adsorption onto
a polymeric adsorbent column is not needed when, e.g., the peptide
adsorbed is substantially free of water-soluble salts (e.g.,
phosphates or acetates). In this context "substantially" free of
water-soluble salts means that the salt content of the peptide is
preferably less than 5%, less than 4.5%, less than 4.25%, less than
4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%,
less than 1.5%, less than 1%, less than 0.5%, less than 0.25%, or
less than 0.1%, of the total weight of the peptide.
[0029] In one embodiment, the polymeric adsorbent column comprises
a polystyrene resin. In particular, the resin is selected so that
the purified peptide eluted or desorbed is not less than 80% of the
peptide amount adsorbed on the resin, e.g., not less than 85%, not
less than 90%, or not less than 95%. In one embodiment, the resin
is formed of crosslinked polystyrene with an average pore diameter
greater than 5 nm, e.g., about 6-8 nm, 10-15 nm, 15-20 nm, or 25-30
nm.
[0030] In one embodiment, the solvent mixture used for eluting the
peptide comprises water, e.g., a mixture of water and one or more
second solvents which can form azeotrope with water such as
ethanol, isopropanol, tert-butanol, 2-butanol, 1-chloro-2-propanol,
1-methoxy-2-propanol, 2-methoxy-ethanol, 2-methyl-2-propanol,
acetic acid, methyl acetate, and propyl acetate. The one or more
second solvents can be Class 3 solvents (or low-toxicity solvents)
as defined in the ICH guideline. In another embodiment, the solvent
mixture used for eluting comprises ether, such as a mixture of
ethanol/ether or isopropanol/ether
[0031] In one embodiment, the alcohol aqueous solution comprises
isopropanol, propanol, tert-butanol, 2-butanol, or ethanol.
[0032] In one embodiment, the ether comprises diethyl ether or
MTBE.
[0033] In one embodiment, the process further includes salt
exchanging the peptide by washing the peptide with an aqueous
solution comprising an ammonium salt (e.g., ammonium acetate),
acetic acid, and/or an acetate salt (e.g., sodium acetate) before
the purification. In one embodiment, if the process includes the
salt exchanging step, the process also includes rinsing the peptide
with deionized water after adsorption on the polymeric resin column
to desalt the peptide.
[0034] In one embodiment, the process further includes lyophilizing
the peptide after the salt exchanging step before the
purification.
[0035] In one embodiment, the process further includes drying the
precipitated peptide after adding the ether, e.g., under reduced
pressure.
[0036] In one embodiment, the purified peptide is selected from the
group consisting of SEQ ID NOs: 1, 8, 9, and 56. Preferably, the
purified peptide is SEQ ID NO: 1 or 9.
[0037] The purification methods of this invention unexpectedly
produce GCC agonist peptides of high purities (e.g., >96%) and
high bulk and/or tap densities (e.g., >0.3 g/mL) compared to
conventional purification methods such as lyophilization, where the
bulk and/or tap densities of the purified are at approximately 10
times lower than those of the peptides purified by the methods of
this invention. The purification process of this invention also
requires much less time than conventional lyophilization, which can
potentially lead to reductions in topoisomer content, deamidation
degradation products, and other impurities. The purification
process of this invention is also readily scalable for commercial
production compared to conventional lyophilization.
[0038] In yet another aspect, the invention features the peptides,
e.g., GCC agonist peptides, purified by the process described
herein. The purified peptides may have one or more of the following
features.
[0039] In one embodiment, the purified peptide has a bulk density
of no less than 0.05 g/mL, no less than 0.1 g/mL, no less than 0.2
g/mL, no less than 0.3 g/mL, no less than 0.4 g/mL, or no less than
0.5 g/mL. For example, the purified peptide has a bulk density
ranging between 0.05 g/mL and 2 g/mL.
[0040] In one embodiment, the purified peptide has a tap density of
no less than 0.08 g/mL, no less than 0.1 g/mL, no less than 0.15
g/mL, no less than 0.2 g/mL, no less than 0.3 g/mL, no less than
0.4 g/mL, no less than 0.5 g/mL, or no less than 0.6 g/mL. For
example, the purified peptide has a tap density ranging between
0.08 g/mL and 2 g/mL.
[0041] In one embodiment, the purified peptide has a
chromatographic purity of no less than 96%, no less than 97%, or no
less than 98%. For example, the GCC agonist peptide has
chromatographic impurity content of no greater than 4%, no greater
than 3.5%, no greater than 3%, no greater than 2.5%, no greater
than 2%, no greater than 1.5%, or no greater than 1%. The
chromatographic impurity content is determined as total area
percentages of impurities by HPLC. The chromatographic impurity
content includes topoisomer content. The impurities do not include
any pharmaceutically acceptable excipient used for drug
formulation.
[0042] In one embodiment, the purified peptide is substantially
free of contaminants resulted from the peptide preparation process
such as organic solvents used in the process, e.g., ammonium,
acetonitrile, acetamide, alcohol (e.g., methanol, ethanol, or
isopropanol), TFA, ether or other contaminants. In this context
"substantially" free of contaminants means that the contaminant
content of the peptide at the end of the purification process is
preferably less than 0.5%, less than 0.3%, less than 0.25%, less
than 0.1%, less than 0.05%, less than 0.04%, less than 0.03%, less
than 0.02%, less than 0.01%, less than 0.005%, less than 0.003%, or
less than 0.001% of the total weight of the peptide. For example,
the purified peptide contains <0.01% acetamide, <0.3%
ammonium ion, <0.01% acetonitrile, and/or <0.1% TFA. The
content of contaminants can be determined by conventional methods
such as gas chromatography. Preferably, the residual solvents in
the purified peptide of the invention are less than the limits set
in the ICH guidelines, e.g., IMPURITIES: GUIDELINE FOR RESIDUAL
SOLVENTS Q3C(R5) (available at
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Qual-
ity/Q3C/Step4/Q 3C_R5_Step4.pdf). For example, the purified peptide
contains <410 ppm acetonitrile (e.g., <40 ppm or <20 ppm),
<5000 ppm ethanol (e.g., <140 ppm), <5000 ppm isopropanol,
<5000 ppm ethyl acetate (e.g., <20 ppm), <3000 ppm
methanol (e.g., <250 ppm), <5000 ppm MTBE (e.g., <20 ppm),
<290 ppm hexane, and/or <5000 ppm heptane or pentane.
[0043] In one embodiment, the purified peptide is substantially
free of topoisomers. In this context "substantially" free of
topoisomers means that the topoisomer content of the peptide at the
end of the purification process is preferably less than 2%, less
than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than
0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than
0.4%, less than 0.3%, less than 0.2%, or less than 0.1%, of the
total weight of the peptide.
[0044] In one embodiment, the purified peptide is substantially
free of water. In this context "substantially" free of water means
that the water content of the peptide at the end of the
purification process is preferably less than 7%, less than 6%, less
than 5%, less than 4.5%, less than 4.25%, less than 4%, less than
3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%,
less than 1%, less than 0.5%, less than 0.25%, or less than 0.1%,
of the total weight of the peptide.
[0045] In one embodiment, the purified peptide includes the GCC
agonist sequence of SEQ ID NO: 1.
[0046] In one embodiment, the purified peptide includes the GCC
agonist sequence of SEQ ID NO: 9.
[0047] The invention also relates to a formulation (e.g., an oral
formulation) containing the peptides prepared and/or purified by
the methods described herein and in particular, a low dose
formulation containing 0.05-10 mg (e.g., 0.1 mg, 0.3 mg or 0.5 mg)
of the purified peptides. The low-dose formulation can further have
one or more additional features as described in PCT/US2011/051805
and can be prepared by the methods disclosed therein, such as dry
blending.
[0048] Other features and advantages of the invention will be
apparent from and are encompassed by the following detailed
description and claims.
BRIEF DESCRIPTION OF DRAWINGS
[0049] FIG. 1 is a graph showing particle size distribution by
sieving analysis for lyophilized plecanatide and precipitated
plecanatide.
[0050] FIG. 2 is an optical microscopic image of lyophilized
plecanatide.
[0051] FIG. 3 is an optical microscopic image of precipitated
plecanatide.
DETAILED DESCRIPTION
[0052] The invention provides processes of preparing peptides,
e.g., peptide GCC agonists, in particular a hybrid solution- and
solid-phase process. The process of the invention includes
providing two or more fragments of a peptide of interest via
solid-phase and/or solution-phase syntheses and coupling them via a
solution-phase synthesis to obtain the target peptide. The process
may further include, if needed, oxidative cyclization of cysteine
amino acid residues of a linear peptide formed by the fragment
coupling to produce a cyclized peptide.
[0053] The fragments described above can be prepared by standard
solution phase peptide synthesis or solid phase peptide synthesis
techniques in which a peptide linkage occurs through the direct
condensation of the amino group (i.e., NH.sub.2) of a first amino
acid with the carboxy group (i.e., COOH) of a second amino acid
with the elimination of a water molecule. In one embodiment, at
least one of the fragments is prepared by solid phase peptide
synthesis.
[0054] Peptide bond synthesis by direct condensation, as formulated
above, requires suppression of the reactive character of the amino
group of the first and of the carboxyl group of the second amino
acid. The masking substituents (i.e., protecting groups) must
permit their ready removal, without inducing breakdown of the
labile peptide molecule. The term "protected peptide" or "protected
peptide fragment" refers to a peptide or peptide fragment, in which
all reactive groups on its constituting amino acids, are masked by
protecting groups, unless otherwise specified. The term
"deprotected peptide" or "deprotected peptide fragment" refers to a
peptide or peptide fragment, in which all reactive groups on its
constituting amino acids, are free from being masked by protecting
groups, unless otherwise specified. The term "reactive groups"
refers to the groups forming the peptide bond and those interfering
with the peptide bond formation, such as amino, carboxyl, hydroxyl,
and thiol (as in cysteine) groups. Examples of protecting groups
for amino include and are not limited to
9-fluorenylmethyloxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc),
benzoyl (Bz), acetyl (Ac), and benzyl (Bn). Examples of protecting
groups for carboxyl include trityl (triphenylmethyl, Trt) and
O-tert-butyl (OtBu). Examples of protecting groups for thiol
include acetamidomethyl (Acm), tert-butyl (tBu), 3-nitro-2-pyridine
sulfenyl (NPYS), 2-pyridine-sulfenyl (Pyr), and trityl (Trt).
Additional examples of protecting groups are described in Greene,
T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis, 3'
edition, John Wiley & Sons: New York, 1999, whose context is
incorporated by reference herein.
[0055] In a preferred embodiment, the methods of the invention are
used for preparing SP-304 (plecanatide). In particular, three
peptide fragments, A, B and C are prepared and then a linear
peptide sequence is assembled by the condensation of fragment A, B
and C as follows: preparing fragment A,
Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-OH, by
solid phase from 2-chloro-trityl chloride resin; preparing fragment
B, Fmoc-Cys(Acm)-Val-Asn-Val-Ala-Cys(Trt)-Thr(tBu)-Gly-OH, by solid
phase from 2-chlorotrityl chloride resin; preparing fragment C,
Cys(Acm)-Leu-OtBu, by solution phase synthesis, coupling fragments
B and C in solution phase to yield fragment B-C, and coupling
fragments A and B-C to yield linear peptide A-B-C.
[0056] The side-chain-protected Fragments A (BocAA1-60H) and B
(FmocAA7-140H) can be prepared by Fmoc SPPS using the super
acid-sensitive 2-chlorotrityl chloride (2-ClTrt) resin and
Fmoc-protected amino acid derivatives, as shown in Scheme 1
below.
##STR00001##
[0057] Fragment C (HAA15-16OtBu) can be prepared by the solution
phase synthesis and then be coupled to Fragment B (FmocAA7-140H) in
solution phase to give Fragment B-C (FmocAA7-16OtBu). The Fmoc
protecting group can then be removed from Fragment B-C
(FmocAA7-16OtBu) to give HAA7-16OtBu, which is then coupled to
Fragment A (BocAA1-60H) to yield side-chain-protected linear SP-304
(BocAA1-16OtBu), as shown in Scheme 2 below.
##STR00002##
[0058] The side-chain-protected linear SP-304 (BocAA1-16OtBu) can
be treated with trifluoroacetic
acid/triisopropylsilane/ethanedithiol (TFA/TIS/EDT) to give the
partially protected SP-304 (HAA1-160H) in which the 2 S-Acm groups
(as shown in Scheme 2 above) are intact. The partially protected
linear SP-304 (HAA1-160H) can be oxidized by H.sub.2O.sub.2,
followed by simultaneous removal of the S-Acm groups and disulfide
formation with iodine to give crude dicyclic SP-304, as shown in
Schemes 3 and 4 below.
##STR00003##
##STR00004##
[0059] The solution of crude dicyclic SP-304 can then be purified
and concentrated, as shown in Scheme 3 above, by loading the
solution to a polystyrenic adsorbent resin (e.g., D101 (Anhui
Sanxing (China); crosslinked polystyrene; surface area 500-550
m.sup.2/g; average pore diameter: 9-10 nm; pore volume: 1.18-1.24
ml/g; bulk density: 0.65-0.70 g/ml; specific density: 1.03-1.07
g/ml; moisture: 67-75%; particle size: 0.315.about.1.25 mm
.gtoreq.95%; effective diameter: 0.4.about.0.7 mm; uniformity
coefficient: .ltoreq.1.6%), DA201C, DA201H, ADS-8, and ADS-5)
column, eluting the dicyclic SP-304 from the column with an eluent
(e.g., a 90% ethanol aqueous solution), concentrating the collected
SP-304 solution under reduced pressure, and precipitating SP-304
with methyl t-butyl ether (MTBE). The precipitate can then be
collected by filtration or centrifugation, dried under high vacuum
to give SP-304 in solid form.
[0060] As illustrated in Scheme 4 above, the solution of crude
dicyclic SP-304 can also be purified directly on preparative HPLC
C18 column with acetonitrile (ACN), methanol, and/or water in
various buffer system. The crude dicyclic SP-304 can also be
purified via other methods known to a skilled person in the
art.
[0061] Those of ordinary skill in the art will recognize that, in
solid phase synthesis, deprotection, and coupling reactions must go
to completion and the side-chain blocking groups must be stable
throughout the synthesis.
[0062] Acetylation of the N-terminal can be accomplished by
reacting the final peptide with acetic anhydride before cleavage
from the resin. C-amidation is accomplished using an appropriate
resin such as methylbenzhydrylamine resin using the Boc
technology.
[0063] In solution phase synthesis, a wide variety of coupling
methods and protecting groups may be used (See, Gross and
Meienhofer, eds., "The Peptides: Analysis, Synthesis, Biology,"
Vol. 1-4 (Academic Press, 1979); Bodansky and Bodansky, "The
Practice of Peptide Synthesis," 2d ed. (Springer Verlag, 1994)). In
addition, intermediate purification and linear scale up are
possible. Those of ordinary skill in the art will appreciate that
solution synthesis requires consideration of main chain and side
chain protecting groups and activation method. In addition, careful
fragment selection is necessary to minimize racemization during
fragment condensation. For example, racemization is minimized when
fragments contain C-terminal Gly or Pro. Solubility considerations
are also a factor. Solid phase peptide synthesis uses an insoluble
polymer for support during organic synthesis. The polymer-supported
peptide chain permits the use of simple washing and filtration
steps instead of laborious purifications at intermediate steps.
Solid-phase peptide synthesis may generally be performed according
to the method of Merrifield et al., J. Am. Chem. Soc., 1963,
85:2149, which involves assembling a linear peptide chain on a
resin support using protected amino acids. Solid phase peptide
synthesis typically utilizes either the Boc or Fmoc strategy, both
of which are well known in the art.
[0064] The processes of the present invention can be used to make
any peptide-based GCC agonist known in the art, such as analogs of
uroguanylin, guanylin, lymphoguanylin, linaclotide, ST peptides,
SP-304, and SP-333. Non-limiting examples of such analogs of
uroguanylin, guanylin, lymphoguanylin, linaclotide, SP-304, SP-333,
and bacterial ST peptides are described in Section 1.1 below. In
certain embodiments, the methods are used to prepare a peptide
consisting essentially of an amino acid sequence selected from SEQ
ID NOs: 1-249. In a preferred embodiment, the peptide thusly made
consists essentially of an amino acid sequence selected from SEQ ID
NOs: 1, 8, 9, 55, and 56. The term "consists essentially of" refers
to a peptide that is identical to the reference peptide in its
amino acid sequence or to a peptide that does not differ
substantially in terms of either structure or function from the
reference peptide. A peptide differs substantially from the
reference peptide if its primary amino acid sequence varies by more
than three amino acids from the reference peptide or if its
activation of cellular cGMP production is reduced by more than 50%
compared to the reference peptide. Preferably, substantially
similar peptides differ by no more than two amino acids and not by
more than about 25% with respect to activating cGMP production. In
preferred embodiments, the GCC agonist made by the methods of the
invention is a peptide comprising at least 12 amino acid residues,
and most preferably comprising between 12 and 26 amino acids.
[0065] In another embodiment, the process of the invention is used
for preparing SP-353, which is a bacterial ST peptide analog. The
general strategy for the hybrid synthesis of SP-353 includes solid
phase and solution phase syntheses to produce suitable peptide
fragments (see Schemes 5 and 6 below), subsequent segment
condensation to form the linear crude peptide (see Scheme 7 below),
and natural oxidative folding to form the cyclized final product
(see Scheme 7 below). The same strategy can also be used to produce
other ST peptide analogs (such as SP-354, linaclotide, etc.) of
similar amino acid sequences shown in Table II.
##STR00005##
##STR00006##
##STR00007##
[0066] In yet another embodiment, the process of the invention is
used for preparing SP-333. The general strategy for the hybrid
synthesis of SP-333 includes solid phase and solution phase
syntheses to produce suitable peptide fragments (see Schemes 8 and
9 below), subsequent segment condensation to form the linear crude
peptide (see Scheme 9 below), oxidative folding to form the
cyclized final product, purification, and lyophilization (see
Scheme 10 below).
##STR00008##
##STR00009##
##STR00010##
[0067] In another aspect, the invention also provides a process of
purifying peptides, e.g., peptide GCC agonists. The general
strategy for purifying peptides, including those synthesized by the
hybrid methods disclosed herein, include, e.g., the steps
illustrated in Scheme 11 below. It is understood that certain steps
in Scheme 11 may be repeated (e.g., rinsing column with deionized
water) or absent (e.g., salt exchange or alcohol removal after
dewatering) to optimize the purification process.
##STR00011##
[0068] In yet another aspect, this invention provides a purified
peptide, e.g., peptide GCC agonists, purified by the precipitation
process described herein. Preferably, the purified peptide is
SP-304 (SEQ ID NO:1) or SP-333 (SEQ ID NO:9). In one embodiment,
the purified SP-304 or SP-333 has a bulk density of no less than
0.05 g/mL, no less than 0.1 g/mL, no less than 0.2 g/mL, no less
than 0.3 g/mL, no less than 0.4 g/mL, or no less than 0.5 g/mL. In
a preferred embodiment, the purified SP-304 or SP-333 has a bulk
density of about 0.05-2 g/mL, about 0.2-0.7 g/mL, about 0.3-0.6
g/mL, or about 0.4-0.5 g/mL. In one embodiment, the purified SP-304
or SP-333 has a tap density of no less than 0.08 g/mL, no less than
0.1 g/mL, no less than 0.15 g/mL, no less than 0.2 g/mL, no less
than 0.3 g/mL, no less than 0.4 g/mL, no less than 0.5 g/mL, or no
less than 0.6 g/mL. For example, the purified SP-304 or SP-333 has
a tap density of 0.08-2 g/mL, about 0.4-0.9 g/mL, about 0.5-0.8
g/mL, or about 0.6-0.7 g/mL. In one embodiment, the purified
peptide SP-304 or SP-333 contains <0.01% acetamide (e.g., <28
ppm), <0.3% ammonium ion (e.g., <0.25%), <0.01%
acetonitrile (e.g., <20 ppm), and/or <0.1% TFA (e.g.,
<0.09%). In one embodiment, the purified peptide SP-304 or
SP-333 has a bulk density of 0.4-0.5 g/mL, has a tap density of
0.6-0.7 g/mL, and contains <0.01% acetamide (e.g., <28 ppm),
<0.3% ammonium ion (e.g., <0.25%), <0.01% acetonitrile
(e.g., <20 ppm), and/or <0.1% TFA (e.g., <0.09%).
1.1 GCC Agonists
[0069] The GCC agonists prepared by the processes of the invention
can bind to guanylate cyclase C and stimulate intracellular
production of cGMP. Optionally, the GCC agonists induce apoptosis
and inhibit proliferation of epithelial cells. The term, "guanylate
cyclase C" refers to a transmembrane form of guanylate cyclase that
acts as the intestinal receptor for the heat-stable toxin (ST)
peptides secreted by enteric bacteria. Guanylate cyclase C is also
the receptor for the naturally occurring peptides guanylin and
uroguanylin. The possibility that there may be different receptors
for each of these peptides has not been excluded. Hence, the term
"guanylate cyclase C" may also encompass a class of transmembrane
guanylate cyclase receptors expressed on epithelial cells lining
the gastrointestinal mucosa.
[0070] The term "GCC agonist" refers to both peptides and
non-peptide compounds such as that bind to an intestinal guanylate
cyclase C and stimulate the intracellular production of cGMP. Where
the GCC agonist is a peptide, the term encompasses biologically
active fragments of such peptides and pro-peptides that bind to
guanylate cyclase C and stimulate the intracellular production of
cGMP.
1.1.1 GCC Agonist Peptides
[0071] The GCC agonists prepared by the methods of the invention
are preferably peptides. In some embodiments, the GCC agonist
peptide is less than 30 amino acids in length. In particular
embodiments, the GCC agonist peptide is less than or equal to 30,
25, 20, 15, 14, 13, 12, 11, 10, or 5 amino acids in length.
Examples of GCC agonist peptides for use in the formulations and
methods of the invention include those described in U.S. Pat. Nos.
7,879,802 and 8,034,782, and U.S. Publication Nos. US 2010-0069306
and US 2010-0120694, each of which is incorporated by reference
herein in its entirety.
[0072] Specific examples of GCC agonist peptides that can be
prepared by the methods of the invention include those described in
Tables I-VII below. As used Tables I-VII, the terms "PEG3" or
"3PEG" refer to a polyethylene glycol such as
aminoethyloxy-ethyloxy-acetic acid (AeeA), and polymers thereof.
The term "X.sub.aa" refers to any natural or unnatural amino acid
or amino acid analogue. The term "M.sub.aa" refers to a cysteine
(Cys), penicillamine (Pen) homocysteine, or 3-mercaptoproline. The
term "Xaa.sub.n1" is meant to denote an amino acid sequence of any
natural or unnatural amino acid or amino acid analogue that is one,
two or three residues in length; Xaa.sub.n2 is meant to denote an
amino acid sequence that is zero or one residue in length; and
Xaa.sub.n3 is meant to denote an amino acid sequence zero, one,
two, three, four, five or six residues in length. Additionally, any
amino acid represented by Xaa, Xaa.sub.n1, Xaa.sub.n2, or
Xaa.sub.n3 may be an L-amino acid, a D-amino acid, a methylated
amino acid or any combination of thereof. Optionally, any GCC
agonist peptide represented by Formulas I to XX in the tables may
contain one or more polyethylene glycol residues at the N-terminus,
C-terminus or both.
[0073] In certain embodiments, a GCC agonist prepared by the
methods of the invention comprises a peptide selected from SEQ ID
NOs: 1-249, the sequences of which are set forth below in Tables I
to VII below. In one embodiment, a GCC agonist prepared by the
methods of the invention comprises the peptide designated by SEQ ID
NOs: 1, 8, 9, 55, or 56.
[0074] In certain embodiments, a GCC agonist prepared by the
methods of the invention comprises a peptide that is substantially
equivalent to a peptide selected from SEQ ID NOs: 1-249. The term
"substantially equivalent" refers to a peptide that has an amino
acid sequence equivalent to that of the binding domain where
certain residues may be deleted or replaced with other amino acids
without impairing the peptide's ability to bind to an intestinal
guanylate cyclase receptor and stimulate fluid and electrolyte
transport.
[0075] In certain embodiments, the GCC agonist peptides are
analogues of uroguanylin or a bacterial ST peptide. Uroguanylin is
a circulating peptide hormone with natriuretic activity. An ST
peptide is a member of a family of heat stable enterotoxins (ST
peptides) secreted by pathogenic strains of E. coli and other
enteric bacteria that activate guanylate cyclase receptor and cause
secretory diarrhea. Unlike bacterial ST peptides, the binding of
uroguanylin to guanylate cyclase receptor is dependent on the
physiological pH of the gut. Therefore, uroguanylin is expected to
regulate fluid and electrolyte transport in a pH dependent manner
and without causing severe diarrhea.
[0076] The GCC agonist peptides prepared by the methods of the
invention can be polymers of L-amino acids, D-amino acids, or a
combination of both. For example, in various embodiments, the
peptides are D retro-inverso peptides. The term "retro-inverso
isomer" refers to an isomer of a linear peptide in which the
direction of the sequence is reversed and the chirality of each
amino acid residue is inverted. See, e.g., Jameson et al., Nature,
368, 744-746 (1994); Brady et al., Nature, 368, 692-693 (1994). The
net result of combining D-enantiomers and reverse synthesis is that
the positions of carbonyl and amino groups in each amide bond are
exchanged, while the position of the side-chain groups at each
alpha carbon is preserved. Unless specifically stated otherwise, it
is presumed that any given L-amino acid sequence of the invention
may be made into a D retro-inverso peptide by synthesizing a
reverse of the sequence for the corresponding native L-amino acid
sequence.
[0077] The GCC agonist peptides prepared by the methods of the
invention are able to induce intracellular cGMP production in cells
and tissues expressing guanylate cyclase C. In certain embodiments,
the GCC agonist peptide stimulates 5%, 10%, 20%, 30%, 40%, 50%,
75%, 90% or more intracellular cGMP compared to naturally occurring
GCC agonists such as uroguanylin, guanylin, or ST peptides.
Optionally, the GCC agonist peptide stimulates 5%, 10%, 20%, 30%,
40%, 50%, 75%, 90% or more intracellular cGMP compared to SP-304
(SEQ ID NO:1). In further embodiments, the GCC agonist peptide
stimulates apoptosis, e.g., programmed cell death, or activate the
cystic fibrosis transmembrane conductance regulator (CFTR).
[0078] In some embodiments, the GCC agonist peptides prepared by
the methods of the invention are more stable than naturally
occurring GCC agonists and/or SP-304 (SEQ ID NO:1), SP-339
(linaclotide) (SEQ ID NO: 55) or SP-340 (SEQ ID NO: 56). For
example, the GCC agonist peptide degrades 2%, 3%, 5%, 10%, 15%,
20%, 30%, 40%, 50%, 75%, 90% or less compared to naturally
occurring GCC agonists and/or SP-304, SP-339 (linaclotide) or
SP-340. In certain embodiments, the GCC agonist peptides for use in
the formulations and methods of the invention are more stable to
proteolytic digestion than naturally occurring GCC agonists and/or
SP-304 (SEQ ID NO:1), SP-339 (linaclotide) (SEQ ID NO: 55) or
SP-340 (SEQ ID NO: 56). In one embodiment, a GCC agonist peptide is
pegylated in order to render the peptides more resistant towards
proteolysis by enzymes of the gastrointestinal tract. In a
preferred embodiment, the GCC agonist peptide is pegylated with the
aminoethyloxy-ethyloxy-acetic acid (Aeea) group at its C-terminal
end, at its N-terminal end, or at both termini.
[0079] Specific examples of GCC agonist peptides that can be
prepared by the methods of the invention include a peptide selected
from the group designated by SEQ ID NOs: 1-249.
[0080] In one embodiment, the GCC agonist peptide is a peptide
having the amino acid sequence of any one of Formulas X-XVII (e.g.
SEQ ID NOs: 87-98).
[0081] In some embodiments, GCC agonist peptides include peptides
having the amino acid sequence of Formula I, wherein at least one
amino acid of Formula I is a D-amino acid or a methylated amino
acid and/or the amino acid at position 16 is a serine. Preferably,
the amino acid at position 16 of Formula I is a D-amino acid or a
methylated amino acid. For example, the amino acid at position 16
of Formula I is a d-leucine or a d-serine. Optionally, one or more
of the amino acids at positions 1-3 of Formula I are D-amino acids
or methylated amino acids or a combination of D-amino acids or
methylated amino acids. For example, Asn.sup.1, Asp.sup.2 or
Glu.sup.3 (or a combination thereof) of Formula I is a D-amino acid
or a methylated amino acid. Preferably, the amino acid at position
Xaa.sup.6 of Formula I is a leucine, serine or tyrosine.
[0082] In alternative embodiments, GCC agonist peptides include
peptides having the amino acid sequence of Formula II, wherein at
least one amino acid of Formula II is a D-amino acid or a
methylated amino acid. Preferably, the amino acid denoted by
Xaa.sub.n2 of Formula II is a D-amino acid or a methylated amino
acid. In some embodiments, the amino acid denoted by Xaa.sub.n2 of
Formula II is a leucine, a d-leucine, a serine, or a d-serine.
Preferably, the one or more amino acids denoted by Xaa.sub.n1 of
Formula II is a D-amino acid or a methylated amino acid.
Preferably, the amino acid at position Xaa.sup.6 of Formula II is a
leucine, a serine, or a tyrosine.
[0083] In some embodiments, GCC agonist peptides include peptides
having the amino acid sequence of Formula III, wherein at least one
amino acid of Formula III is a D-amino acid or a methylated amino
acid and/or Maa is not a cysteine. Preferably, the amino acid
denoted by Xaa.sub.n2 of Formula III is a D-amino acid or a
methylated amino acid. In some embodiments the amino acid denoted
by Xaa.sub.n2 of Formula III is a leucine, a d-leucine, a serine,
or a d-serine. Preferably, the one or more amino acids denoted by
Xaa.sub.n1 of Formula III is a D-amino acid or a methylated amino
acid. Preferably, the amino acid at position Xaa.sup.6 of Formula
III is a leucine, a serine, or a tyrosine.
[0084] In other embodiments, GCC agonist peptides include peptides
having the amino acid sequence of Formula IV, wherein at least one
amino acid of Formula IV is a D-amino acid or a methylated amino
acid, and/or Maa is not a cysteine. Preferably, the Xaa.sub.n2 of
Formula IV is a D-amino acid or a methylated amino acid. In some
embodiments, the amino acid denoted by Xaa.sub.n2 of Formula IV is
a leucine, a d-leucine, a serine, or a d-serine. Preferably, the
one or more of the amino acids denoted by Xaa.sub.n1 of Formula IV
is a D-amino acid or a methylated amino acid. Preferably, the amino
acid denoted Xaa.sup.6 of Formula IV is a leucine, a serine, or a
tyrosine.
[0085] In further embodiments, GCC agonist peptides include
peptides having the amino acid sequence of Formula V, wherein at
least one amino acid of Formula V is a D-amino acid or a methylated
amino acid. Preferably, the amino acid at position 16 of Formula V
is a D-amino acid or a methylated amino acid. For example, the
amino acid at position 16 (i.e., Xaa.sup.16) of Formula V is a
d-leucine or a d-serine. Optionally, one or more of the amino acids
at position 1-3 of Formula V are D-amino acids or methylated amino
acids or a combination of D-amino acids or methylated amino acids.
For example, Asn', Asp.sup.2 or Glu.sup.3 (or a combination
thereof) of Formula V is a D-amino acids or a methylated amino
acid. Preferably, the amino acid denoted at Xaa.sup.6 of Formula V
is a leucine, a serine, or a tyrosine.
[0086] In additional embodiments, GCC agonist peptides include
peptides having the amino acid sequence of Formula VI, VII, VIII,
or IX. Preferably, the amino acid at position 6 of Formula VI, VII,
VIII, or IX is a leucine, a serine, or a tyrosine. In some aspects
the amino acid at position 16 of Formula VI, VII, VIII, or IX is a
leucine or a serine. Preferably, the amino acid at position 16 of
Formula V is a D-amino acid or a methylated amino acid.
[0087] In additional embodiments, GCC agonist peptides include
peptides having the amino acid sequence of Formula X, XI, XII,
XIII, XIV, XV, XVI or XVII. Optionally, one or more amino acids of
Formulas X, XI, XII, XIII, XIV, XV, XVI or XVII is a D-amino acid
or a methylated amino acid. Preferably, the amino acid at the
carboxy terminus of the peptides according to Formulas X, XI, XII,
XIII, XIV, XV, XVI or XVII is a D-amino acid or a methylated amino
acid. For example the amino acid at the carboxy terminus of the
peptides according to Formulas X, XI, XII, XIII, XIV, XV, XVI or
XVII is a D-tyrosine.
[0088] Preferably, the amino acid denoted by Xaa.sup.6 of Formula
XIV is a tyrosine, phenylalanine or a serine. Most preferably the
amino acid denoted by Xaa.sup.6 of Formula XIV is a phenylalanine
or a serine. Preferably, the amino acid denoted by Xaa.sup.4 of
Formula XV, XVI or XVII is a tyrosine, a phenylalanine, or a
serine. Most preferably, the amino acid position Xaa.sup.4 of
Formula V, XVI or XVII is a phenylalanine or a serine.
[0089] In some embodiments, GCRA peptides include peptides
containing the amino acid sequence of Formula XVIII. Preferably,
the amino acid at position 1 of Formula XVIII is a glutamic acid,
aspartic acid, glutamine or lysine. Preferably, the amino acid at
position 2 and 3 of Formula XVIII is a glutamic acid, or an
aspartic acid. Preferably, the amino acid at position 5 a glutamic
acid. Preferably, the amino acid at position 6 of Formula XVIII is
an isoleucine, valine, serine, threonine, or tyrosine. Preferably,
the amino acid at position 8 of Formula XVIII is a valine or
isoleucine. Preferably, the amino acid at position 9 of Formula
XVIII is an asparagine. Preferably, the amino acid at position 10
of Formula XVIII is a valine or a methionine. Preferably, the amino
acid at position 11 of Formula XVIII is an alanine. Preferably, the
amino acid at position 13 of Formula XVIII is a threonine.
Preferably, the amino acid at position 14 of Formula XVIII is a
glycine. Preferably, the amino acid at position 16 of Formula XVIII
is a leucine, serine or threonine
[0090] In alternative embodiments, GCRA peptides include peptides
containing the amino acid sequence of Formula XIX. Preferably, the
amino acid at position 1 of Formula XIX is a serine or asparagine.
Preferably, the amino acid at position 2 of Formula XIX is a
histidine or an aspartic acid. Preferably, the amino acid at
position 3 of Formula XIX is a threonine or a glutamic acid.
Preferably, the amino acid at position 5 of Formula XIX is a
glutamic acid. Preferably, the amino acid at position 6 of Formula
XIX is an isoleucine, leucine, valine, or tyrosine. Preferably, the
amino acid at position 8, 10, 11, or 13 of Formula XIX is an
alanine. Preferably, the amino acid at position 9 of Formula XIX is
an asparagine or a phenylalanine. Preferably, the amino acid at
position 14 of Formula XIX is a glycine.
[0091] In further embodiments, GCRA peptides include peptides
containing the amino acid sequence of Formula XX. Preferably, the
amino acid at position 1 of Formula XX is a glutamine. Preferably,
the amino acid at position 2 or 3 of Formula XX is a glutamic acid
or an aspartic acid. Preferably, the amino acid at position 5 of
Formula XX is a glutamic acid. Preferably, the amino acid at
position 6 of Formula XX is threonine, glutamine, tyrosine,
isoleucine, or leucine. Preferably, the amino acid at position 8 of
Formula XX is isoleucine or valine. Preferably, the amino acid at
position 9 of Formula XX is asparagine. Preferably, the amino acid
at position 10 of Formula XX is methionine or valine. Preferably,
the amino acid at position 11 of Formula XX is alanine. Preferably,
the amino acid at position 13 of Formula XX is a threonione.
Preferably, the amino acid at position 1 of Formula XX is a
glycine. Preferably, the amino acid at position 15 of Formula XX is
a tyrosine. Optionally, the amino acid at position 15 of Formula XX
is two amino acid in length and is Cysteine (Cys), Penicillamine
(Pen) homocysteine, or 3-mercaptoproline and serine, leucine or
threonine.
[0092] In certain embodiments, one or more amino acids of the GCC
agonist peptides are replaced by a non-naturally occurring amino
acid or a naturally or non-naturally occurring amino acid analog.
Such amino acids and amino acid analogs are known in the art. See,
for example, Hunt, "The Non-Protein Amino Acids," in Chemistry and
Biochemistry of the Amino Acids, Barrett, Chapman, and Hall, 1985.
In some embodiments, an amino acid is replaced by a
naturally-occurring, non-essential amino acid, e.g., taurine.
Non-limiting examples of naturally occurring amino acids that can
be replaced by non-protein amino acids include the following: (1)
an aromatic amino acid can be replaced by
3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine,
L-thyroxine, phenylglycine (Phg) or nor-tyrosine (norTyr); (2) Phg
and norTyr and other amino acids including Phe and Tyr can be
substituted by, e.g., a halogen, --CH3, --OH, --CH2NH3, --C(O)H,
--CH2CH3, --CN, --CH2CH2CH3, --SH, or another group; (3) glutamine
residues can be substituted with gamma-Hydroxy-Glu or
gamma-Carboxy-Glu; (4) tyrosine residues can be substituted with an
alpha substituted amino acid such as L-alpha-methylphenylalanine or
by analogues such as: 3-Amino-Tyr; Tyr(CH3); Tyr(PO3(CH3)2);
Tyr(SO3H); beta-Cyclohexyl-Ala; beta-(1-Cyclopentenyl)-Ala;
beta-Cyclopentyl-Ala; beta-Cyclopropyl-Ala; beta-Quinolyl-Ala;
beta-(2-Thiazolyl)-Ala; beta-(Triazole-1-yl)-Ala;
beta-(2-Pyridyl)-Ala; beta-(3-Pyridyl)-Ala; Amino-Phe; Fluoro-Phe;
Cyclohexyl-Gly; tBu-Gly; beta-(3-benzothienyl)-Ala;
beta-(2-thienyl)-Ala; 5-Methyl-Trp; and A-Methyl-Trp; (5) proline
residues can be substituted with homopro (L-pipecolic acid);
hydroxy-Pro; 3,4-Dehydro-Pro; 4-fluoro-Pro; or alpha-methyl-Pro or
an N(alpha)-C(alpha) cyclized amino acid analogues with the
structure: n=0, 1, 2, 3; and (6) alanine residues can be
substituted with alpha-substituted or N-methylated amino acid such
as alpha-amino isobutyric acid (aib), L/D-alpha-ethylalanine
(LID-isovaline), L/D-methylvaline, or L/D-alpha-methylleucine or a
non-natural amino acid such as beta-fluoro-Ala. Alanine can also be
substituted with: n=0, 1, 2, 3 Glycine residues can be substituted
with alpha-amino isobutyric acid (aib) or L/D-alpha-ethylalanine
(L/D-isovaline).
[0093] Further examples of non-natural amino acids include: an
unnatural analog of tyrosine; an unnatural analogue of glutamine;
an unnatural analogue of phenylalanine; an unnatural analogue of
serine; an unnatural analogue of threonine; an alkyl, aryl, acyl,
azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl,
alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate,
boronate, phospho, phosphono, phosphine, heterocyclic, enone,
imine, aldehyde, hydroxylamine, keto, or amino substituted amino
acid, or any combination thereof; an amino acid with a
photoactivatable cross-linker; a spin-labeled amino acid; a
fluorescent amino acid; an amino acid with a novel functional
group; an amino acid that covalently or noncovalently interacts
with another molecule; a metal binding amino acid; an amino acid
that is amidated at a site that is not naturally amidated, a
metal-containing amino acid; a radioactive amino acid; a photocaged
and/or photoisomerizable amino acid; a biotin or biotin-analogue
containing amino acid; a glycosylated or carbohydrate modified
amino acid; a keto containing amino acid; amino acids comprising
polyethylene glycol or polyether; a heavy atom substituted amino
acid (e.g., an amino acid containing deuterium, tritium, .sup.13C,
.sup.15N, or .sup.18O); a chemically cleavable or photocleavable
amino acid; an amino acid with an elongated side chain; an amino
acid containing a toxic group; a sugar substituted amino acid,
e.g., a sugar substituted serine or the like; a carbon-linked
sugar-containing amino acid; a redox-active amino acid; an
.alpha.-hydroxy containing acid; an amino thio acid containing
amino acid; an .alpha., .alpha. disubstituted amino acid; a
.beta.-amino acid; a cyclic amino acid other than proline; an
O-methyl-L-tyrosine; an L-3-(2-naphthyl)alanine; a
3-methyl-phenylalanine; a .rho.-acetyl-L-phenylalanine; an
O-4-allyl-L-tyrosine; a 4-propyl-L-tyrosine; a tri-O-acetyl-GlcNAc
.beta.-serine; an L-Dopa; a fluorinated phenylalanine; an
isopropyl-L-phenylalanine; a p-azido-L-phenylalanine; a
p-acyl-L-phenylalanine; a p-benzoyl-L-phenylalanine; an
L-phosphoserine; a phosphonoserine; a phosphonotyrosine; a
p-iodo-phenylalanine; a 4-fluorophenylglycine; a
p-bromophenylalanine; a p-amino-L-phenylalanine; an
isopropyl-L-phenylalanine; L-3-(2-naphthyl)alanine;
D-3-(2-naphthyl)alanine (dNal); an amino-, isopropyl-, or
O-allyl-containing phenylalanine analogue; a dopa,
O-methyl-L-tyrosine; a glycosylated amino acid; a
p-(propargyloxy)phenylalanine; dimethyl-Lysine; hydroxy-proline;
mercaptopropionic acid; methyl-lysine; 3-nitro-tyrosine;
norleucine; pyro-glutamic acid; Z (Carbobenzoxyl);
.epsilon.-Acetyl-Lysine; .beta.-alanine; aminobenzoyl derivative;
aminobutyric acid (Abu); citrulline; aminohexanoic acid;
aminoisobutyric acid (AM); cyclohexylalanine; d-cyclohexylalanine;
hydroxyproline; nitro-arginine; nitro-phenylalanine;
nitro-tyrosine; norvaline; octahydroindole carboxylate; ornithine
(Orn); penicillamine (PEN); tetrahydroisoquinoline; acetamidomethyl
protected amino acids and pegylated amino acids. Further examples
of unnatural amino acids and amino acid analogs can be found in
U.S. 20030108885, U.S. 20030082575, US20060019347 (paragraphs
410-418) and the references cited therein. The polypeptides of the
invention can include further modifications including those
described in US20060019347, paragraph 589. Exemplary GCC agonist
peptides which include a non-naturally occurring amino acid include
for example SP-368 and SP-369.
[0094] In some embodiments, the GCC agonist peptides are cyclic
peptides. GCC agonist cyclic peptides can be prepared by methods
known in the art. For example, macrocyclization is often
accomplished by forming an amide bond between the peptide N- and
C-termini, between a side chain and the N- or C-terminus [e.g.,
with K.sub.3Fe(CN).sub.6 at pH 8.5] (Samson et al., Endocrinology,
137: 5182-5185 (1996)), or between two amino acid side chains, such
as cysteine. See, e.g., DeGrado, Adv Protein Chem, 39: 51-124
(1988). In various embodiments, the GCC agonist peptides are [4,12;
7,15] bicycles.
[0095] In certain embodiments, one or both Cys residues which
normally form a disulfide bond in a GCC agonist peptide are
replaced with homocysteine, penicillamine, 3-mercaptoproline
(Kolodziej et al. 1996 Int. J. Pept. Protein Res. 48:274), .beta.,
.beta. dimethylcysteine (Hunt et al. 1993 Int. J. Pept. Protein
Res. 42:249), or diaminopropionic acid (Smith et al. 1978 J. Med.
Chem. 2 1:117) to form alternative internal cross-links at the
positions of the normal disulfide bonds.
[0096] In certain embodiments, one or more disulfide bonds in a GCC
agonist peptide are replaced by alternative covalent cross-links,
e.g., an amide linkage (--CH.sub.2CH(O)NHCH.sub.2-- or
--CH.sub.2NHCH(O)CH.sub.2--), an ester linkage, a thioester
linkage, a lactam bridge, a carbamoyl linkage, a urea linkage, a
thiourea linkage, a phosphonate ester linkage, an alkyl linkage
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), an alkenyl linkage
(--CH.sub.2CH.dbd.CHCH.sub.2--), an ether linkage
(--CH.sub.2CH.sub.2OCH.sub.2-- or --CH.sub.2OCH.sub.2CH.sub.2--), a
thioether linkage (--CH.sub.2CH.sub.2SCH.sub.2-- or
--CH.sub.2SCH.sub.2CH.sub.2--), an amine linkage
(--CH.sub.2CH.sub.2NHCH.sub.2-- or --CH.sub.2NHCH.sub.2CH.sub.2--)
or a thioamide linkage (--CH.sub.2C(S)NHCH.sub.2-- or --CH.sub.2NHC
(S)CH.sub.2--). For example, Ledu et al. (Proc. Natl. Acad. Sci.
100:11263-78, 2003) describe methods for preparing lactam and amide
cross-links. Exemplary GCC agonist peptides which include a lactam
bridge include, for example, SP-370.
[0097] In certain embodiments, the GCC agonist peptides have one or
more conventional polypeptide bonds replaced by an alternative
bond. Such replacements can increase the stability of the
polypeptide. For example, replacement of the polypeptide bond
between a residue amino terminal to an aromatic residue (e.g. Tyr,
Phe, Trp) with an alternative bond can reduce cleavage by carboxy
peptidases and may increase half-life in the digestive tract. Bonds
that can replace polypeptide bonds include: a retro-inverso bond
(C(O)--NH instead of NH--C(O); a reduced amide bond (NH--CH.sub.2);
a thiomethylene bond (S--CH.sub.2 or CH.sub.2--S); an oxomethylene
bond (O--CH.sub.2 or CH.sub.2--O); an ethylene bond
(CH.sub.2--CH.sub.2); a thioamide bond (C(S)--NH); a trans-olefine
bond (CH.dbd.CH); a fluoro substituted trans-olefine bond
(CF.dbd.CH); a ketomethylene bond (C(O)--CHR or CHR--C(O) wherein R
is H or CH.sub.3; and a fluoro-ketomethylene bond (C(O)--CFR or
CFR--C(O) wherein R is H or F or CH.sub.3.
[0098] In certain embodiments, the GCC agonist peptides are
modified using standard modifications. Modifications may occur at
the amino (N--), carboxy (C--) terminus, internally or a
combination of any of the preceding. In one aspect described
herein, there may be more than one type of modification on the
polypeptide. Modifications include but are not limited to:
acetylation, amidation, biotinylation, cinnamoylation,
farnesylation, formylation, myristoylation, palmitoylation,
phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation,
sulfurylation and cyclisation (via disulfide bridges or amide
cyclisation), and modification by Cys3 or Cys5. The GCC agonist
peptides described herein may also be modified by 2,
4-dinitrophenyl (DNP), DNP-lysine, modification by
7-Amino-4-methyl-coumarin (AMC), flourescein, NBD
(7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide, rhodamine B,
EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), dabcyl,
dabsyl, dansyl, texas red, FMOC, and Tamra (Tetramethylrhodamine).
The GCC agonist peptides described herein may also be conjugated
to, for example, polyethylene glycol (PEG); alkyl groups (e.g.,
C1-C20 straight or branched alkyl groups); fatty acid moieties;
combinations of PEG, alkyl groups and fatty acid moieties (See,
U.S. Pat. No. 6,309,633; Soltero et al., 2001 Innovations in
Pharmaceutical Technology 106-110); BSA and KLH (Keyhole Limpet
Hemocyanin). The addition of PEG and other polymers which can be
used to modify polypeptides of the invention is described in
US20060 19347 section IX.
[0099] A GCC agonist peptide can also be a derivative of a GCC
agonist peptide described herein. For example, a derivative
includes hybrid and modified forms of GCC agonist peptides in which
certain amino acids have been deleted or replaced. A modification
may also include glycosylation. Preferrably, where the modification
is an amino acid substitution, it is a conservative substitution at
one or more positions that are predicted to be non-essential amino
acid residues for the biological activity of the peptide. A
"conservative substitution" is one in which the amino acid residue
is replaced with an amino acid residue having a similar side chain.
Families of amino acid residues having similar side chains have
been defined in the art. These families include amino acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid, glutamic acid), uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, histidine).
[0100] In one embodiment, a GCC agonist peptide prepared by the
methods described herein is subjected to random mutagenesis in
order to identify mutants having biological activity.
[0101] In one embodiment, the methods of the invention can be used
to prepare a GCC agonist peptide that is substantially homologous
to a GCC agonist peptide described herein. Such substantially
homologous peptides can be isolated by virtue of cross-reactivity
with antibodies to a GCC agonist peptide described herein.
[0102] Further examples of GCC agonist peptides that can be
prepared by the methods of the invention are found in Tables I-VII
below.
1.1.2 Alternative Preparation Methods of GCC Agonist Peptides and
their Fragments
[0103] GCC agonist peptides and their fragments can be prepared
using art recognized techniques such as molecular cloning, peptide
synthesis, or site-directed mutagenesis.
[0104] In addition to the conventional solution- or solid-phase
peptide synthesis described above, the GCC agonist peptides or
their fragments can be produced by modern cloning techniques. For
example, the GCC agonist peptides are produced either in bacteria
including, without limitation, E. coli, or in other existing
systems for polypeptide or protein production (e.g., Bacillus
subtilis, baculovirus expression systems using Drosophila Sf9
cells, yeast or filamentous fungal expression systems, mammalian
cell expression systems), or they can be chemically synthesized. If
the GCC agonist peptide or variant peptide is to be produced in
bacteria, e.g., E. coli, the nucleic acid molecule encoding the
polypeptide may also encode a leader sequence that permits the
secretion of the mature polypeptide from the cell. Thus, the
sequence encoding the polypeptide can include the pre sequence and
the pro sequence of, for example, a naturally-occurring bacterial
ST polypeptide. The secreted, mature polypeptide can be purified
from the culture medium.
[0105] The sequence encoding a GCC agonist peptide described herein
can be inserted into a vector capable of delivering and maintaining
the nucleic acid molecule in a bacterial cell. The DNA molecule may
be inserted into an autonomously replicating vector (suitable
vectors include, for example, pGEM3Z and pcDNA3, and derivatives
thereof). The vector nucleic acid may be a bacterial or
bacteriophage DNA such as bacteriophage lambda or M13 and
derivatives thereof. Construction of a vector containing a nucleic
acid described herein can be followed by transformation of a host
cell such as a bacterium. Suitable bacterial hosts include but are
not limited to, E. coli, B. subtilis, Pseudomonas, Salmonella. The
genetic construct also includes, in addition to the encoding
nucleic acid molecule, elements that allow expression, such as a
promoter and regulatory sequences. The expression vectors may
contain transcriptional control sequences that control
transcriptional initiation, such as promoter, enhancer, operator,
and repressor sequences.
[0106] A variety of transcriptional control sequences are well
known to those in the art. The expression vector can also include a
translation regulatory sequence (e.g., an untranslated 5' sequence,
an untranslated 3' sequence, or an internal ribosome entry site).
The vector can be capable of autonomous replication or it can
integrate into host DNA to ensure stability during polypeptide
production.
[0107] The protein coding sequence that includes a GCC agonist
peptide described herein can also be fused to a nucleic acid
encoding a polypeptide affinity tag, e.g., glutathione
S-transferase (GST), maltose E binding protein, protein A, FLAG
tag, hexa-histidine, myc tag or the influenza HA tag, in order to
facilitate purification. The affinity tag or reporter fusion joins
the reading frame of the polypeptide of interest to the reading
frame of the gene encoding the affinity tag such that a
translational fusion is generated. Expression of the fusion gene
results in translation of a single polypeptide that includes both
the polypeptide of interest and the affinity tag. In some instances
where affinity tags are utilized, DNA sequence encoding a protease
recognition site will be fused between the reading frames for the
affinity tag and the polypeptide of interest.
[0108] Genetic constructs and methods suitable for production of
immature and mature forms of the GCC agonist peptides and variants
described herein in protein expression systems other than bacteria,
and well known to those skilled in the art, can also be used to
produce polypeptides in a biological system.
[0109] The peptides disclosed herein may be modified by attachment
of a second molecule that confers a desired property upon the
peptide, such as increased half-life in the body, for example,
pegylation. Such modifications also fall within the scope of the
term "variant" as used herein.
TABLE-US-00001 TABLE I GCRA Peptides (SP-304 and Derivatives) SEQ
Position of ID Name Disulfide bonds Structure NO SP-304 C4: C12,
C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 1 SP-326 C3: C11, C6: C14
Asp.sup.1-Glu.sup.2-Cys.sup.3-Glu.sup.4-Leu.sup.5-Cys.sup.6-Val.sup.7-Asn-
.sup.8-Val.sup.9-Ala.sup.10-Cys.sup.11-Thr.sup.12-Gly.sup.13-Cys.sup.14-Le-
u.sup.15 2 SP-327 C2: C10, C5: C13
Asp.sup.1-Glu.sup.2-Cys.sup.3-Glu.sup.4-Leu.sup.5-Cys.sup.6-Val.sup.7-Asn-
.sup.8-Val.sup.9-Ala.sup.10-Cys.sup.11-Thr.sup.12-Gly.sup.13-Cys.sup.14
3 SP-328 C2: C10, C5: C13
Glu.sup.1-Cys.sup.2-Glu.sup.3-Leu.sup.4-Cys.sup.5-Val.sup.6-Asn.sup.7-Val-
.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13-Leu.sup.14
4 SP-329 C2: C10, C5: C13
Glu.sup.1-Cys.sup.2-Glu.sup.3-Leu.sup.4-Cys.sup.5-Val.sup.6-Asn.sup.7-Val-
.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13 5
SP-330 C1: C9, C4: C12
Cys.sup.1-Glu.sup.2-Leu.sup.3-Cys.sup.4-Val.sup.5-Asd-Val.sup.7-Ala.sup.8-
-Cys.sup.9-Thr.sup.10-Gly.sup.11-Cys.sup.12-Leu.sup.13 6 SP-331 C1:
C9, C4: C12
Cys.sup.1-Glu.sup.2-Leu.sup.3-Cys.sup.4-Val.sup.5-Asd-Val.sup.7-Ala.sup.8-
-Cys.sup.9-Thr.sup.10-Gly.sup.11-Cys.sup.12 7 SP332 C4: C12, C7:
C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-dLeu.sup.16 8 SP-333 C4: C12, C7: C15
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Va-
l.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dLeu.sup.16 9 SP-334 C4: C12, C7: C15
dAsn.sup.1-dAsp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-V-
al.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14--
Cys.sup.15-dLeu.sup.16 10 SP-335 C4: C12, C7: C15
dAsn.sup.1-dAsp.sup.2-dGlu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7--
Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-
-Cys.sup.15-dLeu.sup.16 11 SP-336 C4: C12, C7: C15
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Va-
l.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-Leu.sup.16 12 SP-337 C4: C12, C7: C15
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-dLeu.sup.6-Cys.sup.7-V-
al.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14--
Cys.sup.15-dLeu.sup.16 13 SP-338 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15 14 SP-342 C4: C12, C7: C15
PEG3-Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.-
7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.-
14-Cys.sup.15-dLeu.sup.16-PEG3 15 SP-343 C4: C12, C7: C15
PEG3-dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup-
.7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr13
-Gly.sup.14-Cys.sup.15-dLeu.sup.16-PEG3 16 SP-344 C4: C12, C7: C15
PEG3-dAsn.sup.1-dAsp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.su-
p.7-Var-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dLeu.sup.16-PEG3 17 SP-347 C4: C12, C7: C15
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Va-
l.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dLeu.sup.16-PEG3 18 SP-348 C4: C12, C7: C15
PEG3-Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.-
7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.-
14-Cys.sup.15-dLeu.sup.16 19 SP-350 C4: C12, C7: C15
PEG3-dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup-
.7-Val.sup.8-Asn.sup.9-Val10
Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cys.sup.15-dLeu.sup.16
20 SP-352 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-dLeu.sup.16-PEG3 21 SP-358 C4: C12, C7: C15
PEG3-dAsn.sup.1-dAsp.sup.2-dGlu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.s-
up.7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.s-
up.14-Cys.sup.15-dLeu.sup.16- 22 PEG3 SP-359 C4: C12, C7: C15
PEG3-dAsn.sup.1-dAsp.sup.2-dGlu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.s-
up.7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.s-
up.14-Cys.sup.15-dLeu.sup.16 23 SP-360 C4: C12, C7: C15
dAsn.sup.1-dAsp.sup.2-dGlu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7--
Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-
-Cys.sup.15-dLeu.sup.16-PEG3 24 SP-361 C4: C12, C7: C15
dAsn.sup.1-dAsp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-V-
al.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14--
Cys.sup.15-dLeu.sup.16 -PEG3 25 SP-362 C4: C12, C7: C15
PEG3-dAsn.sup.1-dAsp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.su-
p.7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.su-
p.14-Cys.sup.15-dLeu.sup.16 26 SP-368 C4: C12, C7: C15
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Va-
l.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dNal.sup.16 27 SP-369 C4: C12, C7: C15
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-AI-
B.sup.8-Asn.sup.9-AIB.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dLeu.sup.16 28 SP-370 C4: C12, C7: C15
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Asp[Lactam].-
sup.7-Val.sup.8-Asn.sup.9-Val0-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-
-Orn.sup.15-dLeu.sup.1 29 SP-371 C4: C12, C7: C15
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Va-
l.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dLeu.sup.16 30 SP-372 C4: C12, C7: C15
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Ser.sup.6-Cys.sup.7-Va-
l.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dLeu.sup.16 31 N1 C4: C12, C7: C15
PEG3-dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup-
.7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup-
.14-Cys.sup.15-dLeu.sup.16 -PEG3 32 N2 C4: C12, C7: C15
PEG3-dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup-
.7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup-
.14-Cys.sup.15-dLeu.sup.16 33 N3 C4: C12, C7: C15
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Va-
l.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dLeu.sup.16 PEG3 34 N4 C4: C12, C7: C15
PEG3-dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Ser.sup.6-Cys.sup-
.7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup-
.14-Cys.sup.15-dLeu.sup.16 -PEG3 35 N5 C4: C12, C7: C15
PEG3-dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Ser.sup.6-Cys.sup-
.7-Val.sup.8-Asn.sup.9-Val10
Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cys.sup.15-dLeu.sup.16
36 N6 C4: C12, C7: C15
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Ser.sup.6-Cys.sup.7-Va-
l.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dLeu.sup.16 -PEG3 37 N7 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val10
Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cys.sup.15-Ser.sup.16
38 N8 C4: C12, C7: C15
PEG3-Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.-
7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.-
14-Cys.sup.15-Ser.sup.16-PEG3 39 N9 C4: C12, C7: C15
PEG3-Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.-
7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.-
14-Cys.sup.15-Ser.sup.16 40 N10 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16-PEG3 41 N11 C4: C12, C7: C15
PEG3-Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.-
7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.-
14-Cys.sup.15-dSer.sup.16 -PEG3 42 N12 C4: C12, C7: C15
PEG3-Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.-
7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.-
14-Cys.sup.15-dSer.sup.16 43 N13 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-dSer.sup.16 -PEG3 44 Formula I C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Xaa.sup.5-Xaa.sup.6-Cys.sup.7-Xaa-
.sup.8-Xaa.sup.9-Xaa.sup.10-Xaa.sup.11-Cys.sup.12-Xaa.sup.13-Xaa.sup.14-Cy-
s.sup.15-Xaa.sup.16 45 Formula II C4: C12, C7: C15
Xaa.sub.n1-Cys.sup.4-Xaa.sup.5-Xaa.sup.6-Cys.sup.7-Xaa.sup.8-Xaa.sup.9-Xa-
al
Xaa.sup.11-Cys.sup.12-Xaa.sup.13-Xaa.sup.14-Cys.sup.15-Xaa.sub.n2.sup.1-
6 46 Formula 4: 12, 7: 15
Xaa.sub.n1-Maa.sup.4-Glu.sup.5-Xaa.sup.6-Maa.sup.7-Val.sup.8-Asn.sup.9-Va-
l.sup.10-Ala.sup.11-Maa.sup.12-Thr.sup.13-Gly.sup.14-Maa.sup.15-
Xaa.sub.n2 47 III Formula 4: 12, 7: 15 Xaa.sub.n1 -
Maa.sup.4-Xaa.sup.5-Xaa.sup.6-
Maa.sup.7-Xaa.sup.8-Xaa.sup.9-Xaa.sup.10-Xaa.sup.11-
Maa.sup.12-Xaa.sup.13-Xaa.sup.14- Maa.sup.15-Xaa.sup.16 48 IV
Formula V C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Xaa.sup.5-Xaa.sup.6-Cys.sup.7-Xaa-
.sup.8-Asn.sup.9-Xaa.sup.10-Xaa.sup.11-Cys.sup.12-Xaa.sup.13-Xaa.sup.14-Cy-
s.sup.15-Xaa.sup.16 49 Formula C4: C12, C7: C15
dAsn.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Xaa.sup.5-Xaa.sup.6-Cys.sup.7-X3-
.sup.8-Asn.sup.9-Xaa.sup.10-Xaa.sup.11-Cys.sup.12-Xaa.sup.13-Xaa.sup.14-Cy-
s.sup.15-d-Xaa.sup.16 50 VI Formula C4: C12, C7: C15
dAsn.sup.1-dGlu.sup.2-Asp.sup.3-Cys.sup.4-Xaa.sup.5-Xaa.sup.6-Cys.sup.7-X-
aa.sup.8-Asn.sup.9-Xaa.sup.10-Xaa.sup.11-Cys.sup.12-Xaa.sup.13-Xaa.sup.14--
Cys.sup.15-d-Xaa.sup.16 51 VII Formula C4: C12, C7: C15
dAsn.sup.1-dAsp.sup.2-Glu.sup.3-Cys.sup.4-Xaa.sup.5-Xaa.sup.6-Cys.sup.7-X-
aa.sup.8-Asn.sup.9-Xaa.sup.10-Xaa.sup.11-Cys.sup.12-Xaa.sup.13-Xaa.sup.14--
Cys.sup.15-d-Xaa.sup.16 52 VII Formula C4: C12, C7: C15
dAsn.sup.1-dAsp.sup.2-dGlu.sup.3-Cys.sup.4-Xaa.sup.5-Xaa.sup.6-Cys.sup.7--
Xaa.sup.8-Tyr.sup.9-Xaa.sup.10-Xaa.sup.11-Cys.sup.12-Xaa.sup.13-Xaa.sup.14-
-Cys.sup.15-d-Xaa.sup.16 53 VIII Formula C4: C12, C7: C15
dAsn.sup.1-dGlu.sup.2-dGlu.sup.3-Cys.sup.4-Xaa.sup.5-Xaa.sup.6-Cys.sup.7--
Xaa.sup.8-Tyr.sup.9-Xaa.sup.10-Xaa.sup.11-Cys.sup.12-Xaa.sup.13-Xaa.sup.14-
-Cys.sup.15-d-Xaa.sup.16 54 IX
TABLE-US-00002 TABLE II Linaclotide and Derivatives Position of
Disulfide SEQ ID Name bonds Structure NO: SP-339 C1: C6, C2: C10,
C5: 13
Cys.sup.1-Cys.sup.2-Glu.sup.3-Tyr.sup.4-Cys.sup.5-Cys.sup.6-Asn.sup.7-Pro-
.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13-Tyr.sup.14
55 (linaclotide) SP-340 C1: C6, C2: C10, C5: 13
Cys.sup.1-Cys.sup.2-Glu.sup.3-Tyr.sup.4-Cys.sup.5-Cys.sup.6-Asn.sup.7-Pro-
.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13 56
SP-349 C1: C6, C2: C10, C5: 13
PEG3-Cys.sup.1-Cys.sup.2-Glu.sup.3-Tyr.sup.4-Cys.sup.5-Cys.sup.6-Asn.sup.-
7-Pro.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13-Tyr.sup.-
14-PEG3 57 SP-353 C3: C8, C4: C12, C7: 15
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Ser.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Tyr.sup.16 58 SP-354 C3: C8, C4: C12, C7: 15
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Phe.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Tyr.sup.16 59 SP-355 C1: C6, C2: C10, C5: 13
Cys.sup.1-Cys.sup.2-Glu.sup.3-Tyr.sup.4-Cys.sup.5-Cys.sup.6-Asn.sup.7-Pro-
.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13-dTyr.sup.14
60 SP-357 C1: C6, C2: C10, C5: 13
PEG3-Cys.sup.1-Cys.sup.2-Glu.sup.3-Tyr.sup.4-Cys.sup.5-Cys.sup.6-Asn.sup.-
7-Pro.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13-Tyr.sup.-
14 61 SP-374 C3: C8, C4: C12, C7: 15
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Tyr.sup.16 62 SP-375 C3: C8, C4: C12, C7: 15
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Ser.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-dTyr.sup.16 63 SP-376 C3: C8, C4: C12, C7: 15
dAsn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Ser.sup.6-Cys.sup.7-Cy-
s.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-Tyr.sup.16 64 SP-377 C3: C8, C4: C12, C7: 15
dAsn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Ser.sup.6-Cys.sup.7-Cy-
s.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dTyr.sup.16 65 SP-378 C3: C8, C4: C12, C7: 15
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-dTyr.sup.16 66 SP-379 C3: C8, C4: C12, C7: 15
dAsn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.7-Cy-
s.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-Tyr.sup.16 67 SP-380 C3: C8, C4: C12, C7: 15
dAsn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.7-Cy-
s.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dTyr.sup.16 68 SP-381 C3: C8, C4: C12, C7: 15
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Phe.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-dTyr.sup.16 69 SP-382 C3: C8, C4: C12, C7: 15
dAsn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Phe.sup.6-Cys.sup.7-Cy-
s.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-Tyr.sup.16 70 SP-383 C3: C8, C4: C12, C7: 15
dAsn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Phe.sup.6-Cys.sup.7-Cy-
s.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dTyr.sup.16 71 SP-384 C1: C6, C2: C10, C5: 13
Cys.sup.1-Cys.sup.2-Glu.sup.3-Tyr.sup.4-Cys.sup.5-Cys.sup.6-Asn.sup.7-Pro-
.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13-Tyr.sup.14-PE-
G3 72 N14 C1: C6, C2: C10, C5: 13
PEG3-Cys.sup.1-Cys.sup.2-Glu.sup.3-Tyr.sup.4-Cys.sup.5-Cys.sup.6-Asn.sup.-
7-Pro.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13-PEG3
73 N15 C1: C6, C2: C10, C5: 13
PEG3-Cys.sup.1-Cys.sup.2-Glu.sup.3-Tyr.sup.4-Cys.sup.5-Cys.sup.6-Asn.sup.-
7-Pro.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13
74 N16 C1: C6, C2: C10, C5: 13
Cys.sup.1-Cys.sup.2-Glu.sup.3-Tyr.sup.4-Cys.sup.5-Cys.sup.6-Asn.sup.7-Pro-
.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13-PEG3
75 N17 C3: C8, C4: C12, C7: 15 PEG3-
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Ser.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 76 Tyr.sup.16-PEG3 N18 C3: C8, C4: C12, C7: 15 PEG3-
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Ser.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 77 Tyr.sup.16 N19 C3: C8, C4: C12, C7: 15
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Ser.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Tyr.sup.16- 78 PEG3 N20 C3: C8, C4: C12, C7: 15 PEG3-
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Phe.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 79 Tyr.sup.16-PEG3 N21 C3: C8, C4: C12, C7: 15 PEG3-
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Phe.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 80 Tyr.sup.16 N22 C3: C8, C4: C12, C7: 15
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Phe.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Tyr.sup.16- 81 PEG3 N23 C3: C8, C4: C12, C7: 15 PEG3-
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 82 Tyr.sup.16-PEG3 N24 C3: C8, C4: C12, C7: 15 PEG3-
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 83 Tyr.sup.16 N25 C3: C8, C4: C12, C7: 15
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Tyr.sup.16- 84 PEG3 N26 C1: C6, C2: C10, C5: 13
Cys.sup.1-Cys.sup.2-Glu.sup.3-Ser.sup.4-Cys.sup.5-Cys.sup.6-Asn.sup.7-Pro-
.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13-Tyr.sup.14
85 N27 C1: C6, C2: C10, C5: 13
Cys.sup.1-Cys.sup.2-Glu.sup.3-Phe.sup.4-Cys.sup.5-Cys.sup.6-Asn.sup.7-Pro-
.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13-Tyr.sup.14
86 N28 C1: C6, C2: C10, C5: 13
Cys.sup.1-Cys.sup.2-Glu.sup.3-Ser.sup.4-Cys.sup.5-Cys.sup.6-Asn.sup.7-Pro-
.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13- 87
N29 C1: C6, C2: C10, C5: 13
Cys.sup.1-Cys.sup.2-Glu.sup.3-Phe.sup.4-Cys.sup.5-Cys.sup.6-Asn.sup.7-Pro-
.sup.8-Ala.sup.9-Cys.sup.10-Thr.sup.11-Gly.sup.12-Cys.sup.13 88 N30
1: 6, 2: 10, 5: 13
Pen.sup.1-Pen.sup.2-Glu.sup.3-Tyr.sup.4-Pen.sup.5-Pen.sup.6-Asn.sup.7-Pro-
.sup.8-Ala.sup.9-Pen.sup.10-Thr.sup.11-Gly.sup.12-Pen.sup.13-Tyr.sup.14
89 N31 1: 6, 2: 10, 5: 13
Pen.sup.1-Pen.sup.2-Glu.sup.3-Tyr.sup.4-Pen.sup.5-Pen.sup.6-Asn.sup.7-Pro-
.sup.8-Ala.sup.9-Pen.sup.10-Thr.sup.11-Gly.sup.12-Pen.sup.13 90
Formula X C9: C14 , C10: C18,
Xaa.sup.1-Xaa.sup.2-Xaa.sup.3-Xaa.sup.4-Xaa.sup.5-Xaa.sup.6-
Asn.sup.7-
Tyr.sup.8-Cys.sup.9-Cys.sup.10-Xaa.sup.11-Tyr.sup.12-Cys.sup.13-Cys.sup.1-
4-Xaa.sup.15-Xaa.sup.16- 91 C13: 21 Xaa.sup.17-Cys.sup.18-
Xaa.sup.19-Xaa.sup.20-Cys.sup.21-Xaa.sup.22 Formula XI C9: C14,
C10: C18,
Xaa.sup.1-Xaa.sup.2-Xaa.sup.3-Xaa.sup.4-Xaa.sup.5-Xaa.sup.6-Asn.sup.7-
Phe.sup.8-Cys.sup.9-Cys.sup.10-Xaa.sup.11-Phe.sup.12-Cys.sup.13-Cys.sup.1-
4-Xaa.sup.15-Xaa.sup.16- 92 C13: 21 Xaa.sup.17-Cys.sup.18-
Xaa.sup.19-Xaa.sup.20-Cys.sup.21-Xaa.sup.22 Formula XII C3: C8, C4:
C12, C7: 15 Asn.sup.1- Phe.sup.2-Cys.sup.3-Cys4 -
Xaa.sup.5-Phe.sup.6-Cys.sup.7-Cys8 - Xaa.sup.9-Xaa.sup.10-
Xaa.sup.11-Cys.sup.12- Xaa.sup.13-Xaa.sup.14-Cys.sup.15-Xaa.sup.16
93 Formula 3: 8, 4: 12, C: 15 Asn.sup.1- Phe.sup.2-Pen.sup.3-Cys4 -
Xaa.sup.5-Phe.sup.6-Cys.sup.7-Pen8 - Xaa.sup.9-Xaa.sup.10-
Xaa.sup.11-Cys.sup.12- Xaa.sup.13-Xaa.sup.14-Cys.sup.15- 94 XIII
Xaa.sup.16 Formula 3: 8, 4: 12, 7: 15 Asn.sup.1-
Phe.sup.2-Maa.sup.3-Maa4 - Xaa.sup.5-Xaa.sup.6-Maa.sup.7-Maa8 -
Xaa.sup.9-Xaa.sup.10- Xaa.sup.11-Maa.sup.12-
Xaa.sup.13-Xaa.sup.14-Maa.sup.15- 95 XIV Xaa.sup.16 Formula XV 1:
6, 2: 10, 5: 13 Maa.sup.1-Maa.sup.2-Glu.sup.3-Xaa.sup.4-
Maa.sup.5-Maa.sup.6-Asn.sup.7-Pro.sup.8-Ala.sup.9-Maa.sup.10-Thr.sup.11-G-
ly.sup.12-Maa.sup.13-Tyr.sup.14 96 Formula 1: 6, 2: 10, 5: 13
Maa.sup.1-Maa.sup.2-Glu.sup.3-Xaa.sup.4-
Maa.sup.5-Maa.sup.6-Asn.sup.7-Pro.sup.8-Ala.sup.9-Maa.sup.10-Thr.sup.11-G-
ly.sup.12-Maa.sup.13- 97 XVI Formula 1: 6, 2: 10, 5: 13
Xaa.sub.n3-Maa.sup.1-Maa.sup.2-Xaa.sup.3-Xaa.sup.4-Maa.sup.5-Maa.sup.6-Xa-
a.sup.7-Xaa.sup.8-Xaa.sup.9-Maa.sup.10-Xaa.sup.11-Xaa.sup.12-Maa.sup.13-Xa-
a.sup.n2 98 XVII
TABLE-US-00003 TABLE III GCRA Peptides Position of SEQ Disulfide ID
Name bonds Structure NO: SP-363 C4: C12,
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Va-
l.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dLeu- 99 C7: C15 AMIDE.sup.16 SP-364 C4: C12,
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Va-
l.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dSer.sup.16 100 C7: C15 SP-365 C4: C12,
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Va-
l.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dSer- 101 C7: C15 AMIDE.sup.16 SP-366 C4: C12,
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Va-
l.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dTyr.sup.16 102 C7: C15 SP-367 C4: C12,
dAsn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Va-
l.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-C-
ys.sup.15-dTyr- 103 C7: C15 AMIDE.sup.16 SP-373 C4: C12,
Pyglu.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-V-
al.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14--
Cys.sup.15-dLeu- 104 C7: C15 AMIDE.sup.16 SP-304 C4: C12,
PEG3-Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.-
7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.-
14-Cys.sup.15- 105 di PEG C7: C15 Leu.sup.16-PEG3 SP-304 C4: C12,
PEG3-Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.-
7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.-
14-Cys.sup.15- 106 N-PEG C7: C15 Leu.sup.16 SP-304 C4: C12,
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16- 107 C-PEG C7: C15 PEG3
TABLE-US-00004 TABLE IV SP-304 Analogs, Uroguanylin, and
Uroguanylin Analogs Position of SEQ Disulfide ID Name bonds
Structure NO Formula C4: C12, Xaa.sup.1-
Xaa.sup.2-Xaa.sup.3-Maa.sup.4-Xaa.sup.5-Xaa.sup.6-Maa.sup.7-Xaa.sup.8-Xaa-
.sup.9-Xaa.sup.10-Xaa.sup.11-Maa.sup.12-Xaa.sup.13-Xaa.sup.14-Maa.sup.15-X-
aa.sup.16 108 XVIII C7: C15 Urogua- C4: C12,
Asn.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 109 nylin C7: C15 N32 C4: C12,
Glu.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 110 C7: C15 N33 C4: C12,
Glu.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 111 C7: C15 N34 C4: C12,
Glu.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 112 C7: C15 N35 C4: C12,
Glu.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 113 C7: C15 N36 C4: C12,
Asp.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 114 C7: C15 N37 C4: C12,
Asp.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 115 C7: C15 N38 C4: C12,
Asp.sup.1-Glu.sup.2-Asp.sup.3-Cy.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val.-
sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cys-
.sup.15-Leu.sup.16 116 C7: C15 N39 C4: C12,
Asp.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 117 C7: C15 N40 C4: C12,
Gln.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 118 C7: C15 N41 C4: C12,
Gln.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 119 C7: C15 N42 C4: C12,
Gln.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 120 C7: C15 N43 C4: C12,
Gln.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 121 C7: C15 N44 C4: C12,
Lys.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 122 C7: C15 N45 C4: C12,
Lys.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 123 C7: C15 N46 C4: C12,
Lys.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 124 C7: C15 N47 C4: C12,
Lys.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 125 C7: C15 N48 C4: C12,
Glu.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 126 C7: C15 N49 C4: C12,
Glu.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 127 C7: C15 N50 C4: C12,
Glu.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 128 C7: C15 N51 C4: C12,
Glu.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 129 C7: C15 N52 C4: C12,
Asp.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 130 C7: C15 N53 C4: C12,
Asp.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 131 C7: C15 N54 C4: C12,
Asp.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 132 C7: C15 N55 C4: C12,
Asp.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 133 C7: C15 N56 C4: C12,
Gln.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 134 C7: C15 N57 C4: C12,
Gln.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 135 C7: C15 N58 C4: C12,
Gln.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 136 C7: C15 N59 C4: C12,
Gln.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 137 C7: C15 N60 C4: C12,
Lys.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 138 C7: C15 N61 C4: C12,
Lys.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 139 C7: C15 N62 C4: C12,
Lys.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 140 C7: C15 N63 C4: C12,
Lys.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val-
.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 141 C7: C15 N65 C4C12,
Glu.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys-
.sup.7-Ile.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly-
.sup.14-Cys.sup.15-Leu.sup.16 142 C7: C15 N66 C4: C12,
Glu.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 143 C7: C15 N67 C4: C12,
Glu.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 144 C7: C15 N68 C4: C12,
Glu.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 145 C7: C15 N69 C4: C12,
Asp.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 146 C7: C15 N70 C4: C12,
Asp.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 147 C7: C15 N71 C4: C12,
Asp.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 148 C7: C15 N72 C4: C12,
Asp.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 149 C7: C15 N73 C4: C12,
Gln.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 150 C7: C15 N74 C4: C12,
Gln.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 151 C7: C15 N75 C4: C12,
Gln.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 152 C7: C15 N76 C4: C12,
Gln.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 153 C7: C15 N77 C4: C12,
Lys.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 154 C7: C15 N78 C4: C12,
Lys.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 155 C7: C15 N79 C4: C12,
Lys.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 156 C7: C15 N80 C4: C12,
Lys.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Leu.sup.16 157 C7: C15 N81 C4: C12,
Glu.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 158 C7: C15 N82 C4: C12,
Glu.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 159 C7: C15 N83 C4: C12,
Glu.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 160 C7: C15 N84 C4: C12,
Glu.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 161 C7: C15 N85 C4: C12,
Asp.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 162 C7: C15 N86 C4: C12,
Asp.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 163 C7: C15 N87 C4: C12,
Asp.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 164 C7: C15 N88 C4: C12,
Asp.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 165 C7: C15 N89 C4: C12,
Gln.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 166 C7: C15 N90 C4: C12,
Gln.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 167 C7: C15 N91 C4: C12,
Gln.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 167 C7: C15 N92 C4: C12,
Gln.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 169 C7: C15 N93 C4: C12,
Lys.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 170 C7: C15 N94 C4: C12,
Lys.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 171 C7: C15 N95 C4: C12,
Lys.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 172 C7: C15 N96 C4: C12,
Lys.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15-Ser.sup.16 173 C7: C15
TABLE-US-00005 TABLE V Guanylin and Analogs SEQ Position of ID Name
Disulfide bonds Structure NO Formula 4: 12, 7: 15
Xaa.sup.1-Xaa.sup.2-Xaa.sup.3-Maa.sup.4-Xaa.sup.5-Xaa.sup.6-Maa.sup.7-Xaa-
.sup.8-Xaa.sup.9-Xaa.sup.10-Xaa.sup.11-Maa.sup.12-Xaa.sup.13-Xaa.sup.14-Ma-
a.sup.15 174 XIX Guany- C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ala-
.sup.8-Phe.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 175 lin N97 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 176 N98 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 177 N99 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Val.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 178 N100 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 179 N101 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 180 N102 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 181 N103 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Val.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 182 N104 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 183 N105 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 184 N106 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 185 N107 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Val.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 186 N108 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 187 N109 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 188 N110 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 189 N111 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Val.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 190 N112 C4: C12, C7: C15
Ser.sup.1-His.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 191 N113 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 192 N114 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 193 N115 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Val.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 194 N116 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 195 N117 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 196 N118 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 197 N119 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Val.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 198 N120 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 199 N121 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 200 N122 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 201 N123 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Val.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 202 N124 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 203 N125 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 204 N126 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 205 N127 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Val.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 206 N128 C4: C12, C7: C15
Asn.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ala-
.sup.8-Asn.sup.9-Ala.sup.10-Ala.sup.11-Cys.sup.12-Ala.sup.13-Gly.sup.14-Cy-
s.sup.15 207
TABLE-US-00006 TABLE VI Lymphoguanylin and Analogs Position of SEQ
Disulfide ID Name bonds Structure NO Formula XX 4: 12,
Xaa.sup.1-Xaa.sup.2-Xaa.sup.3-Maa.sup.4-Xaa.sup.5-Xaa.sup.6-Maa.sup.7-Xaa-
.sup.8-Xaa.sup.9-Xaa.sup.10-Xaa.sup.11-Maa.sup.12-Xaa.sup.13-Xaa.sup.14-Xa-
a.sub.n1.sup.15 208 7: 15 Lymphogua- C4: C12
Gln.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 209 nylin N129 C4: C12
Gln.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 210 N130 C4: C12
Gln.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 211 N131 C4: C12
Gln.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 212 N132 C4: C12
Gln.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 213 N133 C4: C12
Gln.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Glu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 214 N134 C4: C12
Gln.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Glu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 215 N135 C4: C12
Gln.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Glu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 216 N136 C4: C12
Gln.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Glu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 217 N137 C4: C12
Gln.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 218 N138 C4: C12
Gln.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 219 N139 C4: C12
Gln.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 220 N140 C4: C12
Gln.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 221 N141 C4: C12
Gln.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 222 N142 C4: C12
Gln.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 223 N143 C4: C12
Gln.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 224 N144 C4: C12
Gln.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Ty-
r.sup.15 225 N145 C4: C12,
Gln.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 226 C7: C15 Ser.sup.16 N146 C4: C12,
Gln.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 227 C7: C15 Ser.sup.16 N147 C4: C12,
Gln.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 228 C7: C15 Ser.sup.16 N148 C4: C12,
Gln.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 229 C7: C15 Ser.sup.16 N149 C4: C12,
Gln.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Glu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 230 C7: C15 Ser.sup.16 N150 C4: C12,
Gln.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Glu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 231 C7: C15 Ser N151 C4: C12,
Gln.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Glu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 232 C7: C15 Ser.sup.16 N152 C4: C12,
Gln.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Glu.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 233 C7: C15 Ser.sup.16 N153 C4: C12,
Gln.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 234 C7: C15 Ser.sup.16 N154 C4: C12,
Gln.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 235 C7: C15 Ser.sup.16 N155 C4: C12,
Gln.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 236 C7: C15 Ser.sup.16 N156 C4: C12,
Gln.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 237 C7: C15 Ser.sup.16 N157 C4: C12,
Gln.sup.1-Glu.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 238 C7: C15 Ser.sup.16 N158 C4: C12,
Gln.sup.1-Asp.sup.2-Glu.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 239 C7: C15 Ser.sup.16 N159 C4: C12,
Gln.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 240 C7: C15 Ser.sup.16 N160 C4: C12,
Gln.sup.1-Glu.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ile-
.sup.8-Asn.sup.9-Met.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cy-
s.sup.15- 241 C7: C15 Ser.sup.16
TABLE-US-00007 TABLE VII ST Peptide and Analogues Position of SEQ
Disulfide ID Name bonds Structure NO ST C3: C8,
Asn.sup.1-Ser.sup.2-Ser.sup.3-Asn.sup.4-Ser.sup.5-Ser.sup.6-Asn-
.sup.7-Tyr.sup.8-Cys.sup.9-Cys.sup.10-Glu.sup.11-Lys.sup.12-Cys.sup.13-Cys-
.sup.14- 242 Peptide C4: C12,
Asn.sup.15-Pro.sup.16-Ala.sup.17-Cys.sup.18-Thr.sup.19-Gly.sup.20-Cys.sup-
.21-Tyr.sup.22 C7: 15 N161 C3: C8,
PEG3-Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.-
7-Cys.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-
243 C4: C12, Gly.sup.14-Cys.sup.15-Tyr.sup.16-PEG3 C7: 15 N162 C3:
C8,
PEG3-Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.-
7-Cys.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-
244 C4: C12, Gly.sup.14-Cys.sup.15-Tyr.sup.16 C7: 15 N163 C3: C8,
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Thr.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-
245 C4: C12, Cys.sup.15-Tyr.sup.16-PEG3 C7: 15 N164 C3: C8,
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-
246 C4: C12, Cys.sup.15-Tyr.sup.16 C7: 15 N165 C3: C8,
dAsn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Cy-
s.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-
247 C4: C12, Cys.sup.15-dTyr.sup.16 C7: 15 N166 C3: C8,
Asn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Cys-
.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-
248 C4: C12, Cys.sup.15-dTyr.sup.16 C7: 15 N167 C3: C8,
dAsn.sup.1-Phe.sup.2-Cys.sup.3-Cys.sup.4-Glu.sup.5-Tyr.sup.6-Cys.sup.7-Cy-
s.sup.8-Asn.sup.9-Pro.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-
249 C4: C12, Cys.sup.15-Tyr.sup.16 C7: 15
1.2 Methods of Use
[0110] The invention provides methods for treating or preventing
gastrointestinal disorders and increasing gastrointestinal motility
in a subject in need thereof by administering an effective amount
of a GCC agonist or a formulation thereof to the subject.
Non-limiting examples of gastrointestinal disorders that can be
treated or prevented according to the methods of the invention
include irritable bowel syndrome (IBS), non-ulcer dyspepsia, H.
pylori infection related ulcers, chronic intestinal
pseudo-obstruction, functional dyspepsia, colonic
pseudo-obstruction, duodenogastric reflux, gastroesophageal reflux
disease (GERD), ileus (e.g., postoperative ileus), gastroparesis,
heartburn (high acidity in the GI tract), constipation (e.g.,
constipation associated with use of medications such as opioids,
osteoarthritis drugs, or osteoporosis drugs); post surgical
constipation, constipation associated with neuropathic disorders,
Crohn's disease, and ulcerative colitis.
[0111] In one embodiment, the invention provides methods for
treating or preventing gastrointestinal motility disorder,
irritable bowel syndrome, a functional gastrointestinal disorder,
gastroesophageal reflux disease, duodenogastric reflux, functional
heartburn, dyspepsia, functional dyspepsia, non-ulcer dyspepsia,
gastroparesis, chronic intestinal pseudo-obstruction, colonic
pseudo-obstruction, obesity, congestive heart failure, or benign
prostatic hyperplasia.
[0112] In one embodiment, the invention provides methods for
treating or preventing constipation and/or increasing
gastrointestinal motility in a subject in need thereof by
administering an effective amount of a GCC agonist or a formulation
thereof to the subject. Clinically accepted criteria that define
constipation range from the frequency of bowel movements, the
consistency of feces and the ease of bowel movement. One common
definition of constipation is less than three bowel movements per
week. Other definitions include abnormally hard stools or
defecation that requires excessive straining (Schiller 2001 Aliment
Pharmacol Ther 15:749-763). Constipation may be idiopathic
(functional constipation or slow transit constipation) or secondary
to other causes including neurologic, metabolic or endocrine
disorders. These disorders include diabetes mellitus,
hypothyroidism, hyperthyroidism, hypocalcaemia, Multiple sclerosis,
Parkinson's disease, spinal cord lesions, Neurofibromatosis,
autonomic neuropathy, Chagas disease, Hirschsprung disease and
cystic fibrosis. Constipation may also be the result of surgery or
due to the use of drugs such as analgesics (like opioids),
antihypertensives, anticonvulsants, antidepressants, antispasmodics
and antipsychotics.
[0113] In various embodiments, the constipation is associated with
use of a therapeutic agent; the constipation is associated with a
neuropathic disorder; the constipation is postsurgical
constipation; the constipation is associated with a
gastrointestinal disorder; the constipation is idiopathic
(functional constipation or slow transit constipation); the
constipation is associated with neuropathic, metabolic or endocrine
disorder (e.g., diabetes mellitus, hypothyroidism, hyperthyroidism,
hypocalcaemia, Multiple Sclerosis, Parkinson's disease, spinal cord
lesions, neurofibromatosis, autonomic neuropathy, Chagas disease,
Hirschsprung disease or cystic fibrosis). Constipation may also be
the result of surgery or due to the use of drugs such as analgesics
(e.g., opioids), antihypertensives, anticonvulsants,
antidepressants, antispasmodics and antipsychotics.
[0114] In one embodiment, the invention provides methods for
treating or preventing chronic idiopathic constipation and
increasing gastrointestinal motility in a subject in need thereof
by administering an effective amount of a GCC agonist or a
formulation thereof to the subject.
[0115] The term "treating" as used herein refers to a reduction, a
partial improvement, amelioration, or a mitigation of at least one
clinical symptom associated with the gastrointestinal disorders
being treated. The term "preventing" refers to an inhibition or
delay in the onset or progression of at least one clinical symptom
associated with the gastrointestinal disorders to be prevented. The
term "effective amount" as used herein refers to an amount that
provides some improvement or benefit to the subject. In certain
embodiments, an effective amount is an amount that provides some
alleviation, mitigation, and/or decrease in at least one clinical
symptom of the gastrointestinal disorder to be treated. In other
embodiments, the effective amount is the amount that provides some
inhibition or delay in the onset or progression of at least one
clinical symptom associated with the gastrointestinal disorder to
be prevented. The therapeutic effects need not be complete or
curative, as long as some benefit is provided to the subject. The
term "subject" preferably refers to a human subject but may also
refer to a non-human primate or other mammal preferably selected
from among a mouse, a rat, a dog, a cat, a cow, a horse, or a
pig.
[0116] The invention also provides methods for treating
gastrointestinal cancer in a subject in need thereof by
administering an effective amount of a GCC agonist or a formulation
thereof to the subject. Non-limiting examples of gastrointestinal
cancers that can be treated according to the methods of the
invention include gastric cancer, esophageal cancer, pancreatic
cancer, colorectal cancer, intestinal cancer, anal cancer, liver
cancer, gallbladder cancer, or colon cancer.
[0117] The invention also provides methods for treating lipid
metabolism disorders, biliary disorders, inflammatory disorders,
lung disorders, cancer, cardiac disorders including cardiovascular
disorders, eye disorders, oral disorders, blood disorders, liver
disorders, skin disorders, prostate disorders, endocrine disorders,
and obesity.
[0118] Lipid metabolism disorders include, but are not limited to,
dyslipidemia, hyperlipidemia, hypercholesterolemia,
hypertriglyceridemia, sitosterolemia, familial
hypercholesterolemia, xanthoma, combined hyperlipidemia, lecithin
cholesterol acyltransferase deficiency, tangier disease,
abetalipoproteinemia, erectile dysfunction, fatty liver disease,
and hepatitis.
[0119] Billary disorders include gallbladder disorders such as for
example, gallstones, gall bladder cancer cholangitis, or primary
sclerosing cholangitis; or bile duct disorders such as for example,
cholecystitis, bile duct cancer or fascioliasis.
[0120] Inflammatory disorders include tissue and organ inflammation
such as kidney inflammation (e.g., nephritis), gastrointestinal
system inflammation (e.g., Crohn's disease and ulcerative colitis);
necrotizing enterocolitis (NEC); pancreatic inflammation (e.g.,
pancreatis), pancreatic insufficiency, lung inflammation (e.g.,
bronchitis or asthma) or skin inflammation (e.g., psoriasis,
eczema).
[0121] Lung Disorders include for example chronic obstructive
pulmonary disease (COPD), and fibrosis.
[0122] Cancer includes tissue and organ carcinogenesis including
metastases such as for example gastrointestinal cancer, (e.g.,
gastric cancer, esophageal cancer, pancreatic cancer colorectal
cancer, intestinal cancer, anal cancer, liver cancer, gallbladder
cancer, or colon cancer; lung cancer; thyroid cancer; skin cancer
(e.g., melanoma); oral cancer; urinary tract cancer (e.g. bladder
cancer or kidney cancer); blood cancer (e.g. myeloma or leukemia)
or prostate cancer.
[0123] Cardiac disorders include for example, congestive heart
failure, trachea cardia hypertension, high cholesterol, or high
triglycerides. Cardiovascular disorders include for example
aneurysm, angina, atherosclerosis, cerebrovascular accident
(stroke), cerebrovasculardisease, congestive heart failure,
coronary artery disease, myocardial infarction (heart attack), or
peripheral vascular disease.
[0124] Liver disorders include for example cirrhosis and fibrosis.
In addition, GC-C agonist may also be useful to facilitate liver
regeneration in liver transplant patients. Eye disorders include
for example increased intra-ocular pressure, glaucoma, dry eyes,
retinal degeneration, disorders of tear glands or eye inflammation.
Skin disorders include for example xerosis. Oral disorders include
for example dry mouth (xerostomia), Sjogren's syndrome, gum
diseases (e.g., periodontal disease), or salivary gland duct
blockage or malfunction. Prostate disorders include for example
benign prostatic hyperplasia (BPH). Endocrine disorders include for
example diabetes mellitus, hyperthyroidism, hypothyroidism, and
cystic fibrosis.
1.2.1 Therapeutically Effective Dosages
[0125] Disorders are treated, prevented or alleviated by
administering to a subject, e.g., a mammal such as a human in need
thereof, a therapeutically effective dose of a GCC agonist peptide.
The present invention is based in part on the unexpected results of
clinical trials in humans which demonstrated that the formulations
of the invention are therapeutically effective at much lower doses
than predicted based on animal studies. In accordance with one
aspect of the invention, the therapeutically effective dose is
between 0.01 milligrams (mg) and 10 mg per unit dose. The term
"unit dose" refers to a single drug delivery entity, e.g., a
tablet, capsule, solution, inhalation, controlled release or
extended release formulation (e.g. MMX.RTM. technology of Cosmo
Pharmaceuticals). In one embodiment, the effective dose is between
0.01 mg and 9 mg. In another embodiment, the effective dose is
between 0.01 mg and 5 mg. In another embodiment, the effective dose
is between 0.01 mg and 3 mg. In another embodiment, the effective
dose is between 0.10 mg and 5 mg. In another embodiment, the
effective dose is between 0.10 mg and 3 mg. In one embodiment, the
unit dose is 0.01 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.5 mg, 1.0
mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 5 mg, or 10 mg. In one
embodiment, the unit dose is 0.3 mg, 1.0 mg, 3.0 mg, 9.0 mg, or 9.5
mg.
[0126] The GCC agonist peptides may be in a pharmaceutical
composition in unit dose form, together with one or more
pharmaceutically acceptable excipients. The amount of peptide
present should be sufficient to have a positive therapeutic effect
when administered to a patient. What constitutes a "positive
therapeutic effect" will depend upon the particular condition being
treated and will include any significant improvement in a condition
readily recognized by one of skill in the art.
[0127] The GCC agonists for use in the methods described above are
preferably administered orally. Dosage forms include solutions,
suspensions, emulsions, tablets, and capsules.
[0128] The total daily dose can be administered to the patient in a
single dose, or in multiple sub-doses. Typically, sub-doses can be
administered two to six times per day, preferably two to four times
per day, and even more preferably two to three times per day.
Preferably, a single daily dose is administered.
[0129] The GCC agonists may be administered as either the sole
active agent or in combination with one or more additional active
agents. In all cases, additional active agents should be
administered at a dosage that is therapeutically effective using
the existing art as a guide. The GCC agonists may be administered
in a single composition or sequentially with the one or more
additional active agents. In one embodiment, the GCC agonist is
administered in combination with one or more inhibitors of cGMP
dependent phosphodiesterase such as suldinac sulfone, zaprinast,
motapizone, vardenafil, or sildenifil. In another embodiment, the
GCC agonist is administered in combination with one or more
chemotherapeutic agents. In another embodiment, the GCC agonist is
administered in combination with one or more or anti-inflammatory
drugs such as steroids or non-steroidal anti-inflammatory drugs
(NSAIDS), such as aspirin.
[0130] Combination therapy can be achieved by administering two or
more agents, e.g., a GCC agonist peptide described herein and
another compound, each of which is formulated and administered
separately, or by administering two or more agents in a single
formulation. Other combinations are also encompassed by combination
therapy. For example, two agents can be formulated together and
administered in conjunction with a separate formulation containing
a third agent. While the two or more agents in the combination
therapy can be administered simultaneously, they need not be. For
example, administration of a first agent (or combination of agents)
can precede administration of a second agent (or combination of
agents) by minutes, hours, days, or weeks. Thus, the two or more
agents can be administered within minutes of each other or within
1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other or within 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other or within 2,
3, 4, 5, 6, 7, 8, 9, or 10 weeks of each other. In some cases even
longer intervals are possible. While in many cases it is desirable
that the two or more agents used in a combination therapy be
present in within the patient's body at the same time, this need
not be so.
[0131] The GCC agonist peptides described herein may be combined
with phosphodiesterase inhibitors, e.g., sulindae sulfone,
Zaprinast, sildenafil, vardenafil or tadalafil to further enhance
levels of cGMP in the target tissues or organs.
[0132] Combination therapy can also include two or more
administrations of one or more of the agents used in the
combination. For example, if agent X and agent Y are used in a
combination, one could administer them sequentially in any
combination one or more times, e.g., in the order X-Y-X, X-X-Y,
Y-X-Y, Y-Y-X, X-X-Y-Y, etc.
1.2.2 Exemplary Agents for Combination Therapy
[0133] The GCC agonist formulations of the invention may be
administered alone or in combination with one or more additional
therapeutic agents as part of a therapeutic regimen for the
treatment or prevention of a gastrointestinal disease or disorder.
In some embodiments, the GCC agonist formulation comprises one or
more additional therapeutic agents. In other embodiments, the GCC
agonist is formulated separately from the one or more additional
therapeutic agents. In accordance with this embodiment, the GCC
agonist is administered either simultaneously, sequentially, or at
a different time than the one or more additional therapeutic
agents. In one embodiment, the GCC agonist formulation is
administered in combination with one or more additional therapeutic
agents selected from the group consisting of phosphodiesterase
inhibitors, cyclic nucleotides (such as cGMP and cAMP), a laxative
(such as SENNA or METAMUCIL), a stool softner, an anti-tumor
necrosis factor alpha therapy for IBD (such as REMICADE, ENBREL, or
HUMIRA), and anti-inflammatory drugs (such as COX-2 inhibitors,
sulfasalazine, 5-ASA derivatives and NSAIDS). In certain
embodiments, the GCC agonist formulation is administered in
combination with an effective dose of an inhibitor of cGMP-specific
phosphodiesterase (cGMP-PDE) either concurrently or sequentially
with said GCC agonist. cGMP-PDE inhibitors include, for example,
suldinac sulfone, zaprinast, motapizone, vardenifil, and
sildenafil. In another embodiment, the GCC agonist formulation is
administered in combination with inhibitors of cyclic nucleotide
transporters. Further examples of therapeutic agents that may be
administered in combination with the GCC agonist formulations of
the invention are given in the following sections.
[0134] 1.2.2.1 Agents to Treat Gastrointestinal Cancers
[0135] The GCC agonist formulations described herein can be used in
combination with one or more antitumor agents including but not
limited to alkylating agents, epipodophyllotoxins, nitrosoureas,
anti-metabolites, vinca alkaloids, anthracycline antibiotics,
nitrogen mustard agents, and the like. Particular antitumor agents
include tamoxifen, taxol, etoposide, and 5-fluorouracil. In one
embodiment, the GCC agonist formulations are used in combination
with an antiviral agent or a monoclonal antibody.
[0136] Non-limiting examples of antitumor agents that can be used
in combination with the GCC agonist formulations of the invention
for the treatment of colon cancer include anti-proliferative
agents, agents for DNA modification or repair, DNA synthesis
inhibitors, DNA/RNA transcription regulators, RNA processing
inhibitors, agents that affect protein expression, synthesis and
stability, agents that affect protein localization or their ability
to exert their physiological action, agents that interfere with
protein-protein or protein-nucleic acid interactions, agents that
act by RNA interference, receptor binding molecules of any chemical
nature (including small molecules and antibodies), targeted toxins,
enzyme activators, enzyme inhibitors, gene regulators, HSP-90
inhibitors, molecules interfering with microtubules or other
cytoskeletal components or cell adhesion and motility, agents for
phototherapy, and therapy adjuncts.
[0137] Representative anti-proliferative agents include
N-acetyl-D-sphingosine (C.sub.2 ceramide), apigenin, berberine
chloride, dichloromethylenediphosphonic acid disodium salt,
loe-emodine, emodin, HA 14-1, N-hexanoyl-D-sphingosine (C.sub.6
ceramide), 7b-hydroxycholesterol, 25-hydroxycholesterol,
hyperforin, parthenolide, and rapamycin.
[0138] Representative agents for DNA modification and repair
include aphidicolin, bleomycin sulfate, carboplatin, carmustine,
chlorambucil, cyclophosphamide monohydrate, cyclophosphamide
monohydrate ISOPAC.RTM., cis-diammineplatinum(II) dichloride
(Cisplatin), esculetin, melphalan, methoxyamine hydrochloride,
mitomycin C, mitoxantrone dihydrochloride, oxaliplatin, and
streptozocin.
[0139] Representative DNA synthesis inhibitors include
(.+-.)amethopterin (methotrexate), 3-amino-1,2,4-benzotriazine
1,4-dioxide, aminopterin, cytosine b-D-arabinofurdnoside (Ara-C),
cytosine b-D-arabinofuranoside (Ara-C) hydrochloride,
2-fluoroadenine-9-b-D-arabinofuranoside (Fludarabine des-phosphate;
F-ara-A), 5-fluoro-5'-deoxyuridinc, 5-fluorouracil, ganciclovir,
hydroxyurea, 6-mercaptopurine, and 6-thioguanine.
[0140] Representative DNA/RNA transcription regulators include
actinomycin D, daunorubicin hydrochloride,
5,6-dichlorobenzimidazole 1-b-D-ribofuranoside, doxorubicin
hydrochloride, homoharringtonine, and idarubicin hydrochloride.
[0141] Representative enzyme activators and inhibitors include
forskolin, DL-aminoglutethimide, apicidin, Bowman-Birk Inhibitor,
butein, (S)-(+)-camptothecin, curcumin, (-)-deguelin,
(-)-depudecin, doxycycline hyclate, etoposide, formestane,
fostriecin sodium salt, hispidin, 2-imino-1-imidazolidineacetic
acid (Cyclocreatine), oxamflatin, 4-phenylbutyric acid,
roscovitine, sodium valproate, trichostatin A, tyrphostin AG 34,
tyrphostin AG 879, urinary trypsin inhibitor fragment, valproic
acid (2-propylpentanoic acid), and XK469.
[0142] Representative gene regulators include
5-aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol (Vitamin
D3), ciglitizone, cyproterone acetate,
15-deoxy-D.sup.12,14-prostaglandin J.sub.2, epitestosterone,
flutamide, glycyrrhizic acid ammonium salt (glycyrrhizin),
4-hydroxytamoxifen, mifepristone, procainamide hydrochloride,
raloxifene hydrochloride, all trans-retinal (vitamin A aldehyde),
retinoic acid (vitamin A acid), 9-cis-retinoic acid,
13-cis-retinoic acid, retinoic acid p-hydroxyanilide, retinol
(Vitamin A), tamoxifen, tamoxifen citrate salt,
tetradecylthioacetic acid, and troglitazone.
[0143] Representative HSP-90 inhibitors include
17-(allylamino)-17-demethoxygeldanamycin and geldanamycin.
[0144] Representative microtubule inhibitors include colchicines,
dolastatin 15, nocodazole, taxanes and in particular paclitaxel,
podophyllotoxin, rhizoxin, vinblastine sulfate salt, vincristine
sulfate salt, and vindesine sulfate salt and vinorelbine
(Navelbine) ditartrate salt.
[0145] Representative agents for performing phototherapy include
photoactive porphyrin rings, hypericin, 5-methoxypsoralen,
8-methoxypsoralen, psoralen and ursodeoxycholic acid.
[0146] Representative agents used as therapy adjuncts include
amifostine, 4-amino-1,8-naphthalimide, brefeldin A, cimetidine,
phosphomycin disodium salt, leuprolide (leuprorelin) acetate salt,
luteinizing hormone-releasing hormone (LH-RH) acetate salt, lectin,
papaverine hydrochloride, pifithrin-a, (-)-scopolamine
hydrobromide, and thapsigargin.
[0147] The agents can also be anti-VEGF (vascular endothelial
growth factor) agents, as such are known in the art. Several
antibodies and small molecules are currently in clinical trials or
have been approved that function by inhibiting VEGF, such as
Avastin (Bevacizumab), SU5416, SU11248 and BAY 43-9006. The agents
can also be directed against growth factor receptors such as those
of the EGF/Erb-B family such as EGF Receptor (Iressa or Gefitinib,
and Tarceva or Erlotinib), Erb-B2, receptor (Herceptin or
Trastuzumab), other receptors (such as Rituximab or
Rituxan/MabThera), tyrosine kinases, non-receptor tyrosine kinases,
cellular serine/threonine kinases (including MAP kinases), and
various other proteins whose deregulation contribute to oncogenesis
(such as small/Ras family and large/heterotrimeric G proteins).
Several antibodies and small molecules targeting those molecules
are currently at various stages of development (including approved
for treatment or in clinical trials).
[0148] In a preferred embodiment, the invention provides a method
for treating colon cancer in a subject in need thereof by
administering to the subject a GCC agonist formulation in
combination with one or more antitumor agent selected from the
group consisting of paclitaxel, docetaxel, tamoxifen, vinorelbine,
gemcitabine, cisplatin, etoposide, topotecan, irinotecan,
anastrozole, rituximab, trastuzumab, fludarabine, cyclophosphamide,
gentuzumab, carboplatin, interferons, and doxorubicin. In a
particular embodiment the antitumor agent is paclitaxel. In a
further embodiment, the method further comprises an antitumor agent
selected from the group consisting of 5-FU, doxorubicin,
vinorelbine, cytoxan, and cisplatin.
[0149] 1.2.2.2 Agents that Treat Crohn's Disease
[0150] In one embodiment, a GCC agonist formulation of the
invention is administered as part of a combination therapy with one
or more additional therapeutic agents for the treatment of Crohn's
disease. Non-limiting examples of the one or more additional
therapeutic agents include sulfasalazine and other
mesalamine-containing drugs, generally known as 5-ASA agents, such
as Asacol, Dipentum, or Pentasa, or infliximab (REMICADE). In
certain embodiments, the one or more additional agents is a
corticosteroid or an immunosuppressive agent such as
6-mercaptopurine or azathioprine. In another embodiment, the one or
more additional agents is an antidiarrheal agent such as
diphenoxylate, loperamide, or codeine.
[0151] 1.2.2.3 Agents that Treat Ulcerative Colitis
[0152] In one embodiment, a GCC agonist formulation of the
invention is administered as part of a combination therapy with one
or more additional therapeutic agents for the treatment of
ulcerative colitis. The agents that are used to treat ulcerative
colitis overlap with those used to treat Chrohn's Disease.
Non-limiting examples of the one or more additional therapeutic
agents that can be used in combination with a GCC agonist
formulation of the invention include aminosalicylates (drugs that
contain 5-aminosalicyclic acid (5-ASA)) such as sulfasalazine,
olsalazine, mesalamine, and balsalazide. Other therapeutic agents
that can be used include corticosteroids, such as prednisone and
hydrocortisone, immunomodulators, such as azathioprine,
6-mercapto-purine (6-MP), cytokines, interleukins, and lymphokines,
and anti-TNF-alpha agents, including the thiazolidinediones or
glitazones such as rosiglitazone and pioglitazone. In one
emobidment, the one or more additional therapeutic agents includes
both cyclosporine A and 6-MP or azathioprine for the treatment of
active, severe ulcerative colitis.
[0153] 1.2.2.4 Agents that Treat Constipation/Irritable Bowel
Syndrome
[0154] In one embodiment, a GCC agonist formulation of the
invention is administered as part of a combination therapy with one
or more additional therapeutic agents for the treatment of
constipation, such as that associated with irritable bowel
syndrome. Non-limiting examples of the one or more additional
therapeutic agents include laxatives such as SENNA, MIRALAX,
LACTULOSE, PEG, or calcium polycarbophil), stool softeners (such as
mineral oil or COLACE), bulking agents (such as METAMUCIL or bran),
agents such as ZELNORM (also called tegaserod), and anticholinergic
medications such as BENTYL and LEVSIN.
[0155] 1.2.2.5 Agents for the Treatment of Postoperative Ileus
[0156] In one embodiment, a GCC agonist formulation of the
invention is administered as part of a combination therapy with one
or more additional therapeutic agents for the treatment of
postoperative ileus. Non-limiting examples of the one or more
additional therapeutic agents include ENTEREG (alvimopan; formerly
called ado lor/ADL 8-2698), conivaptan, and related agents
describes in U.S. Pat. No. 6,645,959.
[0157] 1.2.2.6 Anti-Obesity Agents
[0158] In one embodiment, a GCC agonist formulation of the
invention is administered as part of a combination therapy with one
or more additional therapeutic agents for the treatment of obesity.
Non-limiting examples of the one or more additional therapeutic
agents include 1 l.beta. HSD-I (11-beta hydroxy steroid
dehydrogenase type 1) inhibitors, such as BVT 3498, BVT 2733,
3-(1-adamantyl)-4-ethyl-5-(ethylthio)-4H-1,2,4-triazole,
3-(1-adamantyl)-5-(3,4,5-trimethoxyphenyl)-4-methyl-4H-1,2,4-triazole,
3-adamantanyl-4,5,6,7,8,9,10,11,12,3a-decahydro-1,2,4-triazolo[4,3-a][11]-
annulene, and those compounds disclosed in WO01/90091, WOO 1/90090,
WOO 1/90092 and WO02/072084; 5HT antagonists such as those in
WO03/037871, WO03/037887, and the like; 5HTIa modulators such as
carbidopa, benserazide and those disclosed in U.S. Pat. No.
6,207,699, WO03/031439, and the like; 5HT2c (serotonin receptor 2c)
agonists, such as BVT933, DPCA37215, IK264, PNU 22394, WAY161503,
R-1065, SB 243213 (Glaxo Smith Kline) and YM 348 and those
disclosed in U.S. Pat. No. 3,914,250, WO00/77010, WO02/36596,
WO02/48124, WO02/10169, WO01/66548, WO02/44152, WO02/51844,
WO02/40456, and WO02/40457; 5HT6 receptor modulators, such as those
in WO03/030901, WO03/035061, WO03/039547, and the like;
acyl-estrogens, such as oleoyl-estrone, disclosed in del Mar-Grasa,
M. et al, Obesity Research, 9:202-9 (2001) and Japanese Patent
Application No. JP 2000256190; anorectic bicyclic compounds such as
1426 (Aventis) and 1954 (Aventis), and the compounds disclosed in
WO00/18749, WO01/32638, WO01/62746, WO01/62747, and WO03/015769; CB
1 (cannabinoid-1 receptor) antagonist/inverse agonists such as
rimonabant (Acomplia; Sanofi), SR-147778 (Sanofi), SR-141716
(Sanofi), BAY 65-2520 (Bayer), and SLV 319 (Solvay), and those
disclosed in patent publications U.S. Pat. Nos. 4,973,587,
5,013,837, 5,081,122, 5,112,820, 5,292,736, 5,532,237, 5,624,941,
6,028,084, 6,509,367, 6,509,367, WO96/33159, WO97/29079,
WO98/31227, WO98/33765, WO98/37061, WO98/41519, WO98/43635,
WO98/43636, WO99/02499, WO00/10967, WO00/10968, WO01/09120,
WO01/58869, WO01/64632, WO01/64633, WO01/64634, WO01/70700,
WO01/96330, WO02/076949, WO03/006007, WO03/007887, WO03/020217,
WO03/026647, WO03/026648, WO03/027069, WO03/027076, WO03/027114,
WO03/037332, WO03/040107, WO03/086940, WO03/084943 and EP658546;
CCK-A (cholecystokinin-A) agonists, such as AR-R 15849, GI 181771
(GSK), JMV-180, A-71378, A-71623 and SR146131 (Sanofi), and those
described in U.S. Pat. No. 5,739,106; CNTF (Ciliary neurotrophic
factors), such as GI-181771 (Glaxo-SmithKline), SR1 46131 (Sanofi
Synthelabo), butabindide, PD 170,292, and PD 149164 (Pfizer); CNTF
derivatives, such as Axokine.RTM. (Regeneron), and those disclosed
in WO94/09134, WO98/22128, and WO99/43813; dipeptidyl peptidase IV
(DP-IV) inhibitors, such as isoleucine thiazolidide, valine
pyrrolidide, NVP-DPP728, LAF237, P93/01, P 3298, TSL 225
(tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;
disclosed by Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998)
1537-1540), TMC-2A/2B/2C, CD26 inhibtors, FE 999011, P9310/K364,
VIP 0177, SDZ 274-444, 2-cyanopyrrolidides and 4-cyanopyrrolidides
as disclosed by Ashworth et al, Bioorg. & Med. Chem. Lett.,
Vol. 6, No. 22, pp 1163-1166 and 2745-2748 (1996) and the compounds
disclosed patent publications. WO99/38501, WO99/46272, WO99/67279
(Probiodrug), WO99/67278 (Probiodrug), WO99/61431 (Probiodrug),
WO02/083128, WO02/062764, WO03/000180, WO03/000181, WO03/000250,
WO03/002530, WO03/002531, WO03/002553, WO03/002593, WO03/004498,
WO03/004496, WO03/017936, WO03/024942, WO03/024965, WO03/033524,
WO03/037327 and EP1258476; growth hormone secretagogue receptor
agonists/antagonists, such as NN703, hexarelin, MK-0677 (Merck),
SM-130686, CP-424391 (Pfizer), LY 444,711 (Eli Lilly), L-692,429
and L-163,255, and such as those disclosed in U.S. Ser. No.
09/662,448, U.S. provisional application 60/203,335, U.S. Pat. No.
6,358,951, US2002049196, US2002/022637, WO01/56592 and WO02/32888;
H3 (histamine H3) antagonist/inverse agonists, such as
thioperamide, 3-(1H-imidazol-4-yl)propyl N-(4-pentenyl)carbamate),
clobenpropit, iodophenpropit, imoproxifan, GT2394 (Gliatech), and
A331440, O-[3-(1H-imidazol-4-yl)propanol]carbamates
(Kiec-Kononowicz, K. et al., Pharmazie, 55:349-55 (2000)),
piperidine-containing histamine H3-receptor antagonists (Lazewska,
D. et al., Pharmazie, 56:927-32 (2001), benzophenone derivatives
and related compounds (Sasse, A. et al., Arch. Pharm. (Weinheim)
334:45-52 (2001)), substituted N-phenylcarbamates (Reidemeister, S.
et al., Pharmazie, 55:83-6 (2000)), and proxifan derivatives
(Sasse, A. et al., J. Med. Chem. 43:3335-43 (2000)) and histamine
H3 receptor modulators such as those disclosed in WO02/15905,
WO03/024928 and WO03/024929; leptin derivatives, such as those
disclosed in U.S. Pat. Nos. 5,552,524, 5,552,523, 5,552,522,
5,521,283, WO96/23513, WO96/23514, WO96/23515, WO96/23516,
WO96/23517, WO96/23518, WO96/23519, and WO96/23520; leptin,
including recombinant human leptin (PEG-OB, Hoffman La Roche) and
recombinant methionyl human leptin (Amgen); lipase inhibitors, such
as tetrahydrolipstatin (orlistat/Xenical.RTM.), Triton WRl 339,
RHC80267, lipstatin, teasaponin, diethylumbelliferyl phosphate,
FL-386, WAY-121898, Bay-N-3176, valilactone, esteracin, ebelactone
A, ebelactone B, and RHC 80267, and those disclosed in patent
publications WO01/77094, U.S. Pat. Nos. 4,598,089, 4,452,813,
USUS5512565, U.S. Pat. Nos. 5,391,571, 5,602,151, 4,405,644,
4,189,438, and 4,242,453; lipid metabolism modulators such as
maslinic acid, erythrodiol, ursolic acid uvaol, betulinic acid,
betulin, and the like and compounds disclosed in WO03/011267; Mc4r
(melanocortin 4 receptor) agonists, such as CHIR86036 (Chiron),
ME-10142, ME-10145, and HS-131 (Melacure), and those disclosed in
PCT publication Nos. WO99/64002, WO00/74679, WOO 1/991752, WOO
1/25192, WOO 1/52880, WOO 1/74844, WOO 1/70708, WO01/70337,
WO01/91752, WO02/059095, WO02/059107, WO02/059108, WO02/059117,
WO02/06276, WO02/12166, WO02/11715, WO02/12178, WO02/15909,
WO02/38544, WO02/068387, WO02/068388, WO02/067869, WO02/081430,
WO03/06604, WO03/007949, WO03/009847, WO03/009850, WO03/013509, and
WO03/031410; Mc5r (melanocortin 5 receptor) modulators, such as
those disclosed in WO97/19952, WO00/15826, WO00/15790,
US20030092041; melanin-concentrating hormone 1 receptor (MCHR)
antagonists, such as T-226296 (Takeda), SB 568849, SNP-7941
(Synaptic), and those disclosed in patent publications WOO 1/21169,
WO01/82925, WO01/87834, WO02/051809, WO02/06245, WO02/076929,
WO02/076947, WO02/04433, WO02/51809, WO02/083134, WO02/094799,
WO03/004027, WO03/13574, WO03/15769, WO03/028641, WO03/035624,
WO03/033476, WO03/033480, JP13226269, and JP1437059; mGluR5
modulators such as those disclosed in WO03/029210, WO03/047581,
WO03/048137, WO03/051315, WO03/051833, WO03/053922, WO03/059904,
and the like; serotoninergic agents, such as fenfluramine (such as
Pondimin.RTM. (Benzeneethanamine,
N-ethyl-alpha-methyl-3-(trifluoromethyl)-, hydrochloride),
Robbins), dexfenfluramine (such as Redux.RTM. (Benzeneethanamine,
N-ethyl-alpha-methyl-3-(trifluoromethyl)-, hydrochloride),
Interneuron) and sibutramine ((Meridia.RTM., Knoll/Reductil.TM.)
including racemic mixtures, as optically pure isomers (+) and (-),
and pharmaceutically acceptable salts, solvents, hydrates,
clathrates and prodrugs thereof including sibutramine hydrochloride
monohydrate salts thereof, and those compounds disclosed in U.S.
Pat. Nos. 4,746,680, 4,806,570, and 5,436,272, US20020006964, WOO
1/27068, and WOO 1/62341; NE (norepinephrine) transport inhibitors,
such as GW 320659, despiramine, talsupram, and nomifensine; NPY 1
antagonists, such as BIBP3226, J-115814, MO 3304, LY-357897,
CP-671906, GI-264879A, and those disclosed in U.S. Pat. No.
6,001,836, WO96/14307, WO01/23387, WO99/51600, WO01/85690,
WO01/85098, WO01/85173, and WO01/89528; NPY5 (neuropeptide Y Y5)
antagonists, such as 152,804, GW-569180A, GW-594884A, GW-587081X,
GW-548118X, FR235208, FR226928, FR240662, FR252384, 1229U91,
GI-264879A, CGP71683A, LY-377897, LY-366377, PD-160170, SR-120562A,
SR-120819A, JCF-104, and H409/22 and those compounds disclosed in
patent publications U.S. Pat. Nos. 6,140,354, 6,191,160, 6,218,408,
6,258,837, 6,313,298, 6,326,375, 6,329,395, 6,335,345, 6,337,332,
6,329,395, 6,340,683, EP01010691, EP-01044970, WO97/19682,
WO97/20820, WO97/20821, WO97/20822, WO97/20823, WO98/27063,
WO00/107409, WO00/185714, WO00/185730, WO00/64880, WO00/68197,
WO00/69849, WO/0113917, WO01/09120, WO01/14376, WO01/85714,
WO01/85730, WO01/07409, WO01/02379, WO01/23388, WO01/23389, WOO
1/44201, WO01/62737, WO01/62738, WO01/09120, WO02/20488,
WO02/22592, WO02/48152, WO02/49648, WO02/051806, WO02/094789,
WO03/009845, WO03/014083, WO03/022849, WO03/028726 and Norman et
al, J. Med. Chem. 43:4288-4312 (2000); opioid antagonists, such as
nalmefene (REVEX.RTM.), 3-methoxynaltrexone, methylnaltrexone,
naloxone, and naltrexone (e.g. PT901; Pain Therapeutics, Inc.) and
those disclosed in US20050004155 and WO00/21509; orexin
antagonists, such as SB-334867-A and those disclosed in patent
publications WO01/96302, WO01/68609, WO02/44172, WO02/51232,
WO02/51838, WO02/089800, WO02/090355, WO03/023561, WO03/032991, and
WO03/037847; PDE inhibitors (e.g. compounds which slow the
degradation of cyclic AMP (cAMP) and/or cyclic GMP (cGMP) by
inhibition of the phosphodiesterases, which can lead to a relative
increase in the intracellular concentration of cAMP and cGMP;
possible PDE inhibitors are primarily those substances which are to
be numbered among the class consisting of the PDE3 inhibitors, the
class consisting of the PDE4 inhibitors and/or the class consisting
of the PDE5 inhibitors, in particular those substances which can be
designated as mixed types of PDE3/4 inhibitors or as mixed types of
PDE3/4/5 inhibitors) such as those disclosed in patent publications
DE1470341, DE2108438, DE2123328, DE2305339, DE2305575, DE2315801,
DE2402908, DE2413935, DE2451417, DE2459090, DE2646469, DE2727481,
DE2825048, DE2837161, DE2845220, DE2847621, DE2934747, DE3021792,
DE3038166, DE3044568, EP000718, EP0008408, EP0010759, EP0059948,
EP0075436, EP0096517, EPOl 12987, EPOl 16948, EP0150937, EP0158380,
EP0161632, EP0161918, EP0167121, EP0199127, EP0220044, EP0247725,
EP0258191, EP0272910, EP0272914, EP0294647, EP0300726, EP0335386,
EP0357788, EP0389282, EP0406958, EP0426180, EP0428302, EP0435811,
EP0470805, EP0482208, EP0490823, EP0506194, EP0511865, EP0527117,
EP0626939, EP0664289, EP0671389, EP0685474, EP0685475, EP0685479,
JP92234389, JP94329652, JP95010875, U.S. Pat. Nos. 4,963,561,
5,141,931, WO9117991, WO9200968, WO9212961, WO9307146, WO9315044,
WO9315045, WO9318024, WO9319068, WO9319720, WO9319747, WO9319749,
WO9319751, WO9325517, WO9402465, WO9406423, WO9412461, WO9420455,
WO9422852, WO9425437, WO9427947, WO9500516, WO9501980, WO9503794,
WO9504045, WO9504046, WO9505386, WO9508534, WO9509623, WO9509624,
WO9509627, WO9509836, WO9514667, WO9514680, WO9514681, WO9517392,
WO9517399, WO9519362, WO9522520, WO9524381, WO9527692, WO9528926,
WO9535281, WO9535282, WO9600218, WO9601825, WO9602541, WO9611917,
DE3142982, DEl 116676, DE2162096, EP0293063, EP0463756, EP0482208,
EP0579496, EP0667345 U.S. Pat. No. 6,331,543, US20050004222
(including those disclosed in formulas I-XIII and paragraphs 37-39,
85-0545 and 557-577), WO9307124, EP0163965, EP0393500, EP0510562,
EP0553174, WO9501338 and WO9603399, as well as PDE5 inhibitors
(such as RX-RA-69, SCH-51866, KT-734, vesnarinone, zaprinast,
SKF-96231, ER-21355, BF/GP-385, NM-702 and sildenafil
(Viagra.TM.)), PDE4 inhibitors (such as etazolate, ICI63197,
RP73401, imazolidinone (RO-20-1724), MEM 1414 (R1533/R1500;
Pharmacia Roche), denbufylline, rolipram, oxagrelate, nitraquazone,
Y-590, DH-6471, SKF-94120, motapizone, lixazinone, indolidan,
olprinone, atizoram, KS-506-G, dipamfylline, BMY-43351, atizoram,
arofylline, filaminast, PDB-093, UCB-29646, CDP-840, SKF-107806,
piclamilast, RS-17597, RS-25344-000, SB-207499, TIBENELAST,
SB-210667, SB-211572, SB-211600, SB-212066, SB-212179, GW-3600,
CDP-840, mopidamol, anagrelide, ibudilast, amrinone, pimobendan,
cilostazol, quazinone and
N-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxybenzamide-
, PDE3 inhibitors (such as ICI153, 100, bemorandane (RWJ 22867),
MCI-154, UD-CG 212, sulmazole, ampizone, cilostamide, carbazeran,
piroximone, imazodan, CI-930, siguazodan, adibendan, saterinone,
SKF-95654, SDZ-MKS-492, 349-U-85, emoradan, EMD-53998, EMD-57033,
NSP-306, NSP-307, revizinone, NM-702, WIN-62582 and WIN-63291,
enoximone and milrinone, PDE3/4 inhibitors (such as benafentrine,
trequinsin, ORG-30029, zardaverine, L-686398, SDZ-ISQ-844,
ORG-20241, EMD-54622, and tolafentrine) and other PDE inhibitors
(such as vinpocetin, papaverine, enprofylline, cilomilast,
fenoximone, pentoxifylline, roflumilast, tadalafil (Cialis.RTM.),
theophylline, and vardenafil (Levitra.RTM.); Neuropeptide Y2 (NPY2)
agonists include but are not limited to: polypeptide YY and
fragments and variants thereof (e.g. YY3-36 (PYY3-36) (N. Engl. J.
Med. 349:941, 2003; IKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY (SEQ ID
NO:XXX)) and PYY agonists such as those disclosed in WO02/47712,
WO03/026591, WO03/057235, and WO03/027637; serotonin reuptake
inhibitors, such as, paroxetine, fluoxetine (Prozac.TM.),
fluvoxamine, sertraline, citalopram, and imipramine, and those
disclosed in U.S. Pat. Nos. 6,162,805, 6,365,633, WO03/00663, WOO
1/27060, and WOO 1/162341; thyroid hormone .beta. agonists, such as
KB-2611 (KaroBioBMS), and those disclosed in WO02/15845,
WO97/21993, WO99/00353, GB98/284425, U.S. Provisional Application
No. 60/183,223, and Japanese Patent Application No. JP 2000256190;
UCP-I (uncoupling protein-1), 2, or 3 activators, such as phytanic
acid,
4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1-propeny-
l]benzoic acid (TTNPB), retinoic acid, and those disclosed in
WO99/00123; .beta.3 (beta adrenergic receptor 3) agonists, such as
AJ9677/TAK677 (Dainippon/Takeda), L750355 (Merck), CP331648
(Pfizer), CL-316,243, SB 418790, BRL-37344, L-796568, BMS-196085,
BRL-35135A, CGP12177A, BTA-243, GW 427353, Trecadrine, Zeneca
D7114, N-5984 (Nisshin Kyorin), LY-377604 (Lilly), SR 59119A, and
those disclosed in U.S. Pat. Nos. 5,541,204, 5,770,615, 5,491,134,
5,776,983, US488064, U.S. Pat. Nos. 5,705,515, 5,451,677,
WO94/18161, WO95/29159, WO97/46556, WO98/04526 and WO98/32753,
WO01/74782, WO02/32897, WO03/014113, WO03/016276, WO03/016307,
WO03/024948, WO03/024953 and WO03/037881; noradrenergic agents
including, but not limited to, diethylpropion (such as Tenuate.RTM.
(1-propanone, 2-(diethylamino)-1-phenyl-, hydrochloride), Merrell),
dextroamphetamine (also known as dextroamphetamine sulfate,
dexamphetamine, dexedrine, Dexampex, Ferndex, Oxydess II, Robese,
Spancap #1), mazindol ((or
5-(p-chlorophenyl)-2,5-dihydro-3H-imidazo[2,1-a]isoindol-5-01) such
as Sanorex
.RTM., Novartis or Mazanor.RTM., Wyeth Ayerst), phenylpropanolamine
(or Benzenemethanol, alpha-(1-aminoethyl)-, hydrochloride),
phentermine ((or Phenol,
3-[[4,5-duhydro-1H-imidazol-2-yl)ethyl](4-methylpheny-1)amino],
monohydrochloride) such as Adipex-P.RTM., Lemmon, FASTIN.RTM.,
Smith-Kline Beecham and Ionamin.RTM., Medeva), phendimetrazine ((or
(2S,3S)-3,4-Dimethyl-2phenylmorpholine L-(+)-tartrate (1:1)) such
as Metra.RTM. (Forest), Plegine.RTM. (Wyeth-Ayerst), Prelu-2.RTM.
(Boehringer Ingelheim), and Statobex.RTM. (Lemmon), phendamine
tartrate (such as Thephorin.RTM.
(2,3,4,9-Tetrahydro-2-methyl-9-phenyl-1H-indenol[2,1-c]pyridine
L-(+)-tartrate (1:1)), Hoffmann-LaRoche), methamphetamine (such as
Desoxyn.RTM., Abbot ((S)--N, (alpha)-dimethylbenzeneethanamine
hydrochloride)), and phendimetrazine tartrate (such as Bontril.RTM.
Slow-Release Capsules, Amarin (-3,4-Dimethyl-2-phenylmorpholine
Tartrate); fatty acid oxidation upregulator/inducers such as
Famoxin.RTM. (Genset); monamine oxidase inhibitors including but
not limited to befloxatone, moclobemide, brofaromine, phenoxathine,
esuprone, befol, toloxatone, pirlindol, amiflamine, sercloremine,
bazinaprine, lazabemide, milacemide, caroxazone and other certain
compounds as disclosed by WO01/12176; and other anti-obesity agents
such as 5HT-2 agonists, ACC (acetyl-CoA carboxylase) inhibitors
such as those described in WO03/072197, alpha-lipoic acid
(alpha-LA), AOD9604, appetite suppressants such as those in
WO03/40107, ATL-962 (Alizyme PLC), benzocaine, benzphetamine
hydrochloride (Didrex), bladderwrack (focus vesiculosus), BRS3
(bombesin receptor subtype 3) agonists, bupropion, caffeine, CCK
agonists, chitosan, chromium, conjugated linoleic acid,
corticotropin-releasing hormone agonists, dehydroepiandrosterone,
DGAT1 (diacylglycerol acyltransferase 1) inhibitors, DGAT2
(diacylglycerol acyltransferase 2) inhibitors, dicarboxylate
transporter inhibitors, ephedra, exendin-4 (an inhibitor of glp-1)
FAS (fatty acid synthase) inhibitors (such as Cerulenin and C75),
fat resorption inhibitors (such as those in WO03/053451, and the
like), fatty acid transporter inhibitors, natural water soluble
fibers (such as psyllium, plantago, guar, oat, pectin), galanin
antagonists, galega (Goat's Rue, French Lilac), garcinia cambogia,
germander (teucrium chamaedrys), ghrelin antibodies and ghrelin
antagonists (such as those disclosed in WO01/87335, and
WO02/08250), polypeptide hormones and variants thereof which affect
the islet cell secretion, such as the hormones of the
secretin/gastric inhibitory polypeptide (GIP)/vasoactive intestinal
polypeptide (VIP)/pituitary adenylate cyclase activating
polypeptide (PACAP)/glucagon-like polypeptide II
(GLP-II)/glicentin/glucagon gene family and/or those of the
adrenomedullin/amylin/calcitonin gene related polypeptide (CGRP)
gene family includingGLP-1 (glucagon-like polypeptide 1) agonists
(e.g. (1) exendin-4, (2) those GLP-I molecules described in
US20050130891 including GLP-1(7-34), GLP-1(7-35), GLP-1(7-36) or
GLP-1(7-37) in its C-terminally carboxylated or amidated form or as
modified GLP-I polypeptides and modifications thereof including
those described in paragraphs 17-44 of US20050130891, and
derivatives derived from GLP-1-(7-34)COOH and the corresponding
acid amide are employed which have the following general formula:
R--NH-HAEGTFTSDVSYLEGQAAKEFIAWLVK-CONH.sub.2 wherein R.dbd.H or an
organic compound having from 1 to 10 carbon atoms. Preferably, R is
the residue of a carboxylic acid. Particularly preferred are the
following carboxylic acid residues: formyl, acetyl, propionyl,
isopropionyl, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,
tert-butyl.) and glp-1 (glucagon-like polypeptide-1),
glucocorticoid antagonists, glucose transporter inhibitors, growth
hormone secretagogues (such as those disclosed and specifically
described in U.S. Pat. No. 5,536,716), interleukin-6 (IL-6) and
modulators thereof (as in WO03/057237, and the like), L-carnitine,
Mc3r (melanocortin 3 receptor) agonists, MCH2R (melanin
concentrating hormone 2R) agonist/antagonists, melanin
concentrating hormone antagonists, melanocortin agonists (such as
Melanotan II or those described in WO 99/64002 and WO 00/74679),
nomame herba, phosphate transporter inhibitors, phytopharm compound
57 (CP 644,673), pyruvate, SCD-I (stearoyl-CoA desaturase-1)
inhibitors, T71 (Tularik, Inc., Boulder Colo.), Topiramate
(Topimax.RTM., indicated as an anti-convulsant which has been shown
to increase weight loss), transcription factor modulators (such as
those disclosed in WO03/026576), .beta.-hydroxy steroid
dehydrogenase-1 inhibitors (.beta.-HSD-I),
.beta.-hydroxy-.beta.-methylbutyrate, p57 (Pfizer), Zonisamide
(Zonegran.TM. indicated as an anti-epileptic which has been shown
to lead to weight loss), and the agents disclosed in US20030119428
paragraphs 20-26.
[0159] 1.2.2.7 Phosphodiesterase inhibitors
[0160] In certain embodiments, the regimen of combination therapy
includes the administration of one or more phosphodiesterase
("PDE") inhibitors. PDE inhibitors slow the degradation of cyclic
AMP (cAMP) and/or cyclic GMP (cGMP) by inhibiting
phosphodiesterases, which can lead to a relative increase in the
intracellular concentration of cAMP and/or cGMP. Non-limiting
examples of PDE inhibitors that can be used in combination with the
GCC agonists of the invention include PDE3 inhibitors, PDE4
inhibitors and/or PDE5 inhibitors, in particular those substances
which can be designated as mixed types of PDE3/4 inhibitors or as
mixed types of PDE3/4/5 inhibitors. Non-limiting examples of such
PDE inhibitors are described in the following patent applications
and patents: DE1470341, DE2108438, DE2123328, DE2305339, DE2305575,
DE2315801, DE2402908, DE2413935, DE2451417, DE2459090, DE2646469,
DE2727481, DE2825048, DE2837161, DE2845220, DE2847621, DE2934747,
DE3021792, DE3038166, DE3044568, EP000718, EP0008408, EP0010759,
EP0059948, EP0075436, EP0096517, EPOl 12987, EPOl 16948, EP0150937,
EP0158380, EP0161632, EP0161918, EP0167121, EP0199127, EP0220044,
EP0247725, EP0258191, EP0272910, EP0272914, EP0294647, EP0300726,
EP0335386, EP0357788, EP0389282, EP0406958, EP0426180, EP0428302,
EP0435811, EP0470805, EP0482208, EP0490823, EP0506194, EP0511865,
EP0527117, EP0626939, EP0664289, EP0671389, EP0685474, EP0685475,
EP0685479, JP92234389, JP94329652, JP95010875, U.S. Pat. Nos.
4,963,561, 5,141,931, WO9117991, WO9200968, WO9212961, WO9307146,
WO9315044, WO9315045, WO9318024, WO9319068, WO9319720, WO9319747,
WO9319749, WO9319751, WO9325517, WO9402465, WO9406423, WO9412461,
WO9420455, WO9422852, WO9425437, WO9427947, WO9500516, WO9501980,
WO9503794, WO9504045, WO9504046, WO9505386, WO9508534, WO9509623,
WO9509624, WO9509627, WO9509836, WO9514667, WO9514680, WO9514681,
WO9517392, WO9517399, WO9519362, WO9522520, WO9524381, WO9527692,
WO9528926, WO9535281, WO9535282, WO9600218, WO9601825, WO9602541,
WO9611917, DE3142982, DEl 116676, DE2162096, EP0293063, EP0463756,
EP0482208, EP0579496, EP0667345 U.S. Pat. No. 6,331,543,
US20050004222 (including those disclosed in formulas I--XIII and
paragraphs 37-39, 85-0545 and 557-577) and WO9307124, EP0163965,
EP0393500, EP0510562, EP0553174, WO9501338 and WO9603399. PDE5
inhibitors which may be mentioned by way of example are RX-RA-69,
SCH-51866, KT-734, vesnarinone, zaprinast, SKF-96231, ER-21355,
BF/GP-385, NM-702 and sildenafil (Viagra.RTM.). PDE4 inhibitors
which may be mentioned by way of example are RO-20-1724, MEM 1414
(R1533/R1500; Pharmacia Roche), DENBUFYLLINE, ROLIPRAM, OXAGRELATE,
NITRAQUAZONE, Y-590, DH-6471, SKF-94120, MOTAPIZONE, LIXAZINONE,
INDOLIDAN, OLPRINONE, ATIZORAM, KS-506-G, DIPAMFYLLINE, BMY-43351,
ATIZORAM, AROFYLLINE, FILAMINAST, PDB-093, UCB-29646, CDP-840,
SKF-107806, PICLAMILAST, RS-17597, RS-25344-000, SB-207499,
TIBENELAST, SB-210667, SB-211572, SB-211600, SB-212066, SB-212179,
GW-3600, CDP-840, MOPIDAMOL, ANAGRELIDE, IBUDILAST, AMRINONE,
PIMOBENDAN, CILOSTAZOL, QUAZINONE and
N-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxybenzamide-
. PDE3 inhibitors which may be mentioned by way of example are
SULMAZOLE, AMPIZONE, CILOSTAMIDE, CARBAZERAN, PIROXIMONE, IMAZODAN,
CI-930, SIGUAZODAN, ADIBENDAN, SATERINONE, SKF-95654, SDZ-MKS-492,
349-U-85, EMORADAN, EMD-53998, EMD-57033, NSP-306, NSP-307,
REVIZINONE, NM-702, WIN-62582 and WIN-63291, ENOXIMONE and
MILRINONE. PDE3/4 inhibitors which may be mentioned by way of
example are BENAFENTRINE, TREQUINSIN, ORG-30029, ZARDAVERINE,
L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, and TOLAFENTRINE.
Other PDE inhibitors include: cilomilast, pentoxifylline,
roflumilast, tadalafil (Cialis.RTM.), theophylline, and vardenafil
(Levitra.RTM.), zaprinast (PDE5 specific). GCC AGONIST
[0161] 1.2.2.8 Analgesic Agents
[0162] In certain embodiments, the regimen of combination therapy
includes the administration of one or more analgesic agents, e.g.,
an analgesic compound or an analgesic polypeptide. In some
embodiments, the GCC agonist formulation is administered
simultaneously or sequentially with one or more analgesic agents.
In other embodiments, the GCC agonist is covalently linked or
attached to an analgesic agent to create a therapeutic conjugate.
Non-limiting examples of analgesic agents that can be used include
calcium channel blockers, 5HT receptor antagonists (for example
5HT3, 5HT4 and 5HT1 receptor antagonists), opioid receptor agonists
(loperamide, fedotozine, and fentanyl), NK1 receptor antagonists,
CCK receptor agonists (e.g., loxiglumide), NK1 receptor
antagonists, NK3 receptor antagonists, norepinephrine-serotonin
reuptake inhibitors (NSRI), vanilloid and cannabanoid receptor
agonists, and sialorphin. Further examples of analgesic agents in
the various classes are known in the art.
[0163] In one embodiment, the analgesic agent is an analgesic
polypeptide selected from the group consisting of
sialorphin-related polypeptides, including those comprising the
amino acid sequence QHNPR (SEQ ID NO: 239), including: VQHNPR (SEQ
ID NO: 240); VRQHNPR (SEQ ID NO: 241); VRGQHNPR (SEQ ID NO: 242);
VRGPQHNPR (SEQ ID NO: 243); VRGPRQHNPR (SEQ ID NO: 244);
VRGPRRQHNPR (SEQ ID NO: 245); and RQHNPR (SEQ ID NO: 246).
Sialorphin-related polypeptides bind to neprilysin and inhibit
neprilysin-mediated breakdown of substance P and Met-enkephalin.
Thus, compounds or polypeptides that are inhibitors of neprilysin
are useful analgesic agents which can be administered with the GCC
agonists described herein or covalently linked to a GCC agonist to
form a therapeutic conjugate. Sialorphin and related polypeptides
are described in U.S. Pat. No. 6,589,750; U.S. 20030078200 A1; and
WO 02/051435 A2.
[0164] In another embodiment, a GCC agonist formulation of the
invention is administered as part of a regimen of combination
therapy with an opioid receptor antagonist or agonist. In one
embodiment, the GCC agonist and the opioid receptor antagonist or
agonist are linked via a covalent bond. Non-limiting examples of
opioid receptor antagonists include naloxone, naltrexone, methyl
nalozone, nalmefene, cypridime, beta funaltrexamine, naloxonazine,
naltrindole, nor-binaltorphimine, enkephalin pentapeptide (HOE825;
Tyr-D-Lys-Gly-Phe-L-homoserine), trimebutine, vasoactive intestinal
polypeptide, gastrin, glucagons. Non-limiting examples of opioid
receptor agonists include fedotozine, asimadoline, and
ketocyclazocine, the compounds described in WO03/097051 and
WO05/007626, morphine, diphenyloxylate, frakefamide
(H-Tyr-D-Ala-Phe(F)-Phe-NH 2; WO 01/019849 A1), and loperamide.
[0165] Further non-limiting examples of analgesic agents that can
be used in a regimen of combination therapy along with the GCC
agonist formulations of the invention include the dipeptide Tyr-Arg
(kyotorphin); the chromogranin-derived polypeptide (CgA 47-66; See,
e.g., Ghia et al. 2004 Regulatory polypeptides 119:199); CCK
receptor agonists such as caerulein; conotoxin polypeptides;
peptide analogs of thymulin (FR Application 2830451); CCK (CCKa or
CCKb) receptor antagonists, including loxiglumide and
dexloxiglumide (the R-isomer of loxiglumide) (WO 88/05774); 5-HT4
agonists such as tegaserod (Zelnorm.RTM.), mosapride,
metoclopramide, zacopride, cisapride, renzapride, benzimidazolone
derivatives such as BIMU 1 and BIMU 8, and lirexapride; calcium
channel blockers such as ziconotide and related compounds described
in, for example, EP625162B1, U.S. Pat. Nos. 5,364,842, 5,587,454,
5,824,645, 5,859,186, 5,994,305, 6,087,091, 6,136,786, WO 93/13128
A1, EP 1336409 A1, EP 835126 A1, EP 835126 B1, U.S. Pat. Nos.
5,795,864, 5,891,849, 6,054,429, WO 97/01351 A1; NK-I, receptor
antagonists such as aprepitant (Merck & Co Inc), vofopitant,
ezlopitant (Pfizer, Inc.), R-673 (Hoffmann-La Roche Ltd), SR-48968
(Sanofi Synthelabo), CP-122,721 (Pfizer, Inc.), GW679769 (Glaxo
Smith Kline), TAK-637 (Takeda/Abbot), SR-14033, and related
compounds described in, for example, EP 873753 A1, US 20010006972
A1, US 20030109417 A1, WO 01/52844 A1 (for a review see Giardina et
al. 2003. Drugs 6:758); NK-2 receptor antagonists such as
nepadutant (Menarini Ricerche SpA), saredutant (Sanoft-Synthelabo),
GW597599 (Glaxo Smith Kline), SR-144190 (Sanofi-Synthelabo) and
UK-290795 (Pfizer Inc); NK3 receptor antagonists such as osanetant
(SR-142801; Sanoft-Synthelabo), SSR-241586, talnetant and related
compounds described in, for example, WO 02/094187 A2, EP 876347 A1,
WO 97/21680 A1, U.S. Pat. No. 6,277,862, WO 98/1 1090, WO 95/28418,
WO 97/19927, and Boden et al. (J Med Chem. 39:1664-75, 1996);
norepinephrine-serotonin reuptake inhibitors (NSRI) such as
milnacipran and related compounds described in WO 03/077897; and
vanilloid receptor antagonists such as arvanil and related compouds
described in WO 01/64212 A1.
[0166] In addition to sialorphin-related polypeptides, analgesic
polypeptides include: AspPhe, endomorphin-1, endomorphin-2,
nocistatin, dalargin, lupron, ziconotide, and substance P.
[0167] 1.2.2.9 Insulin and Insulin Modulating Agents
[0168] The GCC agonist peptides described herein can be used in
combination therapy with insulin and related compounds including
primate, rodent, or rabbit insulin including biologically active
variants thereof including allelic variants, more preferably human
insulin available in recombinant form. Sources of human insulin
include pharmaceutically acceptable and sterile formulations such
as those available from Eli Lilly (Indianapolis, Ind. 46285) as
Humulin.TM. (human insulin rDNA origin). See, the THE PHYSICIAN'S
DESK REFERENCE, 55.sup.th Ed. (2001) Medical Economics, Thomson
Healthcare (disclosing other suitable human insulins).
[0169] The GCC peptides described herein can also be used in
combination therapy with agents that can boost insulin effects or
levels of a subject upon administration, e.g. glipizide and/or
rosiglitazone. The polypeptides and agonists described herein can
be used in combitherapy with SYMLIN.RTM. (pramlintide acetate) and
Exenatide.RTM. (synthetic exendin-4; a 39 aa polypeptide).
[0170] 1.2.2.10 Anti-Hypertensive Agents
[0171] The GCC agonist peptides described herein can be used in
combination therapy with an anti-hypertensive agent including but
not limited to: (1) diuretics, such as thiazides, including
chlorthalidone, chlorthiazide, dichlorophenamide,
hydroflumethiazide, indapamide, polythiazide, and
hydrochlorothiazide; loop diuretics, such as bumetanide, ethacrynic
acid, furosemide, and torsemide; potassium sparing agents, such as
amiloride, and triamterene; carbonic anhydrase inhibitors, osmotics
(such as glycerin) and aldosterone antagonists, such as
spironolactone, epirenone, and the like; (2) beta-adrenergic
blockers such as acebutolol, atenolol, betaxolol, bevantolol,
bisoprolol, bopindolol, carteolol, carvedilol, celiprolol, esmolol,
indenolol, metaprolol, nadolol, nebivolol, penbutolol, pindolol,
propanolol, sotalol, tertatolol, tilisolol, and timolol, and the
like; (3) calcium channel blockers such as amlodipine, aranidipine,
azelnidipine, barnidipine, benidipine, bepridil, cinaldipine,
clevidipine, diltiazem, efonidipine, felodipine, gallopamil,
isradipine, lacidipine, lemildipine, lercanidipine, nicardipine,
nifedipine, nilvadipine, nimodepine, nisoldipine, nitrendipine,
manidipine, pranidipine, and verapamil, and the like; (4)
angiotensin converting enzyme (ACE) inhibitors such as benazepril;
captopril; ceranapril; cilazapril; delapril; enalapril; enalopril;
fosinopril; imidapril; lisinopril; losinopril; moexipril;
quinapril; quinaprilat; ramipril; perindopril; perindropril;
quanipril; spirapril; tenocapril; trandolapril, and zofenopril, and
the like; (5) neutral endopeptidase inhibitors such as omapatrilat,
cadoxatril and ecadotril, fosidotril, sampatrilat, AVE7688, ER4030,
and the like; (6) endothelin antagonists such as tezosentan,
A308165, and YM62899, and the like; (7) vasodilators such as
hydralazine, clonidine, minoxidil, and nicotinyl alcohol, and the
like; (8) angiotensin II receptor antagonists such as aprosartan,
candesartan, eprosartan, irbesartan, losartan, olmesartan,
pratosartan, tasosartan, telmisartan, valsartan, and EXP-3137,
FI6828K, and RNH6270, and the like; (9) .alpha./.beta. adrenergic
blockers such as nipradilol, arotinolol and amosulalol, and the
like; (10) alpha 1 blockers, such as terazosin, urapidil, prazosin,
tamsulosin, bunazosin, trimazosin, doxazosin, naftopidil,
indoramin, WHP 164, and XENOlO, and the like; (11) alpha 2 agonists
such as lofexidine, tiamenidine, moxonidine, rilmenidine and
guanobenz, and the like; (12) aldosterone inhibitors, and the like;
and (13) angiopoietin-2-binding agents such as those disclosed in
WO03/030833. Specific anti-hypertensive agents that can be used in
combination with polypeptides and agonists described herein
include, but are not limited to: diuretics, such as thiazides
(e.g., chlorthalidone, cyclothiazide (CAS RN 2259-96-3),
chlorothiazide (CAS RN 72956-09-3, which may be prepared as
disclosed in U.S. Pat. No. 2,809,194), dichlorophenamide,
hydroflumethiazide, indapamide, polythiazide, bendroflumethazide,
methyclothazide, polythiazide, trichlormethazide, chlorthalidone,
indapamide, metolazone, quinethazone, althiazide (CAS RN 5588-16-9,
which may be prepared as disclosed in British Patent No. 902,658),
benzthiazide (CAS RN 91-33-8, which may be prepared as disclosed in
U.S. Pat. No. 3,108,097), buthiazide (which may be prepared as
disclosed in British Patent Nos. 861,367), and
hydrochlorothiazide), loop diuretics (e.g. bumetanide, ethacrynic
acid, furosemide, and torasemide), potassium sparing agents (e.g.
amiloride, and triamterene (CAS Number 396-01-O)), and aldosterone
antagonists (e.g. spironolactone (CAS Number 52-01-7), epirenone,
and the like); .beta.-adrenergic blockers such as Amiodarone
(Cordarone, Pacerone), bunolol hydrochloride (CAS RN 31969-05-8,
Parke-Davis), acebutolol (.+-.N-[3-Acetyl-4-[2-hydroxy-3-[(1
methylethyl)amino]propoxy]phenyl]-butanamide, or
(.+-.)-3'-Acetyl-4'-[2-hydroxy-3-(isopropylamino) propoxy]
butyranilide), acebutolol hydrochloride (e.g. Sectral.RTM.,
Wyeth-Ayerst), alprenolol hydrochloride (CAS RN 13707-88-5 see
Netherlands Patent Application No. 6,605,692), atenolol (e.g.
Tenormin.RTM., AstraZeneca), carteolol hydrochloride (e.g.
Cartrol.RTM. Filmtab.RTM., Abbott), Celiprolol hydrochloride (CAS
RN 57470-78-7, also see in U.S. Pat. No. 4,034,009), cetamolol
hydrochloride (CAS RN 77590-95-5, see also U.S. Pat. No.
4,059,622), labetalol hydrochloride (e.g. Normodyne.RTM.,
Schering), esmolol hydrochloride (e.g. Brevibloc.RTM., Baxter),
levobetaxolol hydrochloride (e.g. Betaxon.TM. Ophthalmic
Suspension, Alcon), levobunolol hydrochloride (e.g. Betagan.RTM.
Liquifilm.RTM. with C CAP.RTM. Compliance Cap, Allergan), nadolol
(e.g. Nadolol, Mylan), practolol (CAS RN 6673-35-4, see also U.S.
Pat. No. 3,408,387), propranolol hydrochloride (CAS RN 318-98-9),
sotalol hydrochloride (e.g. Betapace AF.TM., Berlex), timolol
(2-Propanol,
1-[(1,1-dimethylethyl)amino]-3-[[4-4(4-morpholinyl)-1,2,5-thiadiazol-3-yl-
]oxy]-, hemihydrate, (S)--, CAS RN 91524-16-2), timolol maleate
(S)-1-[(1,1-dimethylethyl)
amino]-3-[[4-(4-morpholinyl)-1,2,5-thiadiazol-3-yl] oxy]-2-propanol
(Z)-2-butenedioate (1:1) salt, CAS RN 26921-17-5), bisoprolol
(2-Propanol,
1-[4-[[2-(1-methylethoxy)ethoxy]-methyl]phenoxyl]-3-[(1-meth-ylethyl)amin-
o]-, (.+-.), CAS RN 66722-44-9), bisoprolol fumarate (such as
(.+-.)-1-[4-[[2-(1-Methylethoxy)
ethoxy]methyl]phenoxy]-3-[(1-methylethyl)amino]-2-propanol
(E)-2-butenedioate (2:1) (salt), e.g., Zebeta.TM., Lederle
Consumer), nebivalol (2H-1-Benzopyran-2-methanol,
.alpha..alpha.'-[iminobis(methylene)]bis[6-fluoro-3,4-dihydro-, CAS
RN 99200-09-6 see also U.S. Pat. No. 4,654,362), cicloprolol
hydrochloride, such 2-Propanol,
1-[4-[2-(cyclopropylmethoxy)ethoxy]phenoxy]-3-[1-methylethyl)amino]-,
hydrochloride, A.A.S. RN 63686-79-3), dexpropranolol hydrochloride
(2-Propanol,
1-[1-methylethy)-amino]-3-(1-naphthalenyloxy)-hydrochloride (CAS RN
13071-11-9), diacetolol hydrochloride (Acetamide,
N-[3-acetyl-4-[2-hydroxy-3-[(1-methyl-ethyl)amino]propoxy]
[phenyl]-, monohydrochloride CAS RN 69796-04-9), dilevalol
hydrochloride (Benzamide,
2-hydroxy-5-[1-hydroxy-2-[1-methyl-3-phenylpropyl)amino]ethyl]-,
monohydrochloride, CAS RN 75659-08-4), exaprolol hydrochloride
(2-Propanol, 1-(2-cyclohexylphenoxy)-3-[(1-methylethyl)amino]-,
hydrochloride CAS RN 59333-90-3), flestolol sulfate (Benzoic acid,
2-fluro-,
3-[[2-[aminocarbonyl)amino]-dimethylethyl]amino]-2-hydroxypropy- l
ester, (+)-sulfate (1:1) (salt), CAS RN 88844-73-9; metalol
hydrochloride (Methanesulfonamide, N-[4-[1-hydroxy-2-(methyl
amino)propyl]phenyl]-, monohydrochloride CAS RN 7701-65-7),
metoprolol 2-Propanol,
1-[4-(2-methoxyethyl)phenoxy]-3-[1-methylethyl)amino]-; CAS RN
37350-58-6), metoprolol tartrate (such as 2-Propanol,
1-[4-(2-methoxyethyl)phenoxy]-3-[(1-methylethyl)amino]-, e.g.,
Lopressor.RTM., Novartis), pamatolol sulfate (Carbamic acid,
[2-[4-[2-hydroxy-3-[(1-methylethyl)amino]propoxyl]phenyl]-ethyl]-,
methyl ester, (.+-.) sulfate (salt) (2:1), CAS RN 59954-01-7),
penbutolol sulfate (2-Propanol,
1-(2-cyclopentylphenoxy)-3-[1,1-dimethyle-thyl)amino] 1, (S)-,
sulfate (2:1) (salt), CAS RN 38363-32-5), practolol (Acetamide,
N-[4-[2-hydroxy-3-[(1-methylethyl)amino]-propoxy]phenyl]-, CAS RN
6673-35-4) tiprenolol hydrochloride (Propanol,
1-[(1-methylethyl)amino]-3-[2-(methylthio)-phenoxy]-,
hydrochloride, (.+-.), CAS RN 39832-43-4), tolamolol (Benzamide,
4-[2-[[2-hydroxy-3-(2-methylphenoxy)-propyl] amino] ethoxyl]-, CAS
RN 38103-61-6), bopindolol, indenolol, pindolol, propanolol,
tertatolol, and tilisolol, and the like; calcium channel blockers
such as besylate salt of amlodipine (such as
3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-
-methyl-3,5-pyridinedicarboxylate benzenesulphonate, e.g.,
Norvasc.RTM., Pfizer), clentiazem maleate
(1,5-Benzothiazepin-4(5H)-one,
3-(acetyloxy)-8-chloro-5-[2-(dimethylamino)ethyl]-2,3-dihydro-2-(4-methox-
yphenyl)-(2S-cis)-, (Z)-2-butenedioate (1:1), see also U.S. Pat.
No. 4,567,195), isradipine (3,5-Pyridinedicarboxylic acid,
4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-, methyl
1-methylethyl ester,
(.+-.)-4(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedi-
carboxylate, see also U.S. Pat. No. 4,466,972); nimodipine (such as
is isopropyl (2-methoxyethyl) 1,
4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-dicarboxylate,
e.g. Nimotop.RTM., Bayer), felodipine (such as ethyl methyl
4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate-
-, e.g. Plendil.RTM. Extended-Release, AstraZeneca LP), nilvadipine
(3,5-Pyridinedicarboxylic acid,
2-cyano-1,4-dihydro-6-methyl-4-(3-nitrophenyl)-, 3-methyl
5-(1-methylethyl) ester, also see U.S. Pat. No. 3,799,934),
nifedipine (such as 3, 5-pyridinedicarboxylic acid,
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester, e.g.,
Procardia XL.RTM. Extended Release Tablets, Pfizer), diltiazem
hydrochloride (such as 1,5-Benzothiazepin-4(5H)-one,
3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,3-dihydro-2(4-methoxyphenyl)-,
monohydrochloride, (.+-.)-cis., e.g., Tiazac.RTM., Forest),
verapamil hydrochloride (such as benzeneacetronitrile,
(alpha)-[[3-[[2-(3,4-dimethoxyphenyl)
ethyl]methylamino]propyl]-3,4-dimethoxy-(alpha)-(1-methylethyl)
hydrochloride, e.g., Isoptin.RTM. SR, Knoll Labs), teludipine
hydrochloride (3,5-Pyridinedicarboxylic acid,
2-[(dimethylamino)methyl]4-[2-[(1E)-3-(1,1-dimethylethoxy)-3-oxo-1-propen-
yl]phenyl]-1,4-dihydro-6-methyl-, diethyl ester, monohydrochloride)
CAS RN 108700-03-4), belfosdil (Phosphonic acid, [2-(2-phenoxy
ethyl)-1,3-propane-diyl]bis-, tetrabutyl ester CAS RN 103486-79-9),
fostedil (Phosphonic acid, [[4-(2-benzothiazolyl)phenyl]methyl]-,
diethyl ester CAS RN 75889-62-2), aranidipine, azelnidipine,
barnidipine, benidipine, bepridil, cinaldipine, clevidipine,
efonidipine, gallopamil, lacidipine, lemildipine, lercanidipine,
monatepil maleate (1-Piperazinebutanamide, N-(6,
11-dihydrodibenzo(b,e)thiepin-11-yl)4-(4-fluorophenyl)-, (+)-,
(Z)-2-butenedioate (1:1)
(.+-.)-N-(6,11-Dihydrodibenzo(b,e)thiep-in-11-yl)-4-(p-fluorophenyl)-1-pi-
perazinebutyramide maleate (1:1) CAS RN 132046-06-1), nicardipine,
nisoldipine, nitrendipine, manidipine, pranidipine, and the like;
T-channel calcium antagonists such as mibefradil; angiotensin
converting enzyme (ACE) inhibitors such as benazepril, benazepril
hydrochloride (such as 3-[[1-(ethoxycarbonyl)-3-phenyl-(1
S)-propyl]amino]-2,3, 4,5-tetrahydro-2-oxo-1H-1-(3
S)-benzazepine-1-acetic acid monohydrochloride, e.g., Lotrel.RTM.,
Novartis), captopril (such as
1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline, e.g., Captopril,
Mylan, CAS RN 62571-86-2 and others disclosed in U.S. Pat. No.
4,046,889), ceranapril (and others disclosed in U.S. Pat. No.
4,452,790), cetapril (alacepril, Dainippon disclosed in Eur.
Therap. Res. 39:671 (1986); 40:543 (1986)), cilazapril
(Hoffman-LaRoche) disclosed in J. Cardiovasc. Pharmacol. 9:39
(1987), indalapril (delapril hydrochloride
(2H-1,2,4-Benzothiadiazine-7-sulfonamide,
3-bicyclo[2.2.1]hept-5-en-2-yl-6-chloro-3,4-dihydro-, 1,1-dioxide
CAS RN 2259-96-3); disclosed in U.S. Pat. No. 4,385,051), enalapril
(and others disclosed in U.S. Pat. No. 4,374,829), enalopril,
enaloprilat, fosinopril, ((such as L-proline,
4-cyclohexyl-1-[[[2-methyl-1-(1-oxopropoxy) propoxy](4-phenylbutyl)
phosphinyl]acetyl]-, sodium salt, e.g., Monopril, Bristol-Myers
Squibb and others disclosed in U.S. Pat. No. 4,168,267), fosinopril
sodium (L-Proline,
4-cyclohexyl-1-[[(R)-[(1S)-2-methyl-1-(1-ox-opropoxy)propox),
imidapril, indolapril (Schering, disclosed in J. Cardiovasc.
Pharmacol. 5:643, 655 (1983)), lisinopril (Merck), losinopril,
moexipril, moexipril hydrochloride (3-Isoquinolinecarboxylic acid,
2-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]-1,-
2,3,4-tetrahydro-6,7-dimethoxy-, monohydrochloride, (3S)-CAS RN
82586-52-5), quinapril, quinaprilat, ramipril (Hoechsst) disclosed
in EP 79022 and Curr. Ther. Res. 40:74 (1986), perindopril erbumine
(such as
2S,3aS,7aS-1-[(S)--N--[(S)-1-CarboxybutyljalanyljhexahydroA-indolinecarbo-
xylic acid, 1-ethyl ester, compound with tert-butylamine (1:1),
e.g., Aceon.RTM., Solvay), perindopril (Servier, disclosed in Eur.
J. clin. Pharmacol. 31:519 (1987)), quanipril (disclosed in U.S.
Pat. No. 4,344,949), spirapril (Schering, disclosed in Acta.
Pharmacol. Toxicol. 59 (Supp. 5): 173 (1986)), tenocapril,
trandolapril, zofenopril (and others disclosed in U.S. Pat. No.
4,316,906), rentiapril (fentiapril, disclosed in Clin. Exp.
Pharmacol. Physiol. 10:131 (1983)), pivopril, YS980, teprotide
(Bradykinin potentiator BPP9a CAS RN 35115-60-7), BRL 36,378 (Smith
Kline Beecham, see EP80822 and EP60668), MC-838 (Chugai, see CA.
102:72588v and Jap. J. Pharmacol. 40:373 (1986), CGS 14824
(Ciba-Geigy, 3-([1-ethoxycarbonyl-3-phenyl-(l
S)-propyl]amino)-2,3,4,5-tetrahydro-2-ox-o-1-(3 S)-benzazepine-1
acetic acid HCl, see U.K. Patent No. 2103614), CGS 16,617
(Ciba-Geigy,
3(S)-[[(1S)-5-amino-1-carboxypentyl]amino]-2,3,4,5-tetrahydro-2-oxo-1H-1--
benzazepine-1-ethanoic acid, see U.S. Pat. No. 4,473,575), Ru 44570
(Hoechst, see Arzneimittelforschung 34:1254 (1985)), R 31-2201
(Hoffman-LaRoche see FEBS Lett. 165:201 (1984)), CI925
(Pharmacologist 26:243, 266 (1984)), WY-44221 (Wyeth, see J. Med.
Chem. 26:394 (1983)), and those disclosed in US2003006922
(paragraph 28), U.S. Pat. Nos. 4,337,201, 4,432,971
(phosphonamidates); neutral endopeptidase inhibitors such as
omapatrilat (Vanlev.RTM.), CGS 30440, cadoxatril and ecadotril,
fasidotril (also known as aladotril or alatriopril), sampatrilat,
mixanpril, and gemopatrilat, AVE7688, ER4030, and those disclosed
in U.S. Pat. Nos. 5,362,727, 5,366,973, 5,225,401, 4,722,810,
5,223,516, 4,749,688, 5,552,397, 5,504,080, 5,612,359, 5,525,723,
EP0599444, EP0481522, EP0599444, EP0595610, EP0534363, EP534396,
EP534492, EP0629627; endothelin antagonists such as tezosentan,
A308165, and YM62899, and the like; vasodilators such as
hydralazine (apresoline), clonidine (clonidine hydrochloride
(1H-Imidazol-2-amine, N-(2,6-dichlorophenyl)4,5-dihydro-,
monohydrochloride CAS RN 4205-91-8), catapres, minoxidil (loniten),
nicotinyl alcohol (roniacol), diltiazem hydrochloride (such as
1,5-Benzothiazepin-4(5H)-one, 3-(acetyloxy)-5
[2-(dimethylamino)ethyl]-2,3-dihydro-2(4-methoxyphenyl)-,
monohydrochloride, (+)-cis, e.g., Tiazac.RTM., Forest), isosorbide
dinitrate (such as 1,4:3,6-dianhydro-D-glucitol 2,5-dinitrate e.g.,
Isordil.RTM. Titradose.RTM., Wyeth-Ayerst), sosorbide mononitrate
(such as 1,4:3,6-dianhydro-D-glucito-1,5-nitrate, an organic
nitrate, e.g., Ismo.RTM., Wyeth-Ayerst), nitroglycerin (such as 2,3
propanetriol trinitrate, e.g., Nitrostat.RTM. Parke-Davis),
verapamil hydrochloride (such as benzeneacetonitrile,
(+)-(alpha)[3-[[2-(3,4 dimethoxypheny 1)ethyl]methyl
amino]propyl]-3,4-dimethoxy-(alpha)-(1-methylethyl) hydrochloride,
e.g., Covera HS.RTM. Extended-Release, Searle), chromonar (which
may be prepared as disclosed in U.S. Pat. No. 3,282,938), clonitate
(Annalen 1870 155), droprenilamine (which may be prepared as
disclosed in DE2521113), lidoflazine (which may be prepared as
disclosed in U.S. Pat. No. 3,267,104); prenylamine (which may be
prepared as disclosed in U.S. Pat. No. 3,152,173), propatyl nitrate
(which may be prepared as disclosed in French Patent No.
1,103,113), mioflazine hydrochloride (1-Piperazineacetamide,
3-(aminocarbonyl)4-[4,4-bis(4-fluorophenyl)butyl]-N-(2,6-dichlorophenyl)--
, dihydrochloride CAS RN 83898-67-3), mixidine (Benzeneethanamine,
3,4-dimethoxy-N-(l-methyl-2-pyrrolidinylidene)-Pyrrolidine,
2-[(3,4-dimethoxyphenethyl)imino]-1-methyl-1-Methyl-2-[(3,
4-dimethoxyphenethyl)imino]pyrrolidine CAS RN 27737-38-8),
molsidomine (1,2,3-Oxadiazolium,
5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, inner salt CAS RN
25717-80-0), isosorbide mononitrate (D-Glucitol,
1,4:3,6-dianhydro-, 5-nitrate CAS RN 16051-77-7), erythrityl
tetranitrate (1,2,3,4-Butanetetrol, tetranitrate, (2R,3S)-rel-CAS
RN 7297-25-8), clonitrate(1,2-Propanediol, 3-chloro-, dinitrate
(7CI, 8CI, 9CI) CAS RN 2612-33-1), dipyridamole Ethanol, 2,2',2'',
2'''-[(4,8-di-1-piperidinylpyrimido[5,4-d]pyrimidine-2,6-diyl)dinitrilo]t-
etrakis-CAS RN 58-32-2), nicorandil (CAS RN 65141-46-0 3-),
pyridinecarboxamide
(N-[2-(nitrooxy)ethyl]-Nisoldipine3,5-Pyridinedicarboxylic acid,
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, methyl 2-methylpropyl
ester CAS RN 63675-72-9), nifedipine3,5-Pyridinedicarboxylic acid,
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester CAS RN
21829-25-4), perhexiline maleate (Piperidine,
2-(2,2-dicyclohexylethyl)-, (2Z)-2-butenedioate (1:1) CAS RN
6724-53-4), oxprenolol hydrochloride (2-Propanol,
1-[(1-methylethyl)amino]-3-[2-(2-propenyloxy)phenoxy]-,
hydrochloride CAS RN 6452-73-9), pentrinitrol (1,3-Propanediol,
2,2-bis[(nitrooxy)methyl]-, mononitrate (ester) CAS RN 1607-17-6),
verapamil (Benzeneacetonitrile,
.alpha.-[3-[[2-(3,4-dimethoxyphenyl)ethyl]-methylamino]propyl]-3,
4-dimethoxy-.alpha.-(1-methylethyl)-CAS RN 52-53-9) and the like;
angiotensin II receptor antagonists such as, aprosartan,
zolasartan, olmesartan, pratosartan, FI6828K, RNH6270, candesartan
(1H-Benzimidazole-7-carboxylic acid,
2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]4-yl]methyl]-CAS
RN 139481-59-7), candesartan cilexetil
((+/-)-1-(cyclohexylcarbonyloxy)ethyl-2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)b-
iphenyl-4-yl]-1H-benzimidazole carboxylate, CAS RN 145040-37-5,
U.S. Pat. Nos. 5,703,110 and 5,196,444), eprosartan
(3-[1-4-carboxyphenylmethyl)-2-n-butyl-imidazol-5-yl]-(2-thienylmethyl)
propenoic acid, U.S. Pat. Nos. 5,185,351 and 5,650,650), irbesartan
(2-n-butyl-3-[[2'-(lh-tetrazol-5-yl)biphenyl-4-yl]methyl]
1,3-diazazspiro[4,4]non-1-en-4-one, U.S. Pat. Nos. 5,270,317 and
5,352,788), losartan
(2-N-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-y-
l)-methyl]imidazole, potassium salt, U.S. Pat. Nos. 5,138,069,
5,153,197 and 5,128,355), tasosartan
(5,8-dihydro-2,4-dimethyl-8-[(2'-(1H-tetrazol-5-yl)[l,r-biphenyl]4-yl)met-
hyl]-pyrido[2,3-d]pyrimidin-7(6H)-one, U.S. Pat. No. 5,149,699),
telmisartan
(4'-[(1,4-dimethyl-2'-propyl-(2,6'-bi-1H-benzimidazol)-r-yl)]-[1,1'-biphe-
nyl]-2-carboxylic acid, CAS RN 144701-48-4, U.S. Pat. No.
5,591,762), milfasartan, abitesartan, valsartan (Diovan.RTM.
(Novartis),
(S)--N-valeryl-N-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]valine,
U.S. Pat. No. 5,399,578), EXP-3137
(2-N-butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidaz-
ole-5-carboxylic acid, U.S. Pat. Nos. 5,138,069, 5,153,197 and
5,128,355),
3-(2'-(tetrazol-5-yl)-l,r-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imid-
azo[4,5-b]pyridine,
4'[2-ethyl-4-methyl-6-(5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-2-yl]-benz-
imidazol-1-yl]-methyl]-l,r-biphenyl]-2-carboxylic acid,
2-butyl-6-(l-methoxy-1-methylethyl)-2-[2'-)1H-tetrazol-5-yl)biphenyl-4-yl-
methyl] guinazolin-4(3H)-one,
3-[2'-carboxybiphenyl-4-yl)methyl]-2-cyclopropyl-7-methyl-3H-imidazo[4,5--
b]pyridine,
2-butyl-4-chloro-1-[(2'-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole-carb-
oxylic acid, 2-butyl-4-chloro-1-[[2'-(1H-tetrazol-5-yl) [1,
1'-biphenyl]-4-yl]methyl]-1H-imidazole-5-carboxylic
acid-1-(ethoxycarbonyl-oxy)ethyl ester potassium salt, dipotassium
2-butyl-4-(methylthio)-1-[[2-[[[(propylamino)carbonyl]amino]-sulfonyl](1,-
1'-biphenyl)-4-yl]methyl]-1H-imidazole-5-carboxylate,
methyl-2-[[4-butyl-2-methyl-6-oxo-5-[[2'-(1H-tetrazol-5-yl)-[1,1'-bipheny-
l]-4-yl]methyl]-1-(6H)-pyrimidinyl]methyl]-3-thiophencarboxylate,
5-[(3,5-dibutyl-1H-1,2,4-triazol-1-yl)methyl]-2-[2-(1H-tetrazol-5-ylpheny-
l)]pyridine, 6-butyl-2-(2-phenylethyl)-5
[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-methyl]pyrimidin-4-(3H)-one
D,L lysine salt,
5-methyl-7-n-propyl-8-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-[1,2,4-
]-triazolo[1,5-c]pyrimidin-2(3H)-one,
2,7-diethyl-5-[[2'-(5-tetrazoly)biphenyl-4-yl]methyl]-5H-pyrazolo[1,5-b][-
1,2,4]triazole potassium salt,
2-[2-butyl-4,5-dihydro-4-oxo-3-[2'-(1H-tetrazol-5-yl)-4-biphenylmethyl]-3-
H-imidazol[4,5-c]pyridine-5-ylmethyl]benzoic acid, ethyl ester,
potassium salt,
3-methoxy-2,6-dimethyl-4-[[2'(1H-tetrazol-5-yl)-1,1'-biphenyl-4-yl]-
methoxy]pyridine,
2-ethoxy-1-[[2'-(5-oxo-2,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]met-
hyl]-1H-benzimidazole-7-carboxylic acid,
1-[N-(2'-(1H-tetrazol-5-yl)biphenyl-4-yl-methyl)-N-valerolylaminomethyl)c-
yclopentane-1-carboxylic acid, 7-methyl-2n-propyl-3-[[2'
1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidazo[4,5-6]pyridine,
2-[5-[(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine-3-yl)methyl]-2-quin-
olinyl]sodium benzoate,
2-butyl-6-chloro-4-hydroxymethyl-5-methyl-3-[[2'-(I
H-tetrazol-5-yl)biphenyl-4-yl]methyl]pyridine,
2-[[[2-butyl-1-[(4-carboxyphenyl)methyl]-1H-imidazol-5-yl]methyl]amino]be-
nzoic acid tetrazol-5-yl)biphenyl-4-yl]methyl]pyrimidin-6-one,
4(S)-[4-(carboxymethyl)phenoxy]-N-[2(R)-[4-(2-sulfobenzamido)imidazol-1-y-
l]octanoyl]-L-proline,
1-(2,6-dimethylphenyl)-4-butyl-1,3-dihydro-3-[[6-[2-(H-tetrazol-5-yl)phen-
yl]-3-pyridinyl]methyl]-2H-imidazol-2-one,
5,8-ethano-5,8-dimethyl-2-n-propyl-5,6,7,8-tetrahydro-1-[[2'(1H-tetrazol--
5-yl)biphenyl-4-yl]methyl]-1H,4H-1,3,4a,8a-tetrazacyclopentanaphthalene-9--
one,
4-[1-[2'-(1,2,3,4-tetrazol-5-yl)biphen-4-yl)methylamino]-5,6,7,8-tetr-
ahydro-2-trifylquinazoline,
2-(2-chlorobenzoyl)imino-5-ethyl-3-[2'-(1H-tetrazole-5-yl)biphenyl-4-yl)m-
ethyl-1,3,4-thiadiazoline,
2-[5-ethyl-3-[2-(1H-tetrazole-5-yl)biphenyl-4-yl]methyl-1,3,4-thiazoline--
2-ylidene]aminocarbonyl-1-cyclopentencarboxylic acid dipotassium
salt, and
2-butyl-4-[N-methyl-N-(3-methylcrotonoyl)amino]-1-[[2'-(1H-tetrazol-5-yl)-
biphenyl-4-yl]methyl]-1H-imidzole-5-carboxylic acid
1-ethoxycarbonyloxyethyl ester, those disclosed in patent
publications EP475206, EP497150, EP539086, EP539713, EP535463,
EP535465, EP542059, EP497121, EP535420, EP407342, EP415886,
EP424317, EP435827, EP433983, EP475898, EP490820, EP528762,
EP324377, EP323841, EP420237, EP500297, EP426021, EP480204,
EP429257, EP430709, EP434249, EP446062, EP505954, EP524217,
EP514197, EP514198, EP514193, EP514192, EP450566, EP468372,
EP485929, EP503162, EP533058, EP467207 EP399731, EP399732,
EP412848, EP453210, EP456442, EP470794, EP470795, EP495626,
EP495627, EP499414, EP499416, EP499415, EP511791, EP516392,
EP520723, EP520724, EP539066, EP438869, EP505893, EP530702,
EP400835, EP400974, EP401030, EP407102, EP411766, EP409332,
EP412594, EP419048, EP480659, EP481614, EP490587, EP467715,
EP479479, EP502725, EP503838, EP505098, EP505111 EP513,979
EP507594, EP510812, EP511767, EP512675, EP512676, EP512870,
EP517357, EP537937, EP534706, EP527534, EP540356, EP461040,
EP540039, EP465368, EP498723, EP498722, EP498721, EP515265,
EP503785, EP501892, EP519831, EP532410, EP498361, EP432737,
EP504888, EP508393, EP508445, EP403159, EP403158, EP425211,
EP427463, EP437103, EP481448, EP488532, EP501269, EP500409,
EP540400, EP005528, EP028834, EP028833, EP411507, EP425921,
EP430300, EP434038, EP442473, EP443568, EP445811, EP459136,
EP483683, EP518033, EP520423, EP531876, EP531874, EP392317,
EP468470, EP470543, EP502314, EP529253, EP543263, EP540209,
EP449699, EP465323, EP521768, EP415594, WO92/14468, WO93/08171,
WO93/08169, WO91/00277, WO91/00281, WO91/14367, WO92/00067,
WO92/00977, WO92/20342, WO93/04045, WO93/04046, WO91/15206,
WO92/14714, WO92/09600, WO92/16552, WO93/05025, WO93/03018,
WO91/07404, WO92/02508, WO92/13853, WO91/19697, WO91/11909,
WO91/12001, WO91/11999, WO91/15209, WO91/15479, WO92/20687,
WO92/20662, WO92/20661, WO93/01177, WO91/14679, WO91/13063,
WO92/13564, WO91/17148, WO91/18888, WO91/19715, WO92/02257,
WO92/04335, WO92/05161, WO92/07852, WO92/15577, WO93/03033,
WO91/16313, WO92/00068, WO92/02510, WO92/09278, WO9210179,
WO92/10180, WO92/10186, WO92/10181, WO92/10097, WO92/10183,
WO92/10182, WO92/10187, WO92/10184, WO92/10188, WO92/10180,
WO92/10185, WO92/20651, WO93/03722, WO93/06828, WO93/03040,
WO92/19211, WO92/22533, WO92/06081, WO92/05784, WO93/00341,
WO92/04343, WO92/04059, U.S. Pat. Nos. 5,104,877, 5,187,168,
5,149,699, 5,185,340, 4,880,804, 5,138,069, 4,916,129, 5,153,197,
5,173,494, 5,137,906, 5,155,126, 5,140,037, 5,137,902, 5,157,026,
5,053,329, 5,132,216, 5,057,522, 5,066,586, 5,089,626, 5,049,565,
5,087,702, 5,124,335, 5,102,880, 5,128,327, 5,151,435, 5,202,322,
5,187,159, 5,198,438, 5,182,288, 5,036,048, 5,140,036, 5,087,634,
5,196,537, 5,153,347, 5,191,086, 5,190,942, 5,177,097, 5,212,177,
5,208,234, 5,208,235, 5,212,195, 5,130,439, 5,045,540, 5,041,152,
and 5,210,204, and pharmaceutically acceptable salts and esters
thereof, a/f3 adrenergic blockers such as nipradilol, arotinolol,
amosulalol, bretylium tosylate (CAS RN: 61-75-6), dihydroergtamine
mesylate (such as ergotaman-3',6', 18-trione,
9,10-dihydro-12'-hydroxy-2'-methyl-5'-(phenylmethyl)-,
(5'(.alpha.))-, monomethanesulfonate, e.g., DHE 45.RTM. Injection,
Novartis), carvedilol (such as
(+)-1-(Carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]
amino]-2-propanol, e.g., Coreg.RTM., SmithKline Beecham), labetalol
(such as 5-[1-hydroxy-2-[(1-methyl-3-phenylpropyl)
amino]ethyljsalicylamide monohydrochloride, e.g., Normodyne.RTM.,
Schering), bretylium tosylate (Benzenemethanaminium,
2-bromo-N-ethyl-N,N-dimethyl-, salt with 4-methylbenzenesulfonic
acid (1:1) CAS RN 61-75-6), phentolamine mesylate (Phenol,
3-[[(4,5-dihydro-1H-imidazol-2-yl)methyl](4-methylphenyl)amino]-- ,
monomethanesulfonate (salt) CAS RN 65-28-1), solypertine tartrate
(5H-1,3-Dioxolo[4,5-f]indole,
7-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-,
(2R,3R)-2,3-dihydroxybutanedioate (1:1) CAS RN 5591-43-5),
zolertine hydrochloride (Piperazine,
1-phenyl4-[2-(1H-tetrazol-5-yl)ethyl]-, monohydrochloride (8Cl,
9Cl) CAS RN 7241-94-3) and the like; a adrenergic receptor
blockers, such as alfuzosin (CAS RN: 81403-68-1), terazosin,
urapidil, prazosin (Minipress.RTM.), tamsulosin, bunazosin,
trimazosin, doxazosin, naftopidil, indoramin, WHP 164, XENOlO,
fenspiride hydrochloride (which may be prepared as disclosed in
U.S. Pat. No. 3,399,192), proroxan (CAS RN 33743-96-3), and
labetalol hydrochloride and combinations thereof; a 2 agonists such
as methyldopa, methyldopa HCL, lofexidine, tiamenidine, moxonidine,
rilmenidine, guanobenz, and the like; aldosterone inhibitors, and
the like; renin inhibitors including Aliskiren (SPPlOO;
Novartis/Speedel); angiopoietin-2-binding agents such as those
disclosed in WO03/030833; anti-angina agents such as ranolazine
(hydrochloride 1-Piperazineacetamide,
N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-,
dihydrochloride CAS RN 95635-56-6), betaxolol hydrochloride
(2-Propanol, 1-[4-[2
(cyclopropylmethoxy)ethyl]phenoxy]-3-[(1-methylethyl)amino]-,
hydrochloride CAS RN 63659-19-8), butoprozine hydrochloride
(Methanone,
[4-[3(dibutylamino)propoxy]phenyl](2-ethyl-3-indolizinyl)-,
monohydrochloride CAS RN 62134-34-3), cinepazet
maleatel-Piperazineacetic acid,
4-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-, ethyl ester,
(2Z)-2-butenedioate (1:1) CAS RN 50679-07-7), tosifen
(Benzenesulfonamide, 4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]
amino] carbonyl]-CAS RN 32295-184), verapamilhydrochloride
(Benzeneacetonitrile,
.alpha.-[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimetho-
xy-.alpha.-(1-methylethyl)-, monohydrochloride CAS RN 152-114),
molsidomine (1,2,3-Oxadiazolium,
5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, inner salt CAS RN
25717-80-0), and ranolazine hydrochloride (1-Piperazineacetamide,
N-(2,6-dimethylphenyl)4-[2-hydroxy-3-(2-meth-oxyphenoxy)propyl]-,
dihydrochloride CAS RN 95635-56-6); tosifen (Benzenesulfonamide,
4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]amino]carbonyl]-CAS RN
32295-184); adrenergic stimulants such as guanfacine hydrochloride
(such as N-amidino-2-(2,6-dichlorophenyl) acetamide hydrochloride,
e.g., Tenex.RTM. Tablets available from Robins);
methyldopa-hydrochlorothiazide (such as
levo-3-(3,4-dihydroxyphenyl)-2-methylalanine) combined with
Hydrochlorothiazide (such as
6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide
1,1-dioxide, e.g., the combination as, e.g., Aldoril.RTM. Tablets
available from Merck), methyldopa-chlorothiazide (such as
6-chloro-2H-1, 2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide and
methyldopa as described above, e.g., Aldoclor.RTM., Merck),
clonidine hydrochloride (such as 2-(2,6-dichlorophenyl
amino)-2-imidazoline hydrochloride and chlorthalidone (such as
2-chloro-5-(1-hydroxy-3-oxo-1-isoindolinyl) benzenesulfonamide),
e.g., Combipres.RTM., Boehringer Ingelheim), clonidine
hydrochloride (such as 2-(2,6-dichlorophenylamino)-2-imidazoline
hydrochloride, e.g., Catapres.RTM., Boehringer Ingelheim),
clonidine (1H-Imidazol-2-amine,
N-(2,6-dichlorophenyl)4,5-dihydro-CAS RN 4205-90-7), Hyzaar (Merck;
a combination of losartan and hydrochlorothiazide), Co-Diovan
(Novartis; a combination of valsartan and hydrochlorothiazide,
Lotrel (Novartis; a combination of benazepril and amlodipine) and
Caduet (Pfizer; a combination of amlodipine and atorvastatin), and
those agents disclosed in US20030069221.
[0172] 1.2.2.11 Agents for the Treatment of Respiratory
Disorders
[0173] The GCC agonist peptides described herein can be used in
combination therapy with one or more of the following agents useful
in the treatment of respiratory and other disorders including but
not limited to: (1) .beta.-agonists including but not limited to:
albuterol (PRO VENTIL.RTM., S ALBUT AMOl.RTM., VENTOLIN.RTM.),
bambuterol, bitoterol, clenbuterol, fenoterol, formoterol,
isoetharine (BRONKOSOL.RTM., BRONKOMETER.RTM.), metaproterenol
(ALUPENT.RTM., METAPREL.RTM.), pirbuterol (MAXAIR.RTM.),
reproterol, rimiterol, salmeterol, terbutaline (BRETHAIRE.RTM.,
BRETHINE.RTM., BRICANYL.RTM.), adrenalin, isoproterenol
(ISUPREL.RTM.), epinephrine bitartrate (PRIMATENE.RTM.), ephedrine,
orciprenline, fenoterol and isoetharine; (2) steroids, including
but not limited to beclomethasone, beclomethasone dipropionate,
betamethasone, budesonide, bunedoside, butixocort, dexamethasone,
flunisolide, fluocortin, fluticasone, hydrocortisone, methyl
prednisone, mometasone, predonisolone, predonisone, tipredane,
tixocortal, triamcinolone, and triamcinolone acetonide; (3)
02-agonist-corticosteroid combinations [e.g.,
salmeterol-fluticasone (AD V AIR.RTM.), formoterol-budesonid (S
YMBICORT.RTM.)]; (4) leukotriene D4 receptor
antagonists/leukotriene antagonists/LTD4 antagonists (i.e., any
compound that is capable of blocking, inhibiting, reducing or
otherwise interrupting the interaction between leukotrienes and the
Cys LTI receptor) including but not limited to: zafhiukast,
montelukast, montelukast sodium (SINGULAIR.RTM.), pranlukast,
iralukast, pobilukast, SKB-106,203 and compounds described as
having LTD4 antagonizing activity described in U.S. Pat. No.
5,565,473; (5) 5-lipoxygenase inhibitors and/or leukotriene
biosynthesis inhibitors [e.g., zileuton and BAY1005 (CA registry
128253-31-6)]; (6) histamine H1 receptor antagonists/antihistamines
(i.e., any compound that is capable of blocking, inhibiting,
reducing or otherwise interrupting the interaction between
histamine and its receptor) including but not limited to:
astemizole, acrivastine, antazoline, azatadine, azelastine,
astamizole, bromopheniramine, bromopheniramine maleate,
carbinoxamine, carebastine, cetirizine, chlorpheniramine,
chloropheniramine maleate, cimetidine clemastine, cyclizine,
cyproheptadine, descarboethoxyloratadine, dexchlorpheniramine,
dimethindene, diphenhydramine, diphenylpyraline, doxylamine
succinate, doxylarnine, ebastine, efletirizine, epinastine,
famotidine, fexofenadine, hydroxyzine, hydroxyzine, ketotifen,
levocabastine, levocetirizine, levocetirizine, loratadine,
meclizine, mepyramine, mequitazine, methdilazine, mianserin,
mizolastine, noberastine, norasternizole, noraztemizole,
phenindamine, pheniramine, picumast, promethazine, pynlamine,
pyrilamine, ranitidine, temelastine, terfenadine, trimeprazine,
tripelenamine, and triprolidine; (7) an anticholinergic including
but not limited to: atropine, benztropine, biperiden, flutropium,
hyoscyamine (e.g. Levsin.RTM.; Levbid.RTM.; Levsin/SL.RTM.,
Anaspaz.RTM., Levsinex Timecaps.RTM., NuLev.RTM.), ilutropium,
ipratropium, ipratropium bromide, methscopolamine, oxybutinin,
rispenzepine, scopolamine, and tiotropium; (8) an anti-tussive
including but not limited to: dextromethorphan, codeine, and
hydromorphone; (9) a decongestant including but not limited to:
pseudoephedrine and phenylpropanolamine; (10) an expectorant
including but not limited to: guafenesin, guaicolsulfate, terpin,
ammonium chloride, glycerol guaicolate, and iodinated glycerol;
(11) a bronchodilator including but not limited to: theophylline
and aminophylline; (12) an anti-inflammatory including but not
limited to: fluribiprofen, diclophenac, indomethacin, ketoprofen,
S-ketroprophen, tenoxicam; (13) a PDE (phosphodiesterase) inhibitor
including but not limited to those disclosed herein; (14) a
recombinant humanized monoclonal antibody [e.g. xolair (also called
omalizumab), rhuMab, and talizumab]; (15) a humanized lung
surfactant including recombinant forms of surfactant proteins SP-B,
SP-C or SP-D [e.g. SURFAXIN.RTM., formerly known as dsc-104
(Discovery Laboratories)], (16) agents that inhibit epithelial
sodium channels (ENaC) such as amiloride and related compounds;
(17) antimicrobial agents used to treat pulmonary infections such
as acyclovir, amikacin, amoxicillin, doxycycline, trimethoprin
sulfamethoxazole, amphotericin B, azithromycin, clarithromycin,
roxithromycin, clarithromycin, cephalosporins (ceffoxitin,
cefmetazole etc), ciprofloxacin, ethambutol, gentimycin,
ganciclovir, imipenem, isoniazid, itraconazole, penicillin,
ribavirin, rifampin, rifabutin, amantadine, rimantidine,
streptomycin, tobramycin, and vancomycin; (18) agents that activate
chloride secretion through Ca++ dependent chloride channels (such
as purinergic receptor (P2Y(2) agonists); (19) agents that decrease
sputum viscosity, such as human recombinant DNase 1,
(Pulmozyme.RTM.); (20) nonsteroidal anti-inflammatory agents
(acemetacin, acetaminophen, acetyl salicylic acid, alclofenac,
alminoprofen, apazone, aspirin, benoxaprofen, bezpiperylon,
bucloxic acid, carprofen, clidanac, diclofenac, diclofenac,
diflunisal, diflusinal, etodolac, fenbufen, fenbufen, fenclofenac,
fenclozic acid, fenoprofen, fentiazac, feprazone, flufenamic acid,
flufenisal, flufenisal, fluprofen, flurbiprofen, flurbiprofen,
furofenac, ibufenac, ibuprofen, indomethacin, indomethacin,
indoprofen, isoxepac, isoxicam, ketoprofen, ketoprofen, ketorolac,
meclofenamic acid, meclofenamic acid, mefenamic acid, mefenamic
acid, miroprofen, mofebutazone, nabumetone oxaprozin, naproxen,
naproxen, niflumic acid, oxaprozin, oxpinac, oxyphenbutazone,
phenacetin, phenylbutazone, phenylbutazone, piroxicam, piroxicam,
pirprofen, pranoprofen, sudoxicam, tenoxican, sulfasalazine,
sulindac, sulindac, suprofen, tiaprofenic acid, tiopinac,
tioxaprofen, tolfenamic acid, tolmetin, tolmetin, zidometacin,
zomepirac, and zomepirac); and (21) aerosolized antioxidant
therapeutics such as S-Nitrosoglutathione.
[0174] 1.2.2.12 Anti-Diabetic Agents
[0175] The GCC agonist peptides described herein can be used in
therapeutic combination with one or more anti-diabetic agents,
including but not limited to: PPAR.gamma. agonists such as
glitazones (e.g., WAY-120,744, AD 5075, balaglitazone, ciglitazone,
darglitazone (CP-86325, Pfizer), englitazone (CP-68722, Pfizer),
isaglitazone (MIT/J&J), MCC-555 (Mitsibishi disclosed in U.S.
Pat. No. 5,594,016), pioglitazone (such as such as Actos.TM.
pioglitazone; Takeda), rosiglitazone (Avandia.TM.; Smith Kline
Beecham), rosiglitazone maleate, troglitazone (Rezulin.RTM.,
disclosed in U.S. Pat. No. 4,572,912), rivoglitazone (CS-Ol 1,
Sankyo), GL-262570 (Glaxo Welcome), BRL49653 (disclosed in
WO98/05331), CLX-0921, 5-BTZD, GW-0207, LG-100641, JJT-501
(JPNT/P&U), L-895645 (Merck), R-119702 (Sankyo/Pfizer), NN-2344
(Dr. Reddy/NN), YM-440 (Yamanouchi), LY-300512, LY-519818, R483
(Roche), T131 (Tularik), and the like and compounds disclosed in
U.S. Pat. Nos. 4,687,777, 5,002,953, 5,741,803, 5,965,584,
6,150,383, 6,150,384, 6,166,042, 6,166,043, 6,172,090, 6,211,205,
6,271,243, 6,288,095, 6,303,640, 6,329,404, 5,994,554, WO97/10813,
WO97/27857, WO97/28115, WO97/28137, WO97/27847, WO00/76488,
WO03/000685, WO03/027112, WO03/035602, WO03/048130, WO03/055867,
and pharmaceutically acceptable salts thereof; biguanides such as
metformin hydrochloride (N,N-dimethylimidodicarbonimidic diamide
hydrochloride, such as Glucophage.TM., Bristol-Myers Squibb);
metformin hydrochloride with glyburide, such as Glucovance.TM.,
Bristol-Myers Squibb); buformin (Imidodicarbonimidic diamide,
N-butyl-); etoformine (1-Butyl-2-ethylbiguanide, Schering A. G.);
other metformin salt forms (including where the salt is chosen from
the group of, acetate, benzoate, citrate, ftimarate, embonate,
chlorophenoxyacetate, glycolate, palmoate, aspartate,
methanesulphonate, maleate, parachlorophenoxyisobutyrate, formate,
lactate, succinate, sulphate, tartrate, cyclohexanecarboxylate,
hexanoate, octanoate, decanoate, hexadecanoate, octodecanoate,
benzenesulphonate, trimethoxybenzoate, paratoluenesulphonate,
adamantanecarboxylate, glycoxylate, glutarnate,
pyrrolidonecarboxylate, naphthalenesulphonate, 1-glucosephosphate,
nitrate, sulphite, dithionate and phosphate), and phenformin;
protein tyrosine phosphatase-IB (PTP-IB) inhibitors, such as
A-401,674, KR 61639, OC-060062, OC-83839, OC-297962, MC52445,
MC52453, ISIS 113715, and those disclosed in WO99/585521,
WO99/58518, WO99/58522, WO99/61435, WO03/032916, WO003/032982,
WO003/041729, WO003/055883, WO02/26707, WO02/26743, JP2002114768,
and pharmaceutically acceptable salts and esters thereof;
sulfonylureas such as acetohexamide (e.g. Dymelor, Eli Lilly),
carbutamide, chlorpropamide (e.g. Diabinese.RTM., Pfizer),
gliamilide (Pfizer), gliclazide (e.g. Diamcron, Servier Canada
Inc), glimepiride (e.g. disclosed in U.S. Pat. No. 4,379,785, such
as Amaryl, Aventis), glipentide, glipizide (e.g. Glucotrol or
Glucotrol XL Extended Release, Pfizer), gliquidone, glisolamide,
glyburide/glibenclamide (e.g. Micronase or Glynase Prestab,
Pharmacia & Upjohn and Diabeta, Aventis), tolazamide (e.g.
Tolinase), and tolbutamide (e.g. Orinase), and pharmaceutically
acceptable salts and esters thereof; meglitinides such as
repaglinide (e.g. Pranidin.RTM., Novo Nordisk), KAD1229
(PF/Kissei), and nateglinide (e.g. Starlix.RTM., Novartis), and
pharmaceutically acceptable salts and esters thereof; a glucoside
hydrolase inhibitors (or glucoside inhibitors) such as acarbose
(e.g. Precose.TM., Bayer disclosed in U.S. Pat. No. 4,904,769),
miglitol (such as GLYSET.TM., Pharmacia & Upjohn disclosed in
U.S. Pat. No. 4,639,436), camiglibose (Methyl
6-deoxy-6-[(2R,3R,4R,5
S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidino]-alpha-D-glucopyranoside,
Marion Merrell Dow), voglibose (Takeda), adiposine, emiglitate,
pradimicin-Q, salbostatin, CKD-711, MDL-25,637, MDL-73,945, and MOR
14, and the compounds disclosed in U.S. Pat. Nos. 4,062,950,
4,174,439, 4,254,256, 4,701,559, 4,639,436, 5,192,772, 4,634,765,
5,157,116, 5,504,078, 5,091,418, 5,217,877, US51091 and WOO 1/47528
(polyamines); .alpha.-amylase inhibitors such as tendamistat,
trestatin, and Al-3688, and the compounds disclosed in U.S. Pat.
Nos. 4,451,455, 4,623,714, and 4,273,765; SGLT2 inhibtors including
those disclosed in U.S. Pat. Nos. 6,414,126 and 6,515,117; an aP2
inhibitor such as disclosed in U.S. Pat. No. 6,548,529; insulin
secreatagogues such as linogliride, A-4166, forskilin, dibutyrl
cAMP, isobutylmethylxanthine (IBMX), and pharmaceutically
acceptable salts and esters thereof; fatty acid oxidation
inhibitors, such as clomoxir, and etomoxir, and pharmaceutically
acceptable salts and esters thereof; A2 antagonists, such as
midaglizole, isaglidole, deriglidole, idazoxan, earoxan, and
fluparoxan, and pharmaceutically acceptable salts and esters
thereof; insulin and related compounds (e.g. insulin mimetics) such
as biota, LP-100, novarapid, insulin detemir, insulin lispro,
insulin glargine, insulin zinc suspension (lente and ultralente),
Lys-Pro insulin, GLP-I (1-36) amide, GLP-I (73-7) (insulintropin,
disclosed in U.S. Pat. No. 5,614,492), LY-315902 (Lilly), GLP-I
(7-36)-NH2), AL-401 (Autoimmune), certain compositions as disclosed
in U.S. Pat. Nos. 4,579,730, 4,849,405, 4,963,526, 5,642,868,
5,763,396, 5,824,638, 5,843,866, 6,153,632, 6,191,105, and WO
85/05029, and primate, rodent, or rabbit insulin including
biologically active variants thereof including allelic variants,
more preferably human insulin available in recombinant form
(sources of human insulin include pharmaceutically acceptable and
sterile formulations such as those available from Eli Lilly
(Indianapolis, Ind. 46285) as Humulin.TM. (human insulin rDNA
origin), also see the THE PHYSICIAN'S DESK REFERENCE, 55.sup.th Ed.
(2001) Medical Economics, Thomson Healthcare (disclosing other
suitable human insulins); non-thiazolidinediones such as JT-501 and
farglitazar (GW-2570/GI-262579), and pharmaceutically acceptable
salts and esters thereof; PPAR.alpha./.gamma. dual agonists such as
AR-HO39242 (Aztrazeneca), GW-409544 (Glaxo-Wellcome), BVT-142,
CLX-0940, GW-1536, GW-1929, GW-2433, KRP-297 (Kyorin Merck;
5-[(2,4-Dioxo thiazolidinyl)methyl]
methoxy-N-[[4-(trifluoromethyl)phenyl] methyljbenzamide), L-796449,
LR-90, MK-0767 (Merck/Kyorin/Banyu), SB 219994, muraglitazar (BMS),
tesaglitzar (Astrazeneca), reglitazar (JTT-501) and those disclosed
in WO99/16758, WO99/19313, WO99/20614, WO99/38850, WO00/23415,
WO00/23417, WO00/23445, WO00/50414, WOO1/00579, WOO1/79150,
WOO2/062799, WOO3/004458, WOO3/016265, WOO3/018010, WOO3/033481,
WOO3/033450, WOO3/033453, WOO3/043985, WO 031053976, U.S.
application Ser. No. 09/664,598, filed Sep. 18, 2000, Murakami et
al. Diabetes 47, 1841-1847 (1998), and pharmaceutically acceptable
salts and esters thereof; other insulin sensitizing drugs; VPAC2
receptor agonists; GLK modulators, such as those disclosed in
WO03/015774; retinoid modulators such as those disclosed in
WO03/000249; GSK 30/GSK 3 inhibitors such as
4-[2-(2-bromophenyl)-4-(4-fluorophenyl-1H-imidazol-5-yl]pyridine
and those compounds disclosed in WO03/024447, WO03/037869,
WO03/037877, WOO3/037891, WOO3/068773, EP1295884, EP1295885, and
the like; glycogen phosphorylase (HGLPa) inhibitors such as
CP-368,296, CP-316,819, BAYR3401, and compounds disclosed in WOO
1/94300, WOO2/20530, WOO3/037864, and pharmaceutically acceptable
salts or esters thereof, ATP consumption promotors such as those
disclosed in WO03/007990; TRB3 inhibitors; vanilloid receptor
ligands such as those disclosed in WOO3/049702; hypoglycemic agents
such as those disclosed in WOO3/015781 and WO03/040114; glycogen
synthase kinase 3 inhibitors such as those disclosed in WOO3/035663
agents such as those disclosed in WO99/51225, US20030134890,
WO01/24786, and WO03/059870; insulin-responsive DNA binding
protein-1 (IRDBP-I) as disclosed in WO03/057827, and the like;
adenosine A2 antagonists such as those disclosed in WOO3/035639,
WO03/035640, and the like; PPAR.delta. agonists such as GW 501516,
GW 590735, and compounds disclosed in JP10237049 and WOO2/14291;
dipeptidyl peptidase IV (DP-IV) inhibitors, such as isoleucine
thiazolidide, NVP-DPP728A
(1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl] amino]
acetyl]-2-cyano-(S)-pyrrolidine, disclosed by Hughes et al,
Biochemistry, 38(36), 11597-11603, 1999), P32/98, NVP-LAF-237,
P3298, TSL225
(tryptophyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid,
disclosed by Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998)
1537-1540), valine pyrrolidide, TMC-2A/2B/2C, CD-26 inhibitors,
FE999011, P9310/K364, VIP 0177, DPP4, SDZ 274-444,
2-cyanopyrrolidides and 4-cyanopyrrolidides as disclosed by
Ashworth et al, Bioorg. & Med. Chem. Lett., Vol. 6, No. 22, pp
1163-1166 and 2745-2748 (1996), and the compounds disclosed in U.S.
Pat. Nos. 6,395,767, 6,573,287, 6,395,767 (compounds disclosed
include BMS-477118, BMS-471211 and BMS 538,305), WO99/38501,
WO99/46272, WO99/67279, WO99/67278, WO99/61431 WO03/004498,
WO03/004496, EP1258476, WO02/083128, WO02/062764, WO03/000250,
WO03/002530, WO03/002531, WO03/002553, WO03/002593, WO03/000180,
and WO03/000181; GLP-I agonists such as exendin-3 and exendin-4
(including the 39 aa polypeptide synthetic exendin-4 called
Exenatide.RTM.), and compounds disclosed in US2003087821 and NZ
504256, and pharmaceutically acceptable salts and esters thereof;
peptides including amlintide and Symlin.RTM. (pramlintide acetate);
and glycokinase activators such as those disclosed in US2002103199
(fused heteroaromatic compounds) and WO02/48106
(isoindolin-1-one-substituted propionamide compounds).
[0176] All patents, patent applications, and publications mentioned
herein are hereby incorporated by reference in their entireties.
However, where a patent, patent application, or publication
containing express definitions is incorporated by reference, those
express definitions should be understood to apply to the
incorporated patent, patent application, or publication in which
they are found, and not to the remainder of the text of this
application. In the case of conflict, the present specification,
including definitions, will control. The references cited herein
are not admitted to be prior art to the invention.
[0177] It is to be understood that while the invention has been
described in conjunction with the preferred specific embodiments
thereof, that the foregoing description is intended to illustrate
and not limit the scope of the invention. It will be understood by
those skilled in the art that various changes may be made and
equivalents may be substituted without departing from the scope of
the invention, and further that other aspects, advantages and
modifications will be apparent to those skilled in the art to which
the invention pertains.
EXAMPLES
Example 1: Preparation of Side-Chain Protected Fragments of
SP-304
[0178] Attachment Fmoc-AA-OH to 2-C1Trt Resin
[0179] [162] 2-ClTrt resin (10 g, substitution=1.0 mmol/g resin)
was suspended in 100 mL of dichloromethane (DCM) for 5 minutes, and
then drained. The esterification was performed using 1.5 equiv. of
Fmoc-amino acid and 1.7 equiv. Diisopropylethylamine (DIEA) in 80
mL of DCM (with minimum quantity of dimethylformamide (DMF) to
dissolve the amino acid completely) for 2 hours. The resulting
resin was washed with 60 mL of DCM and endcapped with 60 mL of
DIEA/methanol (1:9, v/v) solution for 30 minutes. The loaded resin
was then washed with DCM (6 vol.) for 2 times, DMF (6 vol.) for 3
times and methyl t-butylether (MTBE) (6 vol.) for 3 times, and
dried under high vacuum. The substitution of the Fmoc-protected
resin was determined by Fmoc release assay. Finally, the Fmoc group
was deprotected with a mixture of 5% piperidine, 1%
1,8-diazobicyclo[5.4.0]undec-7-ene (DBU) and 1%
N-Hydroxybenzotriazole (HOBt) in DMF (10 vol.) for 2 times and the
resin was washed and dried under high vacuum to give the final
resin for peptide synthesis. The results of the experiments are
listed in Table VIII below.
TABLE-US-00008 TABLE VIII Preparation of H-Gly-2ClTrt and
H-Leu-2ClTrt resin Substitution of Synthesis the loaded Amino acid
2-ClTrt scale resin from Fmoc Yield resin (mmol) release assay
(Weight, % yield) H-Gly-2ClTrt resin 4.16 0.57 mmol/g resin 6 g,
82% H-Leu-2ClTrt resin 5.20 0.81 mmol/g resin 6.4 g, 100%
H-Gly-2ClTrt resin 300 0.80 mmol/g resin 328.6 g, 88% H-Leu-2ClTrt
resin 300 0.75 mmol/g resin 338.5 g, 84%
[0180] Synthesis of Side-Chain-Protected Fragments A and B
[0181] H-Gly-2-C1Trt resin or H-Leu-2-C1Trt resin was suspended in
DMF (10 vol.) for 20 minutes, then drained. The resulting resin was
washed with DMF (10 vol.) for 5 minutes. The chain assembly was
conducted using the standard Fmoc chemistry. Generally, 1.5 equiv.
of Fmoc amino acid and 1.5 equiv. of HOBt were dissolved in DMF
(4.5 vol.), followed by addition of 1.5 equiv. of DIEA. Then, the
resulting solution was cooled to below 5.degree. C. with an ice
water bath, and activated by addition of 1.5 equiv. of HBTU. DCM
(1.5 vol.) was added to the resin, followed by addition of the
activated Fmoc amino acid solution. The resulting mixture was
stirred at room temperature for 2 hours and the completion of the
acylation was monitored by Kaiser Test. If Kaiser Test indicated
the presence of unreacted amine after 2 hours, recoupling with the
same protocol using 1.0 equiv. of Fmoc amino acid, 1.0 equiv. of
HOBt and 1.0 equiv. of DIEA was required. Capping was generally
achieved by acetylating the unreacted amine with a mixture of
acetic anhydride/pyridine/DMF solution. The peptide sequence was
assembled by repeating the above capping procedure with the
corresponding Fmoc-amino acid derivatives in the sequence from C-
to N-terminal. The coupling of Fmoc-Cys(Trt)-OH or Fmoc-Cys(Acm)-OH
residue was achieved by using 2.0 equiv. of Fmoc-Cys(Trt)-OH or
Fmoc-Cys(Acm)-OH, 2.0 of equiv. HOBt and 2.0 equiv. of DIC in situ
activation in DCM/DMF protocol to minimize racemization of
cysteine.
[0182] After completion of the synthetic step, the peptide resin
was thoroughly washed with DMF (10 vol.), MTBE (10 vol.), DMF (10
vol. 3 times) and MTBE (10 vol. 3 times) and subsequently dried in
a vacuum oven to a constant weight.
[0183] The side-chain-protected peptide was cleaved from the resin
using 1% TFA/DCM (10 vol.) for 3 times, 5 minutes for each time and
the cleavage fractions were collected onto pyridine each time (1:1
volume ratio to TFA in each cleavage fraction). The peptide resin
was washed with DCM (7.5 vol.). The fractions were combined and
concentrated under vacuum to 10% of the original volume, and the
resulting solution was reconstituted with ethanol (3 vol.) and
concentrated to 50% of the original volume. Finally, the peptide
was precipitated out by addition of water (1 vol.). The solid was
collected by vacuum filtration or centrifugation and washed with
water twice. The product was dried in vacuum to a constant weight
and subjected to HPLC and ES-MS analysis. The results of the
experiments are presented in Table IX below.
TABLE-US-00009 TABLE IX Preparation of Fragments A and B Final
peptide resin weight and Yield of Synthesis yield from the Fragment
scale weight gain of obtained Purity Fragment Masses Fragment
(mmol) the resin (% yield) (HPLC) Calculated/Found* FmocAA7-14OH
(1) 3.4 9.5 g, 66.4% 3.337 g 87.8% 1599.95/1598.86 (61.3%)
BocAA1-6OH (2) 5.2 11.8 g, 76.3% 5.896 g 94.6% 1474.80/1473.94
(76.9%) FmocAA7-14OH (1') 200 479.7 g, 74.4% 213.7 g 90.1%
1599.95/1598.81 (66.8%) BocAA1-6OH (2') 200 544.0 g, 101.3% 247.0 g
94.2% 1474.80/1473.94 (83.7%) *Calculated = average molecular mass;
Found = mono-isotopic mass by ES-MS
Example 2: Condensation of Fragments of Sp-304 in Solution
[0184] Synthesis of Fragment C: H-AA15-16OtBu (1-1)
[0185] A solution of Fmoc-Cys(Acm)-OH (124.38 gm, 0.3 mol),
H-Leu-OtBu.HCl (67.12 gm, 0.3 mol), and HOBt (40.54 gm, 0.3 mol) in
DMF (600 mL) was cooled to -5.degree. C.
2-[1H-Benzotriazole-1-yl]-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) (113.79 gm, 0.3 mol) was added and
dissolved completely. DIEA (183.1 mL, 1.05 mol) was added dropwise
over a period of 105 minutes at the same temperature with good
stirring, keeping the pH of the mixture between 6 and 7. Stirring
was continued for 15 minutes at 0.degree. C. and the reaction was
monitored with TLC. The reaction mixture was diluted with ethyl
acetate (EtOAc) (600 mL) and 5% H.sub.3PO.sub.4 (300 mL). The
organic layer was separated and the aqueous layer was extracted
with EtOAc (600 mL). The combined extracts were washed with 5%
H.sub.3PO.sub.4 (2 times), H.sub.2O (1 time), saturated NaHCO.sub.3
(3 times), H.sub.2O (2 times), and brine (2 times). The solution
was dried over MgSO.sub.4 anhydrous, filtered, and evaporated in
vacuo to dryness. The product was recrystallized from petroleum
ether/EtOAc (3:1) and dried: 166.75 gm (yield 95.0%, purity
99.0%).
[0186] Fmoc-Cys(Acm)-Leu-OtBu (166.75 gm, 0.277 mol) was dissolved
in a 10% piperidine/DCM solution (810 mL) with stirring. The
reaction was monitored with TLC. After the reaction was completed
in 3 hours, the solvent and the volatile materials were removed
using a rotavap. The oily material obtained was triturated with
petroleum ether to remove the by-products by decantation. The
residue, a syrup, was taken up in EtOAc, and washed with a mixture
solution of NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4 (pH=6), then
saturated NaHCO.sub.3, purified H.sub.2O, and brine. The organic
layer was dried over MgSO.sub.4 anhydrous. Evaporation of the
solvent and the volatile materials yielded an oily product
H-Cys(Acm)-Leu-OtBu (1-1) (73.23 gm, yield: 73.1%, purity:
98.0%).
[0187] Synthesis of Fragment B-C: HAA7-16OtBu (2-3)
[0188] A solution of Fmoc-AA7-140H (1') (198.3 gm, 124.0 mmol),
H-AA15-16OtBu (1-1) (52.3 gm, 148.8 mmol) and Cl-HOBt (21.0 gm,
124.0 mmol) in DMF (2500 mL) was cooled to -5.degree. C. HBTU (51.7
gm, 136.4 mmol) was added and dissolved completely. DIEA (54.1 mL,
310 mmol) was then added with stirring, keeping the pH of the
mixture between 6 and 7. Stirring was continued for 30 minutes at
0-5.degree. C. The reaction mixture was allowed to warm up to
20-27.degree. C. slowly and stirring was continued for one and a
half hours. Then, the mixture was poured into precooled
(10-20.degree. C.) 0.5 N HCl aq. (20 L). The suspension was stored
at 20-25.degree. C. for 45 minutes. The solid was collected by
filtering the suspension through a fritted-glass funnel of medium
porosity and subsequent washing with 0.5 N HCl aq (3500 mL, 2
times), purified water (3500 mL), saturated NaHCO.sub.3 aq. (3500
mL, 2 times) and purified water (3500 mL, 2 times) and diethyl
ether (2000 mL, 2 times). Finally, the wet, crude peptide
FmocAA7-16OtBu (2-3) was dried in a desiccator under high vacuum at
room temperature to yield product 238.22 gm (purity, 85.27%, yield
98.9%).
[0189] Synthesis of the Fully Protected A-B-C: BocAA1-16OtBu
(3-3)
[0190] A solution of HAA7-16OtBu (2-3) (183.67 gm, 106.71 mmol),
BocAA1-6-OH (2') (157.65 gm, 106.71 mmol), and 6-Cl-HOBt (18.141
gm, 106.71 mmol) in DMF (3 L) were cooled to at -3 to 0.degree. C.
HBTU (44.523 gm, 117.38 mmol) was added and dissolved completely.
DIEA (55.8 mL, 320.13 mmol) was then added with stirring, keeping
the pH of the mixture 6. Stirring was continued for 20 minutes at
-5 to 0.degree. C. The reaction mixture was allowed to warm up to
25.degree. C. slowly and stirring was continued for 2.5 hours,
followed by further addition of HBTU (4.048 gm, 10.671 mmol) and
DIEA (2 mL). Stirring was continued for another 1.5 hours. The
resulting mixture was poured into MeOH (15 L), and the precipitate
was collected and washed with MeOH/DMF mixture (5:1, v/v) (2 times,
3 L), 0.1N HCl (3 L, 2 times), saturated NaHCO.sub.3 (2 times),
purified water (3 times), diethyl ether (2 times) and dried in
vacuo to yield the product BocAA1-16OtBu (3-3) (278.0 gm, yield
82.0%. Note: the purity was determined after the deprotection).
[0191] Synthesis of the Partially Protected Linear SP-304:
HAA1-160H (4-4)
[0192] A mixture of TFA/TIS/EDT (8:1:1, 2400 mL) was cooled to
(0-5.degree. C.) under nitrogen and Boc-AA1-16OtBu (3-3) (201 gm)
was added in portions. The resulting suspension was stirred at
0-10.degree. C. for 30 min, then the reaction solution was allowed
to warm up to 20-25.degree. C. with a water bath (10 minutes) and
stirring was continued for additional 1 hr and 50 min at the same
temperature. The reaction mixture was poured into pre-cooled
(10.degree. C.) MTBE (18 L). Some heat was evolved during the
addition of the peptide/TFA solution and the internal temperature
went up to 25.degree. C. The resulting suspension was then stored
in an ice-water bath (5-10.degree. C.) for 40 min. The precipitate
was collected by filtration and washed with MTBE (2000 mL, 4
times), and dried in vacuum over P.sub.2O.sub.5, yielding 148.37 gm
of off-white product, HAA1-160H (4-4) (purity: 62.23%, ES-MS, MW:
calculated=1828.07, found=1826.67).
Example 3: Oxidative Cyclization and Purification of SP-304 by
Polystyrenic Absorbent Resin
[0193] HAA1-160H (4-4) (0.58 gm) was dissolved in 5 mL of
acetonitrile and diluted with 575 mL of purified water. The
solution was adjusted to pH 8-9 with 25% ammonia solution, and 3%
hydrogen peroxide (0.58 mL) was added, then the reaction mixture
was kept for an hour with monitoring the disulfide formation by
HPLC. Nitrogen was then passed through the reaction mixture and the
solution was acidified to pH 3-4 with acetic acid (71.8% HPLC,
recovery 98.5% estimated from peak area). The resulting mixture was
added 1% iodine/ACN dropwise over a period of 10 minutes with good
stirring until the yellow color of the iodine persisted. Stirring
was continued for 30 minutes at 17-20.degree. C. The iodine was
quenched by addition of 0.5 M ascorbic acid aq. until the yellow
disappeared. Then, the pH of the mixture was adjusted to 6-7 with
25% ammonia solution (51.0% HPLC, recovery 50% estimated from peak
area).
[0194] The polystyrenic absorbent resin (D101) was packed to a
3(ID).times.9(L) CM column and well equilibrated with 6 column
volume (CV) of ethanol, 4 CV of purified water, 2 CV of 5% HCl aq.,
4 CV of purified water, 2 CV of 2% NaOH aq. and 4 CV of purified
water at the flow rate of 3 CV/hour. The oxidized peptide solution
was then loaded to the column at 2 CV/h. After loading, the column
was washed with 2 CV purified water at 2 CV/h. The elution was
conducted by applying 80% ethanol aq. to the column at 2 CV/h. The
fractions with UV absorbents at 215 nm, was collected and combined
(125 mL). The combined fractions were then evaporated in vacuum to
10% of the original volume and the suspension was precipitated with
125 mL of cold MTBE (10 vol). The solid was collected by filtration
and dried in vacuum to yield the crude SP-304 (0.282 g, 55.0%
HPLC).
Example 4: Oxidative Cyclization and Purification of SP-304 by
RP-HPLC
[0195] Crude peptide (4-4), prepared as described in Example 2 was
dissolved in 10% ACN aqueous to an approximate concentration of
1.25 g/L with continuous stirring via a mechanical stirrer. The pH
of the peptide solution was adjusted to 8.5-9.0 with 20% ammonia
aqueous and the resulting solution was stirred vigorously open to
the atmosphere. Hydrogen peroxide (3%, 0.25 equiv.) was added and
stirring was continued at room temperature for 60 minutes. The HPLC
analysis showed complete consumption of the linear peptide. Then,
the solution was acidified to pH 3-4 with 10% AcOH aqueous. The
resulting solution was diluted to a concentration of about 1 g/L
with purified water. Iodine (1.3% in ACN) was added in with
vigorous stirring over a period of 10 minutes until the yellow
color of the iodine persisted. At about half-hourly intervals
samples were taken from the mixture and analyzed by RP-HPLC. The
monocyclized peptide peak decreased gradually and a new peak
(dicyclized peptide) emerged. Oxidation was complete when no
monocyclized peptide peak left. The excess iodine was neutralized
by a small amount of ascorbic acid. The resulting solution was
loaded on a C.sub.18 RP-HPLC column packed with Kromasil 100 .ANG.,
10 .mu.m silica gel. After the dicyclized peptide solution was
loaded, a 3 column volume of a solution of 90% mobile phase A (1.0%
TEA, 0.5% H.sub.3PO.sub.4 in H.sub.2O, pH=7) and 10% mobile phase B
(acetonitrile) was loaded to wash out lines. Then, gradient was
operated from 10% B to 30% B in 80 minutes. Fractions were
collected at recorded intervals when main peak began to elute. The
purity of each fraction was monitored by analytical RP-HPLC.
Fractions of purity .ltoreq.95% (not meeting the Main Pool
criteria) were pooled accordingly and forward processed using the
same buffer system and gradient elution parameters stated above.
All fractions with purity .gtoreq.95% were pooled and stored at
2-8.degree. C. The purified peptide solution was diluted in a 1:1
ratio with purified water and then loaded to the same RP-HPLC
column. The counter-ion exchange was accomplished by washing the
column with 2-3 column volumes of 0.5M ammonium acetate aqueous,
followed by gradient elution from 90% C (0.2% AcOH aqueous
solution) and 10% mobile phase D (ACN) to 50% mobile phase C and
50% mobile phase D in 50 minutes. Fractions were collected at
recorded intervals and monitored by analytical RP-HPLC. The
fractions (.gtoreq.95%) were collected and lyophilized to obtain
final dry peptide, 68.0 g (96.1% pure).
Example 5: Desalination and Isolation of SP-304 after Purification
by RP-HPLC
[0196] After plecanatide was purified by RP-HPLC as described in
Example 4, it was desalinated and isolated. Briefly, the purified
plecanatide in ammonium acetate/acetonitrile/water buffer was
loaded onto a column packed with polymeric absorbent (macroporous
adsorption resin) and then eluted by a mixture of alcohol/water.
Finally, the peptide alcohol solution was concentrated under
reduced pressure, precipitated with an ether, e.g., diethyl ether
or MTBE, and dried under vacuum to give the final product.
[0197] Resin (Polymeric Adsorbents) Screening
[0198] Resin Pre-Treatment:
[0199] Polymeric adsorbents, DA201-C(from Jiangsu Suqing, China;
crosslinked polystyrene; surface area 1200-1400 m.sup.2/g; average
pore diameter: 3-4 nm; pore volume: 1.1-1.2 ml/g; bulk density:
0.68-0.75 g/ml; specific density: 1.03-1.1 g/ml; moisture: 50-60%;
particle size: 0.315-1.25 mm .gtoreq.95%; effective diameter:
0.4-0.7 mm; uniformity coefficient: <1.6%), DA201-H (from
Jiangsu Suqing, China; crosslinked polystyrene; surface area
.gtoreq.800 m.sup.2/g; average pore diameter: 6-8 nm; pore volume:
1.5-1.8 ml/g; bulk density: 0.65-0.70 g/ml; specific density:
1.02-1.07 g/ml; moisture: 55-65%; particle size: 0.315-1.25 mm
.gtoreq.95%; effective diameter: 0.4-0.7 mm; uniformity
coefficient: <1.6%), ADS-5 (from Nankai Hecheng, China;
crosslinked polystyrene; surface area 520-600 m.sup.2/g; average
pore diameter: 25-30 nm; bulk density: 0.7-0.8 g/ml; moisture:
60-70%; particle size: 0.315-1.25 mm .gtoreq.95%; uniformity
coefficient: <1.6%), and ADS-8 (from Nankai Hecheng, China;
crosslinked polystyrene; surface area 450-500 m.sup.2/g; average
pore diameter: 12-16 nm; bulk density: 0.65-0.75 g/ml; moisture:
60-70%; particle size: 0.315-1.25 mm 295%; uniformity coefficient:
<1.6%) were suspended in 4-6 volume of ethanol overnight. Decant
or suction off the supernatant from the settled resin. Add 6-8
volume of deionized water and resuspend the resin with gentle
overhead stirring. Again, decant or suction off the supernatant
from the settled resin. Repeat the above water treatment and
decantation steps until fines appearance is minimal.
[0200] Column Packing and Regeneration:
[0201] Resuspend the pre-treated resins above with 1-2 volume of
deionized water to form the resin slurry respectively by using
gentle agitation. Pour the resin slurry slowly down the inside of
the column to prevent air entrapment. After the resin slurry has
been fully transferred to the column, rinse the inside of the
column using a squirt bottle containing deionized water. Open the
column outlet to from a settled resin bed (ID=4 cm, H=10 cm). Then
the resin beds were washed successively at a flow rate of 3 CV per
hour by 4 CV of deionized water, 2 CV of 5% HCl aq, 4 CV of
deionized water, 2 CV of 2% NaOH aq and finally 4 CV of deionized
water till the pH of the elute was around 7.
[0202] Preparation of Loading Samples:
[0203] 2000 mg of lyophilized plecanatide was dissolved in a
mixture of 60 mL of ACN and 150 mL of 0.2% AcOH aq (the pH of the
AcOH aq was adjusted to 4 with 10% ammonia aq.). After filtration
with 1.2 m Nylon membrane, the filtrate was diluted to 250 mL with
0.2% AcOH aq (pH4) and split into 4 parts (62.5 mL each) for
loading.
[0204] Loading the Sample to the Columns:
[0205] 62.5 mL of peptide solution above was loaded onto the above
4 columns at a flow rate of 2 CV/h respectively. The loading elute
was collected and tested by RP-HPLC to evaluate the absorbent
capacity of each resin. The absorbent capacity results of each
resin were demonstrated in Table X below.
TABLE-US-00010 TABLE X Peptide in Resins loading sample Peptide
absorbed Absorbent ratio DA201-C 500 mg 303.5 mg 60.7% DA201-H 500
mg 493.4 mg 98.7% ADS-5 500 mg 450.5 mg 90.1% ADS-8 500 mg 466.1 mg
93.2%
[0206] HPLC Method:
[0207] HPLC machine: Shimadzu LC-10AD vp; column: Kromasil, C18,
4.6.times.250 mm; mobile phase A: 0.1% TFA in water; mobile phase
B: 0.1% TFA in ACN; detect at: 215 nm; column temperature:
40.degree. C.; flow rate: 1.0 mL/min; gradient: 25% B to 45% B in
30 min.
[0208] Absorbent Capacity Calculation:
[0209] The absorbent capacity of each resin was demonstrated by the
absorbent ratio of the peptide loaded onto each column, which was
calculated by the quantity of the peptide absorbed in each resin
column divided by the peptide quantity in each loading sample (500
mg). The quantity of peptide absorbed in each column was calculated
by the formula below:
Quantity of peptide absorbed=Quantity of peptide in the loading
sample-Quantity of peptide in the loading elute=500 mg-62.5
mL.times.(1.6 mg/mL.times.HPLC peak area of the eluate/HPLC peak
area of the peptide standard solution)
[0210] Washing the Column with Deionized Water:
[0211] The loaded columns above were then washed with 2 CV of
deionized water at 2 CV/h to remove the salts. The washing eluates
were collected and analyzed by RP-HPLC to determine the peptide
quantity desorbed by water using the same method above. The
desorbed peptide ratios of each resin were listed in Table XI
below.
TABLE-US-00011 TABLE XI Desorption Resins Peptide absorbed in resin
Peptide desorbed ratio DA201-C 303.5 mg 184.5 mg 60.8% DA201-H
493.4 mg 40.9 mg 8.3% ADS-5 450.5 mg 41.9 mg 9.3% ADS-8 466.1 mg
40.1 mg 8.6%
[0212] Desorbing the Peptide with 90% Ethanol/Water:
[0213] After 2 CV of water washes, the peptide absorbed in each
column was then eluted by 1-2 CV of 90% ethanol in water at 2 CV/h.
The elution was collected and analyzed by RP-HPLC to determine the
peptide quantity desorbed by 90% ethanol using the same method
above. The desorbed peptide ratios of each resin were listed in
Table XII below.
TABLE-US-00012 TABLE XII Peptide absorbed in resin after 2CV water
Desorption Resins washes Peptide desorbed ratio DA201-C 119.0 mg
47.6 mg (by 2CV ethanol) 40% DA201-H 452.5 mg 452.5 mg (by 1.5CV
ethanol) 100% ADS-5 408.6 mg 408.6 mg (by 1.5 CV ethanol) 100%
ADS-8 426.0 mg 426.0 mg (by 1.5 CV ethanol) 100%
[0214] Isolation of the Peptide from the Ethanol Solution:
[0215] The collected peptide/ethanol/water solution from each
column was concentrated under reduced pressure, precipitated with
MTBE, filtered and dried in vacuo to give the final product. The
overall yield of the peptide processed by each column was
demonstrated in Table XIII below.
TABLE-US-00013 TABLE XIII Overall Resins Peptide in loading sample
Final product obtained yield DA201-C 500 mg 52 mg 10.4% DA201-H 500
mg 460 mg 92.0% ADS-5 500 mg 400 mg 80.0% ADS-8 500 mg 430 mg
86.0%
[0216] Resin Screening Conclusion:
[0217] From the data above (Table X to Table XIII), the DA201-H
resin presented the best absorbent capacity and the best desorption
(by ethanol) performance for plecanatide among the resins in the
experiment.
[0218] Desalination and Isolation Process Optimization
[0219] Eluting Solvents Selection:
[0220] Isopropanol and ethanol are two commonly used solvents for
eluting the peptide from the polymeric absorbents. Table XIV shows
the amount of plecanatide that is able to be dissolved in aqueous
ethanol or isopropanol solution depending on the v/v % isopropanol
(or ethanol): water.
TABLE-US-00014 TABLE XIV Solvent Solubility 90% IPA/Water 67.5
mg/mL 75% IPA/Water 596.1 mg/mL 50% IPA/Water 635.0 mg/mL 90%
EtOH/Water 302.0 mg/mL 75% EtOH/Water 700.0 mg/mL
[0221] The water in the peptide/alcohol solution can be removed by
azeotropic distillation. Table XV shows the property of the binary
azeotropes of ethanol/water and isopropanol/water.
TABLE-US-00015 TABLE XV Boiling Boiling Azeotrope Azeotrope
Component A Component B Point A Point B Boiling Point Wt. % A Water
Ethanol 100.degree. C. 78.3.degree. C. 78.2.degree. C. 4% Water
Isopropanol 100.degree. C. 82.3.degree. C. 80.3.degree. C.
12.6%
[0222] Degradation of plecanatide will occur during the long time
storage of the peptide/alcohol/water solution and the concentration
process. Table XVI demonstrates the stability data of plecanatide
in 75% IPA/water solution and 90% EtOH/water at 23.degree. C.
TABLE-US-00016 TABLE XVI Duration Purity (in 90% EtOH aq.)* Purity
(in 75% IPA aq.)* 0 hours 98.5% 98.7% 2 hours 98.4% 98.7% 4 hours
N/A 98.3% 6 hours 98.1% 97.5% 8 hours N/A 96.6% 10 hours 95.5% N/A
24 hours 92.0% N/A 25 hours N/A 96.1% *HPLC Method: HPLC machine:
Shimadzu LC-10AD vp: column: Kromasil, C18, 4.6 .times. 250 mm;
mobile phase A: 0.1% TFA in water; mobile phase B: 0.1% TFA in ACN;
detect at: 215 nm; column temperature: 40.degree. C.; flow rate:
1.0 mL/min; gradient: 25% B to 45% B in 30 min.
[0223] From the testing data obtained, plecanatide was fairly
stable at 23.degree. C. in alcohol/water solution within 6
hours.
[0224] Elution experiments were conducted using isopropanol/water
mixtures with different v/v %. The desorption ratio and water
content of the peptide eluates were listed in Table XVII below.
TABLE-US-00017 TABLE XVII v/v % IPA/Water 75% IPA/water 95%
IPA/water 100% IPA/water Water content in 65.9% 53.0% 49.8% peptide
Desorption ratio 100% 100% 90%
[0225] 75% Isopropanol/Water as Eluting Solution:
[0226] 500 mg of plecanatide (98.1% pure) was dissolved in a
mixture of 16 mL ACN and 49 mL of 0.2% AcOH aqueous (the pH of the
0.2% AcOH solution was adjusted to 4.0 by addition of 10%
NH.sub.4OH aq). After filtration by 1.2 m nylon membrane, the
peptide solution was loaded onto a column packed with DA201-H resin
(ID=4 cm, H=10 cm, packed and pre-treated by the procedure
mentioned previously) at 2 CV/h. After loading, the column was
washed with 2 CV deionized water at 2 CV/h. Then, the peptide was
eluted by 1.5 CV of 75% IPA/water at 2 CV/h. The elution was
monitored by RP-HPLC. The eluate was collected (124 mL, 98.3%
pure). Karl Fisher analysis indicated water (65.9% wt. %). Into a
1-neck, 500-mL round bottom flask was placed 124 mL of
peptide/IPA/water eluate collected above (97.6% pure, slight
degradation occurred during storage at 2-8.degree. C. for 2 days).
The flask was then placed under reduced pressure (60 Pa) on a
rotary evaporator and partially immersed in a 23.degree. C. water
bath. A whitish suspension was formed by feed-stripping
approximately 622 mL of isopropanol in 2 hours to about 1/3 of the
initial volume of the solution (97.7% pure). Karl Fisher analysis
indicated water (0.17% wt. %). To the concentrate was added 350 mL
of pre-chilled diethyl ether, the solid was collected by
centrifugation at 3500 rpm for 3 minutes and dried under vacuum to
yield 333 mg of final product (yield 66.6%, 97.2% HPLC purity).
[0227] 95% Isopropanol/Water as Eluting Solution I:
[0228] 500 mg of plecanatide (98.1% pure) was dissolved in a
mixture of 16 mL ACN and 49 mL of 0.2% AcOH aqueous (the pH of the
0.2% AcOH solution was adjusted to 4.0 by addition of 10%
NH.sub.4OH aq). After filtration by 1.2 m nylon membrane, the
peptide solution was loaded onto a column packed with DA201-H resin
(ID=4 cm, H=10 cm, pre-treated by the procedure mentioned
previously) at 2 CV/h. After loading, the column was washed with 2
CV deionized water at 2 CV/h. Then, the peptide was eluted by 1.5
CV of 95% IPA/water at 2 CV/h. The elution was monitored by
RP-HPLC. The eluate was collected (117 mL, 98.1% pure). Karl Fisher
analysis indicated water (52% wt. %). Into a 1-neck, 500-mL round
bottom flask was placed 117 mL of peptide/IPA/water eluate
collected above. The flask was then placed under reduced pressure
(50 Pa) on a rotary evaporator and partially immersed in a
23.degree. C. water bath. A whitish solid was formed by
feed-stripping approximately 470 mL of isopropanol in 130 min
(97.9% pure). To the solid above was added 50 mL of pre-chilled
diethyl ether to form a suspension and evaporated under reduced
pressure (50 Pa) on a rotary evaporator at 23.degree. C. to
dryness. Yield was 430 mg of final product (86%). HPLC purity was
97.9%.
[0229] 95% Isopropanol/Water as Eluting Solution II:
[0230] 500 mg of plecanatide (98.1% pure) was dissolved in a
mixture of 16 mL ACN and 49 mL of 0.2% AcOH aqueous (the pH of the
0.2% AcOH solution was adjusted to 4.0 by addition of 10%
NH.sub.4OH aq). After filtration by 1.2 m nylon membrane, the
peptide solution was loaded onto a column packed with DA201-H resin
(ID=4 cm, H=10 cm, packed and pre-treated by the procedure
mentioned previously) at 2 CV/h. After loading, the column was
washed with 2 CV deionized water at 2 CV/h. Then, the peptide was
eluted by 1.5 CV of 95% IPA/water at 2 CV/h. The elution was
monitored by RP-HPLC. The eluate was collected (118 mL, 98.2%
pure). Karl Fisher analysis indicated water (53% wt. %). Into a
1-neck, 500-mL round bottom flask was placed 118 mL of
peptide/IPA/water eluate collected above. The flask was then placed
under reduced pressure (50 Pa) on a rotary evaporator and partially
immersed in a 23.degree. C. water bath. A whitish suspension
(.about.40 mL) was formed by feed-stripping approximately 330 mL of
isopropanol in 100 min (97.7% pure). To the suspension above was
added 400 mL of pre-chilled diethyl ether to form a suspension.
After standing at ambient temperature for 1 hour, the solid was
collected by centrifugation at 3500 rpm for 3 minutes, and dried
under vacuum to yield 370 mg of final product. Yield was 74%. HPLC
purity was 97.9%.
[0231] 95% Isopropanol/Water as Eluting Solution III:
[0232] 10 g of plecanatide was desalted and precipitated in a
manner similar to that described above. Interestingly, the
precipitation yield was improved to 93%, which is a significant
increase in yield. HPLC purity after precipitation was 98.47%.
Example 6: Characterization of Lyophilized SP-304 and Precipitated
SP-304
[0233] The plecanatide purified by lyophilization as described in
Example 4 and plecanatide purified by precipitation as described in
Example 5 were analyzed to determine significant chemical impurity
values such as the levels of acetamide, TFA, ammonium ion, and
acetonitrile. The results are listed in the table below.
TABLE-US-00018 Acetamide TFA Ammonium ion Acetonitrile Lyophilized
356 ppm 0.14% 1.58% 40 ppm plecanatide Precipitated <28 ppm (LOQ
0.09% 0.23% Not Detected plecanatide of method) (20 ppm LOQ)
[0234] As demonstrated by results above, the precipitation process
provided significantly reduced levels of undesirable process
impurities.
[0235] The plecanatide purified by lyophilization as described in
Example 4 and plecanatide purified by precipitation as described in
Example 5 were measured to obtain their bulk densities, tap
densities, particle size distribution, and shape.
[0236] Equipment:
[0237] (1) Tap Density Tester Model TD-1020; (2) Sonic Sifter
Separator Model L3P; (3) Optical Microscope LINITRON 2850.
[0238] Methods:
1) Bulk and Tap Density Measurements: Modified USP 1 Method
[0239] a. 100.0 mL graduate cylinder was used for lyophilized
plecanatide
[0240] b. 10.0 mL graduated cylinder was used for precipitated
plecanatide
2) Particle Size Distribution Analysis
[0241] a. Screens used: 200, 140, 100, 60, 40 and 30 meshes.
[0242] b. Sample size: 2 g of lyophilized plecanatide and 6.4 g of
precipitated plecanatide
3) Optical Microscopic Analysis: Particle Size and Shape
[0243] a. Dry powder was manually dispersed onto a microscopic
plate
[0244] b. Magnification: 100.times.
[0245] c. Under normal light condition (no polarized filters)
[0246] Results:
[0247] (1) Physical Appearance: lyophilized plecanatide is a light,
fluffy and white powder. Precipitated plecanatide is a slightly
off-white powder.
[0248] (2) Bulk and Tap Density: Table XVIII provides bulk and tap
density data for Plecanatide samples of both lyophilized and
precipitated:
TABLE-US-00019 TABLE XVIII Plecanatide Sample Bulk Density, g/mL
Tap Density, g/mL Lyophilized, Lot 101221 0.0332 0.0680
Precipitated, Lot 120210 0.486 0.641
[0249] As seen from the data, the precipitated plecanatide is
unexpectedly significantly denser than the lyophilized plecanatide.
The precipitated plecanatide has less tendency of dust generation
during processing, which affords the advantages of increased safety
and reduced processing losses.
[0250] (3) Particle Size Distribution: Table XIX summarizes the
particle size distribution analysis. FIG. 1 presents the data
graphically.
TABLE-US-00020 TABLE XIX # Particle Mesh Size Weight Retained (g)
Percent Retained Size (.mu.m) Lyophilized Precipitated Lyophilized
Precipitated 30 600 0.07 0.45 3.6% 7.1% 40 425 0.26 0.17 13.3% 2.7%
60 250 0.17 0.67 8.7% 10.5% 100 150 0.51 2.08 26.0% 32.7% 140 106
0.65 1.22 33.2% 19.2% 200 75 0.24 0.68 12.2% 10.7% Pan <75 0.06
1.09 3.1% 17.1% Total 1.96 6.36 100.0% 100.0%
[0251] As demonstrated by Table XIX and FIG. 1, the particle size
distributions are different for both types of plecanatide. During
analysis, it was observed that the precipitated plecanatide
contained some larger particles, which could be broken up easily.
It was also noticed that the lyophilized plecanatide appeared to be
flaky and sticking onto top and bottom of sieves, whereas no
sticking was observed for the precipitated plecanatide. It
indicates a better processing property of the precipitated
plecanatide.
[0252] (4) Particle Size and Shape: FIGS. 2 and 3 provide optical
microscopic analysis of samples of lyophilized and precipitated
plecanatide, respectively. As seen in FIG. 2, the lyophilized
plecanatide is in amorphous form and has irregular shapes of
particles. They form physical aggregates with particles lying on
top of each other. In FIG. 3, the precipitated plecanatide shows
distinguishable individual particles. From the particle appearances
and shapes, the precipitated plecanatide will have better flow
property and therefore can facilitate solid processing during
manufacturing.
[0253] The lyophilized and precipitated forms of plecanatide have
shown distinguishable physical appearances and properties by
density, particle size distribution and shape analyses. The
precipitated form is more suitable for solid dosage form processing
during manufacturing in terms of reducing dust generation, less
sticking onto processing equipment, and potentially less processing
losses.
[0254] The precipitated form is more suitable for processing solid
dosage form during manufacture (e.g., a low-dose solid dosage
form). The higher density of the precipitated material will reduce
aerosol or "dust" losses during weighing, transferring, and
blending. The different particle shape has been shown to reduce
loss caused by sticking to screens or sieves. The higher density
should improve content uniformity since the size and density of the
drug particles more closely match those of the excipients.
Example 7: Low-Dose Formulation of Precipitated SP-304
[0255] The plecanatide purified by precipitation as described in
Example 5 is processed further to make low-dose formulations as
described below.
TABLE-US-00021 composition of dry-blending batch Concentration %
Item No. Ingredient w/w 1 SP-304 0.3246 2 Microcrystalline 99.43
cellulose (Avicel PH 102) 3 Magnesium stearate 0.2500 4 HPMC
capsule shells n/a Total 100
Blending:
[0256] Avicel PH 102 is screened through a 60 mesh screen.
V-blenders (1 Qt, 4 Qt, and 16 Qt) are then dusted by the screened
Avicel PH 102. SP-304 is screened through a 200 mesh screen and
loaded into the 1-Qt V-blender. Then, about 80 g Avicel PH 102 is
added into the 1-Qt blender and the mixture is blended for 10
minutes at 25 rpm. The mixture is then transferred to the 4-Qt
V-blender which is pre-dusted by the screened Avicel PH 102. The
1-Qt blender is rinsed with Avicel and the rinse material is
transferred to the 4-Qt blender. The rinsing is repeated until all
SP-304 is transferred to the 4-Qt blender. About 200 g Avicel is
added to the 4-Qt V-blender and the mixture is blended for 10
minutes. The resulting blend is then screened through a 60 mesh
screen and then transferred into the pre-dusted 16-Qt blender
(dusted with 1500 g Avicel). The 4-Qt blender is rinsed with Avicel
and the rinse material is transferred to the 16-Qt blender. The
remaining Avicel is added to the 16-Qt blender and the mixture is
blended for 10 minutes. The resulting blend is passed through
Comil, rinsed with excess of Avicel, and then returned to the 16-Qt
blender and is further blended for 5 minutes. Proper amount of
magnesium stearate is weighed, screened through a 60 mesh screen,
and added into the 16-Qt blender. The resulting mixture is blended
for 2 minutes. The resulting mixture is then either packaged in
capsules or compressed to form tablets.
Encapsulation
[0257] A MG2 Planeta capsule filler is set up. Average weight of
the empty capsule shells is determined and target capsule fill
weight was calculated (.+-.5%). The blend from the above process is
added into the hopper of the capsule filler and encapsulation is
started. Run weight parameters are manually adjusted. Resulting
capsules are then sorted according to the target fill weight.
Compression
[0258] A Fette tablet press is set up. Then the blend mixture is
loaded into the powder hopper and tooling is installed. The weight
of each tablet is set to be 100 mg.+-.5% and hardness to be 4-6 Kp.
The weight, hardness, and thickness of tablets are measured and
recorded every 5 to 10 minutes. Friability measurement is also
performed to ensure satisfactory product.
Sequence CWU 1
1
261116PRTArtificial SequenceChemically Synthesized 1Asn Asp Glu Cys
Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
15215PRTArtificial SequenceChemically Synthesized 2Asp Glu Cys Glu
Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
15314PRTArtificial SequenceChemically Synthesized 3Asp Glu Cys Glu
Leu Cys Val Asn Val Ala Cys Thr Gly Cys1 5 10414PRTArtificial
SequenceChemically Synthesized 4Glu Cys Glu Leu Cys Val Asn Val Ala
Cys Thr Gly Cys Leu1 5 10513PRTArtificial SequenceChemically
Synthesized 5Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys1 5
10613PRTArtificial SequenceChemically Synthesized 6Cys Glu Leu Cys
Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10712PRTArtificial
SequenceChemically Synthesized 7Cys Glu Leu Cys Val Asn Val Ala Cys
Thr Gly Cys1 5 10816PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid 8Asn
Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
15916PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid 9Asn Asp
Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
151016PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(2)..(2)wherein ASP is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid 10Asn Asp
Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
151116PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(2)..(2)wherein ASP is a D-amino
acidMISC_FEATURE(3)..(3)wherein GLU is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid 11Asn Asp
Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
151216PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino acid 12Asn
Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
151316PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(6)..(6)wherein LEU is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid 13Asn Asp
Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
151415PRTArtificial SequenceChemically Synthesized 14Asn Asp Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys1 5 10
151516PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(16)..(16)wherein LEU is
a D-amino acidMISC_FEATURE(16)..(16)wherein LEU at position 16 is
attached to polyethylene glycol 15Asn Asp Glu Cys Glu Leu Cys Val
Asn Val Ala Cys Thr Gly Cys Leu1 5 10 151616PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(1)..(1)wherein ASN at
position 1 is attached to polyethylene
glycolMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU at position 16 is attached to
polyethylene glycol 16Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala
Cys Thr Gly Cys Leu1 5 10 151716PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(1)..(1)wherein ASN is a
D-amino acidMISC_FEATURE(2)..(2)wherein ASP is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU at position 16 is attached to
polyethylene glycol 17Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala
Cys Thr Gly Cys Leu1 5 10 151816PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU at position 16 is attached to
polyethylene glycolMISC_FEATURE(16)..(16)wherein LEU is a D-amino
acid 18Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys
Leu1 5 10 151916PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(16)..(16)wherein LEU is
a D-amino acid 19Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys
Thr Gly Cys Leu1 5 10 152016PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(1)..(1)wherein ASN is a
D-amino acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid
20Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1
5 10 152116PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(16)..(16)wherein LEU at position 16 is
attached to polyethylene glycolMISC_FEATURE(16)..(16)wherein LEU is
a D-amino acid 21Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys
Thr Gly Cys Leu1 5 10 152216PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(1)..(1)wherein ASN is a
D-amino acidMISC_FEATURE(2)..(2)wherein ASP is a D-amino
acidMISC_FEATURE(3)..(3)wherein GLU is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU at position 16 is attached to
polyethylene glycol 22Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala
Cys Thr Gly Cys Leu1 5 10 152316PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(1)..(1)wherein ASN is a
D-amino acidMISC_FEATURE(2)..(2)wherein ASP is a D-amino
acidMISC_FEATURE(3)..(3)wherein GLU is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid 23Asn Asp
Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
152416PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(2)..(2)wherein ASP is a D-amino
acidMISC_FEATURE(3)..(3)wherein GLU is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU at position 16 is attached to
polyethylene glycolMISC_FEATURE(16)..(16)wherein LEU is a D-amino
acid 24Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys
Leu1 5 10 152516PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(2)..(2)wherein ASP is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU at position 16 is attached to
polyethylene glycolMISC_FEATURE(16)..(16)wherein LEU is a D-amino
acid 25Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys
Leu1 5 10 152616PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(1)..(1)wherein ASN is a
D-amino acidMISC_FEATURE(2)..(2)wherein ASP is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid 26Asn Asp
Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
152716PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein x is
3-(2-naphthyl)alanineMISC_FEATURE(16)..(16)wherein x is a D-amino
acid 27Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys
Xaa1 5 10 152816PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMOD_RES(8)..(8)wherein the x is a 2-aminoisobutyric acid,
AibMOD_RES(10)..(10)wherein the x is a 2-aminoisobutyric acid,
AibMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid 28Asn Asp
Glu Cys Glu Leu Cys Xaa Asn Xaa Ala Cys Thr Gly Cys Leu1 5 10
152916PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(7)..(7)wherein ASP at position 7 is attached to a
Lactam bridgeMISC_FEATURE(15)..(15)wherein x at position 15 is
ornithineMOD_RES(15)..(15)wherein x is an ornithine,
OrnMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid 29Asn Asp
Glu Cys Glu Leu Asp Val Asn Val Ala Cys Thr Gly Xaa Leu1 5 10
153016PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid 30Asn Asp
Glu Cys Glu Tyr Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
153116PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid 31Asn Asp
Glu Cys Glu Ser Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
153216PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(1)..(1)wherein ASN is a
D-amino acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU at position 16 is attached to
polyethylene glycol 32Asn Asp Glu Cys Glu Tyr Cys Val Asn Val Ala
Cys Thr Gly Cys Leu1 5 10 153316PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(1)..(1)wherein ASN is a
D-amino acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid
33Asn Asp Glu Cys Glu Tyr Cys Val Asn Val Ala Cys Thr Gly Cys Leu1
5 10 153416PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU at position 16 is attached to
polyethylene glycolMISC_FEATURE(16)..(16)wherein LEU is a D-amino
acid 34Asn Asp Glu Cys Glu Tyr Cys Val Asn Val Ala Cys Thr Gly Cys
Leu1 5 10 153516PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(1)..(1)wherein ASN is a
D-amino acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU at position 16 is attached to
polyethylene glycol 35Asn Asp Glu Cys Glu Ser Cys Val Asn Val Ala
Cys Thr Gly Cys Leu1 5 10 153616PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(1)..(1)wherein ASN is a
D-amino acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid
36Asn Asp Glu Cys Glu Ser Cys Val Asn Val Ala Cys Thr Gly Cys Leu1
5 10 153716PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU at position 16 is attached to
polyethylene glycolMISC_FEATURE(16)..(16)wherein LEU is a D-amino
acid 37Asn Asp Glu Cys Glu Ser Cys Val Asn Val Ala Cys Thr Gly Cys
Leu1 5 10 153816PRTArtificial SequenceChemically Synthesized 38Asn
Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
153916PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(16)..(16)wherein SER at
position 16 is attached to polyethylene glycol 39Asn Asp Glu Cys
Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
154016PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycol 40Asn Asp Glu Cys Glu Leu Cys Val
Asn Val Ala Cys Thr Gly Cys Ser1 5 10 154116PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(16)..(16)wherein SER at
position 16 is attached to polyethylene glycol 41Asn Asp Glu Cys
Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
154216PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(16)..(16)wherein SER is
a D-amino acidMISC_FEATURE(16)..(16)wherein SER at position 16 is
attached to polyethylene glycol 42Asn Asp Glu Cys Glu Leu Cys Val
Asn Val Ala Cys Thr Gly Cys Ser1 5 10 154316PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(1)..(1)wherein ASN at
position 1 is attached to polyethylene
glycolMISC_FEATURE(16)..(16)wherein SER is a D-amino acid 43Asn Asp
Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
154416PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(16)..(16)wherein SER at position 16 is
attached to polyethylene glycolMISC_FEATURE(16)..(16)wherein SER is
a D-amino acid 44Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys
Thr Gly Cys Ser1 5 10 154516PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(5)..(6)wherein x is any natural, or
unnatural amino acid or amino acid analogue, and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(8)..(11)wherein x is any natural, or unnatural
amino acid or amino acid analogue, and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(13)..(14)wherein x is any natural, or unnatural
amino acid or amino acid analogue, and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(16)..(16)wherein x is any natural, or unnatural
amino acid or amino acid analogue, and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino acid 45Asn
Asp Glu Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa1 5 10
154616PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(2)..(2)wherein x is any natural, or unnatural
amino acid or amino acid analogue that is zero or one residue in
length and may be an L-amino acid, or a D-amino acid, or a
methylated or an unmethylated amino acidMISC_FEATURE(3)..(3)wherein
x is any natural, or unnatural amino acid or amino acid analogue
that is zero or one residue in length and may be an L-amino acid,
or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(5)..(6)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(8)..(11)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(13)..(14)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(16)..(16)wherein x is any natural, or unnatural
amino acid, or amino acid analogue that is zero or one residue in
length and may be an L-amino acid, or a D-amino acid, or a
methylated or an unmethylated amino acid 46Xaa Xaa Xaa Cys Xaa Xaa
Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa1 5 10 154716PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(1)..(1)wherein x is any
natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(2)..(2)wherein x is any natural, or unnatural
amino acid or amino acid analogue that is zero or one residue in
length and may be an L-amino acid, or a D-amino acid, or a
methylated or an unmethylated amino acidMISC_FEATURE(3)..(3)wherein
x is any natural, or unnatural amino acid or amino acid analogue
that is zero or one residue in length and may be an L-amino acid,
or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(4)..(4)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(6)..(6)wherein x is
any natural, or unnatural amino acid, or amino acid analogue and
may be an L-amino acid, or a D-amino acid, or a methylated or an
unmethylated amino acidMISC_FEATURE(7)..(7)wherein x is a cysteine,
penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(12)..(12)wherein x is a cysteine,
penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(15)..(15)wherein x is a cysteine,
penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(16)..(16)wherein x is any natural, or
unnatural amino acid or amino acid analogue that is zero or one
residue in length and may be an L-amino acid, or a D-amino acid, or
a methylated or an unmethylated amino acid 47Xaa Xaa Xaa Xaa Glu
Xaa Xaa Val Asn Val Ala Xaa Thr Gly Xaa Xaa1 5 10
154816PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(2)..(2)wherein x is any natural, or unnatural
amino acid or amino acid analogue that is zero or one residue in
length and may be an L-amino acid, or a D-amino acid, or a
methylated or an unmethylated amino acidMISC_FEATURE(3)..(3)wherein
x is any natural, or unnatural amino acid or amino acid analogue
that is zero or one residue in length and may be an L-amino acid,
or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(4)..(4)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(5)..(6)wherein x is
any natural, or unnatural amino acid or amino acid analogue and may
be an L-amino acid, or a D-amino acid, or a methylated or an
unmethylated amino acidMISC_FEATURE(7)..(7)wherein x is a cysteine,
penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(8)..(11)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(12)..(12)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(13)..(14)wherein x
is any natural, or unnatural amino acid or amino acid analogue and
may be an L-amino acid, or a D-amino acid, or a methylated or an
unmethylated amino acidMISC_FEATURE(15)..(15)wherein x is a
cysteine, penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(16)..(16)wherein x is any natural, or
unnatural amino acid, or amino acid analogue that is zero or one
residue in length and may be an L-amino acid, or a D-amino acid, or
a methylated or an unmethylated amino acid 48Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10
154916PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(5)..(6)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(8)..(8)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(10)..(11)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(13)..(14)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(16)..(16)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino acid 49Asn
Asp Asp Cys Xaa Xaa Cys Xaa Asn Xaa Xaa Cys Xaa Xaa Cys Xaa1 5 10
155016PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(5)..(6)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(8)..(8)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(10)..(11)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(13)..(14)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(16)..(16)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino acid 50Asn
Glu Glu Cys Xaa Xaa Cys Xaa Asn Xaa Xaa Cys Xaa Xaa Cys Xaa1 5 10
155116PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(2)..(2)wherein GLU is a D-amino
acidMISC_FEATURE(5)..(6)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(8)..(8)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(10)..(11)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(13)..(14)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(16)..(16)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino acid 51Asn
Glu Asp Cys Xaa Xaa Cys Xaa Asn Xaa Xaa Cys Xaa Xaa Cys Xaa1 5 10
155216PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(2)..(2)wherein ASP is a D-amino
acidMISC_FEATURE(5)..(6)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(8)..(8)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(10)..(11)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(13)..(14)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(16)..(16)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino acid 52Asn
Asp Glu Cys Xaa Xaa Cys Xaa Asn Xaa Xaa Cys Xaa Xaa Cys Xaa1 5 10
155316PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(2)..(2)wherein ASP is a D-amino
acidMISC_FEATURE(3)..(3)wherein GLU is a D-amino
acidMISC_FEATURE(5)..(6)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(8)..(8)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(10)..(11)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(13)..(14)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(16)..(16)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino acid 53Asn
Asp Glu Cys Xaa Xaa Cys Xaa Tyr Xaa Xaa Cys Xaa Xaa Cys Xaa1 5 10
155416PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(2)..(2)wherein GLU is a D-amino
acidMISC_FEATURE(3)..(3)wherein GLU is a D-amino
acidMISC_FEATURE(5)..(6)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, a methylated or an unmethylated amino
acidMISC_FEATURE(8)..(8)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, a methylated or an unmethylated amino
acidMISC_FEATURE(10)..(11)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, a methylated or an unmethylated amino
acidMISC_FEATURE(13)..(14)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, a methylated or an unmethylated amino
acidMISC_FEATURE(16)..(16)wherein x is any natural, or unnatural
amino acid, or amino acid analogue and may be an L-amino acid, or a
D-amino acid, a methylated or an unmethylated amino acid 54Asn Glu
Glu Cys Xaa Xaa Cys Xaa Tyr Xaa Xaa Cys Xaa Xaa Cys Xaa1 5 10
155514PRTArtificial SequenceChemically Synthesized 55Cys Cys Glu
Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 105613PRTArtificial
SequenceChemically Synthesized 56Cys Cys Glu Tyr Cys Cys Asn Pro
Ala Cys Thr Gly Cys1 5 105714PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein CYS at position 1 is
attached to polyethylene glycolMISC_FEATURE(14)..(14)wherein TYR at
position 14 is attached to polyethylene glycol 57Cys Cys Glu Tyr
Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 105816PRTArtificial
SequenceChemically Synthesized 58Asn Phe Cys Cys Glu Ser Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10 155916PRTArtificial
SequenceChemically Synthesized 59Asn Phe Cys Cys Glu Phe Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10 156014PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(14)..(14)wherein TYR is
a D-amino acid 60Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly
Cys Tyr1 5 106114PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein CYS at position 1 is
attached to polyethylene glycol 61Cys Cys Glu Tyr Cys Cys Asn Pro
Ala Cys Thr Gly Cys Tyr1 5 106216PRTArtificial SequenceChemically
Synthesized 62Asn Phe Cys Cys Glu Thr Cys Cys Asn Pro Ala Cys Thr
Gly Cys Tyr1 5 10 156316PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(16)..(16)wherein TYR is a D-amino acid
63Asn Phe Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1
5 10 156416PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino acid 64Asn
Phe Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
156516PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein TYR is a D-amino acid 65Asn Phe
Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
156616PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(16)..(16)wherein TYR is a D-amino acid
66Asn Phe Cys Cys Glu Thr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1
5 10 156716PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino acid 67Asn
Phe Cys Cys Glu Thr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
156816PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein TYR is a D-amino acid 68Asn Phe
Cys Cys Glu Thr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
156916PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(16)..(16)wherein TYR is a D-amino acid
69Asn Phe Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1
5 10 157016PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino acid 70Asn
Phe Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
157116PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein TYR is a D-amino acid 71Asn Phe
Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
157214PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(14)..(14)wherein TYR at position 14 is
attached to polyethylene glycol 72Cys Cys Glu Tyr Cys Cys Asn Pro
Ala Cys Thr Gly Cys Tyr1 5 107313PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein CYS at position 1 is
attached to polyethylene glycolMISC_FEATURE(13)..(13)wherein CYS at
position 13 is attached to polyethylene glycol 73Cys Cys Glu Tyr
Cys Cys Asn Pro Ala Cys Thr Gly Cys1 5 107413PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(1)..(1)wherein CYS at
position 1 is attached to polyethylene glycol 74Cys Cys Glu Tyr Cys
Cys Asn Pro Ala Cys Thr Gly Cys1 5 107513PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(13)..(13)wherein CYS at
position 13 is attached to polyethylene glycol 75Cys Cys Glu Tyr
Cys Cys Asn Pro Ala Cys Thr Gly Cys1 5 107616PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(1)..(1)wherein ASN at
position 1 is attached to polyethylene
glycolMISC_FEATURE(16)..(16)wherein TYR at position 16 is attached
to polyethylene glycol 76Asn Phe Cys Cys Glu Ser Cys Cys Asn Pro
Ala Cys Thr Gly Cys Tyr1 5 10 157716PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(1)..(1)wherein ASN at
position 1 is attached to polyethylene glycol 77Asn Phe Cys Cys Glu
Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
157816PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(16)..(16)wherein TYR at position 16 is
attached to polyethylene glycol 78Asn Phe Cys Cys Glu Ser Cys Cys
Asn Pro Ala
Cys Thr Gly Cys Tyr1 5 10 157916PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(16)..(16)wherein TYR at
position 16 is attached to polyethylene glycol 79Asn Phe Cys Cys
Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
158016PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycol 80Asn Phe Cys Cys Glu Phe Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10 158116PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(16)..(16)wherein TYR at
position 16 is attached to polyethylene glycol 81Asn Phe Cys Cys
Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
158216PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycolMISC_FEATURE(16)..(16)wherein TYR at
position 16 is attached to polyethylene glycol 82Asn Phe Cys Cys
Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
158316PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN at position 1 is
attached to polyethylene glycol 83Asn Phe Cys Cys Glu Tyr Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10 158416PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(16)..(16)wherein TYR at
position 16 is attached to polyethylene glycol 84Asn Phe Cys Cys
Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
158514PRTArtificial SequenceChemically Synthesized 85Cys Cys Glu
Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 108614PRTArtificial
SequenceChemically Synthesized 86Cys Cys Glu Phe Cys Cys Asn Pro
Ala Cys Thr Gly Cys Tyr1 5 108713PRTArtificial SequenceChemically
Synthesized 87Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys1
5 108813PRTArtificial SequenceChemically Synthesized 88Cys Cys Glu
Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys1 5 108914PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(1)..(2)wherein x is
penicillamineMISC_FEATURE(5)..(6)wherein x is
penicillamineMISC_FEATURE(10)..(10)wherein x is
penicillamineMISC_FEATURE(13)..(13)wherein x is penicillamine 89Xaa
Xaa Glu Tyr Xaa Xaa Asn Pro Ala Xaa Thr Gly Xaa Tyr1 5
109013PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(2)wherein x is
penicillamineMISC_FEATURE(5)..(6)wherein x is
penicillamineMISC_FEATURE(10)..(10)wherein x is
penicillamineMISC_FEATURE(13)..(13)wherein x is penicillamine 90Xaa
Xaa Glu Tyr Xaa Xaa Asn Pro Ala Xaa Thr Gly Xaa1 5
109122PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(6)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(11)..(11)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(15)..(17)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(19)..(20)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(22)..(22)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino acid 91Xaa
Xaa Xaa Xaa Xaa Xaa Asn Tyr Cys Cys Xaa Tyr Cys Cys Xaa Xaa1 5 10
15Xaa Cys Xaa Xaa Cys Xaa 209222PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(6)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(11)..(11)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(15)..(17)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(19)..(20)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(22)..(22)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino acid 92Xaa
Xaa Xaa Xaa Xaa Xaa Asn Phe Cys Cys Xaa Phe Cys Cys Xaa Xaa1 5 10
15Xaa Cys Xaa Xaa Cys Xaa 209316PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(5)..(5)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(9)..(11)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(13)..(14)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(16)..(16)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino acid 93Asn
Phe Cys Cys Xaa Phe Cys Cys Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa1 5 10
159416PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(3)..(3)wherein x is
penicillamineMISC_FEATURE(5)..(5)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(8)..(8)wherein x is
penicillamineMISC_FEATURE(9)..(11)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(13)..(14)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(16)..(16)wherein x is any natural, or unnatural
amino acid or amino acid analogue and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino acid 94Asn
Phe Xaa Cys Xaa Phe Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa1 5 10
159516PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(3)..(4)wherein x is a cysteine,
penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(5)..(6)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(7)..(8)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(9)..(11)wherein x is
any natural, or unnatural amino acid or amino acid analogue and may
be an L-amino acid, or a D-amino acid, or a methylated or an
unmethylated amino acidMISC_FEATURE(12)..(12)wherein x is a
cysteine, penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(13)..(14)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(15)..(15)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(16)..(16)wherein x
is any natural, or unnatural amino acid or amino acid analogue and
may be an L-amino acid, or a D-amino acid, or a methylated or an
unmethylated amino acid 95Asn Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 159614PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(1)..(2)wherein x is a
cysteine, penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(4)..(4)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(5)..(6)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(10)..(10)wherein x
is a cysteine, penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(13)..(13)wherein x is a cysteine,
penicillamine homocysteine, or 3-mercaptoproline 96Xaa Xaa Glu Xaa
Xaa Xaa Asn Pro Ala Xaa Thr Gly Xaa Tyr1 5 109713PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(1)..(2)wherein x is a
cysteine, or penicillamine, homocysteine, or
3-mercaptoprolineMISC_FEATURE(4)..(4)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(5)..(6)wherein x is a cysteine, or penicillamine,
homocysteine, or 3-mercaptoprolineMISC_FEATURE(10)..(10)wherein x
is a cysteine, or penicillamine, homocysteine, or
3-mercaptoprolineMISC_FEATURE(13)..(13)wherein x is a cysteine, or
penicillamine, homocysteine, or 3-mercaptoproline 97Xaa Xaa Glu Xaa
Xaa Xaa Asn Pro Ala Xaa Thr Gly Xaa1 5 109820PRTArtificial
SequenceChemically SynthesizedMISC_FEATURE(1)..(1)wherein x is any
natural, or unnatural amino acid or amino acid analogue, and may be
zero or one residue in length, and may be an L-amino acid, or a
D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(2)..(2)wherein x is any natural, or unnatural
amino acid or amino acid analogue, and may be zero or one residue
in length, and may be an L-amino acid, or a D-amino acid, or a
methylated or an unmethylated amino acidMISC_FEATURE(3)..(3)wherein
x is any natural, or unnatural amino acid or amino acid analogue,
and may be zero or one residue in length, and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(4)..(4)wherein x is any natural, or unnatural
amino acid or amino acid analogue, and may be zero or one residue
in length, and may be an L-amino acid, or a D-amino acid, or a
methylated or an unmethylated amino acidMISC_FEATURE(5)..(5)wherein
x is any natural, or unnatural amino acid or amino acid analogue,
and may be zero or one residue in length, and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(6)..(6)wherein x is any natural, or unnatural
amino acid or amino acid analogue, and may be zero or one residue
in length, and may be an L-amino acid, or a D-amino acid, or a
methylated or an unmethylated amino acidMISC_FEATURE(7)..(8)wherein
x is a cysteine, penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(9)..(10)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(11)..(12)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(13)..(15)wherein x
is any natural, or unnatural amino acid or amino acid analogue and
may be an L-amino acid, or a D-amino acid, or a methylated or an
unmethylated amino acidMISC_FEATURE(16)..(16)wherein x is a
cysteine, penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(17)..(18)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(19)..(19)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(20)..(20)wherein x
is any natural, or unnatural amino acid or amino acid analogue, and
may be zero or one residue in length, and may be an L-amino acid,
or a D-amino acid, or a methylated or an unmethylated amino acid
98Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
5 10 15Xaa Xaa Xaa Xaa 209916PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU is a D-amino
acidMISC_FEATURE(16)..(16)wherein LEU is conjugated to an AMIDE
99Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1
5 10 1510016PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein SER is a D-amino acid 100Asn Asp
Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1510116PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein SER is a D-amino
acidMISC_FEATURE(16)..(16)wherein SER is conjugated to an AMIDE
101Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1
5 10 1510216PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein TYR is a D-amino acid 102Asn Asp
Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Tyr1 5 10
1510316PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein TYR is a D-amino
acidMISC_FEATURE(16)..(16)wherein TYR is conjugated to an AMIDE
103Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Tyr1
5 10 1510416PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein x is Pyroglutamic
acidMISC_FEATURE(16)..(16)wherein LEU is conjugated to an
AMIDEMISC_FEATURE(16)..(16)wherein LEU is a D-amino acid 104Xaa Asp
Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1510516PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is attached to
polyethylene glycolMISC_FEATURE(16)..(16)wherein LEU is attached to
polyethylene glycol 105Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala
Cys Thr Gly Cys Leu1 5 10 1510616PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is attached to
polyethylene glycol 106Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala
Cys Thr Gly Cys Leu1 5 10 1510716PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(16)..(16)wherein LEU is attached to
polyethylene glycol 107Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala
Cys Thr Gly Cys Leu1 5 10 1510816PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(3)wherein x is any natural, or
unnatural amino acid or amino acid analogue and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(4)..(4)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(5)..(6)wherein x is
any natural, or unnatural amino acid or amino acid analogue, and
may be an L-amino acid, or a D-amino acid, or a methylated or an
unmethylated amino acidMISC_FEATURE(7)..(7)wherein x is a cysteine,
penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(8)..(11)wherein x is any natural,
or
unnatural amino acid or amino acid analogue, and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(12)..(12)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(13)..(14)wherein x
is any natural, or unnatural amino acid or amino acid analogue, and
may be an L-amino acid, or a D-amino acid, or a methylated or an
unmethylated amino acidMISC_FEATURE(15)..(15)wherein x is a
cysteine, penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(16)..(16)wherein x is any natural, or
unnatural amino acid or amino acid analogue, and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acid 108Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa1 5 10 1510916PRTArtificial SequenceChemically Synthesized
109Asn Asp Asp Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1
5 10 1511016PRTArtificial SequenceChemically Synthesized 110Glu Asp
Asp Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1511116PRTArtificial SequenceChemically Synthesized 111Glu Asp Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1511216PRTArtificial SequenceChemically Synthesized 112Glu Glu Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1511316PRTArtificial SequenceChemically Synthesized 113Glu Glu Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1511416PRTArtificial SequenceChemically Synthesized 114Asp Asp Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1511516PRTArtificial SequenceChemically Synthesized 115Asp Asp Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1511616PRTArtificial SequenceChemically Synthesized 116Asp Glu Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1511716PRTArtificial SequenceChemically Synthesized 117Asp Glu Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1511816PRTArtificial SequenceChemically Synthesized 118Gln Asp Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1511916PRTArtificial SequenceChemically Synthesized 119Gln Asp Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1512016PRTArtificial SequenceChemically Synthesized 120Gln Glu Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1512116PRTArtificial SequenceChemically Synthesized 121Gln Glu Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1512216PRTArtificial SequenceChemically Synthesized 122Lys Asp Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1512316PRTArtificial SequenceChemically Synthesized 123Lys Asp Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1512416PRTArtificial SequenceChemically Synthesized 124Lys Glu Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1512516PRTArtificial SequenceChemically Synthesized 125Lys Glu Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu1 5 10
1512616PRTArtificial SequenceChemically Synthesized 126Glu Asp Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1512716PRTArtificial SequenceChemically Synthesized 127Glu Asp Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1512816PRTArtificial SequenceChemically Synthesized 128Glu Glu Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1512916PRTArtificial SequenceChemically Synthesized 129Glu Glu Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1513016PRTArtificial SequenceChemically Synthesized 130Asp Asp Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1513116PRTArtificial SequenceChemically Synthesized 131Asp Asp Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1513216PRTArtificial SequenceChemically Synthesized 132Asp Glu Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1513316PRTArtificial SequenceChemically Synthesized 133Asp Glu Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1513416PRTArtificial SequenceChemically Synthesized 134Gln Asp Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1513516PRTArtificial SequenceChemically Synthesized 135Gln Asp Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1513616PRTArtificial SequenceChemically Synthesized 136Gln Glu Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1513716PRTArtificial SequenceChemically Synthesized 137Gln Glu Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1513816PRTArtificial SequenceChemically Synthesized 138Lys Asp Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1513916PRTArtificial SequenceChemically Synthesized 139Lys Asp Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1514016PRTArtificial SequenceChemically Synthesized 140Lys Glu Asp
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1514116PRTArtificial SequenceChemically Synthesized 141Lys Glu Glu
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Ser1 5 10
1514216PRTArtificial SequenceChemically Synthesized 142Glu Asp Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1514316PRTArtificial SequenceChemically Synthesized 143Glu Asp Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1514416PRTArtificial SequenceChemically Synthesized 144Glu Glu Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1514516PRTArtificial SequenceChemically Synthesized 145Glu Glu Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1514616PRTArtificial SequenceChemically Synthesized 146Asp Asp Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1514716PRTArtificial SequenceChemically Synthesized 147Asp Asp Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1514816PRTArtificial SequenceChemically Synthesized 148Asp Glu Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1514916PRTArtificial SequenceChemically Synthesized 149Asp Glu Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1515016PRTArtificial SequenceChemically Synthesized 150Gln Asp Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1515116PRTArtificial SequenceChemically Synthesized 151Gln Asp Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1515216PRTArtificial SequenceChemically Synthesized 152Gln Glu Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1515316PRTArtificial SequenceChemically Synthesized 153Gln Glu Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1515416PRTArtificial SequenceChemically Synthesized 154Lys Asp Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1515516PRTArtificial SequenceChemically Synthesized 155Lys Asp Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1515616PRTArtificial SequenceChemically Synthesized 156Lys Glu Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1515716PRTArtificial SequenceChemically Synthesized 157Lys Glu Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Leu1 5 10
1515816PRTArtificial SequenceChemically Synthesized 158Glu Asp Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1515916PRTArtificial SequenceChemically Synthesized 159Glu Asp Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1516016PRTArtificial SequenceChemically Synthesized 160Glu Glu Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1516116PRTArtificial SequenceChemically Synthesized 161Glu Glu Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1516216PRTArtificial SequenceChemically Synthesized 162Asp Asp Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1516316PRTArtificial SequenceChemically Synthesized 163Asp Asp Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1516416PRTArtificial SequenceChemically Synthesized 164Asp Glu Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1516516PRTArtificial SequenceChemically Synthesized 165Asp Glu Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1516616PRTArtificial SequenceChemically Synthesized 166Gln Asp Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1516716PRTArtificial SequenceChemically Synthesized 167Gln Asp Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1516816PRTArtificial SequenceChemically Synthesized 168Gln Glu Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1516916PRTArtificial SequenceChemically Synthesized 169Gln Glu Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1517016PRTArtificial SequenceChemically Synthesized 170Lys Asp Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1517116PRTArtificial SequenceChemically Synthesized 171Lys Asp Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1517216PRTArtificial SequenceChemically Synthesized 172Lys Glu Asp
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1517316PRTArtificial SequenceChemically Synthesized 173Lys Glu Glu
Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1517415PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(3)wherein x is any natural, or
unnatural amino acid, or amino acid analogue, and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(4)..(4)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(5)..(6)wherein x is
any natural, or unnatural amino acid, or amino acid analogue, and
may be an L-amino acid, or a D-amino acid, or a methylated or an
unmethylated amino acidMISC_FEATURE(7)..(7)wherein x is a cysteine,
penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(8)..(11)wherein x is any natural, or
unnatural amino acid, or amino acid analogue, and may be an L-amino
acid, or a D-amino acid, or a methylated or an unmethylated amino
acidMISC_FEATURE(12)..(12)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(13)..(14)wherein x
is any natural, or unnatural amino acid, or amino acid analogue,
and may be an L-amino acid, or a D-amino acid, or a methylated or
an unmethylated amino acidMISC_FEATURE(15)..(15)wherein x is a
cysteine, penicillamine homocysteine, or 3-mercaptoproline 174Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10
1517515PRTArtificial SequenceChemically Synthesized 175Ser His Thr
Cys Glu Ile Cys Ala Phe Ala Ala Cys Ala Gly Cys1 5 10
1517615PRTArtificial SequenceChemically Synthesized 176Ser His Thr
Cys Glu Ile Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1517715PRTArtificial SequenceChemically Synthesized 177Ser His Thr
Cys Glu Leu Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1517815PRTArtificial SequenceChemically Synthesized 178Ser His Thr
Cys Glu Val Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1517915PRTArtificial SequenceChemically Synthesized 179Ser His Thr
Cys Glu Tyr Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1518015PRTArtificial SequenceChemically Synthesized 180Ser His Thr
Cys Glu Ile Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1518115PRTArtificial SequenceChemically Synthesized 181Ser His Thr
Cys Glu Leu Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1518215PRTArtificial SequenceChemically Synthesized 182Ser His Thr
Cys Glu Val Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1518315PRTArtificial SequenceChemically Synthesized 183Ser His Thr
Cys Glu Tyr Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1518415PRTArtificial SequenceChemically Synthesized 184Ser His Thr
Cys Glu Ile Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1518515PRTArtificial SequenceChemically Synthesized 185Ser His Thr
Cys Glu Leu Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1518615PRTArtificial SequenceChemically Synthesized 186Ser His Thr
Cys Glu Val Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1518715PRTArtificial SequenceChemically Synthesized 187Ser His Thr
Cys Glu Tyr Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1518815PRTArtificial SequenceChemically Synthesized 188Ser His Thr
Cys Glu Ile Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1518915PRTArtificial SequenceChemically Synthesized 189Ser His Thr
Cys Glu Leu Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1519015PRTArtificial SequenceChemically Synthesized 190Ser His Thr
Cys Glu Val Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1519115PRTArtificial SequenceChemically Synthesized 191Ser His Thr
Cys Glu Tyr Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1519215PRTArtificial SequenceChemically Synthesized 192Asn Asp Glu
Cys Glu Ile Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1519315PRTArtificial SequenceChemically Synthesized 193Asn Asp Glu
Cys Glu Leu Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1519415PRTArtificial SequenceChemically Synthesized 194Asn Asp Glu
Cys Glu Val Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1519515PRTArtificial SequenceChemically Synthesized 195Asn Asp Glu
Cys Glu Tyr Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1519615PRTArtificial SequenceChemically Synthesized 196Asn Asp Glu
Cys Glu Ile Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1519715PRTArtificial SequenceChemically
Synthesized 197Asn Asp Glu Cys Glu Leu Cys Ala Asn Ala Ala Cys Ala
Gly Cys1 5 10 1519815PRTArtificial SequenceChemically Synthesized
198Asn Asp Glu Cys Glu Val Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5
10 1519915PRTArtificial SequenceChemically Synthesized 199Asn Asp
Glu Cys Glu Tyr Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1520015PRTArtificial SequenceChemically Synthesized 200Asn Asp Glu
Cys Glu Ile Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1520115PRTArtificial SequenceChemically Synthesized 201Asn Asp Glu
Cys Glu Leu Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1520215PRTArtificial SequenceChemically Synthesized 202Asn Asp Glu
Cys Glu Val Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1520315PRTArtificial SequenceChemically Synthesized 203Asn Asp Glu
Cys Glu Tyr Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1520415PRTArtificial SequenceChemically Synthesized 204Asn Asp Glu
Cys Glu Ile Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1520515PRTArtificial SequenceChemically Synthesized 205Asn Asp Glu
Cys Glu Leu Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1520615PRTArtificial SequenceChemically Synthesized 206Asn Asp Glu
Cys Glu Val Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1520715PRTArtificial SequenceChemically Synthesized 207Asn Asp Glu
Cys Glu Tyr Cys Ala Asn Ala Ala Cys Ala Gly Cys1 5 10
1520817PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(3)wherein x is any natural, or
unnatural amino acid, or amino acid analogue, and may be an L-amino
acid, or a D-amino acid, or a methylated, or an unmethylated amino
acidMISC_FEATURE(4)..(4)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(5)..(6)wherein x is
any natural, or unnatural amino acid, or amino acid analogue, and
may be an L-amino acid, or a D-amino acid, or a methylated, or an
unmethylated amino acidMISC_FEATURE(7)..(7)wherein x is a cysteine,
penicillamine homocysteine, or
3-mercaptoprolineMISC_FEATURE(8)..(11)wherein x is any natural, or
unnatural amino acid, or amino acid analogue, and may be an L-amino
acid, or a D-amino acid, or a methylated, or an unmethylated amino
acidMISC_FEATURE(12)..(12)wherein x is a cysteine, penicillamine
homocysteine, or 3-mercaptoprolineMISC_FEATURE(13)..(14)wherein x
is any natural, or unnatural amino acid, or amino acid analogue,
and may be an L-amino acid, or a D-amino acid, or a methylated, or
an unmethylated amino acidMISC_FEATURE(15)..(15)wherein x is any
natural, or unnatural amino acid, or amino acid analogue and may be
an L-amino acid, or a D-amino acid, or a methylated, or an
unmethylated amino acidMISC_FEATURE(16)..(16)wherein x is any
natural, or unnatural amino acid, or amino acid analogue, and may
be zero or one residue in length, and may be an L-amino acid, or a
D-amino acid, or a methylated, or an unmethylated amino
acidMISC_FEATURE(17)..(17)wherein x is any natural, or unnatural
amino acid, or amino acid analogue, and may be zero or one residue
in length, and may be an L-amino acid, or a D-amino acid, or a
methylated, or an unmethylated amino acid 208Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10
15Xaa20915PRTArtificial SequenceChemically Synthesized 209Gln Glu
Glu Cys Glu Leu Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1521015PRTArtificial SequenceChemically Synthesized 210Gln Glu Glu
Cys Glu Thr Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1521115PRTArtificial SequenceChemically Synthesized 211Gln Asp Glu
Cys Glu Thr Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1521215PRTArtificial SequenceChemically Synthesized 212Gln Asp Asp
Cys Glu Thr Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1521315PRTArtificial SequenceChemically Synthesized 213Gln Glu Asp
Cys Glu Thr Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1521415PRTArtificial SequenceChemically Synthesized 214Gln Glu Glu
Cys Glu Glu Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1521515PRTArtificial SequenceChemically Synthesized 215Gln Asp Glu
Cys Glu Glu Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1521615PRTArtificial SequenceChemically Synthesized 216Gln Asp Asp
Cys Glu Glu Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1521715PRTArtificial SequenceChemically Synthesized 217Gln Glu Asp
Cys Glu Glu Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1521815PRTArtificial SequenceChemically Synthesized 218Gln Glu Glu
Cys Glu Tyr Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1521915PRTArtificial SequenceChemically Synthesized 219Gln Asp Glu
Cys Glu Tyr Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1522015PRTArtificial SequenceChemically Synthesized 220Gln Asp Asp
Cys Glu Tyr Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1522115PRTArtificial SequenceChemically Synthesized 221Gln Glu Asp
Cys Glu Tyr Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1522215PRTArtificial SequenceChemically Synthesized 222Gln Glu Glu
Cys Glu Ile Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1522315PRTArtificial SequenceChemically Synthesized 223Gln Asp Glu
Cys Glu Ile Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1522415PRTArtificial SequenceChemically Synthesized 224Gln Asp Asp
Cys Glu Ile Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1522515PRTArtificial SequenceChemically Synthesized 225Gln Glu Asp
Cys Glu Ile Cys Ile Asn Met Ala Cys Thr Gly Tyr1 5 10
1522616PRTArtificial SequenceChemically Synthesized 226Gln Glu Glu
Cys Glu Thr Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1522716PRTArtificial SequenceChemically Synthesized 227Gln Asp Glu
Cys Glu Thr Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1522816PRTArtificial SequenceChemically Synthesized 228Gln Asp Asp
Cys Glu Thr Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1522916PRTArtificial SequenceChemically Synthesized 229Gln Glu Asp
Cys Glu Thr Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1523016PRTArtificial SequenceChemically Synthesized 230Gln Glu Glu
Cys Glu Glu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1523116PRTArtificial SequenceChemically Synthesized 231Gln Asp Glu
Cys Glu Glu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1523216PRTArtificial SequenceChemically Synthesized 232Gln Asp Asp
Cys Glu Glu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1523316PRTArtificial SequenceChemically Synthesized 233Gln Glu Asp
Cys Glu Glu Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1523416PRTArtificial SequenceChemically Synthesized 234Gln Glu Glu
Cys Glu Tyr Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1523516PRTArtificial SequenceChemically Synthesized 235Gln Asp Glu
Cys Glu Tyr Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1523616PRTArtificial SequenceChemically Synthesized 236Gln Asp Asp
Cys Glu Tyr Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1523716PRTArtificial SequenceChemically Synthesized 237Gln Glu Asp
Cys Glu Tyr Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1523816PRTArtificial SequenceChemically Synthesized 238Gln Glu Glu
Cys Glu Ile Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1523916PRTArtificial SequenceChemically Synthesized 239Gln Asp Glu
Cys Glu Ile Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1524016PRTArtificial SequenceChemically Synthesized 240Gln Asp Asp
Cys Glu Ile Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1524116PRTArtificial SequenceChemically Synthesized 241Gln Glu Asp
Cys Glu Ile Cys Ile Asn Met Ala Cys Thr Gly Cys Ser1 5 10
1524222PRTArtificial SequenceChemically Synthesized 242Asn Ser Ser
Asn Ser Ser Asn Tyr Cys Cys Glu Lys Cys Cys Asn Pro1 5 10 15Ala Cys
Thr Gly Cys Tyr 2024316PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is attached to
polyethylene glycolMISC_FEATURE(16)..(16)wherein TYR is attached to
polyethylene glycol 243Asn Phe Cys Cys Glu Thr Cys Cys Asn Pro Ala
Cys Thr Gly Cys Tyr1 5 10 1524416PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is attached to
polyethylene glycol 244Asn Phe Cys Cys Glu Thr Cys Cys Asn Pro Ala
Cys Thr Gly Cys Tyr1 5 10 1524516PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(16)..(16)wherein TYR is attached to
polyethylene glycol 245Asn Phe Cys Cys Glu Thr Cys Cys Asn Pro Ala
Cys Thr Gly Cys Tyr1 5 10 1524616PRTArtificial SequenceChemically
Synthesized 246Asn Phe Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr
Gly Cys Tyr1 5 10 1524716PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino
acidMISC_FEATURE(16)..(16)wherein TYR is a D-amino acid 247Asn Phe
Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
1524816PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(16)..(16)wherein TYR is a D-amino acid
248Asn Phe Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1
5 10 1524916PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein ASN is a D-amino acid 249Asn
Phe Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10
1525034PRTArtificial SequenceChemically Synthesized 250Ile Lys Pro
Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn1 5 10 15Arg Tyr
Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30Arg
Tyr25127PRTArtificial SequenceChemically
SynthesizedMISC_FEATURE(1)..(1)wherein HIS is conjugated to an
AMINE having the general formula R-NH, and wherein R is a hydrogen
or an organic compound having one, two, three, four, five, six,
seven, eight, nine, or ten carbon
atomsMISC_FEATURE(27)..(27)wherein LYS is conjugated to an AMIDE
251His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Tyr Leu Glu Gly Gln1
5 10 15Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys 20
252525PRTArtificial SequenceChemically Synthesized
Sialorphin-related polypeptide 252Gln His Asn Pro Arg1
52536PRTArtificial SequenceChemically Synthesized
Sialorphin-related polypeptide 253Val Gln His Asn Pro Arg1
52547PRTArtificial SequenceChemically Synthesized
Sialorphin-related polypeptide 254Val Arg Gln His Asn Pro Arg1
52558PRTArtificial SequenceChemically Synthesized
Sialorphin-related polypeptide 255Val Arg Gly Gln His Asn Pro Arg1
52569PRTArtificial SequenceChemically Synthesized
Sialorphin-related polypeptide 256Val Arg Gly Pro Gln His Asn Pro
Arg1 525710PRTArtificial SequenceChemically Synthesized
Sialorphin-related polypeptide 257Val Arg Gly Pro Arg Gln His Asn
Pro Arg1 5 1025811PRTArtificial SequenceChemically Synthesized
Sialorphin-related polypeptide 258Val Arg Gly Pro Arg Arg Gln His
Asn Pro Arg1 5 102596PRTArtificial SequenceChemically Synthesized
Sialorphin-related polypeptide 259Arg Gln His Asn Pro Arg1
52605PRTArtificial SequenceChemically Synthesized Enkephalin
PentapeptideMISC_FEATURE(2)..(2)wherein LYS is a D-amino
acidMISC_FEATURE(5)..(5)wherein x is a L-homoserine 260Tyr Lys Gly
Phe Xaa1 52614PRTArtificial SequenceChemically Synthesized
FrakefamideMISC_FEATURE(2)..(2)wherein ALA is a D-amino
acidMOD_RES(3)..(3)wherein PHE is fluorinated at the phenyl group
at 4-positionMISC_FEATURE(4)..(4)wherein PHE is attached to an
AMINE 261Tyr Ala Phe Phe1
* * * * *
References