U.S. patent application number 17/459451 was filed with the patent office on 2021-12-16 for treatments for gastrointestinal disorders.
The applicant listed for this patent is Ironwood Pharmaceuticals, Inc.. Invention is credited to Angelika Fretzen, Marco Kessler, Hong Zhao.
Application Number | 20210388026 17/459451 |
Document ID | / |
Family ID | 1000005807891 |
Filed Date | 2021-12-16 |
United States Patent
Application |
20210388026 |
Kind Code |
A1 |
Fretzen; Angelika ; et
al. |
December 16, 2021 |
Treatments for Gastrointestinal Disorders
Abstract
The present invention features peptides, compositions, and
related methods for treating gastrointestinal disorders and
conditions, including but not limited to, irritable bowel syndrome
(IBS), gastrointestinal motility disorders, functional
gastrointestinal disorders, gastroesophageal reflux disease (GERD),
duodenogastric reflux, Crohn's disease, ulcerative colitis,
inflammatory bowel disease, functional heartburn, dyspepsia,
visceral pain, gastroparesis, chronic intestinal pseudo-obstruction
(or colonic pseudo-obstruction), disorders and conditions
associated with constipation, and other conditions and disorders
are described herein. using peptides and other agents that activate
the guanylate cyclase C (GC-C) receptor.
Inventors: |
Fretzen; Angelika;
(Somerville, MA) ; Zhao; Hong; (Lexington, MA)
; Kessler; Marco; (Danvers, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ironwood Pharmaceuticals, Inc. |
Boston |
MA |
US |
|
|
Family ID: |
1000005807891 |
Appl. No.: |
17/459451 |
Filed: |
August 27, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17140267 |
Jan 4, 2021 |
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17459451 |
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16881198 |
May 22, 2020 |
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17140267 |
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16585139 |
Sep 27, 2019 |
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16881198 |
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16270802 |
Feb 8, 2019 |
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16585139 |
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16009560 |
Jun 15, 2018 |
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16270802 |
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15729875 |
Oct 11, 2017 |
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16009560 |
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14556573 |
Dec 1, 2014 |
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15729875 |
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13579685 |
Mar 29, 2013 |
8933030 |
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PCT/US2011/025274 |
Feb 17, 2011 |
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14556573 |
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61305465 |
Feb 17, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 38/08 20130101; C07K 7/08 20130101; A61K 38/10 20130101; C07K
7/06 20130101 |
International
Class: |
C07K 7/08 20060101
C07K007/08; A61K 38/10 20060101 A61K038/10; A61K 45/06 20060101
A61K045/06; A61K 38/08 20060101 A61K038/08; C07K 7/06 20060101
C07K007/06 |
Claims
1. A peptide or a pharmaceutically acceptable salt thereof, wherein
the peptide comprises the amino acid sequence Cys Cys Glu Tyr Cys
Cys Asn Pro Ala Cys Thr Gly Cys Tyr, wherein at least one carboxyl
group of the peptide is an alkyl ester having the formula (--COOR)
in which R is a C.sub.1-6 alkyl.
2. The peptide or a pharmaceutically acceptable salt thereof
according to claim 1, wherein the peptide comprises the amino acid
structure of: ##STR00045## or a pharmaceutically acceptable salt
thereof, wherein R' is H or a C.sub.1-6 alkyl, and at least one R'
is C.sub.1-6 alkyl.
3. The peptide or pharmaceutically acceptable salt thereof
according to claim 1, wherein the peptide comprises the amino acid
structure of: ##STR00046## wherein R is a C.sub.1-6 alkyl.
4. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1 or 3, wherein R is a C.sub.1-4
alkyl.
5. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1, 3, or 4, wherein R is methyl.
6. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1, 3, or 4, wherein R is ethyl.
7. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1, 3, or 4, wherein R is propyl.
8. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1, 3, 4, or 6 wherein the peptide
comprises the amino acid structure of: ##STR00047##
9. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1, 3, 4, or 7 wherein the peptide
comprises the amino acid structure of: ##STR00048##
10. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1, 3, 4, or 5 wherein the peptide
comprises the amino acid structure of: ##STR00049##
11. The peptide or pharmaceutically acceptable salt thereof
according to claim 1, wherein the peptide comprises the amino acid
structure of: ##STR00050## wherein R is C.sub.1-6 alkyl.
12. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1 or 11, wherein R is a C.sub.1-4
alkyl.
13. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1, 11, or 12, wherein R is
methyl.
14. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1, 11, or 12, wherein R is ethyl.
15. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1, 11, or 12, wherein R is
propyl.
16. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1, 11, 12, or 14, wherein the peptide
comprises the amino acid structure of: ##STR00051##
17. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1, 11, 12, or 15, wherein the peptide
comprises the amino acid structure of: ##STR00052##
18. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1, 11, 12, or 13, wherein the peptide
comprises the amino acid structure of: ##STR00053##
19. A peptide or a pharmaceutically acceptable salt thereof,
wherein the peptide comprises the amino acid structure of:
##STR00054##
20. A peptide or a pharmaceutically acceptable salt thereof,
wherein the peptide consists of the amino acid sequence Cys Cys Glu
Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, wherein at least one
carboxyl group of the peptide is an alkyl ester having the formula
(--COOR) in which R is a C.sub.1-6 alkyl.
21. The peptide or a pharmaceutically acceptable salt thereof
according to claim 20, wherein the peptide consists of the amino
acid structure of: ##STR00055## or a pharmaceutically acceptable
salt thereof, wherein R' is H or a C.sub.1-6 alkyl, and at least
one R' is C.sub.1-6 alkyl.
22. The peptide or pharmaceutically acceptable salt thereof
according to claim 20, wherein the peptide consists of the amino
acid structure of: ##STR00056## wherein R is a C.sub.1-6 alkyl.
23. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20 or 22, wherein R is a C.sub.1-4
alkyl.
24. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20, 22, or 23, wherein R is
methyl.
25. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20, 22, or 23, wherein R is
ethyl.
26. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20, 22, or 23, wherein R is
propyl.
27. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20, 22, 23, or 25, wherein the
peptide consists of the amino acid structure of: ##STR00057##
28. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20, 22, 23, or 26, wherein the
peptide consists of the amino acid structure of: ##STR00058##
29. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20, 22, 23, or 24, wherein the
peptide consists of the amino acid structure of: ##STR00059##
30. A peptide or pharmaceutically acceptable salt thereof according
to claim 20, wherein the peptide consists of the amino acid
structure of: ##STR00060## wherein R is C.sub.1-6 alkyl.
31. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20 or 30, wherein R is a C.sub.1-4
alkyl.
32. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20, 30, or 31, wherein R is
methyl.
33. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20, 30, or 31, wherein R is
ethyl.
34. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20, 30, or 31, wherein R is
propyl.
35. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20, 30, 31, or 33, wherein the
peptide consists of the amino acid structure of: ##STR00061##
36. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20, 30, 31, or 34, wherein the
peptide consists of the amino acid structure of: ##STR00062##
37. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 20, 30, 31, or 32, wherein the
peptide consists of the amino acid structure of: ##STR00063##
38. A peptide or pharmaceutically acceptable salt thereof, wherein
the peptide consists of the amino acid structure of:
##STR00064##
39. The peptide or pharmaceutically acceptable salt thereof
according to any one of claims 1-38, wherein the peptide activates
the guanylate cyclase C receptor.
40. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1-19 or 39, wherein the peptide
comprises 30 or fewer amino acids.
41. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1-19 or 39, wherein the peptide
comprises 20 or fewer amino acids.
42. The peptide or pharmaceutically acceptable salt thereof
according to any one of claim 1-19 or 39, wherein fewer than five
amino acids precede the first Cys residue of the amino acid
sequence.
43. The peptide or pharmaceutically acceptable salt thereof
according to any one of claims 1-42, wherein said peptide or
pharmaceutically acceptable salt thereof is isolated.
44. The peptide or pharmaceutically acceptable salt thereof
according to any one of claims 1-43, wherein said peptide or
pharmaceutically acceptable salt thereof is purified.
45. A pharmaceutical composition comprising a peptide or
pharmaceutically acceptable salt thereof according to any one of
claims 1-44.
46. A pharmaceutical composition comprising two or more peptides or
pharmaceutically acceptable salts thereof according to any one of
claims 1-44.
47. A pharmaceutical composition comprising two or more peptides
selected from: i. a peptide or a pharmaceutically acceptable salt
thereof, wherein the peptide comprises the amino acid structure of:
##STR00065## ii. a peptide or a pharmaceutically acceptable salt
thereof, wherein the peptide comprises an amino acid structure of:
##STR00066## and iii. a peptide or a pharmaceutically acceptable
salt thereof, wherein the peptide comprises the amino acid sequence
Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, wherein at
least one carboxyl group of the peptide is an alkyl ester having
the formula (--COOR) in which R is a C.sub.1-6 alkyl.
48. A pharmaceutical composition comprising linaclotide and a
peptide or a pharmaceutically acceptable salt thereof, wherein the
peptide comprises an amino acid structure of: ##STR00067## and the
peptide or pharmaceutically acceptable salt thereof comprises less
than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight
compared to the weight of linaclotide.
49. A pharmaceutical composition comprising linaclotide and a
peptide or a pharmaceutically acceptable salt thereof, wherein the
peptide comprises an amino acid structure of: ##STR00068## and the
peptide or pharmaceutically acceptable salt thereof comprises less
than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight compared to
the weight of linaclotide.
50. A pharmaceutical composition comprising linaclotide and a
peptide or a pharmaceutically acceptable salt thereof, wherein the
peptide comprises an amino acid structure of: ##STR00069## and the
peptide or pharmaceutically acceptable salt thereof comprises less
than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight
compared to the weight of linaclotide.
51. A pharmaceutical composition comprising linaclotide and a
peptide or a pharmaceutically acceptable salt thereof, wherein the
peptide comprises an amino acid structure of: ##STR00070## and the
peptide or pharmaceutically acceptable salt thereof comprises less
than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight compared to
the weight of linaclotide.
52. A pharmaceutical composition comprising linaclotide and a
peptide or a pharmaceutically acceptable salt thereof, wherein the
peptide comprises an amino acid structure of: ##STR00071## and the
peptide or pharmaceutically acceptable salt thereof comprises less
than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight
compared to the weight of linaclotide.
53. A pharmaceutical composition comprising linaclotide and a
peptide or a pharmaceutically acceptable salt thereof, wherein the
peptide comprises an amino acid structure of: ##STR00072## and the
peptide or pharmaceutically acceptable salt thereof comprises less
than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight compared to
the weight of linaclotide.
54. A pharmaceutical composition comprising a peptide or
pharmaceutically acceptable salt thereof, wherein the peptide
consists of the amino acid structure of: ##STR00073## and the
peptide comprises at least 90% by weight compared to the weight of
linaclotide or another guanylate cyclase C agonist.
55. A pharmaceutical composition consisting essentially of a
peptide or pharmaceutically acceptable salt thereof, wherein the
peptide comprises the amino acid structure of: ##STR00074##
56. The pharmaceutical composition according to any one of claims
45-55, further comprising one or more agents selected from (i) a
cation selected from Mg.sup.2+, Ca.sup.2+, Zn.sup.2+, Mn.sup.2+,
K.sup.+, Na.sup.+ or Al.sup.3+, or (ii) a sterically hindered
primary amine.
57. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier, a peptide according to any one of claims 1-44
and one or more agents selected from (i) a cation selected from
Mg.sup.2+, Ca.sup.2+, Zn.sup.2+, Mn.sup.2+, K.sup.+, Na.sup.+ or
Al.sup.3+, or (ii) a sterically hindered primary amine.
58. The pharmaceutical composition according to claim 56 or 57,
wherein said agent is Mg.sup.2+, Ca.sup.2+, Zn.sup.2+, Mn.sup.2+,
K.sup.+, Na.sup.+ or Al.sup.3+.
59. The pharmaceutical composition according to claim 58, wherein
said Mg.sup.2+, Ca.sup.2+, Zn.sup.2+, Mn.sup.2+, K.sup.+, Na.sup.+
or Al.sup.3+ is provided as magnesium acetate, magnesium chloride,
magnesium phosphate, magnesium sulfate, calcium acetate, calcium
chloride, calcium phosphate, calcium sulfate, zinc acetate, zinc
chloride, zinc phosphate, zinc sulfate, manganese acetate,
manganese chloride, manganese phosphate, manganese sulfate,
potassium acetate, potassium chloride, potassium phosphate,
potassium sulfate, sodium acetate, sodium chloride, sodium
phosphate, sodium sulfate, aluminum acetate, aluminum chloride,
aluminum phosphate or aluminum sulfate.
60. The pharmaceutical composition according to claim 56 or 57,
wherein said agent is a sterically hindered primary amine.
61. The pharmaceutical composition according to claim 60, wherein
the sterically hindered primary amine is an amino acid.
62. The pharmaceutical composition according to claim 61, wherein
the amino acid is a naturally-occurring amino acid, a non-naturally
occurring amino acid or an amino acid derivative.
63. The pharmaceutical composition according to claim 62, wherein
the naturally-occurring amino acid is histidine, phenylalanine,
alanine, glutamic acid, aspartic acid, glutamine, leucine,
methionine, asparagine, tyrosine, threonine, isoleucine, tryptophan
or valine or the non-naturally occurring amino acid is
1-aminocyclohexane carboxylic acid, lanthanine or theanine.
64. The pharmaceutical composition according to claim 60, wherein
the sterically hindered primary amine has the formula: ##STR00075##
wherein R.sub.1, R.sub.2 and R.sub.3 are independently selected
from: H, C(O)OH, C1-C6 alkyl, C1-C6 alkyl ether, C1-C6
alkylthioether, C1-C6 alkyl carboxylic acid, C1-C6 alkyl
carboxylamide and alkylaryl, wherein any group can be singly or
multiply substituted with: halogen or amino, and provided that no
more than one of R.sub.1, R.sub.2 and R.sub.3 is H.
65. The pharmaceutical composition according to claim 64, wherein
the sterically hindered primary amine is cyclohexylamine or
2-methylbutylamine.
66. The pharmaceutical composition according to claim 60, wherein
the sterically hindered primary amine is a polymeric amine.
67. The pharmaceutical composition according to claim 66, wherein
the polymeric amine is chitosan.
68. The pharmaceutical composition according to any one of claims
60-67, wherein said pharmaceutical composition further comprises
Mg.sup.2+, Ca.sup.2+, Zn.sup.2+, Mn.sup.2+, K.sup.+, Na.sup.+ or
Al.sup.3+.
69. The pharmaceutical composition according to claim 68, wherein
said Mg.sup.2+, Ca.sup.2+, Zn.sup.2+, Mn.sup.2+, K.sup.+, Na.sup.+
or Al.sup.3+ is provided as magnesium acetate, magnesium chloride,
magnesium phosphate, magnesium sulfate, calcium acetate, calcium
chloride, calcium phosphate, calcium sulfate, zinc acetate, zinc
chloride, zinc phosphate, zinc sulfate, manganese acetate,
manganese chloride, manganese phosphate, manganese sulfate,
potassium acetate, potassium chloride, potassium phosphate,
potassium sulfate, sodium acetate, sodium chloride, sodium
phosphate, sodium sulfate, aluminum acetate, aluminum chloride,
aluminum phosphate or aluminum sulfate.
70. The pharmaceutical composition according to any one of claims
45-69, further comprising an antioxidant.
71. The pharmaceutical composition according to claim 70, wherein
said antioxidant is BHA, vitamin E or propyl gallate.
72. The pharmaceutical composition according to any one of claims
45-71, further comprising a pharmaceutically acceptable binder or
additive.
73. The pharmaceutical composition according to claim 72, wherein
the pharmaceutically acceptable binder or additive is selected from
polyvinyl alcohol, polyvinylpyrrolidone (povidone), a starch,
maltodextrin or a cellulose ether.
74. The pharmaceutical composition according to claim 73, wherein
the pharmaceutically acceptable binder or additive is polyvinyl
alcohol.
75. The pharmaceutical composition of claim 73, wherein the
pharmaceutically acceptable binder or additive is a cellulose
ether.
76. The pharmaceutical composition of claim 75, wherein the
cellulose ether is selected from: methylcellulose, ethylcellulose,
carboxymethylcellulose, hydroxyethyl cellulose, hydroxyethyl
methylcellulose, hydroxypropyl cellulose and hydroxypropyl
methylcellulose.
77. The pharmaceutical composition of any of claims 45-76, further
comprising a pharmaceutically acceptable filler.
78. The pharmaceutical composition according to claim 77, wherein
the pharmaceutically acceptable filler is cellulose, isomalt,
mannitol or dibasic calcium phosphate.
79. The pharmaceutical composition of claim 78, wherein the
cellulose is selected from microfine cellulose and microcrystalline
cellulose.
80. The pharmaceutical composition according to any one of claims
45-79, further comprising an additional therapeutic agent.
81. The pharmaceutical composition according to claim 80, wherein
said additional therapeutic agent is selected from one or more of
an analgesic agent, an antidepressant, a promotility or prokinetic
agent, an antiemetic, an antibiotic, a proton pump inhibitor, an
acid blocker, a PDE5 inhibitor, an acid pump antagonist, a GABA-B
agonist, a bile acid sequestrant or a mucosal protecting agent.
82. A dosage unit comprising a pharmaceutical composition according
to any one of claims 45-81.
83. The dosage unit according to claim 82, wherein said dosage unit
is a capsule or tablet.
84. The dosage unit according to claim 83, wherein each of said
dosage units comprises 5 .mu.g to 1 mg of said peptide.
85. A method for treating a gastrointestinal disorder comprising
administering the pharmaceutical composition according to any one
of claims 45-81.
86. The method of claim 85, wherein the gastrointestinal disorder
is selected from the group consisting of: irritable bowel syndrome
(IBS), constipation, a functional gastrointestinal disorder,
gastroesophageal reflux disease, functional heartburn, dyspepsia,
visceral pain, gastroparesis, chronic intestinal
pseudo-obstruction, colonic pseudo-obstruction, Crohn's disease,
ulcerative colitis, and inflammatory bowel disease.
87. The method of claim 86, wherein the gastrointestinal disorder
is constipation.
88. The method of claim 87, wherein the constipation is chronic
constipation, idiopathic constipation, due to post-operative ileus,
or caused by opiate use.
89. The method of claim 86, wherein the gastrointestinal disorder
is irritable bowel syndrome (IBS).
90. The method of claim 89, wherein the irritable bowel syndrome is
constipation-predominant irritable bowel syndrome (c-IBS),
diarrhea-predominant irritable bowel syndrome (d-IBS) or
alternating between the two irritable bowel syndromes (a-IBS).
91. The method of claim 86, wherein the gastrointestinal disorder
is dyspepsia.
92. The method of claim 86, wherein the gastrointestinal disorder
is gastroparesis.
93. The method according to claim 92, wherein said gastroparesis is
idiopathic, diabetic or post-surgical gastroparesis.
94. The method of claim 86, wherein the gastrointestinal disorder
is chronic intestinal pseudo obstruction.
95. The method of claim 86, wherein the gastrointestinal disorder
is Crohn's disease.
96. The method of claim 86, wherein the gastrointestinal disorder
is ulcerative colitis.
97. The method of claim 86, wherein the gastrointestinal disorder
is inflammatory bowel disease.
98. The method of claim 86, wherein the gastrointestinal disorder
is visceral pain.
99. A method for increasing intestinal motility in a patient, the
method comprising administering to the patient an effective amount
of the pharmaceutical composition according to any one of claims
45-81.
100. A method of increasing guanylate cyclase C (GC-C) receptor
activity in a biological sample or organism, comprising contacting
said biological sample or organism with a peptide according to any
one of claims 1-44.
Description
PRIORITY CLAIM
[0001] This application is a continuation of, and claims priority
to U.S. patent application Ser. No. 17/140,267 filed Jan. 4, 2021,
which is a continuation of U.S. patent application Ser. No.
16/881,198 filed May 22, 2020, which is a continuation of U.S.
patent application Ser. No. 16/585,139 filed Sep. 27, 2019, which
is a continuation of U.S. patent application Ser. No. 16/270,802
filed Feb. 8, 2019, which is a continuation of U.S. patent
application Ser. No. 16/009,560 filed Jun. 15, 2018, which is a
continuation of U.S. patent application Ser. No. 15/729,875 filed
Oct. 11, 2017, which is a continuation of U.S. patent application
Ser. No. 14/556,573 filed Dec. 1, 2014, which is a continuation of
U.S. patent application Ser. No. 13/579,685 filed Aug. 17, 2012,
which is the United States National Phase application of
PCT/US2011/025274 filed Feb. 17, 2011. This application also claims
priority to U.S. Provisional Application Ser. No. 61/305,465 filed
Feb. 17, 2010. The entire contents of the aforementioned
applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to peptides, compositions and methods
for treating gastrointestinal disorders.
SEQUENCE LISTING
[0003] This application incorporates by reference in its entirety
the Sequence Listing entitled "IW087PCT1US1CON1_ST25.txt" which is
630 bytes in size and last modified on Dec. 1, 2014 and filed
electronically herewith.
BACKGROUND
[0004] Gastrointestinal disorders (GI) include irritable bowel
syndrome (IBS) which is a common chronic disorder of the intestine
that affects 20 to 60 million individuals in the US alone (Lehman
Brothers, Global Healthcare-Irritable bowel syndrome industry
update, September 1999). IBS is the most common disorder diagnosed
by gastroenterologists and accounts for 12% of visits to primary
care physicians (Camilleri 2001, Gastroenterology 120:652-668). In
the US, the economic impact of IBS is estimated at $25 billion
annually, through direct costs of health care use and indirect
costs of absenteeism from work (Talley 1995, Gastroenterology
109:1736-1741). Patients with IBS have three times more absenteeism
from work and report a reduced quality of life. There is a
tremendous unmet medical need for patients suffering for IBS since
few prescription options exist to treat IBS.
[0005] Patients with IBS suffer from abdominal pain and a disturbed
bowel pattern. Three subgroups of IBS patients have been defined
based on the predominant bowel habit: constipation-predominant
irritable bowel syndrome (c-IBS), diarrhea-predominant irritable
bowel syndrome (d-IBS) or alternating between the two irritable
bowel syndromes (a-IBS). Estimates of individuals who suffer from
c-IBS range from 20-50% of the IBS patients with 30% frequently
cited. In contrast to the other two subgroups that have a similar
gender ratio, c-IBS is more common in women (ratio of 3:1) (Talley
et al. 1995, Am J Epidemiol 142:76-83).
[0006] The definition and diagnostic criteria for IBS have been
formalized in the "Rome Criteria" (Drossman et al. 1999, Gut
45:Suppl II: 1-81), which are well accepted in clinical practice.
Recently, there has been increasing evidence for a role of
inflammation in etiology of IBS. Reports indicate that subsets of
IBS patients have small but significant increases in colonic
inflammatory and mast cells, increased inducible nitric oxide (NO)
and synthase (iNOS) and altered expression of inflammatory
cytokines (reviewed by Talley 2000, Medscape Coverage of DDW
week).
[0007] Gastrointestinal disorders can also include constipation
wherein as many as 34 million Americans suffer from symptoms
associated with chronic constipation (CC) and 8.5 million patients
have sought treatment. Patients with CC often experience hard and
lumpy stools, straining during defecation, a sensation of
incomplete evacuation, and fewer than three bowel movements per
week. The discomfort and bloating of CC significantly affects
patients' quality of life by impairing their ability to work and
participate in typical daily activities.
[0008] Half of CC patients are not satisfied with currently
available treatments for CC. Thus, there remains a need for new
compounds and methods of treating CC.
[0009] U.S. Pat. Nos. 7,304,036 and 7,371,727 disclose peptides
that act as agonists of the guanylate cyclase C (GC-C) receptor for
the treatment of gastrointestinal disorders. One particular peptide
disclosed is linaclotide, which consists of the following amino
acid sequence: Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys
Tyr. These patents also disclose methods for preparing linaclotide
and related peptides.
[0010] Linaclotide has the amino acid structure of:
##STR00001##
[0011] Linaclotide is orally administered and currently in clinical
trials for treatment of irritable bowel syndrome with constipation
(IBS-c) and chronic constipation (CC), has numerous effects on GI
physiology including: (1) reduced visceral pain, (2) reduced
bloating, and (3) increased GI transit, which can lead to increased
stool frequency and improved stool consistency. Orally administered
linaclotide acts locally by activating GC-C at the luminal surface;
there are no detectable levels of linaclotide seen systemically
after oral administration at therapeutic dose levels. Thus, the
results from clinical trials of linaclotide, as well as preclinical
studies that have been done with linaclotide and related peptides,
suggest that GC-C peptide agonists may be used therapeutically.
[0012] The contents of the U.S. Pat. Nos. 7,304,036 and 7,371,727
are incorporated herein by reference in their entirety.
[0013] The present invention feature peptides which may be modified
at their amine groups into imidazolidinone derivatives and/or
modified at their carboxyl groups into alkyl esters that are
capable of activating and/or binding the guanylate cyclase-C (GC-C)
receptors at different affinities. GC-C is a key regulator in
mammals of intestinal function, although low levels of GC-C have
been detected in other tissues. GC-C responds to the endogenous
hormones, guanylin and uroguanylin, and to enteric bacterial
peptides from the heat stable enterotoxin family (ST peptide). When
agonists bind to GC-C, there is an elevation of the second
messenger, cyclic GMP (c-GMP), and an increase in chloride and
bicarbonate secretion, resulting in an increase in intestinal fluid
secretion. In some examples of the present invention, the peptides
described herein may produce increased elevation of c-GMP levels
and provide a therapeutic option for treating gastrointestinal
disorders.
SUMMARY
[0014] The present invention features peptides, compositions, and
related methods for treating gastrointestinal disorders and
conditions, including but not limited to, irritable bowel syndrome
(MS) gastrointestinal motility disorders, constipation, functional
gastrointestinal disorders, gastroesophageal reflux disease (GERD),
duodenogastric reflux, Crohn's disease, ulcerative colitis,
inflammatory bowel disease, functional heartburn, dyspepsia,
visceral pain, gastroparesis, chronic intestinal pseudo-obstruction
(or colonic pseudo-obstruction), and other conditions and disorders
described herein using peptides and compositions that activate the
guanylate cyclase C (GC-C) receptor.
[0015] One aspect of the present invention provides a peptide or a
pharmaceutically acceptable salt thereof, wherein the peptide
comprises the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro
Ala Cys Thr Gly Cys Tyr, wherein at least one carboxyl group of the
peptide is an alkyl ester having the formula (--COOR) in which R is
a C.sub.1-6 alkyl.
[0016] In one embodiment, the peptide comprises the amino acid
structure of:
##STR00002##
or a pharmaceutically acceptable salt thereof, wherein R' is H or a
C.sub.1-6 alkyl, and at least one R' is C.sub.1-6 alkyl.
[0017] A second aspect of the present invention provides a peptide
that comprises the amino acid structure of:
##STR00003##
or a pharmaceutically acceptable salt thereof, wherein R is a
C.sub.1-6 alkyl; or
[0018] a peptide that comprises the amino acid structure of:
##STR00004##
[0019] or a pharmaceutically acceptable salt thereof, wherein R is
C.sub.1-6 alkyl.
[0020] A third aspect of the present invention provides a peptide
or pharmaceutically acceptable salt thereof, wherein the peptide
comprises the amino acid structure of:
##STR00005##
[0021] A fourth aspect of the present invention provides a
pharmaceutical composition comprising a peptide of the present
invention.
[0022] A fifth aspect of the present invention provides a method
for treating a gastrointestinal disorder, which includes
administering a pharmaceutical composition according to the present
invention.
[0023] The details of one or more embodiments of the invention are
set forth in the accompanying description.
BRIEF DESCRIPTION OF THE FIGURE
[0024] FIG. 1 shows the dose response of exemplary peptides of the
present invention in a T84 cell c-GMP assay.
[0025] FIG. 2 demonstrates an example of an analysis of exemplary
peptides by RP-HPLC, wherein "Cys.sub.1-IMD" refers to the
linaclotide imidazolidinone derivative modified on its N-terminal
amine group.
[0026] FIG. 3 shows specific binding of linaclotide and
Cys.sub.1-IMD to cell-surface GC-C receptors on T84 cells in a
competitive radioligand binding assay.
[0027] FIG. 4 shows the dose response of exemplary peptides of the
present invention in a T84 cell c-GMP assay.
[0028] The figures are provided by way of example and are not
intended to limit the scope of the present invention.
DETAILED DESCRIPTION
[0029] Guanylate cyclase C (GC-C) is a transmembrane receptor that
is located on the apical surface of epithelial cells in the stomach
and intestine. The receptor has an extracellular ligand-binding
domain, a single transmembrane region and a C-terminal guanylyl
cyclase domain. When a ligand binds to the extracellular domain of
GC-C, the intracellular catalytic domain catalyzes the production
of cGMP from GTP. In vivo, this increase in intracellular cGMP
initiates a cascade of events that leads to increased secretion of
chloride and bicarbonate into the intestinal lumen, increased
luminal pH, decreased luminal sodium absorption, increased fluid
secretion, and acceleration of intestinal transit. cGMP is secreted
bidirectionally from the epithelium into the mucosa and lumen. The
peptides and compositions of the present invention bind to the
intestinal GC-C receptor which is a regulator of fluid and
electrolyte balance in the intestine.
[0030] In some circumstances it can be desirable to treat patients
with a variant or modified peptide that binds to and activates
intestinal GC-C receptors, but is less active or more active than
the non-variant form of a peptide. Reduced activity can arise from
reduced affinity for the receptor or a reduced ability to activate
the receptor once bound or reduced stability of the peptide.
Increased activity can arise from increased affinity for the
receptor or an increased ability to activate the receptor once
bound or increased stability of the peptide.
Description of Exemplary Peptides:
[0031] In various embodiments, a peptide may be modified wherein at
least one carboxyl group of the amino acid residue of the peptide
is modified to an alkyl ester. This modification may be produced,
for example, by treating a carboxylic acid with an alcohol in the
presence of a dehydrating agent wherein the dehydrating agent can
include but is not limited to a strong acid such as sulfuric acid.
Other methods of producing alkyl esters from carboxyl groups are
readily known in those skilled in the arts and are incorporated
herein.
[0032] As used herein, a carboxyl group has the formula:
(--COOH).
[0033] As used herein, the term "alkyl", refers to a saturated
linear or branched-chain monovalent hydrocarbon radical.
[0034] As used herein, a group is terminal or terminus when the
group is present at the end of the amino acid sequence.
[0035] As used herein, an amine group on a peptide has the
formula:
##STR00006##
wherein R.sup.2 is the rest of the peptide.
[0036] As used herein, an imine group on a peptide has the
formula:
##STR00007##
wherein R.sup.2 is the rest of the peptide.
[0037] In some embodiments, the carboxylic acid of the side chain
of a glutamate amino acid in a peptide sequence is modified into an
alkyl ester.
[0038] In further embodiments, the carboxylic acid on the side
chain of a glutamate amino acid a peptide sequence is modified into
an ethyl ester.
[0039] In other embodiments, the C-terminus carboxylic acid of a
tyrosine amino acid in a peptide sequence is modified into an alkyl
ester.
[0040] In further embodiments, the C-terminus carboxylic acid of a
tyrosine amino acid of a peptide sequence is modified into an ethyl
ester.
[0041] In several embodiments, the present invention provides a
peptide or a pharmaceutically acceptable salt thereof, wherein the
peptide comprises the amino acid sequence Cys Cys Glu Tyr Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr, wherein at least one carboxyl
group of the peptide is an alkyl ester having the formula (--COOR)
in which R is a C.sub.1-6 alkyl.
[0042] In several embodiments, the peptide comprises an amino acid
structure of:
##STR00008##
or a pharmaceutically acceptable salt thereof, wherein R' is H or a
C.sub.1-6 alkyl, and at least one R' is C.sub.1-6 alkyl.
[0043] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof comprises a peptide having an amino acid
structure of:
##STR00009##
[0044] wherein R is a C.sub.1-6 alkyl ("Glu.sub.3-alkyl
ester").
[0045] In other embodiments, R is a C.sub.1-4 alkyl.
[0046] In further embodiments, R is methyl, ethyl, or propyl.
[0047] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof comprises an amino acid structure of
("Glu.sub.3-ethyl ester"):
##STR00010##
[0048] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof comprises an amino acid structure of:
##STR00011##
[0049] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof comprises an amino acid structure of:
##STR00012##
[0050] In some embodiments, the C-terminal tyrosine of the
Glu.sub.3-alkyl ester or pharmaceutically acceptable salt is
absent.
[0051] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof comprises an amino acid structure of:
##STR00013##
[0052] wherein R is C.sub.1-6 alkyl.
[0053] In other embodiments, R is a C.sub.1-4 alkyl.
[0054] In further embodiments, R is methyl, ethyl, or propyl.
[0055] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof comprises an amino acid structure of
("Tyr.sub.14-ethyl ester"):
##STR00014##
[0056] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof comprises an amino acid structure of:
##STR00015##
[0057] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof comprises an amino acid structure of:
##STR00016##
[0058] Without wishing to be bound by any theory, a peptide may be
modified wherein at least one amine group of the amino acid
residues of the peptide is modified into an imine. This
modification may be produced, for example, by treating an amine
group with a carbonyl, such as an aldehyde or ketone, in the
presence of acid catalyst. Other methods of producing imines from
amine groups are readily known to those skilled in the arts and are
incorporated herein.
[0059] In some embodiments, the imine modification may be produced
by a formaldehyde mediated reaction in the presence of acid
catalyst.
[0060] In further embodiments, the imine carbon may be cross-linked
to another amine group of the peptide.
[0061] In other embodiments, a peptide may be modified into an
imine at the .alpha.-amine group of the N-terminal amino acid,
wherein the imine carbon is cross-linked with an amine group of the
second amino acid residue of the peptide forming a five membered
ring.
[0062] In other embodiments, a peptide comprising the amino acid
sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr
may be modified with the addition of methylene at the .alpha.-amine
group of the N-terminal Cys.sub.1 which is cross-linked to the
amine group of Cys.sub.2 to form an imidazolidinone 5 membered ring
at the N-terminus of the peptide ("Cys.sub.1-IMD").
[0063] In one aspect, the invention provides novel GC-C peptide
agonists useful for the treatment of gastrointestinal
disorders.
[0064] In several embodiments, the present invention provides a
peptide or a pharmaceutically acceptable salt thereof, wherein the
peptide comprises the amino acid sequence Cys Cys Glu Tyr Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr, wherein at least one amine group
of the peptide is an imine having the formula
##STR00017##
wherein R.sup.2 is the rest of the peptide.
[0065] In some embodiments, the peptide or a pharmaceutically
acceptable salt comprises a peptide wherein the N-terminal amine
group of the peptide is an imine having the formula
##STR00018##
wherein R.sup.2 is the rest of the peptide.
[0066] In further embodiments, the peptide or pharmaceutically
acceptable salt thereof comprises an amino acid structure of:
##STR00019##
[0067] In several embodiments, the peptide or pharmaceutically
acceptable salt thereof comprises an amino acid structure of:
##STR00020##
[0068] In some embodiments, the C-terminal tyrosine of the
Cys.sub.1-IMD peptide or pharmaceutically acceptable salt thereof
is absent. In some embodiments, the Cys.sub.1-IMD peptide or
pharmaceutically acceptable salt thereof further comprises one or
more peptide modifications, wherein at least one carboxyl group of
the peptide is an alkyl ester having the formula (--COOR) in which
R is a C.sub.1-6 alkyl.
[0069] In several embodiments, the present invention provides a
peptide or a pharmaceutically acceptable salt thereof, wherein the
peptide consists of the amino acid sequence Cys Cys Glu Tyr Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr, wherein at least one carboxyl
group of the peptide is an alkyl ester having the formula (--COOR)
in which R is a C.sub.1-6 alkyl.
[0070] In several embodiments, the peptide consists of an amino
acid structure of:
##STR00021##
or a pharmaceutically acceptable salt thereof, wherein R' is H or a
C.sub.1-6 alkyl, and at least one R' is C.sub.1-6 alkyl.
[0071] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof consists of an amino acid structure of:
##STR00022##
[0072] wherein R is a C.sub.1-6 alkyl. In further embodiments, the
C-terminal tyrosine is absent.
[0073] In other embodiments, R is a C.sub.1-4 alkyl.
[0074] In further embodiments, R is methyl, ethyl, or propyl.
[0075] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof consists of a peptide having an amino acid
structure of:
##STR00023##
[0076] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof consists of a peptide having an amino acid
structure of:
##STR00024##
[0077] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof consists of a peptide having an amino acid
structure of:
##STR00025##
[0078] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof consists of a peptide having an amino acid
structure of:
##STR00026##
[0079] wherein R is C.sub.1-6 alkyl.
[0080] In other embodiments, R is a C.sub.1-4 alkyl.
[0081] In further embodiments, R is methyl, ethyl, or propyl.
[0082] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof consists of an amino acid structure of:
##STR00027##
[0083] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof consists of an amino acid structure of:
##STR00028##
[0084] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof consists of an amino acid structure of:
##STR00029##
[0085] In several embodiments, the present invention provides a
peptide or a pharmaceutically acceptable salt thereof, wherein the
peptide consists of the amino acid sequence Cys Cys Glu Tyr Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr, wherein at least one amine group
of the peptide is an imine having the formula
##STR00030##
wherein R.sup.2 is the rest of the peptide.
[0086] In some embodiments, the peptide or a pharmaceutically
acceptable salt consists of a peptide wherein the N-terminal amine
group of the peptide is an imine having the formula
##STR00031##
wherein R.sup.2 is the rest of the peptide.
[0087] In further embodiments, the peptide or pharmaceutically
acceptable salt thereof consists of an amino acid structure of:
##STR00032##
[0088] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof consists of an amino acid structure of:
##STR00033##
[0089] In some embodiments, the C-terminal tyrosine of the
Cys.sub.1-IMD peptide or pharmaceutically acceptable salt thereof
is absent. In some embodiments, the Cys.sub.1-IMD peptide or
pharmaceutically acceptable salt thereof further comprises one or
more peptide modifications, e.g., wherein at least one carboxyl
group of the peptide is an alkyl ester having the formula (--COOR),
wherein R is a C.sub.1-6 alkyl.
[0090] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof activates the guanylate cyclase C
receptor.
[0091] In other embodiments, the peptide or pharmaceutically
acceptable salt thereof comprises 30 or fewer amino acids.
[0092] In further embodiments, the peptide or pharmaceutically
acceptable salt thereof comprises 20 or fewer amino acids.
[0093] In other embodiments, the peptide or pharmaceutically
acceptable salt thereof comprises a peptide wherein fewer than five
amino acids precede the first Cys residue of the amino acid
sequence.
[0094] In some embodiments, the peptide or pharmaceutically
acceptable salt thereof is isolated.
[0095] In other embodiments, the peptide or pharmaceutically
acceptable salt thereof is purified.
[0096] In some embodiments, a pharmaceutically acceptable salt of
the peptide is provided. In some instances, the pharmaceutically
acceptable salt is a chloride salt.
Variant or Modified Peptides
[0097] In various embodiments, the peptide includes two Cys that
form one disulfide bond, the peptide includes four Cys that form
two disulfide bonds, or the peptide includes six Cys that form
three disulfide bonds.
[0098] In some peptides one or both members of one or both pairs of
Cys residues which normally form a disulfide bond can be replaced
by homocysteine, penicillamine, 3-mercaptoproline (Kolodziej et al.
1996 Int J Pept Protein Res 48:274); (3, (3 dimethylcysteine (Hunt
et al. 1993 Int J Pept Protein Res 42:249) or diaminopropionic acid
(Smith et al. 1978 J Med Chem 21:117) to form alternative internal
cross-links at the positions of the normal disulfide bonds. In
other embodiments, the disulfide bonds may be replaced by
hydrocarbon crosslinking (Schafmeister et al. 2000 J Am Chem Soc
122:5891, Patgiri et al. 2008 Acc Chem Res 41:1289, Henchey et al.
2008 Curr Opin Chem Biol 12:692).
Production of Peptides
[0099] In one embodiment, peptides or precursor peptides of the
invention can be produced recombinantly in any known protein
expression system, including, without limitation, bacteria (e.g.,
E. coli or Bacillus subtilis), insect cell systems (e.g.,
Drosophila 519 cell systems), yeast cell systems (e.g., S.
cerevisiae, S. saccharomyces) or filamentous fungal expression
systems, or animal cell expression systems (e.g., mammalian cell
expression systems). Peptides or precursor peptides of the
invention may also be chemically synthesized.
[0100] If the peptide or variant peptide is to be produced
recombinantly, e.g., E. coli, the nucleic acid molecule encoding
the peptide may also encode a leader sequence that permits the
secretion of the mature peptide from the cell. Thus, the sequence
encoding the peptide can include the pre sequence and the pro
sequence of, for example, a naturally-occurring bacterial ST
peptide. The secreted, mature peptide can be purified from the
culture medium.
[0101] The sequence encoding a 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 and 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. 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 peptide
production.
[0102] The protein coding sequence that includes a peptide
described herein can also be fused to a nucleic acid encoding a
peptide 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
peptide 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
peptide that includes both the peptide 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 peptide of
interest.
[0103] Genetic constructs and methods suitable for production of
immature and mature forms of the 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
peptides in a biological system.
[0104] In other embodiments, peptides containing amino acids not
normally incorporated by the translation machinery and described
above (e.g.--.beta.-carboxylated Asp, .gamma.-carboxylated Glu,
Asu, Aad and Apm) may be recombinantly produced by tRNA
modification methods. Methods for modifying tRNA including, but not
limited to, modifying the anti-codon, the amino acid attachment
site, and/or the accepter stem to allow incorporation of unnatural
and/or arbitrary amino acids are known in the art (Biochem.
Biophys. Res. Comm. (2008) 372: 480-485; Chem. Biol. (2009)
16:323-36; Nat. Methods (2007) 4:239-44; Nat. Rev. Mol. Cell Biol.
(2006) 7:775-82; Methods (2005) 36:227-238; Methods (2005)
36:270-278; Annu. Rev. Biochem. (2004) 73:147-176; Nuc. Acids Res.
(2004) 32:6200-6211; Proc. Natl. Acad. Sci. USA (2003)
100:6353-6357; Royal Soc. Chem. (2004) 33:422-430).
[0105] In some embodiments, peptides may be chemically produced.
Peptides can be synthesized by a number of different methods
including solution and solid phase synthesis using traditional BOC
or FMOC protection. For example, the peptide can be synthesized on
2-Chlorotrityl or Wang resin using consecutive amino acid
couplings. The following protecting groups can be used:
Fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl (alpha-amino
groups, N-terminus); trityl or tert-butyl (thiol groups of Cys);
tert-butyl (.gamma.-carboxyl of glutamic acid and the hydroxyl
group of threonine, if present); and trityl (.beta.-amid function
of the asparagine side chain and the phenolic group of tyrosine, if
present). Coupling can be effected with DIC and HOBt in the
presence of a tertiary amine, and the peptide can be deprotected
and cleaved from the solid support in using cocktail K
(trifluoroacetic acid 81%, phenol 5%, thioanisole 5%,
1,2-ethanedithiol 2.5%, water 3%, dimethylsulphide 2%, ammonium
iodide 1.5% w/w). After removal of trifluoroacetic acid and other
volatiles the peptide can be precipitated using an organic solvent.
Disulfide bonds between Cys residues can be formed using dimethyl
sulfoxide (Tam et al. (1991) J. Am. Chem. Soc. 113:6657-62) or
using an air oxidation strategy. The resulting peptide can be
purified by reverse-phase chromatography and lyophilized.
[0106] These peptides can be made, isolated or used either in form
of the base or as pharmaceutically acceptable salts thereof.
Examples of salts include, without limitation, acetate, chloride,
sulfate and phosphate salts of the peptide.
Compositions of Peptides and GC-C Receptor Agonists
[0107] In another aspect, pharmaceutical compositions are provided
wherein the peptides, alone or in combination, can be combined with
any pharmaceutically acceptable carrier or medium.
[0108] In several embodiments, the pharmaceutical composition
comprises a peptide or pharmaceutically acceptable salt thereof as
described herein. The pharmaceutical composition may comprise two
or more peptides or pharmaceutically acceptable salts thereof
described herein.
[0109] In some embodiments, the pharmaceutical composition
comprises two or more peptides selected from:
i. a peptide or a pharmaceutically acceptable salt thereof, wherein
the peptide comprises the amino acid structure of:
##STR00034##
[0110] ii. a peptide or a pharmaceutically acceptable salt thereof,
wherein the peptide comprises an amino acid structure of:
##STR00035##
and
[0111] iii. a peptide or a pharmaceutically acceptable salt
thereof, wherein the peptide comprises the amino acid sequence Cys
Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, wherein at
least one carboxyl group of the peptide is an alkyl ester having
the formula (--COOR) in which R is a C.sub.1-6 alkyl.
[0112] In other embodiments, the pharmaceutical composition
comprises linaclotide and a peptide or a pharmaceutically
acceptable salt thereof, wherein the peptide comprises an amino
acid structure of:
##STR00036##
and the peptide or pharmaceutically acceptable salt thereof
comprises less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%
by weight compared to the weight of linaclotide.
[0113] In further embodiments, the pharmaceutical composition
comprises linaclotide and a peptide or a pharmaceutically
acceptable salt thereof, wherein the peptide comprises an amino
acid structure of:
##STR00037##
[0114] and the peptide or pharmaceutically acceptable salt thereof
comprises less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight
compared to the weight of linaclotide.
[0115] In some embodiments, the imidazolidinone derivative of
linaclotide comprises less than about 15% by weight of the
composition, less than about 10% by weight of the composition, less
than about 7% by weight of the composition or less than about 5% by
weight of the composition. In other exemplary embodiments, the
imidazolidinone derivative of linaclotide comprises from about
0.01% to about 15% by weight of the composition, about 0.05% to
about 10% by weight of the composition, about 0.05% to about 7% by
weight of the composition or about 0.05% to about 5% by weight of
the composition.
[0116] In other embodiments, the pharmaceutical composition
comprises linaclotide and a peptide or a pharmaceutically
acceptable salt thereof, wherein the peptide comprises an amino
acid structure of:
##STR00038##
and the peptide or pharmaceutically acceptable salt thereof
comprises less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%
by weight compared to the weight of linaclotide.
[0117] In further embodiments, the pharmaceutical composition
comprises linaclotide and a peptide or a pharmaceutically
acceptable salt thereof, wherein the peptide comprises an amino
acid structure of:
##STR00039##
and the peptide or pharmaceutically acceptable salt thereof
comprises less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight
compared to the weight of linaclotide.
[0118] In some embodiments, the Tyr.sub.14-ethyl ester peptide
comprises less than about 15% by weight of the composition, less
than about 10% by weight of the composition, less than about 7% by
weight of the composition or less than about 5% by weight of the
composition. In other exemplary embodiments, the Tyr.sub.14-ethyl
ester comprises from about 0.01% to about 15% by weight of the
composition, about 0.05% to about 10% by weight of the composition,
about 0.05% to about 7% by weight of the composition or about 0.05%
to about 5% by weight of the composition.
[0119] In other embodiments, the pharmaceutical composition
comprising linaclotide and a peptide or a pharmaceutically
acceptable salt thereof, wherein the peptide comprises an amino
acid structure of:
##STR00040##
[0120] and the peptide or pharmaceutically acceptable salt thereof
comprises less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%
by weight compared to the weight of linaclotide.
[0121] In further embodiments, the pharmaceutical composition
comprises linaclotide and a peptide or a pharmaceutically
acceptable salt thereof, wherein the peptide comprises an amino
acid structure of:
##STR00041##
and the peptide or pharmaceutically acceptable salt thereof
comprises less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight
compared to the weight of linaclotide.
[0122] In some embodiments, the Glu.sub.3-ethyl ester peptide
comprises less than about 15% by weight of the composition, less
than about 10% by weight of the composition, less than about 7% by
weight of the composition or less than about 5% by weight of the
composition. In other exemplary embodiments, the Glu.sub.3-ethyl
ester comprises from about 0.01% to about 15% by weight of the
composition, about 0.05% to about 10% by weight of the composition,
about 0.05% to about 7% by weight of the composition or about 0.05%
to about 5% by weight of the composition.
[0123] In some embodiments, the pharmaceutical composition
comprises a peptide or pharmaceutically acceptable salt, wherein
the peptide consists of the amino acid structure of:
##STR00042##
[0124] In other embodiments, the pharmaceutical composition
consists essentially of a peptide or pharmaceutically acceptable
salt thereof, wherein the peptide comprises the amino acid
structure of:
##STR00043##
[0125] The term "consisting essentially of", and variants thereof,
when used to refer to the composition, are used herein to mean that
the composition includes a sole active peptide and other desired
pharmaceutically inactive additives, excipients, and/or components
(e.g., polymers, sterically hindered primary amines, cations,
filling agents, binders, carriers, excipients, diluents,
disintegrating additives, lubricants, solvents, dispersants,
coating additives, absorption promoting additives, controlled
release additives, anti-caking additives, anti-microbial additives,
preservatives, sweetening additives, colorants, flavors,
desiccants, plasticizers, dyes, or the like), and no other active
pharmaceutical ingredient(s).
[0126] The peptides described herein can be combined with any
pharmaceutically tolerable carrier or medium, e.g. solvents,
dispersants, coatings, absorption promoting agents, controlled
release agents, and one or more inert excipients (which include
starches, polyols, granulating agents, microcrystalline cellulose
(e.g. celphere, Celphere Beads.RTM.), diluents, lubricants,
binders, disintegrating agents, and the like), etc. If desired,
tablet dosages of the disclosed compositions may be coated by
standard aqueous or nonaqueous techniques.
[0127] Examples of excipients for use as the pharmaceutically
acceptable carriers and the pharmaceutically acceptable inert
carriers and the aforementioned additional ingredients include, but
are not limited to binders, fillers, disintegrants, lubricants,
anti-microbial agents, and coating agents.
[0128] As used herein, the term "binder" refers to any
pharmaceutically acceptable binder that may be used in the practice
of the invention. Examples of pharmaceutically acceptable binders
include, without limitation, a starch (e.g., corn starch, potato
starch and pre-gelatinized starch (e.g., STARCH 1500.RTM. and
STARCH 1500 LM.RTM., sold by Colorcon, Ltd.) and other starches),
maltodextrin, gelatin, natural and synthetic gums such as acacia,
powdered tragacanth, guar gum, cellulose and its derivatives (e.g.,
methylcellulose, hydroxyethyl cellulose, hydroxyethyl
methylcellulose, hydroxypropyl cellulose and hydroxypropyl
methylcellulose (hypromellose), ethyl cellulose, cellulose acetate,
carboxymethyl cellulose calcium, sodium carboxymethyl cellulose,
carboxymethylcellulose, microcrystalline cellulose (e.g. AVICEL.TM.
such as, AVICEL-PH-101.TM., -103.TM. and -105.TM., sold by FMC
Corporation, Marcus Hook, Pa., USA)), polyvinyl alcohol, polyvinyl
pyrrolidone (e.g., polyvinyl pyrrolidone K30), and mixtures
thereof.
[0129] As used herein, the term "filler" refers to any
pharmaceutically acceptable filler that may be used in the practice
of the invention. Examples of pharmaceutically acceptable fillers
include, without limitation, talc, calcium carbonate (e.g.,
granules or powder), dibasic calcium phosphate, tribasic calcium
phosphate, calcium sulfate (e.g., granules or powder),
microcrystalline cellulose (e.g., Avicel PH101 or Celphere CP-305),
powdered cellulose, dextrates, kaolin, mannitol, silicic acid,
sorbitol, starch (e.g., Starch 1500), pre-gelatinized starch,
lactose, glucose, fructose, galactose, trehalose, sucrose, maltose,
isomalt, raffinose, maltitol, melezitose, stachyose, lactitol,
palatinite, xylitol, myoinositol, and mixtures thereof.
[0130] Examples of pharmaceutically acceptable fillers that may be
particularly used for coating the peptides include, without
limitation, talc, microcrystalline cellulose (e.g., Avicel PH101 or
Celphere CP-305), powdered cellulose, dextrates, kaolin, mannitol,
silicic acid, sorbitol, starch, pre-gelatinized starch, lactose,
glucose, fructose, galactose, trehalose, sucrose, maltose, isomalt,
dibasic calcium phosphate, raffinose, maltitol, melezitose,
stachyose, lactitol, palatinite, xylitol, mannitol, myoinositol,
and mixtures thereof.
[0131] As used herein, the term "additives" refers to any
pharmaceutically acceptable additive. Pharmaceutically acceptable
additives include, without limitation, disintegrants, dispersing
additives, lubricants, glidants, antioxidants, coating additives,
diluents, surfactants, flavoring additives, humectants, absorption
promoting additives, controlled release additives, anti-caking
additives, anti-microbial agents (e.g., preservatives), colorants,
desiccants, plasticizers and dyes. As used herein, an "excipient"
is any pharmaceutically acceptable additive, filler, binder or
agent.
[0132] Compositions of the present invention may also optionally
include other therapeutic ingredients, anti-caking agents,
preservatives, sweetening agents, colorants, flavors, desiccants,
plasticizers, dyes, glidants, anti-adherents, anti-static agents,
surfactants (wetting agents), antioxidants, film-coating agents,
and the like. Any such optional ingredient must be compatible with
the compound described herein to insure the stability of the
formulation. The composition may contain other additives as needed,
including for example lactose, glucose, fructose, galactose,
trehalose, sucrose, maltose, raffinose, maltitol, melezitose,
stachyose, lactitol, palatinite, starch, xylitol, mannitol,
myoinositol, and the like, and hydrates thereof, and amino acids,
for example alanine, glycine and betaine, and peptides and
proteins, for example albumen.
[0133] The compositions can include, for example, various
additional solvents, dispersants, coatings, absorption promoting
additives, controlled release additives, and one or more inert
additives (which include, for example, starches, polyols,
granulating additives, microcrystalline cellulose, diluents,
lubricants, binders, disintegrating additives, and the like), etc.
If desired, tablet dosages of the disclosed compositions may be
coated by standard aqueous or non-aqueous techniques. Compositions
can also include, for example, anti-caking additives,
preservatives, sweetening additives, colorants, flavors,
desiccants, plasticizers, dyes, and the like.
[0134] Suitable disintegrants include, for example, agar-agar,
calcium carbonate, microcrystalline cellulose, croscarmellose
sodium, crospovidone, povidone, polacrilin potassium, sodium starch
glycolate, potato or tapioca starch, other starches,
pre-gelatinized starch, clays, other algins, other celluloses,
gums, and mixtures thereof.
[0135] Suitable lubricants include, for example, calcium stearate,
magnesium stearate, mineral oil, light mineral oil, glycerin,
sorbitol, mannitol, polyethylene glycol, other glycols, stearic
acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil
(e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive
oil, corn oil and soybean oil), zinc stearate, ethyl oleate, ethyl
laurate, agar, syloid silica gel (AEROSIL 200, W.R. Grace Co.,
Baltimore, Md. USA), a coagulated aerosol of synthetic silica
(Evonik Degussa Co., Plano, Tex. USA), a pyrogenic silicon dioxide
(CAB-O-SIL, Cabot Co., Boston, Mass. USA), and mixtures
thereof.
[0136] Suitable glidants include, for example, leucine, colloidal
silicon dioxide, magnesium trisilicate, powdered cellulose, starch,
talc, and tribasic calcium phosphate.
[0137] Suitable anti-caking additives include, for example, calcium
silicate, magnesium silicate, silicon dioxide, colloidal silicon
dioxide, talc, and mixtures thereof.
[0138] Suitable anti-microbial additives that may be used, e.g., as
a preservative for the peptides compositions, include, for example,
benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl
alcohol, butyl paraben, cetylpyridinium chloride, cresol,
chlorobutanol, dehydroacetic acid, ethylparaben, methylparaben,
phenol, phenylethyl alcohol, phenoxyethanol, phenylmercuric
acetate, phenylmercuric nitrate, potassium sorbate, propylparaben,
sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic
acid, thimersol, thymo, and mixtures thereof.
[0139] Suitable antioxidants include, for example, BHA (butylated
hydroxyanisole), BHT (butylated hydroxytoluene), vitamin E, propyl
gallate, ascorbic acid and salts or esters thereof, tocopherol and
esters thereof, alpha-lipoic acid and beta-carotene.
[0140] Suitable coating additives include, for example, sodium
carboxymethyl cellulose, cellulose acetate phthalate,
ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropyl
cellulose, hydroxypropyl methylcellulose, hydroxypropyl methyl
cellulose phthalate, methylcellulose, polyethylene glycol,
polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide,
carnauba wax, microcrystalline wax, and mixtures thereof. Suitable
protective coatings include Aquacoat (e.g. Aquacoat Ethylcellulose
Aquaeous Dispersion, 15% w/w, FMC Biopolymer, ECD-30), Eudragit
(e.g. Eudragit E PO PE-EL, Roehm Pharma Polymers) and Opadry (e.g
Opadry AMB dispersion, 20% w/w, Colorcon).
[0141] In certain embodiments, suitable additives for the peptides
composition include one or more of sucrose, talc, magnesium
stearate, crospovidone or BHA.
[0142] The compositions of the present invention can also include
other excipients, agents, and categories thereof including but not
limited to L-histidine, Pluronic.RTM., Poloxamers (such as
Lutrol.RTM. and Poloxamer 188), ascorbic acid, glutathione,
permeability enhancers (e.g. lipids, sodium cholate, acylcarnitine,
salicylates, mixed bile salts, fatty acid micelles, chelators,
fatty acid, surfactants, medium chain glycerides), protease
inhibitors (e.g. soybean trypsin inhibitor, organic acids), pH
lowering agents and absorption enhancers effective to promote
bioavailability (including but not limited to those described in
U.S. Pat. Nos. 6,086,918 and 5,912,014), materials for chewable
tablets (like dextrose, fructose, lactose monohydrate, lactose and
aspartame, lactose and cellulose, maltodextrin, maltose, mannitol,
microcrystalline cellulose and guar gum, sorbitol crystalline);
parenterals (like mannitol and povidone); plasticizers (like
dibutyl sebacate, plasticizers for coatings, polyvinylacetate
phthalate); powder lubricants (like glyceryl behenate); soft
gelatin capsules (like sorbitol special solution); spheres for
coating (like sugar spheres); spheronization agents (like glyceryl
behenate and microcrystalline cellulose); suspending/gelling agents
(like carrageenan, gellan gum, mannitol, microcrystalline
cellulose, povidone, sodium starch glycolate, xanthan gum);
sweeteners (like aspartame, aspartame and lactose, dextrose,
fructose, honey, maltodextrin, maltose, mannitol, molasses,
sorbitol crystalline, sorbitol special solution, sucrose); wet
granulation agents (like calcium carbonate, lactose anhydrous,
lactose monohydrate, maltodextrin, mannitol, microcrystalline
cellulose, povidone, starch), caramel, carboxymethylcellulose
sodium, cherry cream flavor and cherry flavor, citric acid
anhydrous, citric acid, confectioner's sugar, D&C Red No. 33,
D&C Yellow #10 Aluminum Lake, disodium edetate, ethyl alcohol
15%, FD& C Yellow No. 6 aluminum lake, FD&C Blue #1
Aluminum Lake, FD&C Blue No. 1, FD&C blue no. 2 aluminum
lake, FD&C Green No. 3, FD&C Red No. 40, FD&C Yellow
No. 6 Aluminum Lake, FD&C Yellow No. 6, FD&C Yellow No. 10,
glycerol palmitostearate, glyceryl monostearate, indigo carmine,
lecithin, manitol, methyl and propyl parabens, mono ammonium
glycyrrhizinate, natural and artificial orange flavor,
pharmaceutical glaze, poloxamer 188, Polydextrose, polysorbate 20,
polysorbate 80, polyvidone, pregelatinized corn starch,
pregelatinized starch, red iron oxide, saccharin sodium, sodium
carboxymethyl ether, sodium chloride, sodium citrate, sodium
phosphate, strawberry flavor, synthetic black iron oxide, synthetic
red iron oxide, titanium dioxide, and white wax.
[0143] In some embodiments, there is provided a pharmaceutical
composition comprising a peptide described herein and one or more
agents selected from Mg.sup.2+, Ca.sup.2+, Zn.sup.2+, Mn.sup.2+,
K.sup.+, Na.sup.+ or Al.sup.3+, a combination thereof, and/or a
sterically hindered primary amine. In further embodiments, the
agent is Mg.sup.2+, Ca.sup.2+ or Zn.sup.3+ or a combination
thereof. In some embodiments, the cation is provided, without
limitation, as magnesium acetate, magnesium chloride, magnesium
phosphate, magnesium sulfate, calcium acetate, calcium chloride,
calcium phosphate, calcium sulfate, zinc acetate, zinc chloride,
zinc phosphate, zinc sulfate, manganese acetate, manganese
chloride, manganese phosphate, manganese sulfate, potassium
acetate, potassium chloride, potassium phosphate, potassium
sulfate, sodium acetate, sodium chloride, sodium phosphate, sodium
sulfate, aluminum acetate, aluminum chloride, aluminum phosphate or
aluminum sulfate. In further embodiments, the cation is provided as
magnesium chloride, calcium chloride, calcium phosphate, calcium
sulfate, zinc acetate, manganese chloride, potassium chloride,
sodium chloride or aluminum chloride. In other embodiments, the
cation is provided as calcium chloride, magnesium chloride or zinc
acetate.
[0144] In another embodiment, the agent is a sterically hindered
primary amine. In a further embodiment, the sterically hindered
primary amine is an amino acid. In yet a further embodiment, the
amino acid is a naturally-occurring amino acid. In a still further
embodiment, the naturally-occurring amino acid is selected from the
group consisting of: histidine, phenylalanine, alanine, glutamic
acid, aspartic acid, glutamine, leucine, methionine, asparagine,
tyrosine, threonine, isoleucine, tryptophan, glycine and valine;
yet further, the naturally-occurring amino acid is leucine,
isoleucine, alanine or methionine. In a still further embodiment,
the naturally-occurring amino acid is leucine. In another
embodiment, the sterically hindered primary amine is a
non-naturally occurring amino acid (e.g., 1-aminocyclohexane
carboxylic acid). In a further embodiment, the sterically hindered
primary amine is cyclohexylamine, 2-methylbutylamine or chitosan.
In another embodiment, one or more sterically hindered primary
amines may be used in a composition.
[0145] In some cases, the sterically hindered primary amine has the
formula:
##STR00044##
wherein R.sub.1, R.sub.2 and R.sub.3 are independently selected
from: H, C(O)OH, C1-C6 alkyl, C1-C6 alkylether, C1-C6
alkylthioether, C1-C6 alkyl carboxylic acid, C1-C6 alkyl
carboxylamide and alkylaryl, wherein any group can be singly or
multiply substituted with: halogen or amino, and provided that no
more than two of R.sub.1, R.sub.2 and R.sub.3 are H. In another
embodiment, no more than one of R.sub.1, R.sub.2 and R.sub.3 is
H.
[0146] In other embodiments, there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier,
peptide, a cation selected from Mg.sup.2+, Ca.sup.2+, Zn.sup.2+,
Mn.sup.2+, K.sup.+, Na.sup.+ or Al.sup.3+, or a mixture thereof,
and a sterically hindered primary amine. In one embodiment, the
cation is Mg.sup.2+, Ca.sup.2+ or Zn.sup.2+ or a mixture thereof.
In a further embodiment, the pharmaceutical composition further
comprises a pharmaceutically acceptable binder and/or a
pharmaceutically acceptable glidant, lubricant or additive that
acts as both a glidant and lubricant and/or an antioxidant. In some
embodiments, the pharmaceutical composition is applied to a
carrier. In some embodiments, the carrier is a filler.
[0147] In some cases the molar ratio of cation:sterically hindered
primary amine:peptide in the aqueous solution applied to the
carrier is 5-100:5-50:1. In some cases, the molar ratio of
cation:sterically hindered primary amine may be equal to or greater
than 2:1 (e.g., between 5:1 and 2:1). Thus, in some cases the molar
ratio of cation:sterically hindered primary amine:peptide applied
to the carrier is 100:50:1, 100:30:1, 80:40:1, 80:30:1, 80:20:1,
60:30:1, 60:20:1, 50:30:1, 50:20:1, 40:20:1, 20:20:1, 10:10:1,
10:5:1 or 5:10:1. When binder, e.g., methylcellulose, is present in
the GC-C agonist peptide solution applied to the carrier it can be
present at 0.5%-2.5% by weight (e.g., 0.7%-1.7% or 0.7%-1% or 1.5%
or 0.7%).
[0148] In a further embodiment, the pharmaceutical composition
further comprises a pharmaceutically acceptable binder or additive,
and/or a pharmaceutically acceptable glidant, lubricant or additive
that acts as both a glidant and lubricant and/or an
antioxidant.
[0149] Suitable pharmaceutical compositions in accordance with the
invention will generally include an amount of the active
compound(s) with an acceptable pharmaceutical diluent or excipient,
such as a sterile aqueous solution, to give a range of final
concentrations, depending on the intended use. The techniques of
preparation are generally well known in the art, as exemplified by
Remington's Pharmaceutical Sciences (18th Edition, Mack Publishing
Company, 1995).
[0150] The compositions described herein may be administered
systemically or locally, e.g.: orally (e.g. using capsules,
powders, solutions, suspensions, tablets, sublingual tablets and
the like), by inhalation (e.g. with an aerosol, gas, inhaler,
nebulizer or the like), to the ear (e.g. using ear drops),
topically (e.g. using creams, gels, liniments, lotions, ointments,
pastes, transdermal patches, etc), ophthalmically (e.g. with eye
drops, ophthalmic gels, ophthalmic ointments), rectally (e.g. using
enemas or suppositories), nasally, buccally, vaginally (e.g. using
douches, intrauterine devices, vaginal suppositories, vaginal rings
or tablets, etc), via an implanted reservoir or the like, or
parenterally depending on the severity and type of the disease
being treated. The term "parenteral" as used herein includes, but
is not limited to, subcutaneous, intravenous, intramuscular,
intra-articular, intra-synovial, intrasternal, intrathecal,
intrahepatic, intralesional and intracranial injection or infusion
techniques. Preferably, the compositions are administered orally,
intraperitoneally or intravenously.
[0151] For treatment of gastrointestinal disorders, the peptides
described herein are preferably administered orally, e.g., as a
tablet, capsule, sachet containing a predetermined amount of the
active ingredient pellet, gel, paste, syrup, bolus, electuary,
slurry, powder, lyophilized powder, granules, as a solution or a
suspension in an aqueous liquid or a non-aqueous liquid; as an
oil-in-water liquid emulsion or a water-in-oil liquid emulsion, via
a liposomal formulation (see, e.g., EP 736299) or in some other
form. Orally administered compositions can include binders,
lubricants, inert diluents, lubricating, surface active or
dispersing agents, flavoring agents, and humectants. Orally
administered formulations such as tablets may optionally be coated
or scored and may be formulated so as to provide sustained, delayed
or controlled release of the active ingredient therein.
[0152] The peptides can be co-administered with other agents used
to treat gastrointestinal disorders including but not limited to
the agents described herein.
[0153] In another aspect, suitable pharmaceutical compositions may
comprise one or more other therapeutic agents. Such therapeutic
agents include, without limitation, analgesic agents;
anti-secretory agents, including proton pump inhibitors, acid pump
antagonists, H2 receptor antagonists; PDE5 inhibitors; GABA-B
antagonists; bile acid sequestrants; prokinetic and promotility
agents; antidepressants; antibiotics; antiemetics; and
mucosal-protecting agents.
Methods of Treatment
[0154] In various embodiments, the peptides and compositions
described herein are useful for the treatment of patient
gastrointestinal disorder.
[0155] In some embodiments, the gastrointestinal disorder is
selected from the group consisting of irritable bowel syndrome
(IBS), constipation, a functional gastrointestinal disorder,
gastroesophageal reflux disease, functional heartburn, dyspepsia,
visceral pain, gastroparesis, chronic intestinal
pseudo-obstruction, colonic pseudo-obstruction, Crohn's disease,
ulcerative colitis, and inflammatory bowel disease.
[0156] In a further embodiment, the gastrointestinal disorder is
constipation. The constipation can be chronic constipation,
idiopathic constipation, due to post-operative ileus, or caused by
opiate use. Clinically accepted criteria that define constipation
include 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's disease and Cystic
fibrosis. Constipation may also be the result of surgery
(postoperative ileus) or due to the use of drugs such as analgesics
(like opioids), antihypertensives, anticonvulsants,
antidepressants, antispasmodics and antipsychotics.
[0157] In other embodiments, the gastrointestinal disorder is
irritable bowel syndrome (IBS). The irritable bowel syndrome can be
constipation-predominant irritable bowel syndrome (c-IBS),
diarrhea-predominant irritable bowel syndrome (d-IBS) or
alternating between the two irritable bowel syndromes (a-IBS).
[0158] In other embodiments, the gastrointestinal disorder is
dyspepsia.
[0159] In other embodiments, the gastrointestinal disorder is
gastroparesis. The gastroparesis can be selected from idiopathic,
diabetic or post-surgical gastroparesis.
[0160] In still other embodiments, the gastrointestinal disorder is
chronic intestinal pseudo obstruction.
[0161] In other embodiments, the gastrointestinal disorder is
Crohn's disease.
[0162] In some embodiments, the gastrointestinal disorder is
ulcerative colitis.
[0163] In some embodiments, the gastrointestinal disorder is
inflammatory bowel disease.
[0164] In another embodiment, the gastrointestinal disorder is
visceral pain. In a further embodiment, the present invention
features a method for decreasing gastrointestinal pain or visceral
pain in a patient, the method comprising, administering to the
patient a pharmaceutical composition comprising of peptide
described herein. The peptide agonists described herein can be used
alone or in combination therapy for the treatment, prevention or
reduction of visceral pain associated with a gastrointestinal
disorder or pain associated with another disorder.
[0165] In another embodiment, the invention features a method for
treating inflammation, including inflammation of the
gastrointestinal tract, e.g., inflammation associated with a
gastrointestinal disorder or infection or some other disorder, the
method comprising administering to a patient a pharmaceutical
composition comprising a purified peptide described herein.
[0166] In another embodiment, the invention features a method for
treating a gastrointestinal disorder comprising administering an
agonist of the intestinal guanylate cyclase (GC-C) receptor either
orally, by rectal suppository, or parenterally.
[0167] In still another embodiment, the invention features a method
for treating a gastrointestinal disorder comprising administering
an agonist of the intestinal guanylate cyclase (GC-C) receptor.
[0168] In another aspect, the invention features a method of
increasing the level of cyclic guanosine 3'-monophosphate (cGMP) in
a biological sample, tissue (e.g, the intestinal mucosa), or cell
(e.g., a cell bearing GC-A receptor), or whole organism by
contacting the sample, tissue, or organism to a peptides described
herein.
[0169] The peptide GC-C receptor agonists described herein can be
administered in combination with other agents. For example, the
peptides can be administered with an analgesic peptide or compound.
The analgesic peptide or compound can be covalently attached to a
peptide described herein or it can be a separate agent that is
administered together with or sequentially with a peptide described
herein in a combination therapy. The peptides described herein may
also be administered in combination with other agents used to treat
GI disorders including antidepressants, promotility or prokinetic
agents, antiemetics, antibiotics, proton pump inhibitors, acid
blockers (e.g., histamine H2 receptor antagonists), acid pump
antagonists, PDE5 inhibitors, GABA-B agonists, bile acid
sequestrants, and mucosal protecting agents.
[0170] In some embodiments, useful analgesic agents that may be
used with the peptides described herein include Ca channel blockers
(e.g., ziconotide), 5HT receptor antagonists (e.g., 5HT3, 5HT4 and
5HT1 receptor antagonists), 5HT4 agonists (e.g., tegaserod
[Zelnorm.RTM.], mosapride, zacopride, cisapride, renzapride,
prucalopride [Resolor.RTM.], benzimidazolone derivatives such as
BIMU 1 and BIMU 8, and lirexapride), 5HT1 agonists (e.g.,
sumatriptan and buspirone), opioid receptor agonists (e.g.,
loperamide, fedotozine, enkephalin pentapeptide, morphine,
diphenyloxylate, frakefamide, trimebutine and fentanyl), CCK
receptor agonists (e.g., loxiglumide and dexloxiglumide), NK1
receptor antagonists (e.g., aprepitant, vofopitant, ezlopitant,
R-673 (Hoffmann-La Roche Ltd), SR-48968 and SR-14033, (Sanofi
Synthelabo), CP-122,721 (Pfizer, Inc.), GW679769 (Glaxo Smith
Kline) and TAK-637 (Takeda/Abbot)), NK2 receptor antagonists (e.g.,
nepadutant, saredutant, GW597599 (Glaxo Smith Kline), SR-144190
(Sanofi-Synthelabo) and UK-290795 (Pfizer Inc)), NK3 receptor
antagonists (e.g., osanetant (SR-142801; Sanofi-Synthelabo),
SR-241586 and talnetant), norepinephrine-serotonin reuptake
inhibitors (NSRI) (e.g., milnacipran), mixed and selective dopamine
receptor antagonists (e.g.--metoclopramide, itopride, domperidone),
vanilloid and cannabanoid receptor agonists, sialorphin and
sialorphin-related peptides. Analgesics agents in the various
classes are described in the literature.
[0171] In some embodiments, one or more other therapeutic agents
may be used in combination with the peptides described herein. Such
agents include antidepressants, promotility or prokinetic agents,
antiemetics, antibiotics, proton pump inhibitors, acid blockers
(e.g., histamine H2 receptor antagonists), acid pump antagonists,
PDE5 inhibitors, GABA-B agonists, bile acid sequestrants, and
mucosal protecting agents.
[0172] Examples of antidepressants include, without limitation,
tricyclic antidepressants such as amitriptyline desipramine
(Norpramin.RTM.), imipramine (Tofranil.RTM.), amoxapine
(Asendin.RTM.), nortriptyline; the selective serotonin reuptake
inhibitors (SSRI's) such as paroxetine (Paxil.RTM.), fluoxetine
(Prozac.RTM.), sertraline (Zoloft.RTM.), and citralopram
(Celexa.RTM.); and others such as doxepin (Sinequan.RTM.) and
trazodone (Desyrel.RTM.).
[0173] Examples of promotility and prokinetic agents include,
without limitation, itopride, octreotide, bethanechol,
metoclopramide (Reglan.RTM.), domperidone (Motilium.RTM.),
erythromycin (and derivatives thereof) and cisapride
(Propulsid.RTM.). An example of antiemetics includes, without
limitation, prochlorperazine.
[0174] Examples of antibiotics that may be used include those that
may be used to treat Heliobacter pylori infections, such as
amoxicillin, tetracycline, metronidazole, or clarithromycin. Other
antibiotics such as erythromycin and derivatives thereof may also
be used in combination with the peptides described herein.
[0175] Examples of proton pump inhibitors include, without
limitation, omeprazole (Prilosec.RTM.), esomeprazole (Nexium.RTM.),
lansoprazole (Prevacid.RTM.), pantoprazole (Protonix.RTM.) and
rabeprazole (Aciphex.RTM.). Examples of H2 receptor blockers
include, without limitation, including cimetidine, ranitidine,
famotidine and nizatidine. Examples of acid pump antagonists
include, without limitation, revaprazan, CS-526 (J. Pharmacol. Exp.
Ther. (2007) 323:308-317), PF-03716556 (J. Pharmacol. Exp. Ther.
(2009) 328(2):671-9), and YH1885 (Drug Metab. Dispos. (2001)
29(1):54-9).
[0176] Examples of PDE5 inhibitors include, without limitation,
avanafil, lodenafil, mirodenafil, sildenafil citrate, tadalafil,
vardenafil and udenafil. GABA-B agonists include, without
limitation, baclofen and XP19986 (CAS Registry No. 847353-30-4).
Examples of bile acid sequestrants include, without limitation,
GT102-279, cholestyramine, colesevelam, colesevelam hydrochloride,
ursodeoxycholic acid, colestipol, colestilan, sevelamer,
polydiallylamine cross-linked with epichlorohydrin,
dialkylaminoalkyl derivatives of a cross-linked dextran, and
N-(cycloalkyl)alkylamines. Examples of mucosal protecting agents
include, without limitation, sucralfate (Carafate), teprenone,
polaprezinc, cetraxate and bismuth subsalicyclate.
[0177] Combination therapy can be achieved by administering two or
more agents, e.g., a peptide described herein and another
therapeutic peptide or 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.
Dosage
[0178] The dose range for adult humans may be generally from 5
.mu.g to 100 mg/day orally of the peptides described herein.
Tablets, capsules, or other forms of presentation provided in
discrete units may conveniently contain an amount of compound
described herein which is effective at such dosage or as a multiple
of the same, for instance, units containing 25 .mu.g to 2 mg or
around 100 .mu.g to 1 mg. The precise amount of compound prescribed
to a patient will be the responsibility of the attendant physician.
However, the dose employed will depend on a number of factors,
including the age and sex of the patient, the precise disorder
being treated, and its severity.
[0179] In various embodiments, the dosage unit is administered with
food at anytime of the day, without food at anytime of the day,
with food after an overnight fast (e.g. with breakfast), at bedtime
after a low fat snack. In one particular embodiment, the dosage
unit is administered prior to or subsequent to food consumption
(e.g., a meal). In a further embodiment, the dosage unit is
administered approximately 15 minutes to 1 hour prior to food
consumption. In various embodiments, the dosage unit is
administered once a day, twice a day, three times a day, four times
a day, five times a day or six times a day. In certain embodiments
the dosage unit and daily dose are equivalent.
[0180] The precise amount of each of the two or more active
ingredients in a dosage unit will depend on the desired dosage of
each component. Thus, it can be useful to create a dosage unit that
will, when administered according to a particular dosage schedule
(e.g., a dosage schedule specifying a certain number of units and a
particular timing for administration), deliver the same dosage of
each component as would be administered if the patient was being
treated with only a single component. In other circumstances, it
might be desirable to create a dosage unit that will deliver a
dosage of one or more components that is less than that which would
be administered if the patient was being treated only with a single
component. Finally, it might be desirable to create a dosage unit
that will deliver a dosage of one or more components that is
greater than that which would be administered if the patient was
being treated only with a single component.
[0181] The pharmaceutical composition can include additional
ingredients including but not limited to the excipients described
herein. In certain embodiments, one or more therapeutic agents of
the dosage unit may exist in an extended or control release
formulation and additional therapeutic agents may not exist in
extended release formulation. For example, a peptide or agonist
described herein may exist in a controlled release formulation or
extended release formulation in the same dosage unit with another
agent that may or may not be in either a controlled release or
extended release formulation. Thus, in certain embodiments, it may
be desirable to provide for the immediate release of one or more of
the agents described herein, and the controlled release of one or
more other agents.
[0182] The present invention has been described with reference to
certain exemplary embodiments thereof. However, it will be readily
apparent to those skilled in the art that it is possible to embody
the invention in specific forms other than those of the exemplary
embodiments described above. This may be done without departing
from the spirit of the invention. The exemplary embodiments are
merely illustrative and should not be considered restrictive in any
way. The scope of the invention is defined by the appended claims
and their equivalents, rather than by the preceding
description.
EXAMPLES
[0183] The GC-C agonist peptides or pharmaceutically acceptable
salts thereof as described herein were prepared by solid phase
chemical synthesis and natural folding (air oxidation) by American
Peptide Company (Sunnyvale, Calif.). In some cases, the peptides
were modified after synthesis as described herein.
[0184] The Cys.sub.1-IMD peptide was synthesized by mixing 4.6 g
(3.0 mmol) of linaclotide in 200 ml of EtOH. Formaldehyde at 37%
(1.12 ml/5 eq) was added to this mixture. The reaction mixture was
incubated in a water bath (45.degree. C.) for overnight. The
following day the solvent was removed by rota-evaporation. The
peptide was further purified through reverse-phase
chromatography.
[0185] The Glu.sub.3-ethyl ester peptide was synthesized on a 20
mmol Fmoc-Tyr(tBu)-Wang resin. Protecting groups used for amino
acids are: t-Butyl group for Tyr and Thr, Trt group for Asn and
Cys. The peptide chain was assembled on the resin by repetitive
removal of the Fmoc protecting group and coupling of protected
amino acid. DIC and HOBt were used as coupling reagents and NMM was
used as the base for this reaction. 20% piperidine in DMF was used
as de-Fmoc-reagent. After removal of last Fmoc protecting group,
resin was treated with cocktail K for 3 hours to cleave the peptide
from resin and removal of the side chain protecting groups. The
eluted peptide was precipitated in cold ether and dried. The dried
peptide was dissolved in a mixture of TFA/TIS/water (95:3:2 v/v) in
a ratio of 1 to 10 (g/v). This mixture was stirred at room
temperature for 1 hour. The isolated peptide was also precipitated
in cold ether, collected by filtration and dried under high
vacuum.
[0186] The Tyr.sub.14-ethyl ester peptide was synthesized by a
fragment condensation method. Fragment A
(Boc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro--
Ala-Cys(Trt)-Thr(tBu)-Gly-OH) was prepared on 15 mmol CTC resin
using Fmoc chemistry. This peptide chain was also assembled on the
resin by repetitive removal of the Fmoc protecting group and
coupling of protected amino acid. DIC and HOBt were used as
coupling reagents and NMM was used as the base. 20% piperidine in
DMF was used as de-Fmoc-reagent. After removal of last Fmoc
protecting group, Boc was coupled to protect the N-terminal amino
group. The peptide resin was washed, dried, and treated with 1%
TFA/DCM to cleave peptide from resin. Fragment B (Cys(Trt)-Tyr-OEt)
was prepared from coupling of Fmoc-Cys(Trt)-OH and Tyr-OEt. HCl.
The Fmoc group was removed by treating this di-peptide with 20%
piperidine in DMF.
[0187] The Tyr.sub.14-ethyl ester peptide was finally synthesized
by coupling the two fragments in DMF. HBTU/HOBt/NMM was used as the
coupling reagent for this reaction. The protecting groups were
removed by treating the peptide with cocktail K for 2 hours. This
peptide was precipitated in cold ether and dried. The dried peptide
was dissolved in a mixture of TFA/TIS/water (95:3:2 v/v) in a ratio
of 1 to 10 (g/v). This mixture was stirred at room temperature for
1 hour. The isolated peptide was again precipitated in cold ether,
collected by filtration and dried under high vacuum.
Example 1: cGMP Accumulation in T84 Cells for Analysis of GC-C
Activity
[0188] For the cGMP assay, 4.5.times.10.sup.5 cells/mL of T84 cells
were grown overnight in 24 well tissue culture plates. On next day,
the T84 cells were washed twice with 1 mL of DMEM (pH 7). After the
second wash, the cells were incubated with 450 .mu.L of 1 mM
isobutylmethylxanthine (IBMX) in pH 7 buffer for 10 minutes at
37.degree. C. to inhibit any phosphodiesterase activity. The
peptides were then diluted in DMEM buffer (pH 7) to a 10.times.
concentration. The peptide solution of 50 .mu.L was diluted to a
final volume of 500 .mu.L with the T84 cells, bringing each peptide
concentration to 1.times.. The peptides were tested in duplicate at
100 nM.
[0189] There was no peptide control used to determine endogenous
levels of cGMP. Peptides were incubated for 30 minutes at
37.degree. C. After 30 minutes, the supernatants were removed and
the cells were lysed with 0.1 M HCl. The cells were lysed for 30
minutes on ice. After 30 minutes, lysates were pipetted off and
placed into a 96 well HPLC plate and spun at 10,000.times.G for 10
minutes to remove any cell debris. Supernatants from the previous
spin were removed and placed into a fresh 96 well HPLC plate.
[0190] cGMP concentrations were determined from each sample using
the LC/MS conditions (Table 1 below) and calculated standard curve.
EC.sub.50 values were calculated from concentration-response curves
generated with GraphPad Prism Software.
TABLE-US-00001 TABLE 1 LC/MS conditions MS: Thermo Quantum Ion
Mode: ESI.sup.+ Scan Type: MRM Dwell Collision Retention Time
Energy Tube Time Compound: Transition (msec) (V) Lens (min) cGMP
346 > 152 100 28 139 1.0 HPLC: Agilent Technologies 1200 Series
with CTC Analytics HTS PAL Column: Thermo Hypersil Gold 2.1 .times.
50 mm 5 micron particle size Flow Rate: 400 uL/min Column RT
Temperature: Autosampler 6.degree. C. Temperature: Injection
Volume: 20 uL Mobile Phases: A = 98:2 Water:Acetonitrile + 0.1%
Formic Acid B = 2:98 Water:Acetonitrile + 0.1% Formic Acid
Gradient: Time (min) % A % B 0 100 0 0.3 30 70 2.00 30 70 2.01 100
0 4 100 0
Example 2: Relative Binding Affinity of Exemplary Peptides to the
GC-C Receptor of T84 Cells
[0191] The relative binding affinities of linaclotide and
Cys.sub.1-IMD to the guanylate cyclase-C receptor (GC-C) were
determined using a competitive-binding assay in which the peptides
competed with a known GC-C agonist, porcine-derived heat-stable
enterotoxin (pSTa), for binding sites on cell-surface GC-C
receptors on human colonic epithelial (T84) cells. The pSTa was
radiolabeled with .sup.125I to enable measurement of its receptor
binding. The competitive-binding assay was performed by adding
various concentrations of each peptide (0.1 to 3,000 nM) to 0.20 mL
reaction mixtures containing Dulbecco's modified Eagle's medium
(DMEM), 0.5% bovine serum albumin (BSA), 2.0.times.10.sup.5 T84
cells, and 170 pM [.sup.125I]-pSTa (200,000 cpm). The data were
used to construct competitive radioligand-binding curves and
determine the relative binding affinities of linaclotide and
Cys.sub.1-IMD, as measured by their IC.sub.50 and K.sub.i
values.
[0192] Both linaclotide and Cys.sub.1-IMD competitively inhibited
the specific binding of [.sup.125I]-pSTa to cell-surface GC-C
receptors on T84 cells. Their relative binding affinities, as
measured by their inhibition constants (K.sub.i), were as follows:
Linaclotide K.sub.i=3.9.+-.1.6 nM and Cys.sub.1-IMD
K.sub.i=1.4.+-.0.5 nM (FIG. 3).
Example 3: cGMP Response in T84 Cells Induced by Exemplary
Peptides
[0193] Linaclotide and Cys.sub.1-IMD were tested for guanylate
cyclase-C (GC-C) agonist activity in T84 cells as follows. In each
well of a 96-well plate, approximately 200,000 T84 cells/well was
first incubated with 1 mM 3-isobutyl-1-methylxanthine (IBMX) in
0.18 mL of Dulbecco's modified Eagle's medium (DMEM) for 10 minutes
at 37.degree. C. Each peptide was diluted to final concentrations
ranging from 0.1 to 10,000 nM, and 0.02 mL of each dilution was
added in duplicate to a 96-well plate containing the T84 cells, for
a final volume of 0.20 mL per well. The peptide reactions were
incubated for 30 min at 37.degree. C. Following the incubation, the
supernatants were removed and discarded and the cells were lysed
with cold 0.1 M hydrochloric acid (HCl) for 30 min on ice. The cell
debris was removed by centrifugation and the concentration of
guanosine 3',5'-cyclic monophosphate (cyclic GMP) in each lysate
was determined using liquid chromatography with tandem mass
spectrometry. The data were used to construct dose-response curves
and calculate half-maximal effective concentration (EC.sub.50)
values for each test article.
[0194] Linaclotide and Cys.sub.1-IMD showed GC-C agonist activity
in T84 cells, as measured by the increase in intracellular cGMP
(FIG. 4). The EC.sub.50 values for linaclotide and Cys.sub.1-IMD
were 315.+-.105 nM and 172.+-.32 nM, respectively.
Example 4: Measurement of Content and Purity of Exemplary
Peptides
[0195] Content and purity of the peptides of the present invention
may be determined by reverse phase gradient liquid chromatography
using an Agilent Series 1100 LC System with Chemstation Rev A.09.03
software or equivalent. A YMC Pro.TM. C18 column (dimensions:
3.0.times.150 mm, 3.5 um, 120 .ANG.; Waters Corp., Milford, Mass.)
or equivalent is used and is maintained at 40.degree. C. Mobile
phase A (MPA) consists of water with 0.1% trifluoroacetic acid
while mobile phase B (MPB) consists of 95% acetonitrile:5% water
with 0.1% trifluoroacetic acid. Elution of the peptides is
accomplished with a gradient from 0% to 47% MPB in 28 minutes
followed by a ramp to 100% MPB in 4 minutes with a 5 minute hold at
100% MPB to wash the column. Re-equilibration of the column is
performed by returning to 0% MPB in 1 minute followed by a 10
minute hold at 100% MPA. The flow rate is 0.6 mL/min and detection
is accomplished by UV at 220 nm.
[0196] Samples for analysis are prepared by addition of the
contents of capsules of exemplary peptides to 0.1 N HCl to obtain a
target concentration of 20 .mu.g peptide/mL. 100 .mu.L of this
solution is injected onto the column.
Cys.sub.1-IMD Peptide
[0197] The Cys.sub.1-IMD peptide was purified using a 2-inch Waters
C18 column with 0.1% TFA buffer with a linear gradient of 15-45% in
60 minutes of buffer B at flow rate of 100 mL/min. The pooled
fractions with purity around 95% were loaded onto C18 column. After
equilibrating the column with TEAP buffer and AA buffer, the
peptide was purified and eluted out with HAC buffer with a linear
gradient of 15-75% of buffer B in 60 minutes. Pooled fractions with
purified peptide were lyophilized to dryness. An example of an
analysis of linaclotide and Cys.sub.1-IMD product by RP-HPLC is
shown in FIG. 2.
Glu.sub.3-Ethyl Ester Peptide
[0198] The Glu.sub.3-ethyl ester peptide (6.0 g) was dissolved in
12 L of 0.05M ammonium bicarbonate in water, and the oxidation
process was monitored by Ellman's test, MS and analytical HPLC. The
oxidation process took approximately 48 hours for completion.
[0199] The above solution was filtered and loaded onto a 2-inch C18
column, and purified by using 0.05M ammonium acetate buffer with a
linear gradient of 10-40% of buffer B in 60 minutes at flow rate of
100 mL/min.
[0200] The pooled fractions with purity of >95% were lyophilized
to dryness. After the peptides were dried, the peptide was
re-dissolved in acetonitrile-water and acidified to pH around 4-5
by addition of acetic acid and re-lyophilized to dryness.
Tyr.sub.14-Ethyl Ester Peptide
[0201] The Tyr.sub.14-ethyl ester peptide was purified by
dissolving 2.5 g of the isolated peptide in 5 L of 0.05M ammonium
bicarbonate in water, and the oxidation process was monitored by
Ellman's test, MS and analytical HPLC. This oxidation process took
approximately 16 hours for completion.
[0202] The above solution was filtered and loaded onto a 2-inch
Polymer column, and purified by using 0.05M ammonium bicarbonate
buffer with a linear gradient of 15-45% of buffer B in 60 minutes
at flow rate of 100 mL/min. The pooled fractions with the peptide
were lyophilized to dryness. After the peptide was dried, the
peptide was re-dissolved in acetonitrile-water and acidified to pH
4-5 by addition of acetic acid and re-lyophilized to dryness.
[0203] The contents of the purified peptides were measured by
determining the peptide concentration in the prepared sample
against a similarly prepared external peptide standard.
Other Embodiments
[0204] All publications and patents referred to in this disclosure
are incorporated herein by reference to the same extent as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Should the
meaning of the terms in any of the patents or publications
incorporated by reference conflict with the meaning of the terms
used in this disclosure, the meaning of the terms in this
disclosure are intended to be controlling. Furthermore, the
foregoing discussion discloses and describes merely exemplary
embodiments of the present invention. One skilled in the art will
readily recognize from such discussion and from the accompanying
drawings and claims, that various changes, modifications and
variations can be made therein without departing from the spirit
and scope of the invention as defined in the following claims.
* * * * *