U.S. patent application number 12/528737 was filed with the patent office on 2012-02-16 for methods and compositions for the treatment of heart failure and other disorders.
Invention is credited to Mark Currie, Angelika Fretzen, G. Todd Milne, Daniel Zimmer.
Application Number | 20120040892 12/528737 |
Document ID | / |
Family ID | 39721820 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120040892 |
Kind Code |
A9 |
Zimmer; Daniel ; et
al. |
February 16, 2012 |
Methods and Compositions for the Treatment of Heart Failure and
Other Disorders
Abstract
Peptides that act as GC-C receptor agonists and contain at least
one D-cys and are useful for the treatment of diuresis and heart
disease as well as other disorders are described.
Inventors: |
Zimmer; Daniel; (Somerville,
MA) ; Fretzen; Angelika; (Somerville, MA) ;
Currie; Mark; (Sterling, MA) ; Milne; G. Todd;
(Brookline, MA) |
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20110021419 A1 |
January 27, 2011 |
|
|
Family ID: |
39721820 |
Appl. No.: |
12/528737 |
Filed: |
February 26, 2008 |
PCT Filed: |
February 26, 2008 |
PCT NO: |
PCT/US08/54972 PCKC 00 |
371 Date: |
September 28, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60891626 |
Feb 26, 2007 |
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Current U.S.
Class: |
514/4.8 ;
514/15.6; 514/15.7; 514/16.4; 514/19.2; 514/21.4; 514/21.5;
530/326; 530/327 |
Current CPC
Class: |
A61P 1/00 20180101; A61P
9/04 20180101; A61P 1/16 20180101; A61P 9/00 20180101; A61P 3/04
20180101; A61K 38/00 20130101; A61P 1/08 20180101; A61P 29/00
20180101; A61P 1/12 20180101; A61P 35/00 20180101; A61P 1/04
20180101; C07K 7/08 20130101; A61P 9/12 20180101 |
Class at
Publication: |
514/4.8 ;
530/327; 530/326; 514/21.5; 514/21.4; 514/16.4; 514/15.6; 514/15.7;
514/19.2 |
International
Class: |
A61K 38/10 20060101
A61K038/10; A61P 9/04 20060101 A61P009/04; A61P 9/00 20060101
A61P009/00; A61P 9/12 20060101 A61P009/12; A61P 35/00 20060101
A61P035/00; A61P 29/00 20060101 A61P029/00; A61P 1/08 20060101
A61P001/08; A61P 1/12 20060101 A61P001/12; A61P 1/16 20060101
A61P001/16; A61P 1/04 20060101 A61P001/04; A61P 1/00 20060101
A61P001/00; C07K 7/08 20060101 C07K007/08; A61P 3/04 20060101
A61P003/04 |
Claims
1-84. (canceled)
85. A peptide or a pharmaceutically acceptable salt thereof
comprising the amino acid sequence: Xaa.sub.1 Xaa.sub.2 Xaa.sub.3
Cys Glu Xaa.sub.6 Xaa.sub.7 Cys Xaa.sub.9 Pro Ala Cys Thr Gly
Xaa.sub.15 Xaa.sub.16 (SEQ ID NO:7) or a pharmaceutically
acceptable salt thereof, wherein Xaa.sub.1 is any amino acid or is
missing; Xaa.sub.2 is Ala, Gly, Lys, Ser, Val or is missing;
Xaa.sub.3 is Cys or D-Cys; Xaa.sub.6 is any amino acid; Xaa.sub.7
is Cys or D-Cys; Xaa.sub.9 is Asn or Thr; Xaa.sub.15 is Cys or
D-Cys; Xaa.sub.16 is Lys, Tyr or is missing; provided that: (a) one
or more of Xaa.sub.3, Xaa.sub.7 and Xaa.sub.15 is D-Cys when
Xaa.sub.16 is other than Lys; and (b) the peptide does not consist
of the sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr. Gly
Cys.
86. The peptide of claim 85 wherein at least one of Xaa.sub.3,
Xaa.sub.7 and Xaa.sub.15 is D-Cys.
87. The peptide of claim 86 wherein Xaa.sub.3 is D-Cys
88. The peptide of claim 86 wherein Xaa.sub.7 is D-Cys.
89. The peptide of claim 86 wherein Xaa.sub.15 is D-Cys.
90. The peptide of claim 86 wherein Xaa.sub.3, Xaa.sub.7 and
Xaa.sub.15 are all D-Cys.
91. The peptide of claim 85 wherein Xaa.sub.6 is Val, Ile, Leu Tyr,
Phe, or Trp.
92. The peptide of claim 91 wherein Xaa.sub.6 is Tyr, Phe, Trp, or
Leu.
93. The peptide of claim 85 wherein Xaa.sub.1 is Ala or
missing.
94. The peptide of claim 85 wherein Xaa.sub.2 is missing.
95. The peptide of claim 85 wherein Xaa.sub.1 and Xaa.sub.2 are
missing.
96. The peptide of claim 85 wherein Xaa.sub.9 is Thr.
97. The peptide of claim 85 wherein Xaa.sub.9 is Asn.
98. The peptide of claim 85 wherein Xaa.sub.16 is Lys or Tyr.
99. The peptide of claim 85 wherein Xaa.sub.16 is missing.
100. The peptide of claim 85 wherein the peptide is PEGylated.
101. The PEGylated peptide of claim 100 wherein the peptide is
PEGylated at the amino terminus.
102. The peptide of claim 85 wherein the peptide is purified.
103. A peptide comprising of a peptide selected from FIGS. 3a and
3b or a pharmaceutically acceptable salt thereof.
104. A pharmaceutical composition comprising the peptide of claim
85 and a pharmaceutically acceptable carrier.
105. A method for reducing fluid retention, the method comprising
administering the pharmaceutical composition of claim 104 or a
pharmaceutical composition comprising a peptide consisting of the
amino acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr
Gly Cys.
106. A method of treating a disorder comprising administering to a
patient in need thereof an effective amount of the pharmaceutical
composition of claim 104 or a pharmaceutical composition comprising
a peptide consisting of the amino acid sequence D-Cys Cys Glu Leu
Cys Cys Asn Pro Ala Cys Thr Gly Cys wherein said disorder is
selected from: heart failure, hypertension, salt dependent forms of
high blood pressure, hepatic edema, liver cirrhosis; a
gastrointestinal disorder, gastrointestinal pain, visceral pain,
obesity; benign prostatic hyperplasia; or a side effect associated
with opioid administration.
107. The method of claim 106 wherein said heart failure is acute
heart failure or chronic heart failure.
108. The method of claim 106 wherein the gastrointestinal disorder
is selected from: a gastrointestinal motility disorder, chronic
intestinal pseudo-obstruction, colonic pseudo-obstruction, Crohn's
disease, duodenogastric reflux, dyspepsia, functional dyspepsia,
nonulcer dyspepsia, a functional gastrointestinal disorder,
functional heartburn, gastroesophageal reflux disease (GERD),
gastroparesis, irritable bowel syndrome, post-operative ileus,
inflammatory bowel disorder, ulcerative colitis, or
constipation.
109. The method of claim 108 wherein said constipation is chronic
constipation, idiopathic constipation, or chronic idiopathic
constipation.
110. The method of claim 108 wherein said irritable bowel syndrome
is diarrhea-predominant irritable bowel syndrome,
alternating-irritable bowel syndrome, or constipation-predominant
irritable bowel syndrome.
111. The method of claim 108 wherein the gastrointestinal disorder
is inflammatory bowel disorder.
112. The method of claim 108 wherein the gastrointestinal disorder
is Crohn's disease.
113. The method of claim 108 wherein the gastrointestinal disorder
is ulcerative colitis.
114. The method of claim 106 wherein the patient is being treated
with an opioid selected from the group consisting of alfentanil,
buprenorphine, butorphanol, codeine, dezocine, dihydrocodeine,
fentanyl, hydrocodone, hydromorphone, levorphanol, meperidine
(pethidine), methadone, morphine, nalbuphine, oxycodone,
oxymorphone, pentazocine, propiram, propoxyphene, sufentanil and
tramadol.
115. The method of claim 106 wherein the side effect is selected
from the group consisting of constipation, nausea and vomiting.
116. The method of claim 106 further comprising administering an
opioid antagonist.
117. A method for increasing naturesis, diuresis, or
gastrointestinal motility comprising administering an effective
amount of the pharmaceutical composition of claim 104 or a
pharmaceutical composition comprising a peptide consisting of the
amino acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr
Gly Cys to a patient in need thereof.
Description
TECHNICAL FIELD
[0001] This disclosure relates to methods and compositions for the
treatment of heart failure, gastrointestinal disorders and other
disorders.
BACKGROUND
[0002] Heart failure is a hemodynamic disorder resulting from
impairment of the ability of the ventricle to fill with and/or
eject blood. The disorder is commonly characterized by shortness of
breath, fatigue, limited exercise tolerance, and fluid retention
(both pulmonary congestion and peripheral edema). Heart failure is
generally progressive and can result in Class IV heart failure
(NYHA Heart Failure Classification) in which any physical activity
brings on symptoms such as shortness of breath, and symptoms can
occur even when the patient is at rest. Patients with symptoms of
advanced heart failure are treated by tightly controlling fluid
status and are often administered intravenous peripheral
vasodilators and/or positive inotropic agents. Patients suffering
Class IV heart failure should be at complete rest (confined to a
bed or chair). Among the agents that are intravenously administered
for treatment of advanced heart failure are dobutamine (beta
receptor antagonist), milrinone (phosphodiesterase inhibitor), and
nesiritide. Nesiritide is a cardiac derived peptide hormone (human
natriuretic peptide B) that is thought to bind to and activate
guanylate cyclase A (GC-A) receptor.
[0003] The guanylate cyclase-C (GC-C) receptor (reviewed by Lucas
et al. 2000 Pharmacol. Rev 52:375-414 and Vaandrager et al. 2002
Molecular and Cellular Biochemistry 230:73-83) 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 peptides).
When agonists bind to GC-C, there is an elevation of the second
messenger, cyclic GMP, and an increase in chloride and bicarbonate
secretion, resulting in an increase in intestinal fluid
secretion.
SUMMARY
[0004] Described herein are methods for treating other disorders
such as congestive heart failure and benign prostatic hyperplasia
by administering a peptide or small molecule (parenterally or
orally) that acts as an agonist of the GC-C receptor. Such agents
can be used in combination with natriuretic peptides (e.g., atrial
natriuretic peptide, brain natriuretic peptide or C-type
natriuretic peptide), a diuretic, or an inhibitor of angiotensin
converting enzyme.
[0005] The peptides described herein can be used alone or in
combination therapy to prevent and/or treat disorders associated
with fluid and sodium retention, e.g., diseases of the
electrolyte-water/electrolyte transport system within the kidney,
gut and urogenital system, heart failure (e.g., congestive heart
failure or acute heart failure), hypertension, salt dependent forms
of high blood pressure, hepatic edema, and liver cirrhosis. In
addition they can be used to facilitate diuresis or control
intestinal fluid. The peptides and agonists described herein can
also be used to treat disorders where there is abnormal
proliferation of epithelial cells within the kidney (e.g. as in the
case of renal cancer).
[0006] The peptides and agonists described herein can be used alone
or in combination therapy to prevent and/or treat kidney disease.
"Kidney disease" includes renal failure (including acute renal
failure), renal insufficiency, nephrotic edema, glomerulonephritis,
pyelonephritis, kidney failure, chronic renal failure, nephritis,
nephrosis, azotemia, uremia, immune renal disease, acute nephritic
syndrome, rapidly progressive nephritic syndrome, nephrotic
syndrome, Berger's Disease, chronic nephritic/proteinuric syndrome,
tubulointerstital disease, nephrotoxic disorders, renal infarction,
atheroembolic renal disease, renal cortical necrosis, malignant
nephroangiosclerosis, renal vein thrombosis, renal tubular
acidosis, renal glucosuria, nephrogenic diabetes insipidus,
Bartter's Syndrome, Liddle's Syndrome, polycystic kidney disease,
medullary cystic disease, medullary sponge kidney, hereditary
nephritis, and nail-patella syndrome, along with any disease or
disorder that relates to the renal system and related disorders, as
well as symptoms indicative of or related to, renal or kidney
disease and related disorders.
[0007] The peptides and agonists described herein can be used alone
or in combination therapy to prevent or treat polycystic kidney
disease. Polycystic kidney disease" "PKD" (also called "polycystic
renal disease") refers to a group of disorders characterized by a
large number of cysts distributed throughout dramatically enlarged
kidneys. The resultant cyst development leads to impairment of
kidney function and can eventually cause kidney failure. "PKD"
specifically includes autosomal dominant polycystic kidney disease
(ADPKD) and recessive autosomal recessive polycystic kidney disease
(ARPKD), in all stages of development, regardless of the underlying
cause.
[0008] The peptides and agonists described herein can be used for
treating heart failure, including heart failure at any of stages
I-IV according to New York Heart Association (NYHA) Functional
Classification.
[0009] The peptides can also be used for treating IBS and other
gastrointestinal disorders and conditions (e.g., gastrointestinal
motility disorders, chronic intestinal pseudo-obstruction, colonic
pseudo-obstruction, Crohn's disease, duodenogastric reflux,
dyspepsia, functional dyspepsia, nonulcer dyspepsia, a functional
gastrointestinal disorder, functional heartburn, gastroesophageal
reflux disease (GERD), gastroparesis, irritable bowel syndrome
(IBS, e.g., constipation predominant-IBS, diarrhea predominat-IBS,
and/or alternating-IBS)), post-operative ileus, ulcerative colitis,
chronic constipation, and disorders and conditions associated with
constipation (e.g. constipation associated with use of opiate pain
killers, post-surgical constipation, and constipation associated
with neuropathic disorders as well as other conditions and
disorders are described herein
[0010] Without being bound by any particular theory, in the case of
heart failure, salt retention, fluid retention disorders and
combinations thereof the peptides are also useful because they may
elicit one or more of diuresis, naturesis and/or kaliuresis. Thus,
the peptides described herein may be diuretics.
[0011] Without being bound by any particular theory, in the case of
IBS and other gastrointestinal disorders the peptides are useful
because they may increase gastrointestinal motility. The peptides
may also decrease inflammation and may decrease gastrointestinal
pain, visceral pain, chronic visceral hypersensitivity, or
hypersensitivity to colorectal distension.
[0012] Described herein are pharmaceutical compositions comprising
certain peptides that are capable of activating the
guanylate-cyclase C (GC-C) receptor. Also described herein are
pharmaceutical compositions comprising a peptide or GC-C agonist
described herein and one or more additional therapeutic agents
including, without limitation, the agents described herein. The
other agents can be administered with the peptides described herein
(simultaneously or sequentially). They can also be linked to a
peptide described herein to create therapeutic conjugates.
[0013] Described herein are methods for treating various disorders
by administering a peptide that acts as a partial or complete
agonist of the GC-C receptor. In certain embodiments, the peptide
includes at least six cysteines that can form three disulfide
bonds. In certain embodiments the disulfide bonds are replaced by
other covalent cross-links and in some cases the cysteines are
substituted by other residues to provide for alternative covalent
cross-links. The peptides may also include at least one trypsin or
chymotrypsin cleavage site and/or an amino or carboxy-terminal
analgesic peptide or small molecule, e.g., AspPhe or some other
analgesic peptide. When present within the peptide, the analgesic
peptide or small molecule may be preceded by a chymotrypsin or
trypsin cleavage site that allows release of the analgesic peptide
or small molecule. Certain peptides include a functional
chymotrypsin or trypsin cleavage site located so as to allow
inactivation of the peptide upon cleavage. Certain peptides having
a functional cleavage site undergo cleavage and gradual
inactivation in the digestive tract, and this is desirable in some
circumstances. In certain peptides, a functional chymotrypsin site
is altered, increasing the stability of the peptide in vivo.
[0014] The methods described herein include a method for increasing
intestinal motility comprising administering a GC-C receptor
agonist, e.g., a peptide described herein, to a patient in need
thereof; a method for treating a disorder associated with reduced
gastrointestinal transit rates or reduced gastrointestinal motility
comprising administering a GC-C receptor agonist, e.g., a peptide
described herein, to a patient in need thereof; a method for
treating a gastrointestinal hypomotility disorder comprising
administering a GC-C receptor agonist, e.g., a peptide described
herein, to a patient in need thereof; a method for treating a
non-inflammatory gastrointestinal disorder comprising administering
a GC-C receptor agonist, e.g., a peptide described herein, to a
patient in need thereof; a method for treating a gastrointestinal
disorder other than Crohn's disease and ulcerative colitis
comprising administering a GC-C receptor agonist to a patient in
need thereof; and methods and compositions for increasing
intestinal motility comprising administering a GC-C receptor
agonist to a patient in need thereof. The disorders which can be
treated by administering a GC-C receptor agonist include, for
example, constipation, constipation dominant irritable bowel
syndrome and pelvic floor dyssynergia. In certain embodiments the
patient has been diagnosed as suffering from IBS according to the
Rome criteria. In certain embodiments the patient is female.
[0015] In certain embodiments the peptides include either one or
two or more contiguous negatively charged amino acids (e.g., Asp or
Glu) or one or two or more contiguous positively charged residues
(e.g., Lys or Arg) or one or two or more contiguous positively or
negatively charged amino acids at the carboxy terminus. In these
embodiments all of the flanking amino acids at the carboxy terminus
are either positively or negatively charged. In other embodiments
the carboxy terminal charged amino acids are preceded by a Leu. For
example, any of the following amino acid sequences can be added to
the carboxy terminus of the peptide: Asp; Asp Lys; Lys Lys Lys Lys
Lys Lys; Asp Lys Lys Lys Lys Lys Lys; Leu Lys Lys; and Leu Asp. It
is also possible to simply add Leu at the carboxy terminus.
[0016] Described herein is a peptide or a pharmaceutically
acceptable salt thereof comprising the amino acid sequence:
Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Cys Glu Xaa.sub.6 Xaa.sub.7 Cys
Xaa.sub.9 Pro Ala Cys Thr Gly Xaa.sub.15 Xaa.sub.16 (SEQ ID NO:7)
or a pharmaceutically acceptable salt thereof, [0017] wherein
[0018] Xaa.sub.1 is any amino acid or is missing; [0019] Xaa.sub.2
is Ala, Gly, Lys, Ser, Val or is missing; [0020] Xaa.sub.3 is Cys
or D-Cys; [0021] Xaa.sub.6 is any amino acid; [0022] Xaa.sub.7 is
Cys or D-Cys; [0023] Xaa.sub.9 is Asn or Thr; [0024] Xaa.sub.15 is
Cys or D-Cys; [0025] Xaa.sub.16 is Lys, Tyr or is missing; provided
that: (a) one or more of Xaa.sub.3, Xaa.sub.7 and Xaa.sub.15 is
D-Cys when Xaa.sub.16 is other than Lys; and (b) the peptide does
not consist of the sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala
Cys Thr Gly Cys.
[0026] In various embodiments: Xaa.sub.3 is D-Cys; Xaa.sub.7 is
D-Cys; Xaa.sub.15 is D-Cys; Xaa.sub.3 is D-Cys; Xaa.sub.7 is Cys;
Xaa.sub.15 is D-Cys; Xaa.sub.6 is Val, Ile, Leu Tyr, Phe, or Trp;
Xaa.sub.6 is Val, Ile, or Leu; Xaa.sub.6 is Val; Xaa.sub.6 is Ile;
Xaa.sub.6 is Leu; Xaa.sub.6 is Tyr, Phe, Trp; Xaa.sub.1 is any
amino acid; Xaa.sub.1 is Gly or Ala; Xaa.sub.1 is Gly; Xaa.sub.1 is
Ala; Xaa.sub.1 is missing; Xaa.sub.6 is Tyr; Xaa.sub.6 is Phe;
Xaa.sub.6 is Trp; at least one of Xaa.sub.3, Xaa.sub.7 and
Xaa.sub.15 is D-Cys; at least two of Xaa.sub.3, Xaa.sub.7 and
Xaa.sub.15 are D-Cys; Xaa.sub.3, Xaa.sub.7 and Xaa.sub.15 are all
D-Cys; Xaa.sub.9 is Asn; Xaa.sub.9 is Thr; Xaa.sub.16 is Lys;
Xaa.sub.16 is Tyr; Xaa.sub.16 is missing; the peptide is a peptide
in any of FIGS. 3a and 3b; and the peptide is purified.
[0027] Also described is a pharmaceutical composition comprising
any of the aforementioned peptides and a pharmaceutically
acceptable carrier.
[0028] Also described is a method for reducing fluid retention, the
method comprising administering the pharmaceutical composition
comprising any of the aforementioned peptides and a
pharmaceutically acceptable carrier or a pharmaceutical composition
comprising a peptide consisting of the amino acid sequence D-Cys
Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys.
[0029] Also described is a method of treating a disorder selected
from: heart failure, hypertension, salt dependent forms of high
blood pressure, hepatic edema, or liver cirrhosis comprising
administering a pharmaceutical composition comprising any of the
aforementioned peptides and a pharmaceutically acceptable carrier
or a pharmaceutical composition comprising a peptide consisting of
the amino acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys
Thr Gly Cys.
[0030] The peptides can be used to treat chronic or acute heart
failure. In acute heart failure the patient appears to be in good
health, but suddenly develops a large myocardial infarction or
rupture of a cardiac valve. The acute heart failure is usually
largely systolic and the sudden reduction in cardiac output often
results in systemic hypotension without peripheral edema. Chronic
heart failure is typically observed in patients with dilated
cardiomyopathy or multivalvular heart disease that develops or
progresses slowly. In chronic heart failure, arterial pressure
tends to be well maintained until very late in the course, but
there is often accumulation of peripheral edema
[0031] Also described is a method for increasing naturesis
comprising administering the pharmaceutical composition comprising
any of the aforementioned peptides and a pharmaceutically
acceptable carrier or a pharmaceutical composition comprising a
peptide consisting of the amino acid sequence D-Cys Cys Glu Leu Cys
Cys Asn Pro Ala Cys Thr Gly Cys.
[0032] Also described is a method for increasing diuresis
comprising administering the pharmaceutical composition comprising
any of the aforementioned peptides and a pharmaceutically
acceptable carrier or a pharmaceutical composition comprising a
peptide consisting of the amino acid sequence D-Cys Cys Glu Leu Cys
Cys Asn Pro Ala Cys Thr Gly Cys.
[0033] Also described is a method of treating a gastrointestinal
disorder comprising administering the pharmaceutical composition
comprising any of the aforementioned peptides and a
pharmaceutically acceptable carrier or a pharmaceutical composition
comprising a peptide consisting of the amino acid sequence D-Cys
Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys.
[0034] In various embodiments the gastrointestinal disorder is
selected from: a gastrointestinal motility disorder, chronic
intestinal pseudo-obstruction, colonic pseudo-obstruction, Crohn's
disease, duodenogastric reflux, dyspepsia, functional dyspepsia,
nonulcer dyspepsia, a functional gastrointestinal disorder,
functional heartburn, gastroesophageal reflux disease (GERD),
gastroparesis, irritable bowel syndrome, post-operative ileus,
inflammatory bowel disorder, ulcerative colitis, constipation,
chronic constipation, chronic idiopathic constipation.
[0035] Also described is a method for treating obesity comprising
administering the pharmaceutical composition comprising any of the
aforementioned peptides and a pharmaceutically acceptable carrier
or a pharmaceutical composition comprising a peptide consisting of
the amino acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys
Thr Gly Cys.
[0036] Also described is a method for treating benign prostatic
hyperplasia comprising administering the pharmaceutical composition
comprising any of the aforementioned peptides and a
pharmaceutically acceptable carrier or a pharmaceutical composition
comprising a peptide consisting of the amino acid sequence D-Cys
Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys.
[0037] Also described is a method for treating constipation
comprising administering the pharmaceutical composition comprising
any of the aforementioned peptides and a pharmaceutically
acceptable carrier or a pharmaceutical composition comprising a
peptide consisting of the amino acid sequence D-Cys Cys Glu Leu Cys
Cys Asn Pro Ala Cys Thr Gly Cys.
[0038] In various embodiments: the constipation is idiopathic
constipation; the constipation is chronic idiopathic constipation;
the gastrointestinal disorder is irritable bowel syndrome; the
irritable bowel syndrome is diarrhea-predominant irritable bowel
syndrome; the irritable bowel syndrome is constipation-predominant
irritable bowel syndrome; the irritable bowel syndrome is
alternating-irritable bowel syndrome; the gastrointestinal disorder
is inflammatory bowel disorder; the gastrointestinal disorder is
Crohn's disease; and the gastrointestinal disorder is ulcerative
colitis.
[0039] Also described is a method for increasing gastrointestinal
motility comprising administering the pharmaceutical composition
comprising any of the aforementioned peptides and a
pharmaceutically acceptable carrier or a pharmaceutical composition
comprising a peptide consisting of the amino acid sequence D-Cys
Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys.
[0040] Also described is a method for decreasing gastrointestinal
pain or visceral pain comprising administering the pharmaceutical
composition comprising any of the aforementioned peptides and a
pharmaceutically acceptable carrier or a pharmaceutical composition
comprising a peptide consisting of the amino acid sequence D-Cys
Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys.
[0041] Also described is a method of preventing or treating a
side-effect associated with opioid administration, the method
comprising administering to a patient that is being treated with an
opioid a pharmaceutical composition comprising any of the
aforementioned peptides and a pharmaceutically acceptable carrier
or a pharmaceutical composition comprising a peptide consisting of
the amino acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys
Thr Gly Cys. In various embodiments: the patient is being treated
with an opioid selected from the group consisting of alfentanil,
buprenorphine, butorphanol, codeine, dezocine, dihydrocodeine,
fentanyl, hydrocodone, hydromorphone, levorphanol, meperidine
(pethidine), methadone, morphine, nalbuphine, oxycodone,
oxymorphone, pentazocine, propiram, propoxyphene, sufentanil and
tramadol; the patient is being treated with an opioid selected from
the group consisting of: morphine, codeine, oxycodone, hydrocodone,
dihydrocodeine, propoxyphene, fentanyl and tramadol; the side
effect is selected from the group consisting of constipation,
nausea and vomiting; and the method further comprises administering
an opioid antagonist (e.g., naloxone or naltrexone).
[0042] Also described is a pharmaceutical composition comprising an
opioid and any forgoing peptide or a peptide consisting of the
amino acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr
Gly Cys. In various embodiments: the opioid is selected from the
group consisting of alfentanil, buprenorphine, butorphanol,
codeine, dezocine, dihydrocodeine, fentanyl, hydrocodone,
hydromorphone, levorphanol, meperidine (pethidine), methadone,
morphine, nalbuphine, oxycodone, oxymorphone, pentazocine,
propiram, propoxyphene, sufentanil and tramadol; and the opioid is
selected from the group consisting of: morphine, codeine,
oxycodone, hydrocodone, dihydrocodeine, propoxyphene, fentanyl and
tramadol.
[0043] Also described is a pharmaceutical kit comprising:
(a) a first container containing pharmaceutical dosage units
comprising an effective amount of an opioid; and (b) a second
container containing pharmaceutical dosage units comprising an
effective amount of a forgoing peptide or a peptide consisting of
the amino acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys
Thr Gly Cys. In various embodiments: the opioid is selected from
the group consisting of alfentanil, buprenorphine, butorphanol,
codeine, dezocine, dihydrocodeine, fentanyl, hydrocodone,
hydromorphone, levorphanol, meperidine (pethidine), methadone,
morphine, nalbuphine, oxycodone, oxymorphone, pentazocine,
propiram, propoxyphene, sufentanil and tramadol; and the opioid is
selected from the group consisting of: morphine, codeine,
oxycodone, hydrocodone, dihydrocodeine, propoxyphene, fentanyl and
tramadol.
[0044] Also described is a method for preparing a pharmaceutical
composition comprising admixing a forgoing peptide and a
pharmaceutically acceptable carrier.
[0045] Also described herein are purified peptides comprising,
consisting of, or consisting essentially of the amino acid sequence
of SEQ ID NO: 7 and those peptides depictured in FIG. 3a and FIG.
3b.
[0046] Also described herein are pharmaceutical compositions
comprising peptides comprising, consisting of, or consisting
essentially of the amino acid sequence of SEQ ID NO:7 and those
peptides depicted in FIG. 3a and FIG. 3b.
[0047] In certain embodiments, for example, when fully folded, the
peptide includes disulfide bonds between Cys.sub.3 and Cys.sub.8,
between Cys.sub.4 and Cys.sub.12 and between Cys.sub.7 and
Cys.sub.15. In other embodiments, the peptide is a reduced peptide
having no disulfide bonds. In still other embodiments the peptide
has one or two disulfide bonds chosen from: a disulfide bond
between Cys.sub.3 and Cys.sub.8, a disulfide bond between Cys.sub.4
and Cys.sub.12 and a disulfide bond between Cys.sub.7 and
Cys.sub.15. In other embodiments, one or more of Cys.sub.3,
Cys.sub.7, or Cys.sub.15 is a D-Cys residue and the D-Cys residues
can form disulfide bonds in the same manner as the Cys residues.
Thus, the peptide may include, for example, one or more disulfide
bonds between D-Cys.sub.3 and Cys.sub.8, between Cys.sub.4 and
Cys.sub.12, between D-Cys.sub.7 and Cys.sub.15, between Cys.sub.7
and D-Cys.sub.15, between D-Cys.sub.7 and D-Cys.sub.15.
[0048] In some embodiments the peptide is 13, 14, 15, or 16 amino
acids long.
[0049] In certain embodiments, one or more amino acids can be
replaced by a non-naturally occurring amino acid or a naturally or
non-naturally occurring amino acid analog. In certain embodiments,
one or more L-amino acids can be substituted with a D-amino acid.
There are many amino acids beyond the standard 20 amino acids (Ala,
Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,
Pro, Ser, Thr, Trp, Tyr, and Val). Some are naturally-occurring
others are not (see, for example, Hunt, The Non-Protein Amino
Acids: In Chemistry and Biochemistry of the Amino Acids, Barrett,
Chapman and Hall, 1985). For example, an aromatic amino acid can be
replaced by 3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine,
triiodothyronine, L-thyroxine, phenylglycine (Phg) or nor-tyrosine
(norTyr). Phg and norTyr and other amino acids including Phe and
Tyr can be substituted by, e.g., a halogen, --CH3, --OH,
--CH.sub.2NH.sub.3, --C(O)H, --CH.sub.2CH.sub.3, --CN,
--CH.sub.2CH.sub.2CH.sub.3, --SH, or another group. Any amino acid
can be substituted by the D-form of the amino acid. Thus, for
example, a cysteine residue can be substituted by a D-cysteine
residue.
[0050] With regard to non-naturally occurring amino acids or
naturally and non-naturally occurring amino acid analogs, a number
of substitutions in the peptide of SEQ ID NO:7 or the peptides of
FIG. 3a and FIG. 3b are possible alone or in combination.
[0051] Glu can be replaced by gamma-Hydroxy-Glu or
gamma-Carboxy-Glu.
[0052] Ala can be replaced by an alpha substituted amino acid such
as L-alpha-methylphenylalanine or by analogues such as:
3-Amino-Tyr; Tyr(CH.sub.3); Tyr(PO.sub.3(CH.sub.3).sub.2);
Tyr(SO.sub.3H); beta-Cyclohexyl-Ala; beta-(1-Cyclopentenyl)-Ala;
beta-Cyclopentyl-Ala; beta-Cyclopropyl-Ala; beta-Quinolyl-Ala;
beta-(2-Thiazolyl)-Ala; beta-(Triazole-1-yl)-Ala;
beta-(2-Pyridyl)-Ala; beta-(3-Pyridyl)-Ala; Amino-Phe; Fluoro-Phe;
Cyclohexyl-Gly; tBu-Gly; beta-(3-benzothienyl)-Ala;
beta-(2-thienyl)-Ala; 5-Methyl-Trp; and 4-Methyl-Trp.
[0053] Pro can be an N(alpha)-C(alpha) cyclized amino acid
analogues with the structure:
##STR00001##
Pro can also be homopro (L-pipecolic acid); hydroxy-Pro;
3,4-Dehydro-Pro; 4-fluoro-Pro; or alpha-methyl-Pro.
[0054] Val or Leu can also be an alpha-substituted or N-methylated
amino acid such as alpha-amino isobutyric acid (aib),
L/D-alpha-ethylalanine (L/D-isovaline), L/D-methylvaline, or
L/D-alpha-methylleucine or a non-natural amino acid such as
beta-fluoro-Ala.
[0055] Gly can be alpha-amino isobutyric acid (aib) or
L/D-alpha-ethylalanine (L/D-isovaline).
[0056] Further examples of unnatural amino acids include: an
unnatural analogue of tyrosine; an unnatural analogue of glutamine;
an unnatural analogue of phenylalanine; an unnatural analogue of
serine; an unnatural analogue of threonine; an alkyl, aryl, acyl,
azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl,
alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate,
boronate, phospho, phosphono, phosphine, heterocyclic, enone,
imine, aldehyde, hydroxylamine, keto, or amino substituted amino
acid, or any combination thereof; an amino acid with a
photoactivatable cross-linker; a spin-labeled amino acid; a
fluorescent amino acid; an amino acid with a novel functional
group; an amino acid that covalently or noncovalently interacts
with another molecule; a metal binding amino acid; an amino acid
that is amidated at a site that is not naturally amidated, a
metal-containing amino acid; a radioactive amino acid; a photocaged
and/or photoisomerizable amino acid; a biotin or biotin-analogue
containing amino acid; a glycosylated or carbohydrate modified
amino acid; a keto containing amino acid; amino acids comprising
polyethylene glycol or polyether; a heavy atom substituted amino
acid (e.g., an amino acid containing deuterium, tritium, .sup.13C,
.sup.15N, or .sup.18O); a chemically cleavable or photocleavable
amino acid; an amino acid with an elongated side chain; an amino
acid containing a toxic group; a sugar substituted amino acid,
e.g., a sugar substituted serine or the like; a carbon-linked
sugar-containing amino acid; a redox-active amino acid; an
.alpha..-hydroxy containing acid; an amino thio acid containing
amino acid; an .alpha., .alpha. disubstituted amino acid; a
.beta.-amino acid; a cyclic amino acid other than proline; an
O-methyl-L-tyrosine; an L-3-(2-naphthyl)alanine; a
3-methyl-phenylalanine; a p-acetyl-L-phenylalanine; an
0-4-allyl-L-tyrosine; a 4-propyl-L-tyrosine; a
tri-O-acetyl-GlcNAc.beta.-serine; an L-Dopa; a fluorinated
phenylalanine; an isopropyl-L-phenylalanine; a
p-azido-L-phenylalanine; a p-acyl-L-phenylalanine; a
p-benzoyl-L-phenylalanine; an L-phosphoserine; a phosphonoserine; a
phosphonotyrosine; a p-iodo-phenylalanine; a 4-fluorophenylglycine;
a p-bromophenylalanine; a p-amino-L-phenylalanine; an
isopropyl-L-phenylalanine; L-3-(2-naphthyl)alanine; an amino-,
isopropyl-, or O-allyl-containing phenylalanine analogue; a dopa,
O-methyl-L-tyrosine; a glycosylated amino acid; a
p-(propargyloxy)phenylalanine; dimethyl-Lysine; hydroxy-proline;
mercaptopropionic acid; methyl-lysine; 3-nitro-tyrosine;
norleucine; pyro-glutamic acid; Z (Carbobenzoxyl);
.epsilon.-Acetyl-Lysine; .beta.-alanine; aminobenzoyl derivative;
aminobutyric acid (Abu); citrulline; aminohexanoic acid;
aminoisobutyric acid; cyclohexylalanine; d-cyclohexylalanine;
hydroxyproline; nitro-arginine; nitro-phenylalanine;
nitro-tyrosine; norvaline; octahydroindole carboxylate; ornithine;
penicillamine; tetrahydroisoquinoline; acetamidomethyl protected
amino acids and pegylated amino acids. Further examples of
unnatural amino acids and amino acid analogs can be found in U.S.
20030108885, U.S. 20030082575, US20060019347 (paragraphs 410-418)
and the references cited therein. The peptides described herein can
include further modifications including those described in
US20060019347, paragraph 589.
[0057] In some embodiments, an amino acid can be replaced by a
naturally-occurring, non-essential amino acid, e.g., taurine.
[0058] Methods to manfacture peptides containing unnatural amino
acids can be found in, for example, US20030108885, US20030082575,
US20060019347, Deiters et al., J Am Chem. Soc. (2003) 125:11782-3,
Chin et al., Science (2003) 301:964-7, and the references cited
therein.
[0059] Peptides that include non-natural amino acids can also be
prepared using the methods described in WO02086075
[0060] The peptides described herein can have one or more
conventional peptide bonds replaced by an alternative bond. Such
replacements can increase the stability of the peptide. For
example, replacement of the peptide bond between D-Cys.sub.15 or
Cys.sub.15 and Xaa.sub.16 with an alternative bond can reduce
cleavage by carboxy peptidases and may increase half-life in the
digestive tract. Bonds that can replace peptide bonds include: a
retro-inverso bonds (C(O)--NH instead of NH--C(O); a reduced amide
bond (NH--CH.sub.2); a thiomethylene bond (S--CH.sub.2 or
CH.sub.2--S); an oxomethylene bond (O--CH.sub.2 or CH.sub.2--O); an
ethylene bond (CH.sub.2--CH.sub.2); a thioamide bond (C(S)--NH); a
trans-olefine bond (CH.dbd.CH); an fluoro substituted trans-olefine
bond (CF.dbd.CH); a ketomethylene bond (C(O)--CHR or CHR--C(O)
wherein R is H or CH.sub.3; and a fluoro-ketomethylene bond
(C(O)--CFR or CFR--C(O) wherein R is H or F or CH.sub.3.
[0061] The peptides described herein can be modified using standard
modifications. Modifications may occur at the amino (N--), carboxy
(C--) terminus, internally or a combination of any of the
preceeding. In one aspect described herein, there may be more than
one type of modification of the peptide. Modifications include but
are not limited to: acetylation, amidation, biotinylation,
cinnamoylation, farnesylation, formylation, myristoylation,
palmitoylation, phosphorylation (Ser, Tyr or Thr), stearoylation,
succinylation, sulfurylation and cyclisation (via disulfide bridges
or amide cyclisation), and modification by Cy3 or Cy5. The peptides
described herein may also be modified by 2,4-dinitrophenyl (DNP),
DNP-lysin, modification by 7-Amino-4-methyl-coumarin (AMC),
flourescein, NBD (7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide,
rhodamine B, EDANS (5-((2-aminoethyl)amino)naphthalene-1sulfonic
acid), dabcyl, dabsyl, dansyl, Texas Red, FMOC, and Tamra
(Tetramethylrhodamine). The peptides described herein may also be
conjugated to, for example, polyethylene glycol (PEG); alkyl groups
(e.g., C1-C20 straight or branched alkyl groups); fatty acid
radicals; combinations of PEG, alkyl groups and fatty acid radicals
(see U.S. Pat. No. 6,309,633; Soltero et al., 2001 Innovations in
Pharmaceutical Technology 106-110); BSA and KLH (Keyhole Limpet
Hemocyanin). The addition of PEG and other polymers which can be
used to modify peptides described herein is described in
US2006019347 section IX.
[0062] The peptides and agonists described herein can be chemically
modified to increase therapeutic activity by synthetically adding
sugar moieties (WO 88/02756; WO 89/09786; DE 3910667 A1, EP 0 374
089 A2; and U.S. Pat. No. 4,861,755), adding cationic anchors
(EP0363589), lipid moieties (WO91/09837; U.S. Pat. No. 4,837,303)
or the substituents described as compounds I, II, and III in U.S.
Pat. No. 5,552,520.
[0063] The peptides described herein bear some sequence similarity
to ST peptides. However, they include amino acid changes and/or
additions that improve functionality. These changes can, for
example, increase or decrease activity (e.g., increase or decrease
the ability of the peptide to stimulate intestinal motility), alter
the ability of the peptide to fold correctly, alter the stability
of the peptide, alter the ability of the peptide to bind the GC-C
receptor and/or decrease toxicity. In some cases the peptides may
function more desirably than wild-type ST peptide. For example,
they may limit undesirable side effects such as diarrhea and
dehydration.
[0064] In some embodiments one or both members of one or more pairs
of Cys residues (including where a Cys residue has been substituted
with a D-cys residue) 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); .beta.,
.beta. 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.
[0065] In addition, one or more disulfide bonds can be replaced by
alternative covalent cross-links, e.g., an amide linkage
(--CH.sub.2CH(O)NHCH.sub.2-- or --CH.sub.2NHCH(O)CH.sub.2--), an
ester linkage, a thioester linkage, a lactam bridge, a carbamoyl
linkage, a urea linkage, a thiourea linkage, a phosphonate ester
linkage, an alkyl linkage (--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--),
an alkenyl linkage (--CH.sub.2CH.dbd.CHCH.sub.2--), an ether
linkage (--CH.sub.2CH.sub.2OCH.sub.2-- or
--CH.sub.2OCH.sub.2CH.sub.2--), a thioether linkage
(--CH.sub.2CH.sub.2SCH.sub.2-- or --CH.sub.2SCH.sub.2CH.sub.2--),
an amine linkage (--CH.sub.2CH.sub.2NHCH.sub.2-- or
--CH.sub.2NHCH.sub.2CH.sub.2--) or a thioamide linkage
(--CH.sub.2CH(S)HNHCH.sub.2-- or --CH.sub.2NHCH(S)CH.sub.2--). For
example, Ledu et al. (Proc Nat'l Acad. Sci. 100:11263-78, 2003)
describe methods for preparing lactam and amide cross-links.
Schafmeister et al. (J. Am. Chem. Soc. 122:5891, 2000) describes
stable, hydrocarbon cross-links. Hydrocarbon cross links can be
produced via metathesis (or methathesis followed by hydrogenation
in the case of saturated hydrocarbons cross-links) using one or
another of the Grubbs catalysts (available from Materia, Inc. and
Sigma-Aldrich and described, for example, in U.S. Pat. Nos.
5,831,108 and 6,111,121). In some cases, the generation of such
alternative cross-links requires replacing the Cys residues with
other residues such as Lys or Glu or non-naturally occurring amino
acids. In addition the lactam, amide and hydrocarbon cross-links
can be used to stabilize the peptide even if they link amino acids
at positions other than those occupied by Cys. Such cross-links can
occur between two amino acids that are separated by two amino acids
or between two amino acids that are separated by six amino acids
(see, e.g., Schafmeister et al. (J. Am. Chem. Soc. 122:5891,
2000)).
[0066] The peptide may contain additional carboxyterminal or amino
terminal amino acids or both. For example, the peptide can include
an amino terminal sequence that facilitates recombinant production
of the peptide and is cleaved prior to administration of the
peptide to a patient. The peptide can also include other amino
terminal or carboxyterminal amino acids. In some cases the
additional amino acids protect the peptide, stabilize the peptide
or alter the activity of the peptide. In some cases some or all of
these additional amino acids are removed prior to administration of
the peptide to a patient. The peptide can include 1, 2, 3, 4, 5,
10, 15, 20, 25, 30, 40, 50, 60, 70 80, 90, 100 or more amino acids
at its amino terminus or carboxy terminus or both. The number of
flanking amino acids need not be the same. For example, there can
be 10 additional amino acids at the amino terminus of the peptide
and none at the carboxy terminus.
[0067] The peptides can be co-administered with or linked, e.g.,
covalently linked to any of a variety of other peptides or
compounds including analgesic peptides or analgesic compounds
including, without limitation, the agents described herein.
[0068] Amino acid, non-amino acid, peptide and non-peptide spacers
can be interposed between a peptide that is a GC-C receptor agonist
and a peptide that has some other biological function, e.g., an
analgesic peptide or a peptide used to treat obesity. The linker
can be one that is cleaved from the flanking peptides in vivo or
one that remains linked to the flanking peptides in vivo. For
example, glycine, beta-alanine, glycyl-glycine,
glycyl-beta-alanine, gamma-aminobutyric acid, 6-aminocaproic acid,
L-phenylalanine, L-tryptophan and glycil-L-valil-L-phenylalanine
can be used as spacers (Chaltin et al. 2003 Helvetica Chimica Acta
86:533-547; Caliceti et al. 1993 FARMCO 48:919-32) as can
polyethylene glycols (Butterworth et al. 1987 J. Med. Chem.
30:1295-302) and maleimide derivatives (King et al. 2002
Tetrahedron Lett. 43:1987-1990). Various other linkers are
described in the literature (Nestler 1996 Molecular Diversity
2:35-42; Finn et al. 1984 Biochemistry 23:2554-8; Cook et al. 1994
Tetrahedron Lett. 35:6777-80; Brokx et al. 2002 Journal of
Controlled Release 78:115-123; Griffin et al. 2003 J. Am. Chem.
Soc. 125:6517-6531; Robinson et al. 1998 Proc. Natl. Acad. Sci. USA
95:5929-5934). Linkers are also described in US20050171014, for
example, amino acid linkers such as FALA, VLALA, ALAL, ALALA,
2-cyclohexyl-L-alanine-LALA,
2-cyclohexyl-L-alanine-2-cyclohexyl-L-alanine-LAL,
1-naphtyl-alanine-ChaLAL and 1-naphtyl-alanine-LALA. Peptides and
agonists described herein can also be conjugated to: an affinity
tag (such as (histidine 6) H6), a HIV tat peptide residues 49-57,
HIV tat peptide residues 49-56, the tat sequence YGRKKRRQRRR, a
polyarginine peptide having from 6 to 20 residues (such as R6) and
the following peptide sequences: YARKARRQARR, YARAAARQARA,
YARAARRAARR, YARAARRAARA, ARRRRRRRRR, and YAAARRRRRRR, which are
disclosed in WO 99/29721 and in U.S. Pat. No. 6,221,355 (seq. id.
nos. 3-8).
[0069] The peptides described herein can be attached to one, two or
more different moieties each providing the same or different
functions. For example, the peptide can be linked to a molecule
that is an analgesic and to a peptide that is used to treat
obesity. The peptide and various moieties can be ordered in various
ways. For example, a peptide described herein can have an analgesic
peptide linked to its amino terminus and an anti-obesity peptide
linked to its carboxy terminus. The additional moieties can be
directly covalently bonded to the peptide or can be bonded via
linkers.
[0070] The peptides described herein can be a cyclic peptide or a
linear peptide. In addition, multiple copies of the same peptide
can be incorporated into a single cyclic or linear peptide.
[0071] The peptides can include the amino acid sequence of a
peptide that occurs naturally in a vertebrate (e.g., mammalian)
species or in a bacterial species. In addition, the peptides can be
partially or completely non-naturally occurring peptides. Also
within the disclosure are peptidomimetics corresponding to the
peptides described herein.
[0072] Described herein is a method for treating congestive heart
failure, the method comprising administering to a patient a
pharmaceutical composition comprising a purified peptide
comprising, consisting of or consisting essentially of the amino
acid sequence of SEQ ID NO:7 (e.g., a peptide in FIG. 3a or FIG.
3b). The peptide can be administered alone or in combination with
another agent for treatment of congestive heart failure, for
example, a natriuretic peptide such as atrial natriuretic peptide,
brain natriuretic peptide or C-type natriuretic peptide, an
inhibitor of angiotensin converting enzyme, a diuretic (e.g.
furesomide (Lasix), bumetanide (Bumex), ethacrynic acid (Edecrin),
torsemide (Demadex), amiloride (Midamor), spironolactone
(Aldactone), chorthiazide (Diuril), metolazone (Zaroxylyn)), an
Angiotension-Converting Enzyme (ACE) inhibitor (e.g. captopril
(Capoten), enalopril (Vasotec), lisinopril (Prinivil, Zestril),
ramipril (Altace)), a Beta blocker (e.g. carvedilol (Coreg) or an
inotropes (e.g. digoxin, dobutaimine, dopamine Milrinone). In
various embodiments the congestive heart failure is categorized as
Class II congestive heart failure; the congestive heart failure is
categorized as Class III congestive heart failure; and the
congestive heart failure is categorized as Class IV congestive
heart failure. The New York Heart Association (NYHA) functional
classification system relates congestive heart failure symptoms to
everyday activities and the patient's quality of life. The NYHA
defines the classes of patient symptoms relating to congestive
heart failure as: Class II--slight limitation of physical activity,
comfortable at rest, but ordinary physical activity results in
fatigue, palpitation, or dyspnea; Class III--marked limitation of
physical activity, comfortable at rest, but less than ordinary
activity causes fatigue, palpitation, or dyspnea and Class
IV--unable to carry out any physical activity without discomfort,
symptoms of cardiac insufficiency at rest, if any physical activity
is undertaken, discomfort is increased. Heart failure treatment
using the peptides and methods described herein can also be
classified according to the ACC/AHA guidelines (Stage A: At risk
for developing heart failure without evidence of cardiac
dysfunction; Stage B: Evidence of cardiac dysfunction without
symptoms; Stage C: Evidence of cardiac dysfunction with symptoms;
and Stage D: Symptoms of heart failure despite maximal
therapy).
[0073] Described herein is a method for treating a gastrointestinal
disorder, the method comprising administering a peptide comprising,
consisting essentially of or consisting of the amino acid sequence
of SEQ ID NO:7, e.g., a peptide in FIG. 3a or FIG. 3b. In various
embodiments: the patient is suffering from a disorder selected from
the group consisting of: gastrointestinal motility disorders,
chronic intestinal pseudo-obstruction, colonic pseudo-obstruction,
Crohn's disease, duodenogastric reflux, dyspepsia, functional
dyspepsia, nonulcer dyspepsia, a functional gastrointestinal
disorder, functional heartburn, gastroesophageal reflux disease
(GERD), gastroparesis, irritable bowel syndrome, post-operative
ileus, ulcerative colitis, chronic constipation, and disorders and
conditions associated with constipation (e.g. constipation
associated with use of opiate pain killers, post-surgical
constipation, and constipation associated with neuropathic
disorders as well as other conditions and disorders are described
herein); the patient is suffering from a gastrointestinal motility
disorder, chronic intestinal pseudo-obstruction, colonic
pseudo-obstruction, Crohn's disease, duodenogastric reflux,
dyspepsia, functional dyspepsia, nonulcer dyspepsia, a functional
gastrointestinal disorder, functional heartburn, gastroesophageal
reflux disease (GERD), gastroparesis, inflammatory bowel disease,
irritable bowel syndrome (e.g. d-IBS, c-IBS, and/or a-IBS),
post-operative ileus, ulcerative colitis, chronic constipation, and
disorders and conditions associated with constipation (e.g.
constipation associated with use of opiate pain killers,
post-surgical constipation, and constipation associated with
neuropathic disorders as well as other conditions and disorders are
described herein); the patient has been diagnosed with a functional
gastrointestinal disorder according to the Rome Criteria (e.g. Rome
II), the patient has been diagnosed with irritable bowel syndrome
(e.g. (e.g. diarrhea predominant-IBS, constipation predominant-IBS,
and/or alternating-IBS), according to the Rome Criteria (e.g. Rome
II); the composition is administered orally; the peptide comprises
30 or fewer amino acids, the peptide comprises 20 or fewer amino
acids, the peptide comprises no more than 5 amino acids prior to
Cys.sub.6; the peptide comprises 14 amino acids, the peptide
comprises 13 amino acids; the peptide comprises 150, 140, 130, 120,
110, 100, 90, 80, 70, 60, 50, 40, or 30 or fewer amino acids. In
other embodiments, the peptide comprises 20 or fewer amino acids.
In other embodiments the peptide comprises no more than 20, 15, 10,
or 5 peptides subsequent to Cys.sub.18. In certain embodiments
Xaa.sub.19 is a chymotrypsin or trypsin cleavage site and an
analgesic peptide is present immediately following Xaa.sub.19.
[0074] Described herein is a method for treating a patient
suffering from constipation. 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. Also disclosed
are methods for increasing gastrointestinal motility in a patient,
the method comprising administering to a patient a pharmaceutical
composition comprising a purified peptide comprising, consisting of
or consisting essentially of the amino acid sequence of SEQ ID
NO:7, e.g., a peptide in FIG. 3a or FIG. 3b.
[0075] Also disclosed are methods for increasing the activity of
(activating) an intestinal guanylate cyclase (GC-C) receptor in a
patient, the method comprising administering to a patient a
pharmaceutical composition comprising a purified peptide
comprising, consisting of or consisting essentially of the amino
acid sequence of SEQ ID NO:7, e.g., a peptide in FIG. 3a or FIG.
3b.
[0076] Also disclosed is an isolated nucleic acid molecule
comprising a nucleotide sequence encoding a peptide comprising,
consisting of or consisting essentially of SEQ ID NO:7, e.g., a
peptide in FIG. 3a or FIG. 3b.
[0077] Also described is a method for treating obesity, the method
comprising administering to a patient a pharmaceutical composition
comprising or consisting essentially of a purified peptide
comprising, consisting of or consisting essentially of the amino
acid sequence of SEQ ID NO:7, e.g., a peptide in FIG. 3a or FIG.
3b.
[0078] Also described is a method for treating benign prostatic
hyperplasia, the method comprising administering to a patient a
pharmaceutical composition comprising a purified peptide
comprising, consisting of or consisting essentially of the amino
acid sequence of SEQ ID NO:7, e.g., a peptide in FIG. 3a or FIG.
3b. The peptide can be administered alone or in combination with
another agent for treatment of BPH, for example, a 5-alpha
reductase inhibitor (e.g., finasteride) or an alpha adrenergic
inhibitor (e.g., doxazosine).
[0079] Also described is a method for treating or reducing pain,
including visceral pain, pain associated with a gastrointestinal
disorder or pain associated with some other disorder, the method
comprising administering to a patient a pharmaceutical composition
comprising or consisting essentially of a purified peptide
comprising, consisting of or consisting essentially of the amino
acid sequence of SEQ ID NO:7, e.g., a peptide in FIG. 3a or FIG.
3b.
[0080] Also described is 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 comprising, consisting of or consisting
essentially of the amino acid sequence of SEQ ID NO: 7, e.g., a
peptide in FIG. 3a or FIG. 3b.
[0081] In prevention and/or treatment disorders associated with
fluid and sodium retention (e.g., heart failure, congestive heart
failure, kidney disease, etc), the agents described herein can be
administered, for example, via a parenteral route, intravenously,
and/or subcutaneously. Intravenous administration may comprise, for
example, (1) one or more successive rounds of a bolus followed by
an infusion or an infusion followed by a bolus, (2) infusion, and
(3) bolus administration. The dosage may vary depending on the
administration schedule. Thus, bolus administrations may involve
dosing from about 0.1-1000 ug/kg, from about 1-100 ug/kg, or from
about 10, 15, 20, 25, or 30 ug/kg. Infusion administrations may
involve dosing from about 0.1-1000 ug/kg/hour, from about 1-100
ug/kg/hour, about 10 ug/kg/hour. During the duration of an infusion
administration, the dosage may vary (for example, decreasing over
time, increasing over time, and combinations thereof) or may remain
constant. Subcutaneous administration may involve dosing from about
0.1-1000 ug/kg, from about 1-100 ug/kg, from about 10, 15, 20, 25,
or 30 ug/kg.
[0082] As noted above, isolated nucleic acid molecules comprising a
sequence encoding a peptide described herein are described. Also
described are vectors, e.g., expression vectors that include such
nucleic acid molecules and can be used to express a peptide
described herein in a cultured cell (e.g., a eukaryotice cell or a
prokaryotic cell). The vector can further include one or more
regulatory elements, e.g., a heterologous promoter or elements
required for translation operably linked to the sequence encoding
the peptide. In some cases the nucleic acid molecule will encode an
amino acid sequence that includes the amino acid sequence of a
peptide described herein. For example, the nucleic acid molecule
can encode a preprotein or a preproprotein that can be processed to
produce a peptide described herein. In cases where unnatural amino
acids are present in the peptides described herein, selector codons
can be utilized in the synthesis of such peptides similar to that
described in US20060019347 (for example, paragraphs 398-408,
457-499, and 576-588) herein incorporated by reference.
[0083] A vector that includes a nucleotide sequence encoding a
peptide described herein or a peptide or peptide comprising a
peptide described herein may be either RNA or DNA, single- or
double-stranded, prokaryotic, eukaryotic, or viral. Vectors can
include transposons, viral vectors, episomes, (e.g., plasmids),
chromosomes inserts, and artificial chromosomes (e.g. BACs or
YACs). Suitable bacterial hosts for expression of the encode
peptide or peptide include, but are not limited to, E. coli.
Suitable eukaryotic hosts include yeast such as S. cerevisiae,
other fungi, vertebrate cells, invertebrate cells (e.g., insect
cells), plant cells, human cells, human tissue cells, and whole
eukaryotic organisms. (e.g., a transgenic plant or a transgenic
animal). Further, the vector nucleic acid can be used to transfect
a virus such as vaccinia or baculovirus (for example using the
Bac-to-Bac.RTM. Baculovirus expression system (Invitrogen Life
Technologies, Carlsbad, Calif.)).
[0084] As noted above the disclosure includes vectors and genetic
constructs suitable for production of a peptide described herein or
a peptide or peptide comprising such a peptide. Generally, 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 and may be functional
in, but are not limited to, a bacterium, yeast, plant, or animal
cell. The expression vector can also include a translation
regulatory sequence (e.g., an untranslated 5' sequence, an
untranslated 3' sequence, a poly A addition site, or an internal
ribosome entry site), a splicing sequence or splicing regulatory
sequence, and a transcription termination sequence. The vector can
be capable of autonomous replication or it can integrate into host
DNA.
[0085] Also described are isolated host cells harboring one of the
forgoing nucleic acid molecules and methods for producing a peptide
by culturing such a cell and recovering the peptide or a precursor
of the peptide. Recovery of the peptide or precursor may refer to
collecting the growth solution and need not involve additional
steps of purification. Proteins of the present disclosure, however,
can be purified using standard purification techniques, such as,
but not limited to, affinity chromatography, thermaprecipitation,
immunoaffinity chromatography, ammonium sulfate precipitation, ion
exchange chromatography, filtration, electrophoresis and
hydrophobic interaction chromatography.
[0086] The peptides can be purified. Purified peptides are peptides
separated from other proteins, lipids, and nucleic acids or from
the compounds from which is it synthesized. The peptide can
constitute at least 10, 20, 50 70, 80 or 95% by dry weight of the
purified preparation.
[0087] Also described is a method of increasing the level of cyclic
guanosine 3'-monophosphate (cGMP) in an organ, tissue (e.g, the
intestinal mucosa), or cell (e.g., a cell bearing GC-A receptor) by
administering to a patient a composition comprising or consisting
essentially of a purified peptide comprising, consisting of or
consisting essentially of the amino acid sequence of SEQ ID NO:7
(e.g., a peptide in FIG. 3a or FIG. 3b).
[0088] Also described is a method for treating hypertension. The
method comprises: administering to the patient a pharmaceutical
composition comprising, consisting essentially of, or consisting of
a peptide or agonist described herein and a pharmaceutically
acceptable carrier. The composition can be administered in
combination with another agent for treatment of hypertension, for
example, a diuretic, an ACE inhibitor, an angiotensin receptor
blocker, a beta-blocker, or a calcium channel blocker.
[0089] Also described is a method for treating secondary
hyperglycemias in connection with pancreatic diseases (chronic
pancreatitis, pancreasectomy, hemochromatosis) or endocrine
diseases (acromegaly, Cushing's syndrome, pheochromocytoma or
hyperthyreosis), drug-induced hyperglycemias (benzothiadiazine
saluretics, diazoxide or glucocorticoids), pathologic glucose
tolerance, hyperglycemias, dyslipoproteinemias, adiposity,
hyperlipoproteinemias and/or hypotensions is described. The method
comprises: administering to the patient a pharmaceutical
composition comprising, consisting essentially of, or consisting of
a peptide or agonist described herein and a pharmaceutically
acceptable carrier.
[0090] The peptides described herein can be present with a
counterion. Useful counterions include salts of: acetate,
benzenesulfonate, benzoate, calcium edetate, camsylate, carbonate,
citrate, edetate (EDTA), edisylate, embonate, esylate, fumarate,
gluceptate, gluconate, glutamate, glycollylarsanilate,
hexylresorcinate, iodide, bromide, chloride, hydroxynaphthoate,
isethionate, lactate, lactobionate, estolate, maleate, malate,
mandelate, mesylate, mucate, napsylate, nitrate, pantothenate,
phosphate, salicylate, stearate, succinate, sulfate, tartarate,
tartrate, hydrochlorate, theoclate, acetamidobenzoate, adipate,
alginate, aminosalicylate, anhydromethylenecitrate, ascorbate,
aspartate, camphorate, caprate, caproate, caprylate, cinnamate,
cyclamate, dichloroacetate, formate, gentisate, glucuronate,
glycerophosphate, glycolate, hippurate, fluoride, malonate,
napadisylate, nicotinate, oleate, orotate, oxalate, oxoglutarate,
palmitate, pectinate, pectinate polymer, phenylethylbarbiturate,
picrate, propionate, pidolate, sebacate, rhodanide, tosylate, and
tannate.
[0091] Also described are methods for producing any of the forgoing
peptides comprising providing a cell harboring a nucleic acid
molecule encoding the peptide, culturing the cell under conditions
in which the peptide is expressed, and isolating the expressed
peptide.
[0092] Also described are methods for producing any of the forgoing
peptides comprising chemically synthesizing the peptide and then
purifying the synthesized peptide. Also described are
pharmaceutical compositions comprising the forgoing peptides. Also
described are nucleic acid molecules encoding any of the forgoing
peptides, vectors (e.g., expression vectors) containing such
nucleic acid molecules and host cells harboring the nucleic acid
molecules or vectors.
Metabolites of Asparagine
[0093] In some cases an asparagine (Asn) of a peptide described
herein can be metabolized to have a different structure and the GC
receptor agonist containing such a metabolite of Asn may retain
activity. Peptides where one or more Asn, e.g., one or more Asn of
an embodiment of SEQ ID NO:7, e.g., a peptide in FIG. 3a or FIG. 3b
described herein are replaced by a metabolite of Asn can be useful
in the methods described herein and can be present in a
pharmaceutical composition that optionally contains one or more
additional active ingredients.
[0094] For example, one or more Asn of a peptide and the peptide
bond carboxy terminal thereto having the structure:
##STR00002##
can replaced by a group having a structure selected from:
##STR00003##
[0095] Thus, the Asn and the peptide bond carboxy terminal there to
can be replaced by a cyclic imide:
##STR00004##
Asp:
##STR00005##
[0097] The Asp can be L-Asp or D-Asp. The isoAsn can be D-isoAsn or
L-isoAsn.
[0098] Considering the asparagine only, one or more Asn having the
structure:
##STR00006## [0099] can be optionally replaced by a group having a
structure selected from (a), (b) and (c):
##STR00007##
[0099] provided that an Asn at the carboxy terminus is not replaced
by structure (a) or structure (c). When the Asn is at the carboxy
terminus of the peptide, structure (a) cannot form. Since structure
(c) is formed through structure (a), structure (c) cannot be formed
when the Asn is at the carboxy terminus.
[0100] The formation of the various metabolites of Asp is depicted
below.
##STR00008##
[0101] The details of one or more embodiments described herein are
set forth in the accompanying description. All of the publications,
patents and patent applications are hereby incorporated by
reference.
FIGURES
[0102] FIG. 1 depicts the results of studies on certain peptides
tested in the intestinal GC-C receptor activity assay.
[0103] FIG. 2 depicts the results of studies on certain peptides
tested in the rat diuresis assay.
[0104] FIGS. 3a and 3b depict certain peptides within SEQ ID
NO:7.
[0105] FIG. 4 depicts various pre, pro, N-terminal non-core, and
C-terminal non-core sequences that can be included in a peptide
comprising SEQ ID NO:7.
DETAILED DESCRIPTION
[0106] The peptides described herein bind to the intestinal
guanylate cyclase (GC-C) receptor, a regulator of fluid and
electrolyte balance. The apical membrane of the intestinal
epithelial surface is a major site of GC-C receptor expression.
Activation of the GC-C receptor in the intestine leads to an
increase in intestinal epithelial cyclic GMP (cGMP). This increase
in cGMP is believed to cause a decrease in water and sodium
absorption and an increase in chloride and potassium ion secretion,
leading to changes in intestinal fluid and electrolyte transport
and increased intestinal motility. The intestinal GC-C receptor
possesses an extracellular ligand binding region, a transmembrane
region, an intracellular protein kinase-like region and a cyclase
catalytic domain. Proposed functions for the GC-C receptor are
fluid and electrolyte homeostasis, the regulation of epithelial
cell proliferation and the induction of apoptosis (Shalubhai 2002
Curr Opin Drug Dis Devel 5:261-268).
[0107] In addition to being expressed in the intestine by
gastrointestinal epithelial cells, GC-C is expressed in
extra-intestinal tissues including kidney, lung, pancreas,
pituitary, adrenal, developing liver and gall bladder (reviewed in
Vaandrager 2002 Mol Cell Biochem 230:73-83, Kulaksiz et al. 2004,
Gastroenterology 126:732-740) and male and female reproductive
tissues (reviewed in Vaandrager 2002 Mol Cell Biochem 230:73-83).
This suggests that the GC-C receptor agonists can be used in the
treatment of disorders outside the GI tract, for example,
congestive heart failure and benign prostatic hyperplasia.
[0108] In humans, the GC-C receptor is activated by guanylin (Gn)
(U.S. Pat. No. 5,969,097), uroguanylin (Ugn) (U.S. Pat. No.
5,140,102) and lymphoguanylin (Forte et al. 1999 Endocrinology
140:1800-1806). Interestingly, these agents are 10-100 fold less
potent than a class of bacterially derived peptides, termed ST
(reviewed in Gianella 1995 J Lab Clin Med 125:173-181). ST peptides
are considered super agonists of GC-C and are very resistant to
proteolytic degradation.
[0109] ST peptide is capable of stimulating the enteric nervous
system (Rolfe et al., 1994, J Physiolo 475: 531-537; Rolfe et al.
1999 Gut 44: 615-619; Nzegwu et al. 1996 Exp Physiol 81: 313-315).
Also, cGMP has been reported to have antinociceptive effects in
multiple animal models of pain (Lazaro Ibanez et al. 2001 Eur J
Pharmacol 426: 39-44; Soares et al. 2001 British J Pharmacol 134:
127-131; Jain et al. 2001 Brain Res 909:170-178; Amarante et al.
2002 Eur J Pharmacol 454:19-23). Thus, GC-C agonists may have both
an analgesic as well an anti-inflammatory effect.
[0110] In bacteria, ST peptides are derived from a preproprotein
that generally has at least 70 amino acids. The pre and pro regions
are cleaved as part of the secretion process, and the resulting
mature protein, which generally includes fewer than 20 amino acids,
is biologically active.
[0111] Among the known bacterial ST peptides are: E. coli ST Ib
(Moseley et al. 1983 Infect. Immun. 39:1167) having the mature
amino acid sequence Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys Cys Asn
Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:_); E. coli ST Ia (So and
McCarthy 1980 Proc. Natl. Acad. Sci. USA 77:4011) having the mature
amino acid sequence Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Asn Pro
Ala Cys Ala Gly Cys Tyr (SEQ ID NO:7). E. coli ST I* (Chan and
Giannella 1981 J. Biol. Chem. 256:7744) having the mature amino
acid sequence Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Tyr Pro Ala
Cys Ala Gly Cys Asn (SEQ ID NO:_); C. freundii ST peptide (Guarino
et al. 1989b Infect. Immun. 57:649) having the mature amino acid
sequence Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys
Ala Gly Cys Tyr (SEQ ID NO:_); Y. enterocolitica ST peptides,
Y-ST(Y-STa), Y-STb, and Y-STc (reviewed in Huang et al. 1997
Microb. Pathog. 22:89) having the following pro-form amino acid
sequences: Gln Ala Cys Asp Pro Pro Ser Pro Pro Ala Glu Val Ser Ser
Asp Trp Asp Cys Cys Asp Val Cys Cys Asn Pro Ala Cys Ala Gly Cys
(SEQ ID NO:_) (as well as a Ser-7 to Leu-7 variant of Y-STa (SEQ ID
NO:_), (Takao et al. 1985 Eur. J. Biochem. 152:199)); Lys Ala Cys
Asp Thr Gln Thr Pro Ser Pro Ser Glu Glu Asn Asp Asp Trp Cys Cys Glu
Val Cys Cys Asn Pro Ala Cys Ala Gly Cys (SEQ ID NO:); Gln Glu Thr
Ala Ser Gly Gln Val Gly Asp Val Ser Ser Ser Thr Ile Ala Thr Glu Val
Ser Glu Ala Glu Cys Gly Thr Gln Ser Ala Thr Thr Gln Gly Glu Asn Asp
Trp Asp Trp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Phe Gly Cys
(SEQ ID NO:_), respectively; Y. kristensenii ST peptide having the
mature amino acid sequence Ser Asp Trp Cys Cys Glu Val Cys Cys Asn
Pro Ala Cys Ala Gly Cys (SEQ ID NO:_); V. cholerae non-01 ST
peptide (Takao et al. (1985) FEBS lett. 193:250) having the mature
amino acid sequence Ile Asp Cys Cys Glu Ile Cys Cys Asn Pro Ala Cys
Phe Gly Cys Leu Asn (SEQ ID NO:_); and V. mimicus ST peptide (Arita
et al. 1991 FEMS Microbiol. Lett. 79:105) having the mature amino
acid sequence Ile Asp Cys Cys Glu Ile Cys Cys Asn Pro Ala Cys Phe
Gly Cys Leu Asn (SEQ ID NO:_). The immature (including pre and pro
regions) form of E. coli ST-1A (ST-P) protein has the sequence:
mkklmlaifisvlsfpsfsqstesldsskekitletkkcdvvknnsekksenmnntfyccelccnpacagcy
(SEQ ID NO:_; see GenBank.RTM. Accession No. P01559 (gi:123711).
The pre sequence extends from aa 1-19. The pro sequence extends
from aa 20-54. The mature protein extends from 55-72. The immature
(including pre and pro regions) form of E. coli ST-1B (ST-H)
protein has the sequence:
mkksilfiflsvlsfspfaqdakpvesskekitleskkcniakksnksgpesmnssnyccelccnpactgcy
(SEQ ID NO:_; see GenBank.RTM. Accession No. P07965 (gi:3915589)).
The immature (including pre and pro regions) form of Y.
enterocolitica ST protein has the sequence:
mkkivfvlvlmlssfgafgqetvsgqfsdalstpitaevykqacdpplppaevssdwdccdvccnpacagc
(SEQ ID NO:_; see GenBank.RTM. Accession No. S25659 (gi:282047)).
The peptides described herein, e.g., a peptide comprising SEQ ID
NO:77 (e.g., a peptide of FIG. 3a or FIG. 3b)can include all or
part of such pre and/or pro sequences.
[0112] FIG. 4 depicts various pre, pro, N-terminal non-core, and
C-terminal non-core sequences that can be included in a peptide
comprising SEQ ID NO:7 (e.g., a peptide of FIG. 3a or FIG. 3b).
Thus, a peptide can include the amino acid sequence: [0113]
A'-B'-C'-D'-E' wherein: [0114] A' is an amino acid sequence
comprising a pre sequence depicted in FIG. 4 or is missing; [0115]
B' is an amino acid sequence comprising a pro sequence depicted in
FIG. 4 or is missing; [0116] C' is an amino acid sequence
comprising an N-terminal non-core sequence depicted in FIG. 4 or is
missing; [0117] D' is an amino acid sequence comprising a GC-C
receptor agonist peptide amino acid sequence (e.g., SEQ ID NO:7, a
peptide in FIG. 3a or a peptide in FIG. 3b); and [0118] E' is an
amino acid sequence comprising a C-terminal non-core sequence
depicted in FIG. 4 or is missing;
[0119] The peptides described herein, like the bacterial ST
peptides, have six Cys (or D-Cys) residues. These six residues form
three disulfide bonds in the mature and active form of the peptide.
If the six Cys (or D-Cys) residues are identified, from the amino
to carboxy terminus of the peptide, as A, B, C, D, E, and F, then
the disulfide bonds form as follows: A-D, B-E, and C-F. The
formation of these bonds is thought to be important for GC-C
receptor binding.
[0120] Certain of the peptides described herein may include
analgesic or antinociceptive tags such as the carboxy-terminal
sequence AspPhe immediately following a Trp, Tyr or Phe that
creates a functional chymotrypsin cleavage site or following Lys or
Arg that creates a functional trypsin cleavage site. Chymotrypsin
in the intestinal tract can potentially cleave such peptides
immediately carboxy terminal to the Trp, Phe or Tyr residue,
releasing the dipeptide, AspPhe. This dipeptide has been shown to
have analgesic activity in animal models (Abdikkahi et al. 2001
Fundam Clin Pharmacol 15:117-23; Nikfar et al 1997, 29:583-6;
Edmundson et al 1998 Clin Pharmacol Ther 63:580-93). In this manner
such peptides can treat both pain and inflammation. Other analgesic
peptides can be present at the amino or carboxy terminus of the
peptide (e.g., following a functional cleavage site) including:
endomorphin-1, endomorphin-2, nocistatin, dalargin, lupron, and
substance P.
[0121] In some cases, the peptides described herein are produced as
a prepro protein that includes the amino terminal leader
sequence:
mkksilfiflsvlsfspfaqdakpvesskekitleskkcniakksnksgpesmn. Where the
peptide is produced by a bacterial cell, e.g., E. coli, the
forgoing leader sequence will be cleaved and the mature peptide
will be efficiently secreted from the bacterial cell. U.S. Pat. No.
5,395,490 describes vectors, expression systems and methods for the
efficient production of ST peptides in bacterial cells and methods
for achieving efficient secretion of mature ST peptides. The
vectors, expression systems and methods described in U.S. Pat. No.
5,395,490 can be used to produce the ST peptides and variant ST
peptides of the present disclosure
Variant Peptides
[0122] The disclosure includes variant peptides which can include
one, two, three, four, five, six, seven, eight, nine, or ten (in
some embodiments fewer than 5 or fewer than 3 or 2 or fewer) amino
acid substitutions and/or deletions compared to the sequences of
SEQ ID NO:7 (e.g., a sequence in FIG. 3a or FIG. 3b) The
substitution(s) can be conservative or non-conservative. The
naturally-occurring amino acids can be substituted by D-isomers of
any amino acid, non-natural amino acids, natural and natural amino
acid analogs and other groups. A conservative amino acid
substitution results in the alteration of an amino acid for a
similar acting amino acid, or amino acid of like charge, polarity,
or hydrophobicity. At some positions, even conservative amino acid
substitutions can alter the activity of the peptide. A conservative
substitution can substitute a naturally-occurring amino acid for a
non-naturally-occurring amino acid. The amino acid substitutions
among naturally-occurring amino acids are listed in Table II.
TABLE-US-00001 TABLE II For Amino Acid Code Replace with any of
Alanine Ala Gly, Cys, Ser Arginine Arg Lys, His Asparagine Asn Asp,
Glu, Gln, Aspartic Acid Asp Asn, Glu, Gln Cysteine Cys Met, Thr,
Ser Glutamine Gln Asn, Glu, Asp Glutamic Acid Glu Asp, Asn, Gln
Glycine Gly Ala Histidine His Lys, Arg Isoleucine Ile Val, Leu, Met
Leucine Leu Val, Ile, Met Lysine Lys Arg, His Methionine Met Ile,
Leu, Val Phenylalanine Phe Tyr, His, Trp Proline Pro Serine Ser
Thr, Cys, Ala Threonine Thr Ser, Met, Val Tryptophan Trp Phe, Tyr
Tyrosine Tyr Phe, His Valine Val Leu, Ile, Met
[0123] In some circumstances it can be desirable to treat patients
with a variant peptide that binds to and activates intestinal GC-C
receptor, but is less active than the non-variant form the peptide.
This 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.
Production of Peptides
[0124] Useful peptides can be produced either in bacteria
including, without limitation, E. coli, or in other existing
systems for peptide or protein production (e.g., Bacillus subtilis,
baculovirus expression systems using Drosophila Sf9 cells, yeast or
filamentous fungal expression systems, mammalian cell expression
systems), or they can be chemically synthesized.
[0125] If the peptide or variant peptide is to be produced in
bacteria, e.g., E. coli, the nucleic acid molecule encoding the
peptide will preferably 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.
[0126] The sequence encoding a peptide described herein is
preferably inserted into a vector capable of delivering and
maintaining the nucleic acid molecule in a bacterial cell. The DNA
molecule may be inserted into an autonomously replicating vector
(suitable vectors include, for example, pGEM3Z and pcDNA3, and
derivatives thereof). The vector nucleic acid may be a bacterial or
bacteriophage DNA such as bacteriophage lambda or M13 and
derivatives thereof. Construction of a vector containing a nucleic
acid described herein can be followed by transformation of a host
cell such as a bacterium. Suitable bacterial hosts include but are
not limited to, E. coli, B. subtilis, Pseudomonas, Salmonella. The
genetic construct also includes, in addition to the encoding
nucleic acid molecule, elements that allow expression, such as a
promoter and regulatory sequences. The expression vectors may
contain transcriptional control sequences that control
transcriptional initiation, such as promoter, enhancer, operator,
and repressor sequences. 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.
[0127] 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.
[0128] 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.
[0129] Mature peptides and variants thereof can be synthesized by
the solid-phase chemical synthesis. For example, the peptide can be
synthesized on Cyc(4-CH.sub.2
Bx1)-OCH.sub.2-4-(oxymethyl)-phenylacetamidomethyl resin using a
double coupling program. Protecting groups must be used
appropriately to create the correct disulfide bond pattern. For
example, the following protecting groups can be used:
t-butyloxycarbonyl (alpha-amino groups); acetamidomethyl (thiol
groups of Cys residues B and E); 4-methylbenzyl (thiol groups of
Cys residues C and F); benzyl (y-carboxyl of glutamic acid and the
hydroxyl group of threonine, if present); and bromobenzyl (phenolic
group of tyrosine, if present). Coupling is effected with
symmetrical anhydride of t-butoxylcarbonylamino acids or
hydroxybenzotriazole ester (for asparagine or glutamine residues),
and the peptide is deprotected and cleaved from the solid support
in hydrogen fluoride, dimethyl sulfide, anisole, and p-thiocresol
using 8/1/1/0.5 ratio (v/v/v/w) at 0.degree. C. for 60 min. After
removal of hydrogen fluoride and dimethyl sulfide by reduced
pressure and anisole and p-thiocresol by extraction with ethyl
ether and ethyl acetate sequentially, crude peptides are extracted
with a mixture of 0.5M sodium phosphate buffer, pH 8.0 and
N,N-dimethyl formamide using 1/1 ratio, v/v. The disulfide bond for
Cys residues B and E is the formed using dimethyl sulfoxide (Tam et
al. (1991) J. Am. Chem. Soc. 113:6657-62). The resulting peptide is
the purified by reverse-phase chromatography. The disulfide bond
between Cys residues C and F is formed by first dissolving the
peptide in 50% acetic acid in water. Saturated iodine solution in
glacial acetic acid is added (1 ml iodine solution per 100 ml
solution). After incubation at room temperature for 2 days in an
enclosed glass container, the solution is diluted five-fold with
deionized water and extracted with ethyl ether four times for
removal of unreacted iodine. After removal of the residual amount
of ethyl ether by rotary evaporation the solution of crude product
is lyophilized and purified by successive reverse-phase
chromatography.
[0130] Peptides can also be synthesized by many other methods
including solid phase synthesis using traditional FMOC protection
(i.e., coupling with DCC--HOBt and deprotection with piperidine in
DMF). Cys thiol groups can be trityl protected. Treatment with TFA
can be used for final deprotection of the peptide and release of
the peptide from the solid-state resin. In many cases air oxidation
is sufficient to achieve proper disulfide bond formation.
Example 1
Preparation of Peptides
[0131] Peptides can be recombinantly produced in bacteria as
follows. T7 expression vectors, pET26b(+) (Novagen) expressing the
peptide of interest are constructed using standard molecular
biology techniques and are transformed into E. coli bacterial host
BL21.lamda.. DE3 (Invitrogen). A single colony is innoculated and
grown shaking overnight at 30.degree. C. in L broth+25 mg/l
kanamycin. The overnight culture is added to 3.2 L of batch medium
(Glucose 25 g/l, Casamino Acids 5 g/l, Yeast Extract 5 g/l,
KH.sub.2PO.sub.4 13.3 g/l, (NH.sub.4).sub.2HPO.sub.4 4 g/l,
MgSO.sub.4-7H.sub.20 1.2 g/l, Citric Acid 1.7 g/l, EDTA 8.4 mg/l,
CoCl.sub.2-6H.sub.2O 2.5 mg/l, MnCl.sub.2-4H.sub.2O 15 mg/l,
CuCl.sub.2-4H.sub.2O 1.5 mg/l, H.sub.3BO.sub.3 3 mg/l,
Na.sub.2MoO.sub.4-2H.sub.20 2.5 mg/l, Zn Acetate-2H.sub.2O 13 mg/l,
Ferric Citrate 100 mg/l, Kanamycin 25 mg/l, Antifoam
DF.sub.20.sub.4 1 ml/l) and fermented using the following process
parameters: pH 6.7--control with base only (28% NH.sub.4OH),
30.degree. C., aeration: 5 liters per minute. After the initial
consumption of batch glucose (based on monitoring dissolved oxygen
(DO) levels), 1.5 L of feed medium (Glucose 700 g/l, Casamino Acids
10 g/l, Yeast Extract 10 g/l, MgSO.sub.4-7H.sub.2O 4 g/l, EDTA 13
mg/l, CoCl.sub.2-6H.sub.2O 4 mg/l, MnCl.sub.2-4H.sub.2O 23.5 mg/l,
CuCl.sub.2-4H.sub.2O 2.5 mg/l, H.sub.3BO.sub.3 5 mg/l,
Na.sub.2MoO4-2H.sub.20 4 mg/l, Zn Acetate-2H.sub.20 16 mg/l, Ferric
Citrate 40 mg/l, Antifoam DF.sub.20.sub.4 1 ml/l) is added at a
feed rate controlled to maintain 20% DO. IPTG is added to 0.2 mM 2
hours post feed start. The total run time is approximately 40-45
hours (until feed exhaustion).
[0132] Cells are collected by centrifugation at 5,000 g for 10
minutes. The cell pellet is discarded and the supernatant is passed
through a 50 Kd ultrafiltration unit. The 50 Kd filtrate (0.6
liters) is loaded onto a 110 ml Q-Sepharose fast Flow column
(Amersham Pharmacia, equilibrated with 20 mM Tris-HCl pH 7.5) at a
flow rate of 400 ml/hour. The column is washed with six volumes of
20 mM Tris-HCl pH 7.5 and proteins are eluted with 50 mM acetic
acid collecting 50 ml fractions. Fractions containing peptide are
pooled and the solvent is removed by rotary evaporation. The dried
proteins are resuspended in 10 ml of 8% acetic acid, 0.1%
trifluoroacetic acid (TFA) and loaded onto a Varian Polaris C18-A
column (250.times.21.2 mm 10 .mu.m, equilibrated in the same
buffer) at a flow rate of 20 ml/min. The column is washed with 100
ml of 8% methanol, 0.1% TFA and developed with a gradient (300 ml)
of 24 to 48% methanol, 0.1% TFA, collecting 5-ml fractions.
Fractions containing peptide are pooled and the solvent is removed
by rotary evaporation. The peptides are dissolved in 0.1% TFA and
lyophilized.
[0133] Peptide fractions are analyzed by standard LCMS and HPLC.
Peptides can also be chemically synthesized by a commercial peptide
synthesis company.
Example 2
Activation of the Intestinal GC-C Receptor by Peptides
[0134] The ability of peptides to activate the intestinal GC-C
receptor was assessed in an assay employing the T84 human colon
carcinoma cell line (American Type Culture Collection (Bethesda,
Md.)). For the assays cells were grown to confluency in 24-well
culture plates with a 1:1 mixture of Ham's F12 medium and
Dulbecco's modified Eagle's medium (DMEM), supplemented with 5%
fetal calf serum and were used at between passages 54 and 60.
[0135] Briefly, monolayers of T84 cells in 24-well plates were
washed twice with 1 ml/well DMEM, then incubated at 37.degree. C.
for 10 min with 0.45 ml DMEM containing 1 mM isobutylmethylxanthine
(1BMX), a cyclic nucleotide phosphodiesterase inhibitor. Test
peptides (50 .mu.l) were then added and incubated for 30 minutes at
37.degree. C. The media was aspirated and the reaction was then
terminated by the addition of ice cold 0.5 ml of 0.1N HCl. The
samples were held on ice for 20 minutes and then evaporated to
dryness using a heat gun or vacuum centrifugation. The dried
samples were resuspended in 0.5 ml of phosphate buffer provided in
the Cayman Chemical Cyclic GMP EIA kit (Cayman Chemical, Ann Arbor,
Mich.). Cyclic GMP was measured by EIA according to procedures
outlined in the Cayman Chemical Cyclic GMP EIA kit. FIG. 1 shows
the activity of chemically synthesized peptide variants (depicted
below) in the GC-C receptor activity assay. EC.sub.50 is defined as
the concentration by which 50% of the maximal activity is seen.
Maximum cGMP level in assay is determined as the activity of the
positive ST control,
Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr SEQ ID NO:6
and set to 100%. The positive control was tested twice in this
assay.
Example 3
Diuresis and Naturesis Assays
[0136] Effect on Diuresis and Natriuresis
[0137] The effect of peptides/GC-agonists described herein on
diuresis and natriuresis can be determined using methodology
similar to that described in WO06/001931 (examples 6 (p. 42) and 8
(p. 45)). Briefly, a peptide described herein (180-pmol) is infused
for 60 min into a group of 5 anesthetized rats. Given an estimated
rat plasma volume of 10 mL, the infusion rate is approximately 3
pmol/mL/min. Blood pressure, urine production, and sodium excretion
are monitored for approximately 40 minutes prior to the infusion,
during the infusion, and for approximately 50 minutes after the
infusion to measure the effect of the peptide/GC-C agonist on
diuresis and natriuresis. For comparison, a control group of five
rats is infused with regular saline. Urine and sodium excretion can
be assessed. Dose response can also be determined. A peptide/GC-C
agonist described herein is infused intravenously into rats over 60
minutes. Urine is collected at 30 minute intervals up to 180
minutes after termination of peptide/GC-C agonist infusion, and
urine volume, sodium excretion, and potassium excretion are
determined for each collection interval. Blood pressure is
monitored continuously. For each dose a dose-response relationship
for urine volume, sodium and potassium excretion can be determined.
Plasma concentration of the peptide/GC-agonist is also determined
before and after iv infusion.
[0138] Rat Diuresis Experiment
[0139] Female Sprague-Dawley rats (>170 g, 2-8 per group), are
given 3.0 mL of iosotonic saline perorally, and then anesthetized
with isoflurane/oxygen. Once an appropriate level of anesthesia has
been achieved, a sterile polyurethane catheter (.about.16 cm, 0.6
mm ID, 0.9 mm OD) is inserted 1.5-2.0 cm into the urethra and
secured using 1-2 drops of veterinary bond adhesive applied to
urethra/catheter junction. Rats are then dosed with either vehicle
or test article via the intravenous or intraperitoneal route. Rats
are then placed in appropriately sized rat restraint tubes, with
the catheter protruding out of the restraint tube into a 10 mL
graduated cylinder. Rats are allowed to regain consciousness, and
the volume of urine excreted over a 1-5 hour duration is recorded
periodically for each rat.
[0140] FIG. 2 shows the results of several peptides described
herein tested in the rat diuresis assay.
Example 4
Kd Determination and Binding Assays
[0141] A competition binding assay can be performed using rat
intestinal epithelial cells to determine the affinity of a peptide
for intestinal GC-G receptor. Epithelial cells from the small
intestine of rats are obtained as described by Kessler et al. (J.
Biol. Chem. 245: 5281-5288 (1970)). Briefly, animals are sacrificed
and their abdominal cavities exposed.
[0142] The small intestine is rinsed with 300 ml ice cold saline or
PBS. 10 cm of the small intestine measured at 10 cm from the
pylorus is removed and cut into 1 inch segments. Intestinal mucosa
is extruded from the intestine by gentle pressure between a piece
of parafilm and a P-1000 pipette tip. Intestinal epithelial cells
are placed in 2 ml PBS and pipetted up and down with a 5 ml pipette
to make a suspension of cells. Protein concentration in the
suspension is measured using the Bradford method (Anal. Biochem.
72: 248-254 (1976)).
[0143] A competition binding assay can be performed based on the
method of Giannella et al. (Am. J. Physiol. 245: G492-G498) between
[.sup.125I] labeled peptide. The assay mixture contains: 0.5 ml of
DME with 20 mM HEPES-KOH pH 7.0, 0.9 mg of the cell suspension
listed above, 21.4 fmol [.sup.125I]-SEQ ID NO:4 (42.8 pM), and
different concentrations of competitor peptide (0.01 to 1000 nM).
The mixture is incubated at room temperature for 1 hour, and the
reaction is stopped by applying the mixture to GF/B glass-fiber
filters (Whitman). The filters are washed with 5 ml ice-cold PBS
and radioactivity is measured. Giannella et al. (Am. J. Physiol.
245: G492-G498) observed that the Kd for wild-type ST peptide in
this same assay is .about.13 nm.
[0144] Similar competition binding assays can be performed in
intestinal epithelial cells from wild-type and guanylate cyclase C
knockout (GC-C KO; Mann et al. 1997 Biochem and Biophysical
Research Communications 239:463) mice. Mouse intestinal epithelial
cells are prepared as described above for rat intestinal epithelial
cells except the cells are homogenized with an Omni homogenizer for
20 seconds on the maximum setting to make a suspension of
cells.
[0145] The binding of peptides to GC-C receptors on the cell
surface of human colonic cells (T84 cells; ATCC Catalog No.
CCL-248) can be characterized in a competitive radioligand-binding
assay at pH conditions of 5, 7 and 8. The radiolabeled tracer used
in these experiments is .sup.125I-labeled control peptide. To
determine binding constants, competitive inhibition of binding is
used. T84 cells are cultured in T-150 plastic flasks in DMEM and
Ham's F-12 medium containing 5% fetal bovine serum. Monolayers at
60-70% confluency (approximately 10.sup.7 cells) are collected by
gentle scraping followed by centrifugation, and washed twice in 50
mL of phosphate-buffered saline (PBS). The cells are resuspended in
1 mL DMEM containing 20 mM
N-(2-hydroxymethyl)piperazine-N'-(2-ethanesulfonic acid) (Hepes),
pH 7.0 and 0.5% bovine serum albumin (BSA). T84 cells are incubated
with a constant amount of .sup.125I-control peptide containing
various concentrations of cold competitor. Free .sup.125I-control
peptide is separated from bound tracer by rapid suction filtration.
The binding reactions are carried out in 1.5 mL microfuge tubes in
0.24 mL of DMEM/20 mM Hepes pH 7.0/0.5% BSA containing:
2.5.times.10.sup.5 T84 cells (0.25 mg protein), 200,000 cpm
.sup.125I-control peptide (41 fmol, 170 pM), and 0.01 to 1,000 nM
competitor. Binding assays at pH 5.0 are done in DMEM/20 mM
2-(N-morpholino) ethanesulfonic acid (Mes), pH 5.0. Binding assays
in pH 8.0 are done in DMEM/20 mM Hepes/50 mM sodium bicarbonate pH
8.0. One sample contains no competitor (B.sub.0) and another
contained no cells. After incubation at 37.degree. C. for 1 h, the
reaction mixtures are applied to Whatman GF/B glass-fiber filters
by suction filtration. The filters are then rinsed with 10 mL
ice-cold PBS buffer, inserted into plastic tubes, and added to 2 mL
scintillation fluid. Radioactivity is measured in a LS 6500 liquid
scintillation counter (Beckman-Coulter). The percent bound in each
sample is calculated by the equation:
% B/B.sub.0=(sample cpm-no cells cpm).times.100/(B.sub.0 cpm-no
cells cpm)
[0146] Nonlinear regression analysis of the binding data is used to
calculate the concentration of competitor that resulted in 50%
radioligand bound (IC.sub.50). The apparent dissociation
equilibrium constant (K.sub.i) for each competitor is obtained from
the IC.sub.50 values and the previously reported estimate of the
dissociation constant for the radioligand, K.sub.d.apprxeq.15 nM
(Hamra et al. 1997 PNAS 2705-10) and the method of Cheng and
Prusoff 1973 Biochem Pharmacol 22:3099-108. Using a two site model,
high and low affinity-binding sites are identified on T84 cells
(K.sub.i1 and K.sub.i2) for all the test agents.
Example 5
Pharmacokinetic Properties of Peptides
[0147] To study the pharmacokinetics of peptides, absorbability
studies in mice are performed by administering a peptide
intravaneously via tail vein injection or orally by gavage to
8-week-old CD1 mice. Serum is collected from the animals at various
time points and tested for the presence of peptide using a
competitive enzyme-linked immunoabsorbent assay.
[0148] A similar bioavailability study can be performed in which
LCMS rather than ELISA is used to detect peptide. Initially, serum
samples are extracted from the whole blood of exposed and control
mice, then injected directly (10 mL) onto an in-line solid phase
extraction (SPE) column (Waters Oasis HLB 25 .mu.m column,
2.0.times.15 mm direct connect) without further processing. The
sample on the SPE column is washed with a 5% methanol, 95%
dH.sub.2O solution (2.1 mL/min, 1.0 minute), then loaded onto an
analytical column using a valve switch that places the SPE column
in an inverted flow path onto the analytical column (Waters Xterra
MS C8 5 .mu.m IS column, 2.1.times.20 mm). The sample is eluted
from the analytical column with a reverse phase gradient (Mobile
Phase A: 10 mM ammonium hydroxide in dH.sub.2O, Mobile Phase B: 10
mM ammonium hydroxide in 80% acetonitrile and 20% methanol; 20% B
for the first 3 minutes then ramping to 95% B over 4 min. and
holding for 2 min., all at a flow rate of 0.4 mL/min.). At 9.1
minutes, the gradient returns to the initial conditions of 20% B
for 1 min. Peptide is eluted from the analytical column, and it is
detected by triple-quadrapole mass spectrometry. Instrument
response is converted into concentration units by comparison with a
standard curve using known amounts of chemically synthesized
peptide prepared and injected in mouse serum using the same
procedure.
[0149] Similarly, oral bioavailabity is determined in rats using
LCMS methodology. Rat plasma samples containing peptide are
extracted using a Waters Oasis MAX 96 well solid phase extraction
(SPE) plate. A 200 .mu.L volume of rat plasma is mixed with 200
.mu.L of .sup.13C, .sup.15N-peptide in the well of a prepared SPE
plate. The samples are drawn through the stationary phase with 15
mm Hg vacuum. All samples are rinsed with 200 .mu.L of 2% ammonium
hydroxide in water followed by 200 .mu.L of 20% methanol in water.
The samples are eluted with consecutive 100 .mu.L volumes of May
20, 1975 formic acid/water/methanol and 100 .mu.L May 15, 1980
formic acid/water/methanol. The samples are dried under nitrogen
and resuspended in 100 .mu.L of 20% methanol in water. Samples are
analyzed by a Waters Quattro Micro mass spectrometer coupled to a
Waters 1525 binary pump with a Waters 2777 autosampler. A 40 .mu.L
volume of each sample is injected onto a Thermo Hypersil GOLD C18
column (2.1.times.50 mm, 5 .mu.m). Peptides are eluted by a
gradient over 3 minutes with acetonitrile and water containing
0.05% trifluoroacetic acid.
[0150] Oral bioavailability can also be determined using a
radioimmunoassay (RIA) detection method. Female CD-1 mice (Charles
River, Wilmington, Mass.) weighing approximately 25 g (7-8 weeks
old) or female CD rats (Charles River, Wilmington, Mass.) weighing
approximately 153 g are included in this study. Monoclonal antibody
recognizing the peptide and .sup.125I labeled-peptide, a labeled
tracer, are used in these experiments. Animals are fasted overnight
before administration of compounds. Blood is drawn from all dosed
animals by retro-orbital eye bleeding at specific intervals and
test compound levels are analyzed by radioimmunoassay (RIA).
Samples (80 .mu.L) are first diluted to 0.5 mL with start buffer
(8% methanol, 0.095% TFA in water) and applied to C18 columns
previously conditioned with 1 mL methanol and equilibrated with 2
mL of start buffer. After washing with 1 mL start buffer, peptide
is eluted with 0.8 mL of 80% methanol, 0.05% TFA and dried down in
a centrifugal evaporator. Samples are reconstituted in 0.194 mL
assay buffer (PBS buffer, pH 7.4, containing 10% fetal bovine
serum). Standard dilutions of peptide are made in rat plasma. To
perform RIA analysis, samples from dosed animal and standards are
mixed with 5 .mu.L diluted antibody (in RIA wash buffer:
phosphate-buffered saline (PBS) containing 0.1% bovine serum
albumin (BSA), 1:40,000 final dilution, 0.0022 .mu.g), and
incubated 1 to 4 h at 4.degree. C. One tube contains the zero
standard (B.sub.0) and another no standard and no antibody
(non-specific binding, NSB). Labeled tracer (0.018 .mu.Ci, diluted
in RIA wash buffer) is then added and incubated at 4.degree. C. for
12 to 18 h. The antibody bound fraction containing peptide is
collected by magnetic separation using 10 .mu.L of sheep anti-mouse
IgG beads previously washed twice in 10 volumes RIA assay buffer.
The beads are then washed twice with 1 mL of RIA wash buffer,
collected by magnetic separation, resuspended in 0.1 mL of RIA wash
buffer, and added to 2 mL scintillation fluid. Radioactivity is
measured in a LS 6500 scintillation counter (Beckman-Coulter). The
binding efficiency is defined as the percent radioactivity in the
B.sub.0 sample compared to the input counts. The percent bound in
each sample is calculated by the equation:
% B/B.sub.0=(sample cpm-NSB cpm).times.100/(B.sub.0 cpm-NSB
cpm)
[0151] A standard curve is prepared by plotting % B/B.sub.0 as a
function of the log peptide concentration. A concentration vs. time
plot is generated from the data in GraphPad Prism or Summit
Software PK Solutions 2.0 to generate oral and i.v. PK curves. The
area under the curve from T=0 to 4 hours (AUC.sub.0-4 h) is
calculated by the software for both p.o. and i.v. dosed animals. If
the values are below the lower limit of detection (LOD) than the
LOD is used to estimate the value (in this experiment 2 nM). Oral
Bioavailabilty (F) is calculated using the equation:
F=(AUC.sub.p.o.,(0-4 h)*D.sub.i.v.)/(AUC.sub.i.v.,(0-4
h)*D.sub.p.o.)
where D.sub.i.v. and D.sub.p.o. equal the intravenous and oral
dose, respectively.
Example 6
In Vitro Proteolytic Stability of Peptides
[0152] The stability of petides in the presence of several
mammalian digestive enzymes can be determined. Peptides are exposed
to a variety of in vitro conditions including digestive enzymes and
low ph environments designed to simulate gastric fluid. Peptides
are incubated with chymotrypsin, trypsin, pepsin, aminopeptidase,
carboxypeptidase A, or simulated gastric fluid (sgf) at ph 1.0.
Samples are collected at 0, 3, and 24 h for all conditions except
pepsin digestion and the SGF. For the latter two conditions,
samples are obtained at 0, 1, and 3 h. Negative control samples are
prepared for initial and final time points. A separate, positive
activity control is run in parallel to test peptide. All samples
are analyzed by LC/MS
Chymotrypsin
[0153] 500 .mu.l samples of 0.01 mg/mL peptide and guanylin
(Sigma-Aldrich, G116; positive control) are prepared in the
chymotrypsin reaction buffer (100 mM Tris-HCl, 10 mM CaCl.sub.2, pH
7.5) in 2 mL eppendorf tubes. Zero and 24 h control samples are
prepared by adding 5 .mu.L of a 10 mM chymostatin (Sigma-Alrich,
C7268; a chymotrypsin inhibitor) stock for a final concentration of
100 .mu.M. All samples are incubated at 37.degree. C. for 5 min. 20
.mu.L of a 0.01 mg/mL chymotrypsin stock (.alpha.-chympotrypsin
from bovine pancreas; Sigma-Aldrich, C6423) are added to each
sample for a 0.0004 mg/mL final concentration. Samples are returned
to the 37.degree. C. water bath. The reaction is quenched with 5
.mu.L of a 10 mM chymostatin stock at each time point for a final
concentration of 100 .mu.M. No extra chymostatin is added to the
control samples as they already had inhibitor. Samples are
subsequently flash frozen in liquid nitrogen, and stored at
-80.degree. C. until analysis. Upon analysis, samples are thawed
and transferred to a 1 mL 96-well plate. Standards of peptide and
guanylin are prepared in chymotrypsin reaction buffer at 0.625,
1.25, 2.50, 5.00, and 10.00 .mu.g/mL concentrations. These
standards are used to generate a standard curve for quantification
of samples. When necessary, the standard curves are also used to
calculate the concentration of the corresponding digestion product.
10 .mu.L injections are made of each sample and standard.
Trypsin
[0154] 500 .mu.L samples of 0.01 mg/mL peptide and BAEE
(N.sub.alpha Benzoyl-L-arginine ethyl ester hydrochloride;
Signa-Aldrich, B4500; positive control) are prepared with trypsin
reaction buffer (100 mM Tris-HCl, pH 7.5) in 2 mL eppendorf tubes.
Zero and 24 h time point control samples are prepared (N=1) with 5
.mu.L of a 100 mg/mL AEBSF (4-(2-Aminoethyl)benzenesulfonyl
fluoride hydrochloride; a trypsin inhibitor) stock for a final
concentration of 1 mg/mL. All control and test samples (0, 3, and
24 h) are incubated at 37.degree. C. for 5 min. Twenty (20) .mu.L
of a 0.01 mg/mL trypsin (Sigma-Aldrich, T6467) stock are added to
each sample for a final concentration of 0.0004 mg/mL. Samples are
returned to the 37.degree. C. water bath. The reaction is quenched
with 5 .mu.L of a 100 mg/mL AEBSF stock, which is added to each
sample at the indicated timepoint, for a final concentration of 1
mg/mL. No extra AEBSF is added to the control samples as they
already had inhibitor. Samples are subsequently flash frozen in
liquid nitrogen, and stored at -80.degree. C. until analysis. Upon
analysis, samples are thawed and transferred to a 1 mL 96-well
plate. Standards of peptide and BAEE are prepared in trypsin
reaction buffer at 0.625, 1.25, 2.50, 5.00, and 10.00 .mu.g/mL
concentrations. These standards are used to generate a standard
curve for quantification of samples. When necessary, the standard
curves are also used to calculate the concentration of the
corresponding digestion product. Ten (10) .mu.L injections are made
of each sample and standard.
Pepsin
[0155] 500 .mu.L samples of 100 U/mL pepsin (Pepsin porcine gastric
mucosa; Sigma-Aldrich, P68871; U=release of 0.01 absorbance at 280
nM (A280) as TCA soluble hydrolysis products per min at 37.degree.
C. of hemoglobin) are prepared in the pepsin reaction buffer (100
mM HCl--KCl, pH 2.0) in 5 mL polystyrene round bottom tubes. To the
control samples (0 and 24 h), 500 .mu.L of a 1 M ammonium acetate
(pepsin inhibitor) stock are added, for a final concentration of
0.5 M. All control and test samples (0, 1, and 3 h) are incubated
at 37.degree. C. for 5 min, while shaking. Fifty (50) .mu.L of 0.1
mg/mL peptide and Insulin B chain, oxidized (Sigma-Aldrich, 16383;
positive control), stocks are added to the respective tubes.
Samples are returned to the 37.degree. C. shaking water bath.
Reactions are quenched by the addition of 500 .mu.L of 1 M ammonium
acetate for a final concentration of 0.5 M (except to the control
samples, which already contained 0.5 M ammonium acetate). Samples
are cooled on ice and stored at 4.degree. C. until analysis. Upon
analysis, samples are transferred to a 1 mL 96-well plate.
Standards of peptide and Insulin B chain, oxidized, are prepared in
25 mM Tris-hydrochloric acid, 500 mM sodium chloride, pH 7.5 buffer
at 0.625, 1.25, 2.50, 5.00, and 10.00 .mu.g/mL concentrations.
These standards are used to generate a standard curve for
quantification of samples. Ten (10) .mu.L injections are made of
each sample and standard.
Aminopeptidase
[0156] 500 .mu.L samples of 0.01 mg/mL peptide and chemically
synthesized wild type ST (positive control) are prepared in the
aminopeptidase reaction buffer (5 mM Tris-HCl, 5 mM MgCl.sub.2, pH
7.5) in 2 mL eppendorf tubes. 5 .mu.L of a 5 mg/mL Bestatin
hydrochloride (BioChemika, 08170; an aminopeptidase inhibitor)
stock is added to each control sample (0 and 24 h), for a final
concentration of 0.05 mg/mL. All control and test samples (0, 3,
and 24 h) are incubated at 37.degree. C. for 5 min. 0.02 U
aminopeptidase (Aminopeptidase M, amino acid aryl amidase (Roche,
102768; U=hydrolysis of 1.0 umol of L-leucinamide to leucine and
NH3 per min at pH 8.5 at 25.degree. C.) are added to each sample.
Samples are returned to the 37.degree. C. water bath. The reaction
is quenched with 5 .mu.L of a 5 mg/mL Bestatin hydrochloride stock
at the proper time point. No extra Bestatin hydrochloride is added
to the control samples since they already had inhibitor present.
Samples are subsequently flash frozen in liquid nitrogen, and
stored at -80.degree. C. until analysis. Upon analysis, samples are
thawed and transferred to a 1 mL 96-well plate. Standards of
peptide are prepared in aminopeptidase reaction buffer at 0.625,
1.25, 2.50, 5.00, and 10.00 .mu.g/mL concentrations. These
standards are used to generate a standard curve for quantification
of samples. When necessary, the standard curves are also used to
calculate the concentration of the corresponding digestion product.
Ten (10) .mu.L injections are made of each sample and standard.
Carboxypeptidase A
[0157] 500 .mu.L samples of 0.01 mg/mL peptide and
N--CBZ-Glycine-Glycine-Leucine (Z-Gly-Gly-Leu; Sigma-Aldrich,
C8501; positive control) are prepared in the carboxypeptidase A
reaction buffer (25 mM Tris-HCl, 500 mM NaCl, pH 7.5) in 2 mL
eppendorf tubes. Five (5) .mu.L of a 40 .mu.g/mL carboxypeptidase
inhibitor (carboxypeptidase inhibitor from potato tuber
(Signa-Aldrich, C0279) stock is added to each control sample (0 and
24 h), for a final concentration of 0.4 .mu.g/mL. All control and
test (0, 3 and 24 h) samples are incubated at 37.degree. C. for 5
min. Twenty (20) .mu.L of a 0.01 mg/mL carboxypeptidase A
(Carboxypeptidase A from human pancreas; Sigma-Aldrich, C5358)
stock is added to each sample. The samples are returned to the
37.degree. C. water bath. The reaction is quenched with 5 .mu.L of
a 40 .mu.g/mL carboxypeptidase inhibitor at the proper time point.
No extra carboxypeptidase inhibitor is added to the control samples
since there is already inhibitor present. Samples are subsequently
flash frozen in liquid nitrogen, and stored at -80.degree. C. until
analysis. Upon analysis, samples are thawed and transferred to a 1
mL 96-well deep microtiter plate. Standards of peptide and
Z-Gly-Gly-Leu are prepared in carboxypeptidase A reaction buffer at
0.625, 1.25, 2.50, 5.00, and 10.00 .mu.g/mL concentrations. These
standards are used to generate a standard curve for quantification
of samples. When necessary, the standard curves are also used to
calculate the concentration of the corresponding digestion product.
Ten (10) .mu.L injections are made of each sample and standard.
Carboxypeptidase A--Identification of Proteolysis Product
[0158] To analyze carboxypeptidase A digestion product, samples of
0.01 mg/mL peptide are prepared in the carboxypeptidase A reaction
buffer at a total volume of 500 .mu.L in 2 mL eppendorf tubes.
Triplicate samples are prepared for the following time points: 0,
15, 30, 60, 120, 180 and 240 min. The samples are incubated at
37.degree. C. for 5 min. Twenty (20) .mu.L of a 0.01 mg/mL
carboxypeptidase A stock are added to each sample and returned to
the 37.degree. C. water bath. The reactions are quenched with 5
.mu.L of a 40 .mu.g/mL carboxypeptidase inhibitor at the proper
time points. Samples are subsequently flash frozen in liquid
nitrogen, and stored at -80.degree. C. until analysis. Upon
analysis, samples are thawed and transferred to a 1 mL 96-well
plate. Standards of peptide prepared in carboxypeptidase A reaction
buffer at 0.625, 1.25, 2.50, 5.00, and 10.00 .mu.g/mL
concentrations. These standards are used to generate a standard
curve for quantification of samples. When necessary, the standard
curves are also used to calculate the concentration of the
corresponding digestion product. Ten (10) .mu.L injections are made
of each sample and standard. If the formation of a digestion
product is evident, then a spectral analysis used to determine the
mass of the digestion product, and predict its possible
identity.
Simulated Gastric Fluid (SGF)
[0159] Samples of peptide are prepared in the simulated gastric
fluid buffer (0.2% NaCl (w/v), 0.7% HCl (v/v), pH 1) to a total
volume of 500 .mu.L in 2 mL eppendorf tubes. The reference control
and test samples (0, 1 and 3 h) are incubated at 37.degree. C. for
the time point indicated. The reference control sample is diluted
10-fold (1000 .mu.L volume) in distilled water for a final
concentration of 10 .mu.M and chilled on ice. At each time point,
samples are diluted 10-fold (1000 .mu.L volume) in distilled water
for an expected concentration of 10 .mu.M, and chilled on ice,
until analysis. Upon analysis, samples are transferred to a 1 mL
96-well plate. Standards of peptide are prepared in distilled water
at 0.625, 1.25, 2.50, 5.00, and 10.00 .mu.M concentrations. These
standards are used to generate a standard curve for quantification
of samples. Ten (10) .mu.L injections are made of each sample and
standard.
Example 7
Rodent Intestinal Transit Assays
[0160] In order to determine whether a peptide increases the rate
of gastrointestinal transit, the peptide and controls are tested
using a murine gastrointestinal transit (GIT) assay (Moon et al.
Infection and Immunity 25:127, 1979). In this assay, charcoal,
which can be readily visualized in the gastrointestinal tract, is
administered to mice after the administration of a test compound.
The distance traveled by the charcoal is measured and expressed as
a percentage of the total length of the colon.
[0161] Mice are fasted with free access to water for 12 to 16 hours
before the treatment with peptide or control buffer. The peptides
are orally administered at 1 .mu.g/kg-1 mg/kg of peptide in buffer
(20 mM Tris pH 7.5) 7 minutes before being given an oral dose of 5%
Activated Carbon (Aldrich 242276-250G). Control mice are
administered buffer only before being given a dose of Activated
Carbon. After 15 minutes, the mice are sacrificed and their
intestines from the stomach to the cecum are dissected. The total
length of the intestine as well as the distance traveled from the
stomach to the charcoal front is measured for each animal and the
results are expressed as the percent of the total length of the
intestine traveled by the charcoal front. All results are reported
as the average of 10 mice.+-.standard deviation. A comparison of
the distance traveled by the charcoal between the mice treated with
peptide versus the mice treated with vehicle alone is performed
using a Student's t test and a statistically significant difference
is considered for P<0.05. P-values are calculated using a
two-sided T-Test assuming unequal variances. Controls include
vehicle alone (e.g. Tris buffer) and Zelnorm.RTM..
[0162] An identical experiment can be performed to determine if
peptides are effective in a chronic dosing treatment regimen.
Briefly, 8 week old CD1 female mice are dosed orally once a day for
5 days with either peptide (0.06 mg/kg or 0.25 mg/kg in 20 mM Tris
pH 7.5) or vehicle alone (20 mM Tris pH 7.5). On the 5.sup.th day,
a GIT assay is performed identical to that, above except 200 .mu.l
of a 10% charcoal solution is administered.
[0163] The gastrointestinal transit assay can also performed in
male and female CD rats (Charles River; Wilmington, Mass.). The
assay is performed as described above for mice except an average of
5-8 animals are used for each test group and test peptide and 5%
activated carbon are administered simultaneously (versus 7 minutes
apart). In addition, the animals are sacrificed 10 minutes after
the administration of peptide and test compound. The experiment can
be performed in male and female rats.
[0164] The gastrointestinal transit assay can also performed in
wild-type mice and mice lacking the guanylatc cyclase C receptor
(GC-C KO; Mann et al 1997 Biochem and Biophysical Research
Communications 239:463). Wild type and GC-C KO mice are fasted
overnight and test peptide or vehicle alone are orally administered
10 minutes prior to an oral dose of a 10% Activated Carbon/10% Gum
Arabic suspension. Animals are sacrificed 5 minutes after peptide
or vehicle administration. is is
Example 8
Intestinal Secretion Assay in Suckling Mice (SuMi Assay)
[0165] Peptides are tested for their ability to increase intestinal
secretion using a suckling mouse model of intestinal secretion. In
this model a test compound is administered to suckling mice that
are between 7 and 9 days old. After the mice are sacrificed, the
gastrointestinal tract from the stomach to the cecum is dissected
("guts"). The remains ("carcass") as well as the guts are weighed
and the ratio of guts to carcass weight is calculated. If the ratio
is above 0.09, one can conclude that the test compound increases
intestinal secretion. Wild type ST peptide can be used as a control
in this assay.
Example 9
Colonic Hyperalgesia Animal Models
[0166] Hypersensitivity to colorectal distension is common in
patients with IBS and may be responsible for the major symptom of
pain. Both inflammatory and non-inflammatory animal models of
visceral hyperalgesia to distension have been developed to
investigate the effect of compounds on visceral pain in IBS.
[0167] I. Trinitrobenzenesulphonic Acid (TNBS)--Induced Rectal
Allodynia in Two Rodent Models
[0168] TNBS Visceral Hypersensitivity Rat Model
[0169] Male Wistar rats (220-250 g) are premedicated with 0.5 mg/kg
of acepromazine injected intraperitoneally (IP) and anesthetized by
intramuscular administration of 100 mg/kg of ketamine. Pairs of
nichrome wire electrodes (60 cm in length and 80 .mu.m in diameter)
are implanted in the striated muscle of the abdomen, 2 cm laterally
from the white line. The free ends of electrodes are exteriorized
on the back of the neck and protected by a plastic tube attached to
the skin. Electromyographic (EMG) recordings are started 5 days
after surgery. Electrical activity of abdominal striated muscle is
recorded with an electroencephalograph machine (Mini VIII, Alvar,
Paris, France) using a short time constant (0.03 sec.) to remove
low-frequency signals (<3 Hz).
[0170] Ten days post surgical implantation,
trinitrobenzenesulphonic acid (TNBS) is administered to induce
rectal inflammation. TNBS (80 mg kg.sup.-1 in 0.3 ml 50% ethanol)
is administered intrarectally through a silicone rubber catheter
introduced at 3 cm from the anus under light diethyl-ether
anesthesia, as previously described (Morteau et al. 1994 Dig Dis
Sci 39:1239). Following TNBS administration, rats are placed in
plastic tunnels where they are severely limited in mobility for
several days before colorectal distension (CRD). Experimental
compound is administered one hour before CRD which is performed by
insertion into the rectum, at 1 cm of the anus, a 4 cm long balloon
made from a latex condom (Gue et al, 1997 Neurogastroenterol.
Motil. 9:271). The balloon is fixed on a rigid catheter taken from
an embolectomy probe (Fogarty). The catheter attached balloon is
fixed at the base of the tail. The balloon, connected to a
barostat, is inflated progressively by steps of 15 mmHg, from 0 to
60 mmHg, each step of inflation lasting 5 min. Evaluation of rectal
sensitivity, as measured by EMG, is performed before (1-2 days) and
3 days following rectal instillation of TNBS.
[0171] The number of spike bursts that corresponds to abdominal
contractions is determined per 5 min periods. Statistical analysis
of the number of abdominal contractions and evaluation of the
dose-effects relationships is performed by a one way analysis of
variance (ANOVA) followed by a post-hoc (Student or Dunnett tests)
and regression analysis for ED50 if appropriate.
[0172] TNBS Visceral Hypersensitivity Model in Wild-Type (Wt) Mice
and Mice Lacking the Guanylate-Cyclase C Receptor (GC-C KO)
[0173] TNBS induced visceral hypersensitivity can be assessed in WT
and GC-C KO mice. Two groups (WT and GC-C KO) of male mice (22-25
g) are surgically prepared for electromyographic (EMG) recordings.
Three electrodes are implanted in the striated muscles of the
abdomen for EMG recording of abdominal contractions. Colorectal
distension (CRD) is performed with a balloon inflated by 10 s steps
of 0.02 ml from 0 to 0.12 ml. Under basal conditions mice are
submitted to control CRD (time 0) followed by oral administration
of test peptide (0.01 and 0.3 .mu.g/kg) or vehicle only (distilled
water, 1 ml) at 3 hours. One hour post dosing the CRD procedure is
repeated. Abdominal EMG contractile response to colorectal
distension in basal conditions in both WT and GC-C KO mice (12-14
mice per group) is determined in the absence of vehicle and test
peptide, and the mean+/-standard error of the mean (SEM) are
determined.
[0174] For TNBS induced visceral hypersensitivity conditions, mice
are submitted to control CRD (time 0) and TNBS (20 mg/kg) is
administered at 3 days. Three days post intracolonic TNBS-induction
animals are orally administered test peptide (0.01 and 0.3
.mu.g/kg) or vehicle (distilled water, 1 ml) 1 hour before CRD. The
effect of test peptide (0.01 .mu.g/kg) on abdominal response to
colorectal distension after TNBS in WT and GC-C KO mice (12-14 per
group) at a volume distension of 0.8 ml is determined and the
mean+/-standard error of the mean (SEM) is determined.
[0175] II. Partial Restraint Stress-Induced Hyperalgesia Model
[0176] Five groups of female Wistar rats (weighing 200-250 g each),
are surgically prepared for electromyography as described (Morteau
et al. 1994 Dig Dis Sci 39:1239-48) and can be used to evaluate the
effects of a test peptide on colorectal sensitivity and compliance
after a 2 hour partial restraint stress session. Partial restraint
stress (PRS), a relatively mild stress, is induced as previously
described (Morteau et al. 1994 Dig Dis Sci 39:1239-48). Female rats
are lightly anesthetized with diethyl ether and their shoulders,
upper forelimbs and thoracic trunk are wrapped in a confining
harness of paper tape to restrict, but not prevent body movements.
Control sham-stress animals are anesthitized but not wrapped.
Animals receive isobaric colorectal distensions (CRD) directly
prior to (control CRD) and 15 minutes after two hours of partial
restraint induced stress. Rats are treated orally with test peptide
(0.3, 3, 30 ug/kg) or vehicle only (distilled water 1 mL) one hour
before the CRD procedure. For the CRD procedure, rats are
acclimatized to restraint in polypropylene tunnels (diameter: 7 cm;
length: 20 cm) periodically for several days before CRD in order to
minimize recording artifacts. The balloon used for distension is 4
cm long and made from a latex condom. It is fixed on a rigid
catheter taken from an embolectomy probe (Fogarty). CRD is
performed by insertion of the balloon in the rectum at 1 cm from
the anus. The tube is fixed at the base of the tail. Isobaric
distensions are performed from 0 to 60 mmHg, with each distension
step lasting 5 minutes. The first distension is performed at a
pressure of 15 mmHg and an increment of 15 mmHg is added at each
following step, until a maximal pressure of 60 mmHg is attained.
Electromyographic recordings commence 5 days after surgery.
Electrical activity is recorded with an electroencephalograph (Mini
VIII, Alvar, Paris, France) using a short time constant (0.03 sec.)
to remove low-frequency signals (<3 Hz) and a paper speed of 3.6
cm/minute. Isobaric distensions of the colon are performed by
connecting the balloon to a computerized barostat. Colonic pressure
and balloon volume are continuously monitored on a potentiometric
recorder (L6514, Linseis, Selb, Germany) with a paper speed of 1.0
cm/minute. The number of spike bursts, corresponding to abdominal
contractions, is evaluated per 5-minute period. Colorectal volumes
are determined as the maximal volume obtained for each stage of
distension using the potentiometric recorder. Statistical analysis
of these two parameters is performed using a one way analysis of
variance (ANOVA) followed by an unpaired two-tailed Student's t
test using GraphPad Prism 4.0. p values<0.05 are considered
significantly different. The values are expressed as
mean.+-.SEM.
[0177] III. Water Avoidance Stress-Induced Hyperalgesia Model
[0178] The effect of peptides on basal visceral nociception can be
tested using a model of water avoidance stress-induced visceral
hyperalgesia in adult male Wistar rats. The stress involves
confining rats, to a platform surrounded by water for a period of 1
hour and then measuring their visceromotor response to colonic
distension using electromyography (EMG).
[0179] At least 7 days prior to stress measurements, animals are
deeply anesthetized with pentobarbital sodium (45 mg/kg) and
equipped with electrodes implanted into the external oblique
musculature, just superior to the inguinal ligament. Electrode
leads are then tunneled subcutaneously and externalized laterally
for future access. Following surgery, rats are housed in pairs and
allowed to recover for at least 7 days. On the day of the
experiment, animals are lightly anesthetized with halothane, and a
lubricated latex balloon (6 cm) is inserted intra-anally into the
descending colon. Animals are allowed to recover for 30 minutes,
and colorectal distension (CRD) is initiated. The CRD procedure
consists of graded intensities of phasic CRD (10, 20, 40, 60 mmHg;
20 s duration; 4 min inter-stimulus interval). Visceromotor
response (VMR) to CRD is quantified by measuring EMG activity.
[0180] To determine the effect of a test peptide in a model of
water avoidance stress-induced visceral hyperalgesia, a baseline
CRD is recorded and then the animals are subjected to 1 hour of
water avoidance stress. For water avoidance stress, the test
apparatus consists of a Plexiglas tank with a block affixed to the
center of the floor. The tank is filled with fresh room temperature
water (25.degree. C.) to within 1 cm of the top of the block. The
animals are placed on the block for a period of 1 h. The sham water
avoidance stress entails placing the rats on the same platform in a
waterless container.
Phenylbenzoquinone-Induced Writhing Model
[0181] The PBQ-induced writhing model can be used to assess pain
control activity of the peptides and GC-C receptor agonists. This
model is described by Siegmund et al. (1957 Proc. Soc. Exp. Bio.
Med. 95:729-731). Briefly, one hour after oral dosing with a test
compound, e.g., a peptide, morphine or vehicle, 0.02%
phenylbenzoquinone (PBQ) solution (12.5 mL/kg) is injected by
intraperitoneal route into the mouse. The number of stretches and
writhings are recorded from the 5.sup.th to the 10.sup.th minute
after PBQ injection, and can also be counted between the 35.sup.th
and 40.sup.th minute and between the 60.sup.th and 65.sup.th minute
to provide a kinetic assessment. The results are expressed as the
number of stretches and writhings (mean.+-.SEM) and the percentage
of variation of the nociceptive threshold calculated from the mean
value of the vehicle-treated group. The statistical significance of
any differences between the treated groups and the control group is
determined by a Dunnett's test using the residual variance after a
one-way analysis of variance (P<0.05) using SigmaStat
Software.
Example 10
Measuring the Effect of Peptides on Bowel Habits
[0182] Single doses of 30, 100, 300, 1000 or 3000 .mu.g of peptide
are given to healthy males and postmenopausal females. At each dose
level (peptide or placebo (vehicle) is administered orally in 5.0
mL 50 mM phosphate buffer (pH 6.0) plus 3.times.20 mL water rinses
and 175 mL water after at least a 10-hour fast. In each dosing
group, subjects are randomized to receive either placebo or
peptide. Bowel habits (including Bristol Stool Form Scale score
(BSFS), stool frequency, and stool weight) are evaluated for each
collected bowel movement 48 hours prior to dose and up to
approximately 48 hours postdose. The BSFS scale is as follows: (1)
Separate hard lumps, like nuts; (2) Sausage-shaped but lumpy, (3)
Like a sausage or snake but with cracks on its surface, (4) Like a
sausage or snake, smooth and soft, (5) Soft blobs with clear-cut
edges, (6) Fluffy pieces with ragged edges, a mushy stool, and (7)
Watery, no solid pieces.
Example 11
Examination of the Effect of Peptides on the Consistency and Timing
of Bowel Movements in Humans after a Seven-Day Dosing Period
[0183] Seven daily doses of 30, 100, 300, or 1000 .mu.g of peptide
are given to healthy subjects. Peptide or placebo (vehicle) is
administered orally in 5.0 mL 50 mM phosphate buffer (pH 6.0) plus
3.times.20 mL water rinses and 175 mL water after at least a
10-hour fast. In each dosing group, subjects are randomized to
receive peptide or receive placebo. Daily mean BSFS scores, mean
stool weight and mean case of passage for the different dosing
groups during the seven days prior to and the seven days during
dosing with peptide are collected. The Mean Ease of Passage Scale
is as follows: (1) Manual disimpaction, (2) Enema needed, (3)
Straining needed, (4) Normal, (5) Urgent without pain, (6) Urgent
with pain, and (7) Incontinent.
Example 12
Peptide Effects in a Rat Model of Postoperative Ileus
[0184] Female CD rats are used to test the effect of peptide on
delayed transit induced by abdominal surgery and manual
manipulation of the small intestine. Groups of at least nine rats
undergo abdominal surgery under isoflurane anesthesia. Surgery
consists of laparotomy and 5 minutes of gentle manual intestinal
massage. Following recovery from anesthesia, rats are dosed orally
with either 10 .mu.g/kg peptide 3 or vehicle (20 mM Tris) in a
volume of 300 .mu.l hour after dosing, intestinal transit rate is
measured. Animals are again dosed with 300 .mu.l of the peptide
followed immediately by 500 .mu.l of a charcoal meal (10% charcoal,
10% gum arabic in water). To calculate the distance of the small
intestine traveled by the charcoal front, after 20 minutes, the
total length of the intestine as well as the distance traveled from
the stomach to the charcoal front are measured for each animal.
Example 13
Peptide Effect on cGMP Levels and Secretion in Ligated Loops Rodent
Models
[0185] The effect of peptide on cGMP levels and secretion are
studied by injecting peptide directly into an isolated loop in
either wild-type or GC-C KO mice. This done by surgically ligating
a loop in the small intestine of the mouse. The methodology for
ligated loop formation is a similar to that described in London et
al. 1997 Am J Physiol p. G93-105. The loop is roughly centered and
is a length of 1-3 cm. The loops are injected with 100 .mu.l of
either peptide (5 .mu.g) or vehicle (20 mM Tris, pH 7.5 or Krebs
Ringer, 10 mM Glucose, HEPES buffer (KRGH)). Following a recovery
time of 90 minutes the loops are excised. Weights are recorded for
each loop before and after removal of the fluid contained therein.
The length of each loop is also recorded. A weight to length ratio
(W/L) for each loop is calculated to determine the effects of
peptide on secretion.
[0186] To determine the effect of peptide on cGMP activity, fluid
from the loop is collected in ice-cold trichloracetic acid (TCA)
and stored at -80.degree. C. for use in an assay to measure cGMP
levels in the fluid. Intestinal fluid samples are TCA extracted,
and cyclic GMP is measured by EIA according to procedures outlined
in the Cayman Chemical Cyclic GMP EIA kit (Cayman Chemical, Ann
Arbor, Mich.) to determine cyclic GMP levels in the intestinal
fluid of the mouse in the presence of either peptide or vehicle.
The effects of peptide on cGMP levels and secretion in ligated
loops in female CD rats can also be determined using protocols
similar to those described above. In the case of the rat, however
four loops of intestine are surgically ligated. The first three
loops are distributed equally in the small intestine and the fourth
loop is located in colon. Loops are 1 to 3 centimeters, and are
injected with 200 .mu.L of either peptide (5 .mu.g) or vehicle
(Krebs Ringer, 10 mM glucose, HEPES buffer (KRGH)).
Example 14
Peptide Effects on Opioid Induced Constipation
[0187] The effect of peptide on opioid induced constipation is
studied by dosing female rats (.about.160 g each) with 300 .mu.l of
the opiate, morphine (2.5 mg/kg) via intra-peritoneal injection.
Thirty minutes post dosing, animals are treated with 300 .mu.l of
SEQ ID NO:3 or vehicle only. Ten minutes later, the animals are
orally dosed with 500 .mu.l 10% charcoal, 10% gum arabic meal.
After ten minutes, the animals are sacrificed and gastrointestinal
transit is measured as in Example 3 above.
Example 15
Mass Spectrometry Characterization of Disulfide Bonds in
Peptide
[0188] The position of disulfide bonds in a test peptide can be
determined as follows. To identify the optimal conditions required
to partially reduce a test peptide, chemically synthesized peptide
is alkylated with iodoacetamide after TCEP (tris(2-carboxyethyl)
phosphine) treatment (0.1 to 10 mM for 20 minutes at room
temperature). After TCEP reduction, the reaction is adjusted to pH
8.0 with Tris and iodoacetamide is added to 50 mM. The reaction
products are analyzed by LC-MS. Partially reduced peptide is then
cyanylated, cleaved with base and completely reduced to separate
fragments. After partial reduction, both cyanylation and cleavage
of peptide are performed either in a test tube or in an HPLC
column. A modified method of Wu and Watson ((2002) Methods Mol.
Biol. 194: 1-22) is used to determine the position of the disulfide
bonds. The steps are carried out manually, with isolation of the
alkylation products by solid phase extraction (SPE), or in-line
(automated), with reactions occurring in an SPE column. Briefly,
the manual procedure comprised the following. Chemically
synthesized peptide is partially reduced with 1 mM
tris(2-carboxyethyl) phosphine (TCEP) at pH 3. The sulfhydryl
groups of partially reduced peptide are cyanylated with 2.1
.mu.moles of 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate
(CDAP) for 15 minutes. The reaction mixture is then diluted to 0.5
mL with 10 mM ammonium acetate pH 5.8 and applied to an Amprep
octadecyl C18 minicolumn (100 mg, GE HealthTech). The minicolumn is
washed with 1 mL of 10 mM ammonium acetate pH 5.8 and peptides
eluted with 0.6 mL methanol. After drying, the peptides are cleaved
in 1 M NH4OH and fully reduced with 0.1 M TCEP. After drying, the
peptide fragments are reconstituted in 0.1% formic acid and
analyzed by LC-MS. Briefly, the automated procedure comprised the
following. Peptide is loaded onto an Oasis HLB 2.times.15 mm column
(Waters). Reactions are carried out by filling a 5 mL sample loop
with 1.2 mM TCEP, 2.4 mM CDAP, 2 M NH.sub.4OH or 6 mM TCEP and
pushing each reagent through the column with 0.1% formic acid in 5%
methanol at a flow rate of 0.3 mL/min. The column is then
back-flushed and the cleaved peptides analyzed by LC-MS.
[0189] LC-MS analysis can be conduced using an Atlantis dC18
2.1.times.50 mm column (Waters) equilibrated in 98% buffer A (0.1%
formic acid), 2% buffer B (0.1% formic acid: 85% methanol, 15%
CH3CN) at a flow rate of 0.3 mL/min. After a 4 min wash with the
same buffers, peptides are eluted with a linear gradient of 2%
buffer B to 40% buffer B over 38 min with a constant flow rate of
0.3 mL/min. Cleaved peptide masses are determined using a Micromass
Q-T of II instrument equipped with an electrospray ionization (ESI)
source operating in positive ion mode. The instrument is programmed
to scan in the mass range of m/z 100 to 1000. Molecular weight
predictions and data analysis are carried out with MassLynx version
4.0 software. Based on the method of Wu and Watson (supra), a list
of possible fragments resulting from CN-induced cleavage of singly
reduced and cyanylated species of peptide with all possible
disulfide linkage combinations is generated. The list included the
signature fragments for each possible structure and is used to
predict the disulfide bonding pattern of the test peptide.
PEGylated Peptides
[0190] The in vivo half-life of peptides can be extended by
conjugating the peptide to a water soluble polymer, such as
polyethylene glycol (PEG) to create a PEGylated peptide. The
polyethylene glycol molecules are usually connected to the peptide
via a reactive group found on the peptide, e.g., an amino group
found within a lysine or at the amino terminus of the peptide.
[0191] Various methods are known for attaching polyethylene glycol
to a peptide (see, for example, U.S. Pat. No. 4,002,531; U.S. Pat.
No. 4,904,584; U.S. Pat. No. 5,834,594; U.S. Pat. No. 5,824,784 and
U.S. Pat. No. 5,985,265).
Administration of Peptides and GC-C Receptor Agonists
[0192] For therapeutic and preventive treatment of disorders
described herein, the peptides and agonists described herein can be
administered orally, e.g., as a tablet or cachet containing a
predetermined amount of the active ingredient, pellet, gel, paste,
syrup, bolus, electuary, slurry, sachet; capsule; 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. The peptides and agonists
can be co-administered with other agents used to treat
gastrointestinal disorders including but not limited to the agents
described herein. The peptides and agonists can also be
administered by rectal suppository. For the treatment of disorders
outside the gastrointestinal tract such as congestive heart failure
and benign prostatic hypertrophy, peptides and agonists are
preferably administered parenterally or orally.
[0193] The peptides described herein can be administered alone or
in combination with other agents. For example, the peptides can be
administered together 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.
[0194] Combination therapy can be achieved by administering two or
more agents, e.g., a peptide described herein and an analgesic
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.
[0195] Combination therapy can also include two or more
administrations of one or more of the agents used in the
combination. For example, if agent X and agent Y are used in a
combination, one could administer them sequentially in any
combination one or more times, e.g., in the order X-Y-X, X-X-Y,
Y-X-Y, Y-Y-X, X-X-Y-Y, etc.
[0196] Combination therapy can also include the administration of
two or more agents via different routes or locations. For example,
(a) one agent is administered orally and another agent is
administered intravenously or (b) one agent is administered orally
and another is administered locally. In each case, the agents can
either simultaneously or sequentially. Approximated dosages for
some of the combination therapy agents described herein are found
in the "BNF Recommended Dose" column of tables on pages 11-17 of
WO01/76632 (the data in the tables being attributed to the March
2000 British National Formulary) and can also be found in other
standard formularies and other drug prescribing directories. For
some drugs, the customary prescribed dose for an indication will
vary somewhat from country to country.
[0197] The agents, alone or in combination, can be combined with
any pharmaceutically acceptable carrier or medium. Thus, they can
be combined with materials that do not produce an adverse, allergic
or otherwise unwanted reaction when administered to a patient. The
carriers or mediums used can include 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.
[0198] Compositions of the present disclosure 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), anti-oxidants, 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.
[0199] 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.
[0200] 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, disintegants, lubricants,
anti-microbial agents, and coating agents such as:
BINDERS: corn starch, potato starch, other starches, gelatin,
natural and synthetic gums such as acacia, xanthan, sodium
alginate, alginic acid, other alginates, powdered tragacanth, guar
gum, cellulose and its derivatives (e.g., ethyl cellulose,
cellulose acetate, carboxymethyl cellulose calcium, sodium
carboxymethyl cellulose), polyvinyl pyrrolidone (e.g., povidone,
crospovidone, copovidone, etc), methyl cellulose, Methocel,
pre-gelatinized starch (e.g., STARCH 1500.RTM. and STARCH 1500
LM.RTM., sold by Colorcon, Ltd.), hydroxypropyl methyl cellulose,
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), or mixtures thereof, FILLERS: talc, calcium
carbonate (e.g., granules or powder), dibasic calcium phosphate,
tribasic calcium phosphate, calcium sulfate (e.g., granules or
powder), microcrystalline cellulose, powdered cellulose, dextrates,
kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized
starch, dextrose, fructose, honey, lactose anhydrate, lactose
monohydrate, lactose and aspartame, lactose and cellulose, lactose
and microcrystalline cellulose, maltodextrin, maltose, mannitol,
microcrystalline cellulose & guar gum, molasses, sucrose, or
mixtures thereof, DISINTEGRANTS: agar-agar, alginic acid, calcium
carbonate, microcrystalline cellulose, croscarmellose sodium,
crospovidone, polacrilin potassium, sodium starch glycolate, potato
or tapioca starch, other starches, pre-gelatinized starch, clays,
other algins, other celluloses, gums (like gellan), low-substituted
hydroxypropyl cellulose, or mixtures thereof, LUBRICANTS: calcium
stearate, magnesium stearate, mineral oil, light mineral oil,
glycerin, sorbitol, mannitol, polyethylene glycol, other glycols,
stearic acid, sodium lauryl sulfate, sodium stearyl fumarate,
vegetable based fatty acids lubricant, 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
(Deaussa Co., Plano, Tex. USA), a pyrogenic silicon dioxide
(CAB-O-SIL, Cabot Co., Boston, Mass. USA), or mixtures thereof,
ANTI-CAKING AGENTS: calcium silicate, magnesium silicate, silicon
dioxide, colloidal silicon dioxide, talc, or mixtures thereof,
ANTIMICROBIAL AGENTS: 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, or mixtures thereof, and
COATING AGENTS: sodium carboxymethyl cellulose, cellulose acetate
phthalate, ethylcellulose, gelatin, pharmaceutical glaze,
hydroxypropyl cellulose, hydroxypropyl methylcellulose
(hypromellose), hydroxypropyl methyl cellulose phthalate,
methylcellulose, polyethylene glycol, polyvinyl acetate phthalate,
shellac, sucrose, titanium dioxide, carnauba wax, microcrystalline
wax, gellan gum, maltodextrin, methacrylates, microcrystalline
cellulose and carrageenan or mixtures thereof.
[0201] The formulation can also include other excipients 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. No. 6,086,918 and U.S. Pat. No.
5,912,014), creams and lotions (like maltodextrin and
carrageenans); 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.
[0202] Solid oral dosage forms may optionally be treated with
coating systems (e.g. Opadry.RTM. fx film coating system, for
example Opadry.RTM. blue (OY-LS-20921), Opadry.RTM. white
(YS-2-7063), Opadry.RTM. white (YS-1-7040), and black ink
(S-1-8106).
[0203] The agents either in their free form or as a salt can be
combined with a polymer such as polylactic-glycoloic acid (PLGA),
poly-(I)-lactic-glycolic-tartaric acid (P(I)LGT) (WO 01/12233),
polyglycolic acid (U.S. Pat. No. 3,773,919), polylactic acid (U.S.
Pat. No. 4,767,628), poly(E-caprolactone) and poly(alkylene oxide)
(U.S. 20030068384) to create a sustained release formulation. Such
formulations can be used to implants that release a peptide or
another agent over a period of a few days, a few weeks or several
months depending on the polymer, the particle size of the polymer,
and the size of the implant (see, e.g., U.S. Pat. No. 6,620,422).
Other sustained release formulations and polymers for use in are
described in EP 0 467 389 A2, WO 93/24150, U.S. Pat. No. 5,612,052,
WO 97/40085, WO 03/075887, WO 01/01964A2, U.S. Pat. No. 5,922,356,
WO 94/155587, WO 02/074247A2, WO 98/25642, U.S. Pat. No. 5,968,895,
U.S. Pat. No. 6,180,608, U.S. 20030171296, U.S. 20020176841, U.S.
Pat. No. 5,672,659, U.S. Pat. No. 5,893,985, U.S. Pat. No.
5,134,122, U.S. Pat. No. 5,192,741, U.S. Pat. No. 5,192,741, U.S.
Pat. No. 4,668,506, U.S. Pat. No. 4,713,244, U.S. Pat. No.
5,445,832 U.S. Pat. No. 4,931,279, U.S. Pat. No. 5,980,945, WO
02/058672, WO 9726015, WO 97/04744, and. US20020019446. In such
sustained release formulations microparticles (Delie and
Blanco-Prieto 2005 Molecule 10:65-80) of peptide are combined with
microparticles of polymer. One or more sustained release implants
can be placed in the large intestine, the small intestine or both.
U.S. Pat. No. 6,011,011 and WO 94/06452 describe a sustained
release formulation providing either polyethylene glycols (i.e. PEG
300 and PEG 400) or triacetin. WO 03/053401 describes a formulation
which may both enhance bioavailability and provide controlled
release of the agent within the GI tract. Additional controlled
release formulations are described in U.S. Pat. No. 6,734,188, WO
02/38129, EP 326 151, U.S. Pat. No. 5,236,704, WO 02/30398, WO
98/13029; U.S. 20030064105, U.S. 20030138488A1, U.S. 20030216307A1,
U.S. Pat. No. 6,667,060, WO 01/49249, WO 01/49311, WO 01/49249, WO
01/49311, and U.S. Pat. No. 5,877,224.
[0204] The agents can be administered, e.g., by intravenous
injection, intramuscular injection, subcutaneous injection,
intraperitoneal injection, topical, sublingual, intraarticular (in
the joints), intradermal, buccal, ophthalmic (including
intraocular), intranasaly (including using a cannula),
intraspinally, intrathecally, or by other routes. The agents can be
administered orally, e.g., as a tablet or cachet containing a
predetermined amount of the active ingredient, gel, pellet, paste,
syrup, bolus, electuary, slurry, capsule, powder, lyophilized
powder, granules, sachet, 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 micellar
formulation (see, e.g. WO 97/11682) via a liposomal formulation
(see, e.g., EP 736299, WO 99/59550 and WO 97/13500), via
formulations described in WO 03/094886, via bilosome (bile-salt
based vesicular system), via a dendrimer, 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. The agents can also be
administered transdermally (i.e. via reservoir-type or matrix-type
patches, microneedles, thermal poration, hypodermic needles,
iontophoresis, electroporation, ultrasound or other forms of
sonophoresis, jet injection, or a combination of any of the
preceding methods (Prausnitz et al. 2004, Nature Reviews Drug
Discovery 3:115-124)). The agents can be administered using
high-velocity transdermal particle injection techniques using the
hydrogel particle formulation described in U.S. 20020061336.
Additional particle formulations are described in WO 00/45792, WO
00/53160, and WO 02/19989. An example of a transdermal formulation
containing plaster and the absorption promoter dimethylisosorbide
can be found in WO 89/04179. WO 96/11705 provides formulations
suitable for transdennal adminisitration. The agents can be
administered in the form a suppository or by other vaginal or
rectal means. The agents can be administered in a transmembrane
formulation as described in WO 90/07923. The agents can be
administed non-invasively via the dehydrated particicles described
in U.S. Pat. No. 6,485,706. The agent can be administered in an
enteric-coated drug formulation as described in WO 02/49621. The
agents can be administered intranassaly using the formulation
described in U.S. Pat. No. 5,179,079. Formulations suitable for
parenteral injection are described in WO 00/62759. The agents can
be administered using the casein formulation described in U.S.
20030206939 and WO 00/06108. The agents can be administered using
the particulate formulations described in U.S. 20020034536.
[0205] The agents, alone or in combination with other suitable
components, can be administered by pulmonary route utilizing
several techniques including but not limited to intratracheal
instillation (delivery of solution into the lungs by syringe),
intratracheal delivery of liposomes, insufflation (administration
of powder formulation by syringe or any other similar device into
the lungs) and aerosol inhalation. Aerosols (e.g., jet or
ultrasonic nebulizers, metered-dose inhalers (MDIs), and dry-powder
inhalers (DPIs)) can also be used in intranasal applications.
Aerosol formulations are stable dispersions or suspensions of solid
material and liquid droplets in a gaseous medium and can be placed
into pressurized acceptable propellants, such as hydrofluoroalkanes
(HFAs, i.e. HFA-134a and HFA-227, or a mixture thereof),
dichlorodifluoromethane (or other chlorofluocarbon propellants such
as a mixture of Propellants 11, 12, and/or 114), propane, nitrogen,
and the like. Pulmonary formulations may include permeation
enhancers such as fatty acids, saccharides, chelating agents,
enzyme inhibitors (e.g., protease inhibitors), adjuvants (e.g.,
glycocholate, surfactin, span 85, and nafamostat), preservatives
(e.g., benzalkonium chloride or chlorobutanol), and ethanol
(normally up to 5% but possibly up to 20%, by weight). Ethanol is
commonly included in aerosol compositions as it can improve the
function of the metering valve and in some cases also improve the
stability of the dispersion. Pulmonary formulations may also
include surfactants which include but are not limited to bile salts
and those described in U.S. Pat. No. 6,524,557 and references
therein. The surfactants described in U.S. Pat. No. 6,524,557,
e.g., a C8-C16 fatty acid salt, a bile salt, a phospholipid, or
alkyl saccaride are advantageous in that some of them also
reportedly enhance absorption of the peptide in the formulation.
Also suitable in the disclosure are dry powder formulations
comprising a therapeutically effective amount of active compound
blended with an appropriate carrier and adapted for use in
connection with a dry-powder inhaler. Absorption enhancers which
can be added to dry powder formulations of the present disclosure
include those described in U.S. Pat. No. 6,632,456. WO 02/080884
describes new methods for the surface modification of powders.
Aerosol formulations may include U.S. Pat. No. 5,230,884, U.S. Pat.
No. 5,292,499, WO 017/8694, WO 01/78696, U.S. 2003019437, U.S.
20030165436, and WO 96/40089 (which includes vegetable oil).
Sustained release formulations suitable for inhalation are
described in U.S. 20010036481A1, 20030232019A1, and U.S.
20040018243A1 as well as in WO 01/13891, WO 02/067902, WO
03/072080, and WO 03/079885. Pulmonary formulations containing
microparticles are described in WO 03/015750, U.S. 20030008013, and
WO 00/00176. Pulmonary formulations containing stable glassy state
powder are described in U.S. 20020141945 and U.S. Pat. No.
6,309,671. Other aerosol formulations are described in EP 1338272A1
WO 90/09781, U.S. Pat. No. 5,348,730, U.S. Pat. No. 6,436,367, WO
91/04011, and U.S. Pat. No. 6,294,153 and U.S. Pat. No. 6,290,987
describes a liposomal based formulation that can be administered
via aerosol or other means. Powder formulations for inhalation are
described in U.S. 20030053960 and WO 01/60341. The agents can be
administered intranasally as described in U.S. 20010038824. The
agents can be incorporated into microemulsions, which generally are
thermodynamically stable, isotropically clear dispersions of two
immiscible liquids, such as oil and water, stabilized by an
interfacial film of surfactant molecules (Encyclopedia of
Pharmaceutical Technology (New York: Marcel Dekker, 1992), volume
9). For the preparation of microemulsions, surfactant (emulsifier),
co-surfactant (co-emulsifier), an oil phase and a water phase are
necessary. Suitable surfactants include any surfactants that are
useful in the preparation of emulsions, e.g., emulsifiers that are
typically used in the preparation of creams. The co-surfactant (or
"co-emulsifer") is generally selected from the group of
polyglycerol derivatives, glycerol derivatives and fatty alcohols.
Preferred emulsifier/co-emulsifier combinations are generally
although not necessarily selected from the group consisting of
glyceryl monostearate and polyoxyethylene stearate; polyethylene
glycol and ethylene glycol palmitostearate; and caprilic and capric
triglycerides and oleoyl macrogolglycerides. The water phase
includes not only water but also, typically, buffers, glucose,
propylene glycol, polyethylene glycols, preferably lower molecular
weight polyethylene glycols (e.g., PEG 300 and PEG 400), and/or
glycerol, and the like, while the oil phase will generally
comprise, for example, fatty acid esters, modified vegetable oils,
silicone oils, mixtures of mono- di- and triglycerides, mono- and
di-esters of PEG (e.g., oleoyl macrogol glycerides), etc.
[0206] The agents described herein can be incorporated into
pharmaceutically-acceptable nanoparticle, nanosphere, and
nanocapsule formulations (Delie and Blanco-Prieto 2005 Molecule
10:65-80). Nanocapsules can generally entrap compounds in a stable
and reproducible way (Henry-Michelland et al., 1987;
Quintanar-Guerrero et al., 1998; Douglas et al., 1987). To avoid
side effects due to intracellular polymeric overloading, ultrafine
particles (sized around 0.1 .mu.m) can be designed using polymers
able to be degraded in vivo (e.g. biodegradable
polyalkyl-cyanoacrylate nanoparticles). Such particles are
described in the prior art (Couvreur et al, 1980; 1988; zur Muhlen
et al., 1998; Zambaux et al. 1998; Pinto-Alphandry et al., 1995 and
U.S. Pat. No. 5,145,684).
[0207] The agents described herein can be formulated with pH
sensitive materials which may include those described in WO04041195
(including the seal and enteric coating described therein) and
pH-sensitive coatings that achieve delivery in the colon including
those described in U.S. Pat. No. 4,910,021 and WO9001329. U.S. Pat.
No. 4,910,021 describes using a pH-sensitive material to coat a
capsule. WO9001329 describes using pH-sensitive coatings on beads
containing acid, where the acid in the bead core prolongs
dissolution of the pH-sensitive coating. U.S. Pat. No. 5,175,003
discloses a dual mechanism polymer mixture composed of pH-sensitive
enteric materials and film-forming plasticizers capable of
conferring permeability to the enteric material, for use in
drug-delivery systems; a matrix pellet composed of a dual mechanism
polymer mixture permeated with a drug and sometimes covering a
pharmaceutically neutral nucleus; a membrane-coated pellet
comprising a matrix pellet coated with a dual mechanism polymer
mixture envelope of the same or different composition; and a
pharmaceutical dosage form containing matrix pellets. The matrix
pellet releases acid-soluble drugs by diffusion in acid pH and by
disintegration at pH levels of nominally about 5.0 or higher. The
agents described herein may be formulated in the pH triggered
targeted control release systems described in WO04052339. The
agents described herein may be formulated according to the
methodology described in any of WO03105812 (extruded hyrdratable
polymers); WO0243767 (enzyme cleavable membrane translocators);
WO03007913 and WO03086297 (mucoadhesive systems); WO02072075
(bilayer laminated formulation comprising pH lowering agent and
absorption enhancer); WO04064769 (amidated peptides); WO05063156
(solid lipid suspension with pseudotropic and/or thixotropic
properties upon melting); WO03035029 and WO03035041 (erodible,
gastric retentive dosage forms); U.S. Pat. No. 5,007,790 and U.S.
Pat. No. 5,972,389 (sustained release dosage forms); WO04112711
(oral extended release compositions); WO05027878, WO02072033, and
WO02072034 (delayed release compositions with natural or synthetic
gum); WO05030182 (controlled release formulations with an ascending
rate of release); WO05048998 (microencapsulation system); U.S. Pat.
No. 5,952,314 (biopolymer); U.S. Pat. No. 5,108,758 (glassy amylose
matrix delivery); U.S. Pat. No. 5,840,860 (modified starch based
delivery). JP10324642 (delivery system comprising chitosan and
gastric resistant material such as wheat gliadin or zein); U.S.
Pat. No. 5,866,619 and U.S. Pat. No. 6,368,629 (saccharide
containing polymer); U.S. Pat. No. 6,531,152 (describes a drug
delivery system containing a water soluble core (Ca pectinate or
other water-insoluble polymers) and outer coat which bursts (eg
hydrophobic polymer-Eudragrit)); U.S. Pat. No. 6,234,464; U.S. Pat.
No. 6,403,130 (coating with polymer containing casein and high
methoxy pectin; WO0174175 (Maillard reaction product); WO05063206
(solubility increasing formulation); WO04019872 (transferring
fusion proteins). The agents described herein may be formulated
using gastrointestinal retention system technology (GIRES; Merrion
Pharmaceuticals). GIRES comprises a controlled-release dosage form
inside an inflatable pouch, which is placed in a drug capsule for
oral administration. Upon dissolution of the capsule, a
gas-generating system inflates the pouch in the stomach where it is
retained for 16-24 hours, all the time releasing agents described
herein.
[0208] The agents described herein can be formulated in an osmotic
device including the ones disclosed in U.S. Pat. No. 4,503,030,
U.S. Pat. No. 5,609,590 and U.S. Pat. No. 5,358,502. U.S. Pat. No.
4,503,030 discloses an osmotic device for dispensing a drug to
certain pH regions of the gastrointestinal tract. More
particularly, the disclosure relates to an osmotic device
comprising a wall formed of a semi-permeable pH sensitive
composition that surrounds a compartment containing a drug, with a
passageway through the wall connecting the exterior of the device
with the compartment. The device delivers the drug at a controlled
rate in the region of the gastrointestinal tract having a pH of
less than 3.5, and the device self-destructs and releases all its
drug in the region of the gastrointestinal tract having a pH
greater than 3.5, thereby providing total availability for drug
absorption. U.S. Pat. Nos. 5,609,590 and 5,358,502 disclose an
osmotic bursting device for dispensing a beneficial agent to an
aqueous environment. The device comprises a beneficial agent and
osmagent surrounded at least in part by a semi-permeable membrane.
The beneficial agent may also function as the osmagent. The
semi-permeable membrane is permeable to water and substantially
impermeable to the beneficial agent and osmagent. A trigger means
is attached to the semi-permeable membrane (e.g., joins two capsule
halves). The trigger means is activated by a pH of from 3 to 9 and
triggers the eventual, but sudden, delivery of the beneficial
agent. These devices enable the pH-triggered release of the
beneficial agent core as a bolus by osmotic bursting.
[0209] The agents described herein may be formulated based on the
disclosure described in U.S. Pat. No. 5,316,774 which discloses a
composition for the controlled release of an active substance
comprising a polymeric particle matrix, where each particle defines
a network of internal pores. The active substance is entrapped
within the pore network together with a blocking agent having
physical and chemical characteristics selected to modify the
release rate of the active substance from the internal pore
network. In one embodiment, drugs may be selectively delivered to
the intestines using an enteric material as the blocking agent. The
enteric material remains intact in the stomach but degrades under
the pH conditions of the intestines. In another embodiment, the
sustained release formulation employs a blocking agent, which
remains stable under the expected conditions of the environment to
which the active substance is to be released. The use of
pH-sensitive materials alone to achieve site-specific delivery is
difficult because of leaking of the beneficial agent prior to the
release site or desired delivery time and it is difficult to
achieve long time lags before release of the active ingredient
after exposure to high pH (because of rapid dissolution or
degradation of the pH-sensitive materials).
[0210] The agents may also be formulated in a hybrid system which
combines pH-sensitive materials and osmotic delivery systems. These
hybrid devices provide delayed initiation of sustained-release of
the beneficial agent. In one device a pH-sensitive matrix or
coating dissolves releasing osmotic devices that provide sustained
release of the beneficial agent see U.S. Pat. Nos. 4,578,075,
4,681,583, and 4,851,231. A second device consists of a
semipermeable coating made of a polymer blend of an insoluble and a
pH-sensitive material. As the pH increases, the permeability of the
coating increases, increasing the rate of release of beneficial
agent see U.S. Pat. Nos. 4,096,238, 4,503,030, 4,522,625, and
4,587,117.
[0211] The agents described herein may be formulated in terpolumers
according to U.S. Pat. No. 5,484,610 which discloses terpolymers
which are sensitive to pH and temperature which are useful carriers
for conducting bioactive agents through the gastric juices of the
stomach in a protected form. The terpolymers swell at the higher
physiologic pH of the intestinal tract causing release of the
bioactive agents into the intestine. The terpolymers are linear and
are made up of 35 to 99 wt % of a temperature sensitive component,
which imparts to the terpolymer LCST (lower critical solution
temperature) properties below body temperatures, 1 to 30 wt % of a
pH sensitive component having a pKa in the range of from 2 to 8
which functions through ionization or deionization of carboxylic
acid groups to prevent the bioactive agent from being lost at low
pH but allows bioactive agent release at physiological pH of about
7.4 and a hydrophobic component which stabilizes the LCST below
body temperatures and compensates for bioactive agent effects on
the terpolymers. The terpolymers provide for safe bioactive agent
loading, a simple procedure for dosage form fabrication and the
terpolymer functions as a protective carrier in the acidic
environment of the stomach and also protects the bioactive agents
from digestive enzymes until the bioactive agent is released in the
intestinal tract.
[0212] The agents described herein may be formulated in pH
sensitive polymers according to those described in U.S. Pat. No.
6,103,865. U.S. Pat. No. 6,103,865 discloses pH-sensitive polymers
containing sulfonamide groups, which can be changed in physical
properties, such as swellability and solubility, depending on pH
and which can be applied for a drug-delivery system, bio-material,
sensor, and the like, and a preparation method therefore. The
pH-sensitive polymers are prepared by introduction of sulfonamide
groups, various in pKa, to hydrophilic groups of polymers either
through coupling to the hydrophilic groups of polymers, such as
acrylamide, N,N-dimethylacrylamide, acrylic acid,
N-isopropylacrylamide and the like or copolymerization with other
polymerizable monomers. These pH-sensitive polymers may have a
structure of linear polymer, grafted copolymer, hydrogel or
interpenetrating network polymer.
[0213] The agents described herein may be formulated according U.S.
Pat. No. 5,656,292 which discloses a composition for pH dependent
or pH regulated controlled release of active ingredients especially
drugs. The composition consists of a compactable mixture of the
active ingredient and starch molecules substituted with acetate and
dicarboxylate residues. The preferred dicarboxylate acid is
succinate. The average substitution degree of the acetate residue
is at least 1 and 0.2-1.2 for the dicarboxylate residue. The starch
molecules can have the acetate and dicarboxylate residues attached
to the same starch molecule backbone or attached to separate starch
molecule backbones. The present disclosure also discloses methods
for preparing said starch acetate dicarboxylates by
transesterification or mixing of starch acetates and starch
dicarboxylates respectively.
[0214] The agents described herein may be formulated according to
the methods described in U.S. Pat. Nos. 5,554,147, 5,788,687, and
6,306,422 which disclose a method for the controlled release of a
biologically active agent wherein the agent is released from a
hydrophobic, pH-sensitive polymer matrix. The polymer matrix swells
when the environment reaches pH 8.5, releasing the active agent. A
polymer of hydrophobic and weakly acidic comonomers is disclosed
for use in the controlled release system. Also disclosed is a
specific embodiment in which the controlled release system may be
used. The pH-sensitive polymer is coated onto a latex catheter used
in ureteral catheterization. A ureteral catheter coated with a
pH-sensitive polymer having an antibiotic or urease inhibitor
trapped within its matrix will release the active agent when
exposed to high pH urine.
[0215] The agents described herein may be formulated in/with
bioadhesive polymers according to U.S. Pat. No. 6,365,187.
Bioadhesive polymers in the form of, or as a coating on,
microcapsules containing drugs or bioactive substances which may
serve for therapeutic, or diagnostic purposes in diseases of the
gastrointestinal tract, are described in U.S. Pat. No. 6,365,187.
The polymeric microspheres all have a bioadhesive force of at least
11 mN/cm.sup.2 (110 N/m2) Techniques for the fabrication of
bioadhesive microspheres, as well as a method for measuring
bioadhesive forces between microspheres and selected segments of
the gastrointestinal tract in vitro are also described. This
quantitative method provides a means to establish a correlation
between the chemical nature, the surface morphology and the
dimensions of drug-loaded microspheres on one hand and bioadhesive
forces on the other, allowing the screening of the most promising
materials from a relatively large group of natural and synthetic
polymers which, from theoretical consideration, should be used for
making bioadhesive microspheres. Solutions of medicament in
buffered saline and similar vehicles are commonly employed to
generate an aerosol in a nebulizer. Simple nebulizers operate on
Bernoulli's principle and employ a stream of air or oxygen to
generate the spray particles. More complex nebulizers employ
ultrasound to create the spray particles. Both types are well known
in the art and are described in standard textbooks of pharmacy such
as Sprowls' American Pharmacy and Remington's The Science and
Practice of Pharmacy. Other devices for generating aerosols employ
compressed gases, usually hydrofluorocarbons and
chlorofluorocarbons, which are mixed with the medicament and any
necessary excipients in a pressurized container, these devices are
likewise described in standard textbooks such as Sprowls and
Remington.
[0216] The agents can be a free acid or base, or a
pharmacologically acceptable salt thereof. Solids can be dissolved
or dispersed immediately prior to administration or earlier. In
some circumstances the preparations include a preservative to
prevent the growth of microorganisms. The pharmaceutical forms
suitable for injection can include sterile aqueous or organic
solutions or dispersions which include, e.g., water, an alcohol, an
organic solvent, an oil or other solvent or dispersant (e.g.,
glycerol, propylene glycol, polyethylene glycol, and vegetable
oils). The formulations may contain antioxidants, buffers,
bacteriostats, and solutes that render the formulation isotonic
with the blood of the intended recipient, and aqueous and
non-aqueous sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives.
Pharmaceutical agents can be sterilized by filter sterilization or
by other suitable means. The agent can be fused to immunoglobulins
or albumin, albumin variants or fragments thereof, or incorporated
into a liposome to improve half-life. Thus the peptides described
herein may be fused directly or via a peptide linker, water soluble
polymer, or prodrug linker to albumin or an analog, fragment, or
derivative thereof. Generally, the albumin proteins that are part
of the fusion proteins of the present disclosure may be derived
from albumin cloned from any species, including human. Human serum
albumin (HSA) consists of a single non-glycosylated peptide chain
of 585 amino acids with a formula molecular weight of 66,500. The
amino acid sequence of human HSA is known [See Meloun, et al.
(1975) FEBS Letters 58:136; Behrens, et al. (1975) Fed. Proc.
34:591; Lawn, et al. (1981) Nucleic Acids Research 9:6102-6114;
Minghetti, et al. (1986) J. Biol. Chem. 261:6747, each of which are
incorporated by reference herein]. A variety of polymorphic
variants as well as analogs and fragments of albumin have been
described. [See Weitkamp, et al., (1973) Ann. Hum. Genet. 37:219].
For example, in EP 322,094, various shorter forms of HSA. Some of
these fragments of HSA are disclosed, including HSA(1-373),
HSA(1-388), HSA(1-389), HSA(1-369), and HSA(1-419) and fragments
between 1-369 and 1-419. EP 399,666 discloses albumin fragments
that include HSA(1-177) and HSA(1-200) and fragments between
HSA(1-177) and HSA(1-200). Methods related to albumin fusion
proteins can be found in U.S. Pat. No. 7,056,701, U.S. Pat. No.
6,994,857, U.S. Pat. No. 6,946,134, U.S. Pat. No. 6,926,898, and
U.S. Pat. No. 6,905,688 and the related priority documents and
references cited therein. The agent can also be conjugated to
polyethylene glycol (PEG) chains. Methods for pegylation and
additional formulations containing PEG-conjugates (i.e. PEG-based
hydrogels, PEG modified liposomes) can be found in Harris and
Chess, Nature Reviews Drug Discovery 2: 214-221 and the references
therein. Peptides can also be modified with alkyl groups (e.g.,
C1-C20 straight or branched alkyl groups); fatty acid radicals; and
combinations of PEG, alkyl groups and fatty acid radicals (see U.S.
Pat. No. 6,309,633; Soltero et al., 2001 Innovations in
Pharmaceutical Technology 106-110). The agent can be administered
via a nanocochleate or cochleate delivery vehicle (BioDelivery
Sciences International). The agents can be delivered transmucosally
(i.e. across a mucosal surface such as the vagina, eye or nose)
using formulations such as that described in U.S. Pat. No.
5,204,108. The agents can be formulated in microcapsules as
described in WO 88/01165. The agent can be administered
intra-orally using the formulations described in U.S. 20020055496,
WO 00/47203, and U.S. Pat. No. 6,495,120. The agent can be
delivered using nanoemulsion formulations described in WO
01/91728A2.
Controlled Release Formulations
[0217] In general, one can provide for controlled release of the
agents described herein through the use of a wide variety of
polymeric carriers and controlled release systems including
erodible and non-erodible matrices, osmotic control devices,
various reservoir devices, enteric coatings and multiparticulate
control devices.
[0218] Matrix devices are a common device for controlling the
release of various agents. In such devices, the agents described
herein are generally present as a dispersion within the polymer
matrix, and are typically formed by the compression of a
polymer/drug mixture or by dissolution or melting. The dosage
release properties of these devices may be dependent upon the
solubility of the agent in the polymer matrix or, in the case of
porous matrices, the solubility in the sink solution within the
pore network, and the tortuosity of the network. In one instance,
when utilizing an erodible polymeric matrix, the matrix imbibes
water and forms an aqueous-swollen gel that entraps the agent. The
matrix then gradually erodes, swells, disintegrates or dissolves in
the GI tract, thereby controlling release of one or more of the
agents described herein. In non-erodible devices, the agent is
released by diffusion through an inert matrix.
[0219] Agents described herein can be incorporated into an erodible
or non-erodible polymeric matrix controlled release device. By an
erodible matrix is meant aqueous-erodible or water-swellable or
aqueous-soluble in the sense of being either erodible or swellable
or dissolvable in pure water or requiring the presence of an acid
or base to ionize the polymeric matrix sufficiently to cause
erosion or dissolution. When contacted with the aqueous environment
of use, the erodible polymeric matrix imbibes water and forms an
aqueous-swollen gel or matrix that entraps the agent described
herein. The aqueous-swollen matrix gradually erodes, swells,
disintegrates or dissolves in the environment of use, thereby
controlling the release of a compound described herein to the
environment of use.
[0220] The erodible polymeric matrix into which an agent described
herein can be incorporated may generally be described as a set of
excipients that are mixed with the agent following its formation
that, when contacted with the aqueous environment of use imbibes
water and forms a water-swollen gel or matrix that entraps the drug
form. Drug release may occur by a variety of mechanisms, for
example, the matrix may disintegrate or dissolve from around
particles or granules of the agent or the agent may dissolve in the
imbibed aqueous solution and diffuse from the tablet, beads or
granules of the device. One ingredient of this water-swollen matrix
is the water-swellable, erodible, or soluble polymer, which may
generally be described as an osmopolymer, hydrogel or
water-swellable polymer. Such polymers may be linear, branched, or
crosslinked. The polymers may be homopolymers or copolymers. In
certain embodiments, they may be synthetic polymers derived from
vinyl, acrylate, methacrylate, urethane, ester and oxide monomers.
In other embodiments, they can be derivatives of naturally
occurring polymers such as polysaccharides (e.g. chitin, chitosan,
dextran and pullulan; gum agar, gum arabic, gum karaya, locust bean
gum, gum tragacanth, carrageenans, gum ghatti, guar gum, xanthan
gum and scleroglucan), starches (e.g. dextrin and maltodextrin),
hydrophilic colloids (e.g. pectin), phosphatides (e.g. lecithin),
alginates (e.g. ammonium alginate, sodium, potassium or calcium
alginate, propylene glycol alginate), gelatin, collagen, and
cellulosics. Cellulosics are cellulose polymer that has been
modified by reaction of at least a portion of the hydroxyl groups
on the saccharide repeat units with a compound to form an
ester-linked or an ether-linked substituent. For example, the
cellulosic ethyl cellulose has an ether linked ethyl substituent
attached to the saccharide repeat unit, while the cellulosic
cellulose acetate has an ester linked acetate substituent. In
certain embodiments, the cellulosics for the erodible matrix
comprises aqueous-soluble and aqueous-erodible cellulosics can
include, for example, ethyl cellulose (EC), methylethyl cellulose
(MEC), carboxymethyl cellulose (CMC), CMEC, hydroxyethyl cellulose
(HEC), hydroxypropyl cellulose (HPC), cellulose acetate (CA),
cellulose propionate (CP), cellulose butyrate (CB), cellulose
acetate butyrate (CAB), CAP, CAT, hydroxypropyl methyl cellulose
(HPMC), HPMCP, HPMCAS, hydroxypropyl methyl cellulose acetate
trimellitate (HPMCAT), and ethylhydroxy ethylcellulose (EHEC). In
certain embodiments, the cellulosics comprises various grades of
low viscosity (MW less than or equal to 50,000 daltons, for
example, the Dow Methocel.TM. series E5, E15LV, E50LV and K100LY)
and high viscosity (MW greater than 50,000 daltons, for example,
E4MCR, E10MCR, K4M, K15M and K100M and the Methocer K series) HPMC.
Other commercially available types of HPMC include the Shin Etsu
Metolose 90SH series.
[0221] The choice of matrix material can have a large effect on the
maximum drug concentration attained by the device as well as the
maintenance of a high drug concentration. The matrix material can
be a concentration-enhancing polymer, for example, as described in
WO05/011634.
[0222] Other materials useful as the erodible matrix material
include, but are not limited to, pullulan, polyvinyl pyrrolidone,
polyvinyl alcohol, polyvinyl acetate, glycerol fatty acid esters,
polyacrylamide, polyacrylic acid, copolymers of ethacrylic acid or
methacrylic acid (EUDRAGITO, Rohm America, Inc., Piscataway, N.J.)
and other acrylic acid derivatives such as homopolymers and
copolymers of butylmethacrylate, methylmethacrylate,
ethylmethacrylate, ethylacrylate, (2-dimethylaminoethyl)
methacrylate, and (trimethylaminoethyl) methacrylate chloride.
[0223] The erodible matrix polymer may contain a wide variety of
the same types of additives and excipients known in the
pharmaceutical arts, including osmopolymers, osmagens,
solubility-enhancing or -retarding agents and excipients that
promote stability or processing of the device.
[0224] Alternatively, the agents of the present disclosure may be
administered by or incorporated into a non-erodible matrix device.
In such devices, an agent described herein is distributed in an
inert matrix. The agent is released by diffusion through the inert
matrix. Examples of materials suitable for the inert matrix include
insoluble plastics (e.g methyl acrylate-methyl methacrylate
copolymers, polyvinyl chloride, polyethylene), hydrophilic polymers
(e.g. ethyl cellulose, cellulose acetate, crosslinked
polyvinylpyrrolidone (also known as crospovidone)), and fatty
compounds (e.g. carnauba wax, microcrystalline wax, and
triglycerides). Such devices are described further in Remington:
The Science and Practice of Pharmacy, 20th edition (2000).
[0225] Matrix controlled release devices may be prepared by
blending an agent described herein and other excipients together,
and then forming the blend into a tablet, caplet, pill, or other
device formed by compressive forces. Such compressed devices may be
formed using any of a wide variety of presses used in the
fabrication of pharmaceutical devices. Examples include
single-punch presses, rotary tablet presses, and multilayer rotary
tablet presses, all well known in the art. See for example,
Remington: The Science and Practice of Pharmacy, 20th Edition,
2000. The compressed device may be of any shape, including round,
oval, oblong, cylindrical, or triangular. The upper and lower
surfaces of the compressed device may be flat, round, concave, or
convex.
[0226] In certain embodiments, when formed by compression, the
device has a strength of at least 5 Kiloponds (Kp)/cm.sup.2 (for
example, at least 7 Kp/cm.sup.2). Strength is the fracture force,
also known as the tablet hardness required to fracture a tablet
formed from the materials, divided by the maximum cross-sectional
area of the tablet normal to that force. The fracture force may be
measured using a Schleuniger Tablet Hardness Tester, Model 6D. The
compression force required to achieve this strength will depend on
the size of the tablet, but generally will be greater than about 5
kP/cm.sup.2. Friability is a well-know measure of a device's
resistance to surface abrasion that measures weight loss in
percentage after subjecting the device to a standardized agitation
procedure. Friability values of from 0.8 to 1.0% are regarded as
constituting the upper limit of acceptability. Devices having a
strength of greater than 5 kP/cm.sup.2 generally are very robust,
having a friability of less than 0.5%. Other methods for forming
matrix controlled-release devices are well known in the
pharmaceutical arts. See for example, Remington: The Science and
Practice of Pharmacy, 20th Edition, 2000.
[0227] As noted above, the agents described herein may also be
incorporated into an osmotic control device. Such devices generally
include a core containing one or more agents as described herein
and a water permeable, non-dissolving and non-eroding coating
surrounding the core which controls the influx of water into the
core from an aqueous environment of use so as to cause drug release
by extrusion of some or all of the core to the environment of use.
In certain embodiments, the coating is polymeric,
aqueous-permeable, and has at least one delivery port. The core of
the osmotic device optionally includes an osmotic agent which acts
to imbibe water from the surrounding environment via such a
semi-permeable membrane. The osmotic agent contained in the core of
this device may be an aqueous-swellable hydrophilic polymer or it
may be an osmogen, also known as an osmagent. Pressure is generated
within the device which forces the agent(s) out of the device via
an orifice (of a size designed to minimize solute diffusion while
preventing the build-up of a hydrostatic pressure head).
[0228] Osmotic agents create a driving force for transport of water
from the environment of use into the core of the device. Osmotic
agents include but are not limited to water-swellable hydrophilic
polymers, and osmogens (or osmagens). Thus, the core may include
water-swellable hydrophilic polymers, both ionic and nonionic,
often referred to as osmopolymers and hydrogels. The amount of
water-swellable hydrophilic polymers present in the core may range
from about 5 to about 80 wt % (including for example, 10 to 50 wt
%). Nonlimiting examples of core materials include hydrophilic
vinyl and acrylic polymers, polysaccharides such as calcium
alginate, polyethylene oxide (PEO), polyethylene glycol (PEG),
polypropylene glycol (PPG), poly (2-hydroxyethyl methacrylate),
poly (acrylic) acid, poly (methacrylic) acid, polyvinylpyrrolidone
(PVP) and crosslinked PVP, polyvinyl alcohol (PVA), PVA/PVP
copolymers and PVA/PVP copolymers with hydrophobic monomers such as
methyl methacrylate, vinyl acetate, and the like, hydrophilic
polyurethanes containing large PEO blocks, sodium croscarmellose,
carrageenan, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose
(HPC), hydroxypropyl methyl cellulose (HPMC), carboxymethyl
cellulose (CMC) and carboxyethyl cellulose (CEC), sodium alginate,
polycarbophil, gelatin, xanthan gum, and sodium starch glycolat.
Other materials include hydrogels comprising interpenetrating
networks of polymers that may be formed by addition or by
condensation polymerization, the components of which may comprise
hydrophilic and hydrophobic monomers such as those just mentioned.
Water-swellable hydrophilic polymers include but are not limited to
PEO, PEG, PVP, sodium croscarmellose, HPMC, sodium starch
glycolate, polyacrylic acid and crosslinked versions or mixtures
thereof.
[0229] The core may also include an osmogen (or osmagent). The
amount of osmogen present in the core may range from about 2 to
about 70 wt % (including, for example, from 10 to 50 wt %). Typical
classes of suitable osmogens are water-soluble organic acids, salts
and sugars that are capable of imbibing water to thereby effect an
osmotic pressure gradient across the barrier of the surrounding
coating. Typical useful osmogens include but are not limited to
magnesium sulfate, magnesium chloride, calcium chloride, sodium
chloride, lithium chloride, potassium sulfate, sodium carbonate,
sodium sulfite, lithium sulfate, potassium chloride, sodium
sulfate, mannitol, xylitol, urea, sorbitol, inositol, raffinose,
sucrose, glucose, fructose, lactose, citric acid, succinic acid,
tartaric acid, and mixtures thereof. In certain embodiments, the
osmogen is glucose, lactose, sucrose, mannitol, xylitol, sodium
chloride, including combinations thereof.
[0230] The core may include a wide variety of additives and
excipients that enhance the performance of the dosage form or that
promote stability, tableting or processing. Such additives and
excipients include tableting aids, surfactants, water-soluble
polymers, pH modifiers, fillers, binders, pigments, disintegrants,
antioxidants, lubricants and flavorants. Nonlimiting examples of
additives and excipients include but are not limited to those
described elsewhere herein as well as microcrystalline cellulose,
metallic salts of acids (e.g. aluminum stearate, calcium stearate,
magnesium stearate, sodium stearate, zinc stearate), pH control
agents (e.g. buffers, organic acids, organic acid salts, organic
and inorganic bases), fatty acids, hydrocarbons and fatty alcohols
(e.g. stearic acid, palmitic acid, liquid paraffin, stearyl
alcohol, and palmitol), fatty acid esters (e.g. glyceryl (mono- and
di-) stearates, triglycerides, glyceryl (palmiticstearic) ester,
sorbitan esters (e.g. sorbitan monostearate, saccharose
monostearate, saccharose monopalmitate, sodium stearyl fumarate),
polyoxyethylene sorbitan esters), surfactants (e.g. alkyl sulfates
(e.g. sodium lauryl sulfate, magnesium lauryl sulfate), polymers
(e.g. polyethylene glycols, polyoxyethylene glycols,
polyoxyethylene, polyoxypropylene ethers, including copolymers
thereof), polytetrafluoroethylene), and inorganic materials (e.g.
talc, calcium phosphate), cyclodextrins, sugars (e.g. lactose,
xylitol), sodium starch glycolate). Nonlimiting examples of
disintegrants are sodium starch glycolate (e.g., Explotab.TM. CLV,
(microcrystalline cellulose (e.g., Avicel.TM.), microcrystalline
silicified cellulose (e.g., ProSolv.TM.), croscarmellose sodium
(e.g., Ac-Di-Sol.TM.). When the agent described herein is a solid
amorphous dispersion formed by a solvent process, such additives
may be added directly to the spray-drying solution when forming an
agent described herein/concentration-enhancing polymer dispersion
such that the additive is dissolved or suspended in the solution as
a slurry, Alternatively, such additives may be added following the
spray-drying process to aid in forming the final controlled release
device.
[0231] A nonlimiting example of an osmotic device consists of one
or more drug layers containing an agent described herein, such as a
solid amorphous drug/polymer dispersion, and a sweller layer that
comprises a water-swellable polymer, with a coating surrounding the
drug layer and sweller layer. Each layer may contain other
excipients such as tableting aids, osmagents, surfactants,
water-soluble polymers and water-swellable polymers.
[0232] Such osmotic delivery devices may be fabricated in various
geometries including bilayer (wherein the core comprises a drug
layer and a sweller layer adjacent to each other), trilayer
(wherein the core comprises a swelter layer sandwiched between two
drug layers) and concentric (wherein the core comprises a central
sweller agent surrounded by the drug layer). The coating of such a
tablet comprises a membrane permeable to water but substantially
impermeable to drug and excipients contained within. The coating
contains one or more exit passageways or ports in communication
with the drug-containing layer(s) for delivering the drug agent.
The drug-containing layer(s) of the core contains the drug agent
(including optional osmagents and hydrophilic water-soluble
polymers), while the sweller layer consists of an expandable
hydrogel, with or without additional osmotic agents.
[0233] When placed in an aqueous medium, the tablet imbibes water
through the membrane, causing the agent to form a dispensable
aqueous agent, and causing the hydrogel layer to expand and push
against the drug-containing agent, forcing the agent out of the
exit passageway. The agent can swell, aiding in forcing the drug
out of the passageway. Drug can be delivered from this type of
delivery system either dissolved or dispersed in the agent that is
expelled from the exit passageway.
[0234] The rate of drug delivery is controlled by such factors as
the permeability and thickness of the coating, the osmotic pressure
of the drug-containing layer, the degree of hydrophilicity of the
hydrogel layer, and the surface area of the device. Those skilled
in the art will appreciate that increasing the thickness of the
coating will reduce the release rate, while any of the following
will increase the release rate: increasing the permeability of the
coating; increasing the hydrophilicity of the hydrogel layer;
increasing the osmotic pressure of the drug-containing layer; or
increasing the device's surface area.
[0235] Other materials useful in forming the drug-containing agent,
in addition to the agent described herein itself, include HPMC, PEO
and PVP and other pharmaceutically acceptable carriers. In
addition, osmagents such as sugars or salts, including but not
limited to sucrose, lactose, xylitol, mannitol, or sodium chloride,
may be added. Materials which are useful for forming the hydrogel
layer include sodium CMC, PEO (e.g. polymers having an average
molecular weight from about 5,000,000 to about 7,500,000 daltons),
poly (acrylic acid), sodium (polyacrylate), sodium croscarmellose,
sodium starch glycolat, PVP, crosslinked PVP, and other high
molecular weight hydrophilic materials.
[0236] In the case of a bilayer geometry, the delivery port(s) or
exit passageway(s) may be located on the side of the tablet
containing the drug agent or may be on both sides of the tablet or
even on the edge of the tablet so as to connect both the drug layer
and the sweller layer with the exterior of the device. The exit
passageway(s) may be produced by mechanical means or by laser
drilling, or by creating a difficult-to-coat region on the tablet
by use of special tooling during tablet compression or by other
means.
[0237] The osmotic device can also be made with a homogeneous core
surrounded by a semipermeable membrane coating, as in U.S. Pat. No.
3,845,770. The agent described herein can be incorporated into a
tablet core and a semipermeable membrane coating can be applied via
conventional tablet-coating techniques such as using a pan coater.
A drug delivery passageway can then be formed in this coating by
drilling a hole in the coating, either by use of a laser or
mechanical means. Alternatively, the passageway may be formed by
rupturing a portion of the coating or by creating a region on the
tablet that is difficult to coat, as described above. In one
embodiment, an osmotic device comprises: (a) a single-layer
compressed core comprising: (i) an agent described herein, (ii) a
hydroxyethylcellulose, and (iii) an osmagent, wherein the
hydroxyethylcellulose is present in the core from about 2.0% to
about 35% by weight and the osmagent is present from about 15% to
about 70% by weight; (b) a water-permeable layer surrounding the
core; and (c) at least one passageway within the water-permeable
layer (b) for delivering the drug to a fluid environment
surrounding the tablet. In certain embodiments, the device is
shaped such that the surface area to volume ratio (of a
water-swollen tablet) is greater than 0.6 min.sup.-1 (including,
for example, greater than 1.0 mm.sup.-1). The passageway connecting
the core with the fluid environment can be situated along the
tablet band area. In certain embodiments, the shape is an oblong
shape where the ratio of the tablet tooling axes, i.e., the major
and minor axes which define the shape of the tablet, are between
1.3 and 3 (including, for example, between 1.5 and 2.5). In one
embodiment, the combination of the agent described herein and the
osmagent have an average ductility from about 100 to about 200 Mpa,
an average tensile strength from about 0.8 to about 2.0 Mpa, and an
average brittle fracture index less than about 0.2. The
single-layer core may optionally include a disintegrant, a
bioavailability enhancing additive, and/or a pharmaceutically
acceptable excipient, carrier or diluent.
[0238] In certain embodiments, entrainment of particles of agents
described herein in the extruding fluid during operation of such
osmotic device is desirable. For the particles to be well
entrained, the agent drug form is dispersed in the fluid before the
particles have an opportunity to settle in the tablet core. One
means of accomplishing this is by adding a disintegant that serves
to break up the compressed core into its particulate components.
Nonlimiting examples of standard disintegrants include materials
such as sodium starch glycolate (e.g., Explotab.TM. CLV),
microcrystalline cellulose (e.g., Avicel.TM.), microcrystalline
silicified cellulose (e.g., ProSoIv.TM.) and croscarmellose sodium
(e.g., Ac-Di-Sol.TM.), and other disintegrants known to those
skilled in the art. Depending upon the particular formulation, some
disintegants work better than others. Several disintegants tend to
form gels as they swell with water, thus hindering drug delivery
from the device. Non-gelling, non-swelling disintegrants provide a
more rapid dispersion of the drug particles within the core as
water enters the core. In certain embodiments, non-gelling,
non-swelling disintegrants are resins, for example, ion-exchange
resins. In one embodiment, the resin is Amberlite.TM. IRP 88
(available from Rohm and Haas, Philadelphia, Pa.). When used, the
disintegrant is present in amounts ranging from about 50-74% of the
core agent.
[0239] Water-soluble polymers are added to keep particles of the
agent suspended inside the device before they can be delivered
through the passageway(s) (e.g., an orifice). High viscosity
polymers are useful in preventing settling. However, the polymer in
combination with the agent is extruded through the passageway(s)
under relatively low pressures. At a given extrusion pressure, the
extrusion rate typically slows with increased viscosity. Certain
polymers in combination with particles of the agent described
herein form high viscosity solutions with water but are still
capable of being extruded from the tablets with a relatively low
force. In contrast, polymers having a low weight-average, molecular
weight (<about 300,000) do not form sufficiently viscous
solutions inside the tablet core to allow complete delivery due to
particle settling. Settling of the particles is a problem when such
devices are prepared with no polymer added, which leads to poor
drug delivery unless the tablet is constantly agitated to keep the
particles from settling inside the core. Settling is also
problematic when the particles are large and/or of high density
such that the rate of settling increases.
[0240] In certain embodiments, the water-soluble polymers for such
osmotic devices do not interact with the drug. In certain
embodiments the water-soluble polymer is a non-ionic polymer. A
nonlimiting example of a non-ionic polymer forming solutions having
a high viscosity yet still extrudable at low pressures is
Natrosol.TM. 250H (high molecular weight hydroxyethylcellulose,
available from Hercules Incorporated, Aqualon Division, Wilmington,
Del.; MW equal to about 1 million daltons and a degree of
polymerization equal to about 3,700). Natrosol 250H.TM. provides
effective drug delivery at concentrations as low as about 3% by
weight of the core when combined with an osmagent. Natrosol
250H.TM. NF is a high-viscosity grade nonionic cellulose ether that
is soluble in hot or cold water. The viscosity of a 1% solution of
Natrosol 250H using a Brookfield LVT (30 rpm) at 25.degree. C. is
between about 1, 500 and about 2,500 cps.
[0241] In certain embodiments, hydroxyethylcellulose polymers for
use in these monolayer osmotic tablets have a weight-average,
molecular weight from about 300,000 to about 1.5 million. The
hydroxyethylcellulose polymer is typically present in the core in
an amount from about 2.0% to about 35% by weight.
[0242] Another example of an osmotic device is an osmotic capsule.
The capsule shell or portion of the capsule shell can be
semipermeable. The capsule can be filled either by a powder or
liquid consisting of an agent described herein, excipients that
imbibe water to provide osmotic potential, and/or a water-swellable
polymer, or optionally solubilizing excipients. The capsule core
can also be made such that it has a bilayer or multilayer agent
analogous to the bilayer, trilayer or concentric geometries
described above.
[0243] Another class of osmotic device useful in this disclosure
comprises coated swellable tablets, for example, as described in
EP378404. Coated swellable tablets comprise a tablet core
comprising an agent described herein and a swelling material,
preferably a hydrophilic polymer, coated with a membrane, which
contains holes, or pores through which, in the aqueous use
environment, the hydrophilic polymer can extrude and carry out the
agent. Alternatively, the membrane may contain polymeric or low
molecular weight water-soluble porosigens. Porosigens dissolve in
the aqueous use environment, providing pores through which the
hydrophilic polymer and agent may extrude. Examples of porosigens
are water-soluble polymers such as HPMC, PEG, and low molecular
weight compounds such as glycerol, sucrose, glucose, and sodium
chloride. In addition, pores may be formed in the coating by
drilling holes in the coating using a laser or other mechanical
means. In this class of osmotic devices, the membrane material may
comprise any film-forming polymer, including polymers which are
water permeable or impermeable, providing that the membrane
deposited on the tablet core is porous or contains water-soluble
porosigens or possesses a macroscopic hole for water ingress and
drug release. Embodiments of this class of sustained release
devices may also be multilayered, as described, for example, in
EP378404.
[0244] When an agent described herein is a liquid or oil, such as a
lipid vehicle formulation, for example as described in WO05/011634,
the osmotic controlled-release device may comprise a soft-gel or
gelatin capsule formed with a composite wall and comprising the
liquid formulation where the wall comprises a barrier layer formed
over the external surface of the capsule, an expandable layer
formed over the barrier layer, and a semipermeable layer formed
over the expandable layer. A delivery port connects the liquid
formulation with the aqueous use environment. Such devices are
described, for example, in U.S. Pat. No. 6,419,952, U.S. Pat. No.
6,342,249, U.S. Pat. No. 5,324,280, U.S. Pat. No. 4,672,850, U.S.
Pat. No. 4,627,850, U.S. Pat. No. 4,203,440, and U.S. Pat. No.
3,995,631.
[0245] The osmotic controlled release devices of the present
disclosure can also comprise a coating. In certain embodiments, the
osmotic controlled release device coating exhibits one or more of
the following features: is water-permeable, has at least one port
for the delivery of drug, and is non-dissolving and non-eroding
during release of the drug formulation, such that drug is
substantially entirely delivered through the delivery port(s) or
pores as opposed to delivery primarily via permeation through the
coating material itself. Delivery ports include any passageway,
opening or pore whether made mechanically, by laser drilling, by
pore formation either during the coating process or in situ during
use or by rupture during use. In certain embodiments, the coating
is present in an amount ranging from about 5 to 30 wt % (including,
for example, 10 to 20 wt %) relative to the core weight.
[0246] One form of coating is a semipermeable polymeric membrane
that has the port(s) formed therein either prior to or during use.
Thickness of such a polymeric membrane may vary between about 20
and 800 .mu.m (including, for example, between about 100 to 500
.mu.m). The diameter of the delivery port (s) may generally range
in size from 0.1 to 3000 .mu.m or greater (including, for example,
from about 50 to 3000 .mu.m in diameter). Such port(s) may be
formed post-coating by mechanical or laser drilling or may be
formed in situ by rupture of the coatings; such rupture may be
controlled by intentionally incorporating a relatively small weak
portion into the coating. Delivery ports may also be formed in situ
by erosion of a plug of water-soluble material or by rupture of a
thinner portion of the coating over an indentation in the core. In
addition, delivery ports may be formed during coating, as in the
case of asymmetric membrane coatings of the type disclosed in U.S.
Pat. No. 5,612,059 and U.S. Pat. No. 5,698,220. The delivery port
may be formed in situ by rupture of the coating, for example, when
a collection of beads that may be of essentially identical or of a
variable agent are used. Drug is primarily released from such beads
following rupture of the coating and, following rupture, such
release may be gradual or relatively sudden. When the collection of
beads has a variable agent, the agent may be chosen such that the
beads rupture at various times following administration, resulting
in the overall release of drug being sustained for a desired
duration.
[0247] Coatings may be dense, microporous or asymmetric, having a
denser region supported by a thick porous region such as those
disclosed in U.S. Pat. No. 5,612,059 and U.S. Pat. No. 5,698,220.
When the coating is dense the coating can be composed of a
water-permeable material. When the coating is porous, it may be
composed of either a water-permeable or a water-impermeable
material. When the coating is composed of a porous
water-impermeable material, water permeates through the pores of
the coating as either a liquid or a vapor. Nonlimiting examples of
osmotic devices that utilize dense coatings include U.S. Pat. No.
3,995,631 and U.S. Pat. No. 3,845,770. Such dense coatings are
permeable to the external fluid such as water and may be composed
of any of the materials mentioned in these patents as well as other
water-permeable polymers known in the art.
[0248] The membranes may also be porous as disclosed, for example,
in U.S. Pat. No. 5,654,005 and U.S. Pat. No. 5,458,887 or even be
formed from water-resistant polymers. U.S. Pat. No. 5,120,548
describes another suitable process for forming coatings from a
mixture of a water-insoluble polymer and a leachable water-soluble
additive. The porous membranes may also be formed by the addition
of pore-formers as disclosed in U.S. Pat. No. 4,612,008. In
addition, vapor-permeable coatings may even be formed from
extremely hydrophobic materials such as polyethylene or
polyvinylidene difluorid that, when dense, are essentially
water-impermeable, as long as such coatings are porous. Materials
useful in forming the coating include but are not limited to
various grades of acrylic, vinyls, ethers, polyamides, polyesters
and cellulosic derivatives that are water-permeable and
water-insoluble at physiologically relevant pHs, or are susceptible
to being rendered water-insoluble by chemical alteration such as by
crosslinking. Nonlimiting examples of suitable polymers (or
crosslinked versions) useful in forming the coating include
plasticized, unplasticized and reinforced cellulose acetate (CA),
cellulose diacetate, cellulose triacetate, CA propionate, cellulose
nitrate, cellulose acetate butyrate (CAB), CA ethyl carbamate, CAP,
CA methyl carbamate, CA succinate, cellulose acetate trimellitate
(CAT), CA dimethylaminoacetate, CA ethyl carbonate, CA
chloroacetate, CA ethyl oxalate, CA methyl sulfonate, CA butyl
sulfonate, CA p-toluene sulfonate, agar acetate, amylose
triacetate, beta glucan acetate, beta glucan triacetate,
acetaldehyde dimethyl acetate, triacetate of locust bean gum,
hydroxiated ethylene-vinylacetate, EC, PEG, PPG, PEG/PPG
copolymers, PVP, HEC, HPC, CMC, CMEC, HPMC, HPMCP, HPMCAS, HPMCAT,
poly (acrylic) acids and esters and poly-(methacrylic) acids and
esters and copolymers thereof, starch, dextran, dextrin, chitosan,
collagen, gelatin, polyalkenes, polyethers, polysulfones,
polyethersulfones, polystyrenes, polyvinyl halides, polyvinyl
esters and ethers, natural waxes and synthetic waxes. In various
embodiments, the coating agent comprises a cellulosic polymer, in
particular cellulose ethers, cellulose esters and cellulose
ester-ethers, i.e., cellulosic derivatives having a mixture of
ester and ether substituents, the coating materials are made or
derived from poly (acrylic) acids and esters, poly (methacrylic)
acids and esters, and copolymers thereof, the coating agent
comprises cellulose acetate, the coating comprises a cellulosic
polymer and PEG, the coating comprises cellulose acetate and
PEG.
[0249] Coating is conducted in conventional fashion, typically by
dissolving or suspending the coating material in a solvent and then
coating by dipping, spray coating or by pan-coating. In certain
embodiments, the coating solution contains 5 to 15 wt % polymer.
Typical solvents useful with the cellulosic polymers mentioned
above include but are not limited to acetone, methyl acetate, ethyl
acetate, isopropyl acetate, n-butyl acetate, methyl isobutyl
ketone, methyl propyl ketone, ethylene glycol monoethyl ether,
ethylene glycol monoethyl acetate, methylene dichloride, ethylene
dichloride, propylene dichloride, nitroethane, nitropropane,
tetrachloroethane, 1,4-dioxane, tetrahydrofuran, diglyme, water,
and mixtures thereof. Pore-formers and non-solvents (such as water,
glycerol and ethanol) or plasticizers (such as diethyl phthalate)
may also be added in any amount as long as the polymer remains
soluble at the spray temperature. Pore-formers and their use in
fabricating coatings are described, for example, in U.S. Pat. No.
5,612,059. Coatings may also be hydrophobic microporous layers
wherein the pores are substantially filled with a gas and are not
wetted by the aqueous medium but are permeable to water vapor, as
disclosed, for example, in U.S. Pat. No. 5,798,119. Such
hydrophobic but water-vapor permeable coatings are typically
composed of hydrophobic polymers such as polyalkenes, polyacrylic
acid derivatives, polyethers, polysulfones, polyethersulfones,
polystyrenes, polyvinyl halides, polyvinyl esters and ethers,
natural waxes and synthetic waxes. Hydrophobic microporous coating
materials include but are not limited to polystyrene, polysulfones,
polyethersulfones, polyethylene, polypropylene, polyvinyl chloride,
polyvinylidene fluoride and polytetrafluoroethylene. Such
hydrophobic coatings can be made by known phase inversion methods
using any of vapor-quench, liquid quench, thermal processes,
leaching soluble material from the coating or by sintering coating
particles. In thermal processes, a solution of polymer in a latent
solvent is brought to liquid-liquid phase separation in a cooling
step. When evaporation of the solvent is not prevented, the
resulting membrane will typically be porous. Such coating processes
may be conducted by the processes disclosed, for example, in U.S.
Pat. No. 4,247,498, U.S. Pat. No. 4,490,431 and U.S. Pat. No.
4,744,906. Osmotic controlled-release devices may be prepared using
procedures known in the pharmaceutical arts. See for example,
Remington: The Science and Practice of Pharmacy, 20th Edition,
2000.
[0250] As further noted above, the agents described herein may be
provided in the form of microparticulates, generally ranging in
size from about 10 .mu.m to about 2 mm (including, for example,
from about 100 .mu.m to 1 mm in diameter). Such multiparticulates
may be packaged, for example, in a capsule such as a gelatin
capsule or a capsule formed from an aqueous-soluble polymer such as
HPMCAS, HPMC or starch; dosed as a suspension or slurry in a
liquid; or they may be formed into a tablet, caplet, or pill by
compression or other processes known in the art. Such
multiparticulates may be made by any known process, such as wet-
and dry-granulation processes, extrusion/spheronization,
roller-compaction, melt-congealing, or by spray-coating seed cores.
For example, in wet- and dry-granulation processes, the agent
described herein and optional excipients may be granulated to form
multiparticulates of the desired size. Other excipients, such as a
binder (e.g., microcrystalline cellulose), may be blended with the
agent to aid in processing and forming the multiparticulates. In
the case of wet granulation, a binder such as microcrystalline
cellulose may be included in the granulation fluid to aid in
forming a suitable multiparticulate. See, for example, Remington:
The Science and Practice of Pharmacy, 20 Edition, 2000. In any
case, the resulting particles may themselves constitute the
therapeutic composition or they may be coated by various
film-forming materials such as enteric polymers or water-swellable
or water-soluble polymers, or they may be combined with other
excipients or vehicles to aid in dosing to patients. Suitable
pharmaceutical compositions in accordance with the disclosure 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).
Kits
[0251] The agents described herein and combination therapy agents
can be packaged as a kit that includes single or multiple doses of
two or more agents, each packaged or formulated individually, or
single or multiple doses of two or more agents packaged or
formulated in combination. Thus, one or more agents can be present
in first container, and the kit can optionally include one or more
agents in a second container. The container or containers are
placed within a package, and the package can optionally include
administration or dosage instructions. A kit can include additional
components such as syringes or other means for administering the
agents as well as diluents or other means for formulation.
[0252] Thus, the kits can comprise: a) a pharmaceutical composition
comprising a compound described herein and a pharmaceutically
acceptable carrier, vehicle or diluent; and b) a container or
packaging. The kits may optionally comprise instructions describing
a method of using the pharmaceutical compositions in one or more of
the methods described herein (e.g. disorders associated with fluid
and sodium retention (such as diseases of the
electrolyte-water/electrolyte transport system within the kidney,
gut and urogenital system, heart failure (e.g. congestive heart
failure including heart failure at any of stages I-IV according to
New York Heart Association (NYHA) Functional Classification),
hypertension, hypotension, salt dependent forms of high blood
pressure, hepatic edema, liver cirrhosis, kidney disease,
polycystic kidney disease) and gastrointestinal disorders (e.g.
gastrointestinal motility disorders, chronic intestinal
pseudo-obstruction, colonic pseudo-obstruction, Crohn's disease,
duodenogastric reflux, dyspepsia, functional dyspepsia, nonulcer
dyspepsia, a functional gastrointestinal disorder, functional
heartburn, gastroesophageal reflux disease (GERD), gastroparesis,
irritable bowel syndrome, post-operative ileus, ulcerative colitis,
chronic constipation, and disorders and conditions associated with
constipation (e.g. constipation associated with use of opiate pain
killers, post-surgical constipation, and constipation associated
with neuropathic disorders as well as other conditions and
disorders described herein)). The kit may optionally comprise a
second pharmaceutical composition comprising one or more additional
agents including but not limited to those including analgesic
peptides and compounds, an agent used to treat heart failure
(Diuretics (e.g. furesomide (Lasix), bumetanide (Bumex), ethacrynic
acid (Edecrin), torsemide (Demadex), amiloride (Midamor),
spironolactone (Aldactone), chorthiazide (Diuril), metolazone
(Zaroxylyn)), Angiotension-Converting Enzyme (ACE) inhibitors (e.g.
captopril (Capoten), enalopril (Vasotec), lisinopril (Prinivil,
Zestril), ramipril (Altace)), Beta blockers (e.g. carvedilol
(Coreg) and Inotropes (e.g. digoxin, dobutaimine, dopamine
Milrinone)), a phosphodiesterase inhibitor, an agent used to treat
gastrointestinal and other disorders (including those described
herein), an agent used to treat constipation, an antidiarrheal
agent, an insulin or related compound (including those described
herein), an anti-hypertensive agent, an agent useful in the
treatment of respiratory and other disorders, an anti-obesity
agent, an anti-diabetic agents, an agent that activates soluble
guanylate cyclase and a pharmaceutically acceptable carrier,
vehicle or diluent. The pharmaceutical composition comprising the
compound described herein and the second pharmaceutical composition
contained in the kit may be optionally combined in the same
pharmaceutical composition.
[0253] A kit includes a container or packaging for containing the
pharmaceutical compositions and may also include divided containers
such as a divided bottle or a divided foil packet. The container
can be, for example a paper or cardboard box, a glass or plastic
bottle or jar, a re-sealable bag (for example, to hold a "refill"
of tablets for placement into a different container), or a blister
pack with individual doses for pressing out of the pack according
to a therapeutic schedule. It is feasible that more than one
container can be used together in a single package to market a
single dosage form. For example, tablets may be contained in a
bottle which is in turn contained within a box.
[0254] An example of a kit is a so-called blister pack. Blister
packs are well known in the packaging industry and are being widely
used for the packaging of pharmaceutical unit dosage forms
(tablets, capsules, and the like). Blister packs generally consist
of a sheet of relatively stiff material covered with a foil of a
preferably transparent plastic material. During the packaging
process, recesses are formed in the plastic foil. The recesses have
the size and shape of individual tablets or capsules to be packed
or may have the size and shape to accommodate multiple tablets
and/or capsules to be packed. Next, the tablets or capsules are
placed in the recesses accordingly and the sheet of relatively
stiff material is sealed against the plastic foil at the face of
the foil which is opposite from the direction in which the recesses
were formed. As a result, the tablets or capsules are individually
sealed or collectively sealed, as desired, in the recesses between
the plastic foil and the sheet. Preferably the strength of the
sheet is such that the tablets or capsules can be removed from the
blister pack by manually applying pressure on the recesses whereby
an opening is formed in the sheet at the place of the recess. The
tablet or capsule can then be removed via said opening.
[0255] It maybe desirable to provide a written memory aid
containing information and/or instructions for the physician,
pharmacist or subject regarding when the medication is to be taken.
A "daily dose" can be a single tablet or capsule or several tablets
or capsules to be taken on a given day. When the kit contains
separate compositions, a daily dose of one or more compositions of
the kit can consist of one tablet or capsule while a daily dose of
another one or more compositions of the kit can consist of several
tablets or capsules. A kit can take the form of a dispenser
designed to dispense the daily doses one at a time in the order of
their intended use. The dispenser can be equipped with a
memory-aid, so as to further facilitate compliance with the
regimen. An example of such a memory-aid is a mechanical counter
which indicates the number of daily doses that have been dispensed.
Another example of such a memory-aid is a battery-powered
micro-chip memory coupled with a liquid crystal readout, or audible
reminder signal which, for example, reads out the date that the
last daily dose has been taken and/or reminds one when the next
dose is to be taken.
[0256] Methods to increase chemical and/or physical stability of
the agents the described herein are found in U.S. Pat. No.
6,541,606, U.S. Pat. No. 6,068,850, U.S. Pat. No. 6,124,261, U.S.
Pat. No. 5,904,935, and WO 00/15224, U.S. 20030069182 (via the
addition of nicotinamide), U.S. 20030175230A1, U.S. 20030175230A1,
U.S. 20030175239A1, U.S. 20020045582, U.S. 20010031726, WO
02/26248, WO 03/014304, WO 98/00152A1, WO 98100157A1, WO 90/12029,
WO 00/04880, and WO 91/04743, WO 97/04796 and the references cited
therein.
[0257] Methods to increase bioavailability of the agents described
herein are found in U.S. Pat. No. 6,008,187, U.S. Pat. No.
5,424,289, U.S. 20030198619, WO 90/01329, WO 01/49268, WO 00/32172,
and WO 02/064166. Glycyrrhizinate can also be used as an absorption
enhancer (see, e.g., EP397447). WO 03/004062 discusses Ulex
europaeus I (UEA1) and UEAI mimetics which may be used to target
the agents described herein to the GI tract. The bioavailability of
the agents described herein can also be incrased by addition of
oral bioavailability-enhancing agents such as those described in
U.S. Pat. No. 6,818,615 including but not limited to: cyclosporins
(including cyclosporins A through Z as defined in Table 1 of U.S.
Pat. No. 6,818,615), for example, cyclosporin A (cyclosporin),
cyclosporin F, cyclosporin D, dihydro cyclosporin A, dihydro
cyclosporin C, acetyl cyclosporin A, PSC-833,
(Me-Ile-4)-cyclosporin (SDZ-NIM 811) (both from Sandoz
Pharmaceutical Corp.), and related oligopeptides produced by
species in the genus Topycladium); antifungals including but not
limited to ketoconazole; cardiovascular drug including but not
limited to MS-209 (BASF), amiodarone, nifedipine, reserpine,
quinidine, nicardipine, ethacrynic acid, propafenone, reserpine,
amiloride; anti-migraine natural products including but not limited
to ergot alkaloids; antibiotics including but not limited to
cefoperazone, tetracycline, chloroquine, fosfomycin; antiparasitics
including but not limited to ivermectin; multi-drug resistance
reversers including but not limited to VX-710 and VX-853 (Vertex
Pharmaceutical Incorporated); tyrosine kinase inhibitors including
but not limited to genistein and related isoflavonoids, quercetin;
protein kinase C inhibitors including but not limited to
calphostin; apoptosis inducers including but not limited to
ceramides; and agents active against endorphin receptors including
but not limited to morphine, morphine congeners, other opioids and
opioid antagonists including (but not limited to) naloxone,
naltrexone and nalmefene).
[0258] The agents described herein can be fused to a modified
version of the blood serum protein transferrin. U.S. 20030221201,
U.S. 20040023334, U.S. 20030226155, WO 04/020454, and WO 04/019872
discuss the manufacture and use of transferrin fusion proteins.
Transferrin fusion proteins may improve circulatory half life and
efficacy, decrease undesirable side effects and allow reduced
dosage.
[0259] The peptides and agonists described herein can be
recombinantly expressed in bacteria. Bacteria expressing the
peptide or agonists can be administered orally, rectally, mucosally
or in via some other mode of administration including but not
limited to those described herein. Bacterial hosts suitable for
such administration include but are not limited to certain
Lactobacteria (e.g. Lactococcus lactis, Lactobacillus plantarum,
Lact. rhamnosus and Lact. paracasei ssp. Paracasie and other
species found in normal human flora (Ahrne et al. Journal of
Applied Microbiology 1998 85:88)), certain Streptococcus sp. (e.g.
S. gordonii), and certain B. subtilis strains (including pSM539
described in Porzio et al. BMC Biotechnology 2004 4:27). The
peptides and agonists described herein can be administered using
the Heliobacter based preparation methods described in
WO06/015445.
Dosage
[0260] The dose range for adult humans is generally from 0.005 mg
to 10 g/day orally. Tablets 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 5 mg to 500 mg, usually
around 10 mg to 200 mg. The precise amount of compound administered
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.
[0261] A dosage unit (e.g. an oral dosage unit) can include from,
for example, 1 to 30 .mu.g, 1 to 40 .mu.g, 1 to 50 .mu.g, 1 to 100
.mu.g, 1 to 200 .mu.g, 1 to 300 .mu.g, 1 to 400 .mu.g, 1 to 500
.mu.g, 1 to 600 .mu.g, 1 to 700 .mu.g, 1 to 800 .mu.g, 1 to 900
.mu.g, 1 to 1000 .mu.g, 10 to 30 .mu.g, 10 to 40 .mu.g, 10 to 50
.mu.g, 10 to 100 .mu.g, 10 to 200 .mu.g, 10 to 300 .mu.g, 10 to 400
.mu.g, 10 to 500 .mu.g, 10 to 600 .mu.g, 10 to 700 .mu.g, 10 to 800
.mu.g, 10 to 900 .mu.g, 10 to 1000 .mu.g, 100 to 200 .mu.g, 100 to
300 .mu.g, 100 to 400 .mu.g, 100 to 500 .mu.g, 100 to 600 .mu.g,
100 to 700 .mu.g, 100 to 800 .mu.g, 100 to 900 .mu.g, 100 to 1000
.mu.g, 100 to 1250 .mu.g, 100 to 1500 .mu.g, 100 to 1750 .mu.g, 100
to 2000 .mu.g, 100 to 2250 .mu.g, 100 to 2500 .mu.g, 100 to 2750
.mu.g, 100 to 3000 .mu.g, 200 to 300 .mu.g, 200 to 400 .mu.g, 200
to 500 .mu.g, 200 to 600 .mu.g, 200 to 700 .mu.g, 200 to 800 .mu.g,
200 to 900 .mu.g, 200 to 1000 .mu.g, 200 to 1250 .mu.g, 200 to 1500
.mu.g, 200 to 1750 .mu.g, 200 to 2000 .mu.g, 200 to 2250 .mu.g, 200
to 2500 .mu.g, 200 to 2750 .mu.g, 200 to 3000 .mu.g, 300 to 400
.mu.g, 300 to 500 .mu.g, 300 to 600 .mu.g, 300 to 700 .mu.g, 300 to
800 .mu.g, 300 to 900 .mu.g, 300 to 1000 .mu.g, 300 to 1250 .mu.g,
300 to 1500 .mu.g, 300 to 1750 .mu.g, 300 to 2000 .mu.g, 300 to
2250 .mu.g, 300 to 2500 .mu.g, 300 to 2750 .mu.g, 300 to 3000
.mu.g, 400 to 500 .mu.g, 400 to 600 .mu.g, 400 to 700 .mu.g, 400 to
800 .mu.g, 400 to 900 .mu.g, 400 to 1000 .mu.g, 400 to 1250 .mu.g,
400 to 1500 .mu.g, 400 to 1750 .mu.g, 400 to 2000 .mu.g, 400 to
2250 .mu.g, 400 to 2500 .mu.g, 400 to 2750 .mu.g, 400 to 3000
.mu.g, 500 to 600 .mu.g, 500 to 700 .mu.g, 500 to 800 .mu.g, 500 to
900 .mu.g, 500 to 1000 .mu.g, 500 to 1250 .mu.g, 500 to 1500 .mu.g,
500 to 1750 .mu.g, 500 to 2000 .mu.g, 500 to 2250 .mu.g, 500 to
2500 .mu.g, 500 to 2750 .mu.g, 500 to 3000 .mu.g, 600 to 700 .mu.g,
600 to 800 .mu.g, 600 to 900 .mu.g, 600 to 1000 .mu.g, 600 to 1250
.mu.g, 600 to 1500 .mu.g, 600 to 1750 .mu.g, 600 to 2000 .mu.g, 600
to 2250 .mu.g, 600 to 2500 .mu.g, 600 to 2750 .mu.g, 600 to 3000
.mu.g, 700 to 800 .mu.g, 700 to 900 .mu.g, 700 to 1000 .mu.g, 700
to 1250 .mu.g, 700 to 1500 .mu.g, 700 to 1750 .mu.g, 700 to 2000
.mu.g, 700 to 2250 .mu.g, 700 to 2500 .mu.g, 700 to 2750 .mu.g, 700
to 3000 .mu.g, 800 to 900 .mu.g, 800 to 1000 .mu.g, 800 to 1250
.mu.g, 800 to 1500 .mu.g, 800 to 1750 .mu.g, 800 to 2000 .mu.g, 800
to 2250 .mu.g, 800 to 2500 .mu.g, 800 to 2750 .mu.g, 800 to 3000
.mu.g, 900 to 1000 .mu.g, 900 to 1250 .mu.g, 900 to 1500 .mu.g, 900
to 1750 .mu.g, 900 to 2000 .mu.g, 900 to 2250 .mu.g, 900 to 2500
.mu.g, 900 to 2750 .mu.g, 900 to 3000 .mu.g, 1000 to 1250 .mu.g,
1000 to 1500 .mu.g, 1000 to 1750 .mu.g, 1000 to 2000 .mu.g, 1000 to
2250 .mu.g, 1000 to 2500 .mu.g, 1000 to 2750 .mu.g, 1000 to 3000
.mu.g, 2 to 500 .mu.g, 50 to 500 .mu.g, 3 to 100 .mu.g, 5 to 20
.mu.g, 5 to 100 .mu.g, 10 .mu.g, 20 .mu.g, 30 .mu.g, 40 .mu.g, 50
.mu.g, 60 .mu.g, 70 .mu.g, 75 .mu.g, 80 .mu.g, 90 .mu.g, 100 .mu.g,
150 .mu.g, 200 .mu.g, 250 .mu.g, 300 .mu.g, 350 .mu.g, 400 .mu.g,
450 .mu.g, 500 .mu.g, 550 .mu.g, 600 .mu.g, 650 .mu.g, 700 .mu.g,
750 .mu.g, 800 .mu.g, 850 .mu.g, 900 .mu.g, 950 .mu.g, 1000 .mu.g,
1050 .mu.g, 1100 .mu.g, 1150 .mu.g, 1200 .mu.g, 1250 .mu.g, 1300
.mu.g, 1350 .mu.g, 1400 .mu.g, 1450 .mu.g, 1500 .mu.g, 1550 .mu.g,
1600 .mu.g, 1650 .mu.g, 1700 .mu.g, 1750 .mu.g, 1800 .mu.g, 1850
.mu.g, 1900 .mu.g, 1950 .mu.g, 2000 .mu.g, 2050 .mu.g, 2100 .mu.g,
2150 .mu.g, 2200 .mu.g, 2250 .mu.g, 2300 .mu.g, 2350 .mu.g, 2400
.mu.g, 2450 .mu.g, 2500 .mu.g, 2550 .mu.g, 2600 .mu.g, 2650 .mu.g,
2700 .mu.g, 2750 .mu.g, 2800 .mu.g, 2850 .mu.g, 2900 .mu.g, 2950
.mu.g, 3000 .mu.g, 3250 .mu.g, 3500 .mu.g, 3750 .mu.g, 4000 .mu.g,
4250 .mu.g, 4500 .mu.g, 4750 .mu.g, 5000 .mu.g of a peptide or
agonist described herein. 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 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, six
times a day. The dosage unit can optionally comprise other
agents.
[0262] A dosage unit (e.g. an oral dosage unit) can include, for
example, from 1 to 30 .mu.g, 1 to 40 .mu.g, 1 to 50 .mu.g, 1 to 100
.mu.g, 1 to 200 .mu.g, 1 to 300 .mu.g, 1 to 400 .mu.g, 1 to 500
.mu.g, 1 to 600 .mu.g, 1 to 700 .mu.g, 1 to 800 .mu.g, 1 to 900
.mu.g, 1 to 1000 .mu.g, 10 to 30 .mu.g, 10 to 40 .mu.g, 10 to 50
.mu.g, 10 to 100 .mu.g, 10 to 200 .mu.g, 10 to 300 .mu.g, 10 to 400
.mu.g, 10 to 500 .mu.g, 10 to 600 .mu.g, 10 to 700 .mu.g, 10 to 800
.mu.g, 10 to 900 .mu.g, 10 to 1000 .mu.g, 100 to 200 .mu.g, 100 to
300 .mu.g, 100 to 400 .mu.g, 100 to 500 .mu.g, 100 to 600 .mu.g,
100 to 700 .mu.g, 100 to 800 .mu.g, 100 to 900 .mu.g, 100 to 1000
.mu.g, 100 to 1250 .mu.g, 100 to 1500 .mu.g, 100 to 1750 .mu.g, 100
to 2000 .mu.g, 100 to 2250 .mu.g, 100 to 2500 .mu.g, 100 to 2750
.mu.g, 100 to 3000 .mu.g, 200 to 300 .mu.g, 200 to 400 .mu.g, 200
to 500 .mu.g, 200 to 600 .mu.g, 200 to 700 .mu.g, 200 to 800 .mu.g,
200 to 900 .mu.g, 200 to 1000 .mu.g, 200 to 1250 .mu.g, 200 to 1500
.mu.g, 200 to 1750 .mu.g, 200 to 2000 .mu.g, 200 to 2250 .mu.g, 200
to 2500 .mu.g, 200 to 2750 .mu.g, 200 to 3000 .mu.g, 300 to 400
.mu.g, 300 to 500 .mu.g, 300 to 600 .mu.g, 300 to 700 .mu.g, 300 to
800 .mu.g, 300 to 900 .mu.g, 300 to 1000 .mu.g, 300 to 1250 .mu.g,
300 to 1500 .mu.g, 300 to 1750 .mu.g, 300 to 2000 .mu.g, 300 to
2250 .mu.g, 300 to 2500 .mu.g, 300 to 2750 .mu.g, 300 to 3000
.mu.g, 400 to 500 .mu.g, 400 to 600 .mu.g, 400 to 700 .mu.g, 400 to
800 .mu.g, 400 to 900 .mu.g, 400 to 1000 .mu.g, 400 to 1250 .mu.g,
400 to 1500 .mu.g, 400 to 1750 .mu.g, 400 to 2000 .mu.g, 400 to
2250 .mu.g, 400 to 2500 .mu.g, 400 to 2750 .mu.g, 400 to 3000
.mu.g, 500 to 600 .mu.g, 500 to 700 .mu.g, 500 to 800 .mu.g, 500 to
900 .mu.g, 500 to 1000 .mu.g, 500 to 1250 .mu.g, 500 to 1500 .mu.g,
500 to 1750 .mu.g, 500 to 2000 .mu.g, 500 to 2250 .mu.g, 500 to
2500 .mu.g, 500 to 2750 .mu.g, 500 to 3000 .mu.g, 600 to 700 .mu.g,
600 to 800 .mu.g, 600 to 900 .mu.g, 600 to 1000 .mu.g, 600 to 1250
.mu.g, 600 to 1500 .mu.g, 600 to 1750 .mu.g, 600 to 2000 .mu.g, 600
to 2250 .mu.g, 600 to 2500 .mu.g, 600 to 2750 .mu.g, 600 to 3000
.mu.g, 700 to 800 .mu.g, 700 to 900 .mu.g, 700 to 1000 .mu.g, 700
to 1250 .mu.g, 700 to 1500 .mu.g, 700 to 1750 .mu.g, 700 to 2000
.mu.g, 700 to 2250 .mu.g, 700 to 2500 .mu.g, 700 to 2750 .mu.g, 700
to 3000 .mu.g, 800 to 900 .mu.g, 800 to 1000 .mu.g, 800 to 1250
.mu.g, 800 to 1500 .mu.g, 800 to 1750 .mu.g, 800 to 2000 .mu.g, 800
to 2250 .mu.g, 800 to 2500 .mu.g, 800 to 2750 .mu.g, 800 to 3000
.mu.g, 900 to 1000 .mu.g, 900 to 1250 .mu.g, 900 to 1500 .mu.g, 900
to 1750 .mu.g, 900 to 2000 .mu.g, 900 to 2250 .mu.g, 900 to 2500
.mu.g, 900 to 2750 .mu.g, 900 to 3000 .mu.g, 1000 to 1250 .mu.g,
1000 to 1500 .mu.g, 1000 to 1750 .mu.g, 1000 to 2000 .mu.g, 1000 to
2250 .mu.g, 1000 to 2500 .mu.g, 1000 to 2750 .mu.g, 1000 to 3000
.mu.g, 2 to 500 .mu.g, 50 to 500 .mu.g, 3 to 100 .mu.g, 5 to 20
.mu.g, 5 to 100 .mu.g, 10 .mu.g, 20 .mu.g, 30 .mu.g, 40 .mu.g, 50
.mu.g, 60 .mu.g, 70 .mu.g, 75 .mu.g, 80 .mu.g, 90 .mu.g, 100 .mu.g,
150 .mu.g, 200 .mu.g, 250 .mu.g, 300 .mu.g, 350 .mu.g, 400 .mu.g,
450 .mu.g, 500 .mu.g, 550 .mu.g, 600 .mu.g, 650 .mu.g, 700 .mu.g,
750 .mu.g, 800 .mu.g, 850 .mu.g, 900 .mu.g, 950 .mu.g, 1000 .mu.g,
1050 .mu.g, 1100 .mu.g, 1150 .mu.g, 1200 .mu.g, 1250 .mu.g, 1300
.mu.g, 1350 .mu.g, 1400 .mu.g, 1450 .mu.g, 1500 .mu.g, 1550 .mu.g,
1600 .mu.g, 1650 .mu.g, 1700 .mu.g, 1750 .mu.g, 1800 .mu.g, 1850
.mu.g, 1900 .mu.g, 1950 .mu.g, 2000 .mu.g, 2050 .mu.g, 2100 .mu.g,
2150 .mu.g, 2200 .mu.g, 2250 .mu.g, 2300 .mu.g, 2350 .mu.g, 2400
.mu.g, 2450 .mu.g, 2500 .mu.g, 2550 .mu.g, 2600 .mu.g, 2650 .mu.g,
2700 .mu.g, 2750 .mu.g, 2800 .mu.g, 2850 .mu.g, 2900 .mu.g, 2950
.mu.g, 3000 .mu.g, 3250 .mu.g, 3500 .mu.g, 3750 .mu.g, 4000 .mu.g,
4250 .mu.g, 4500 .mu.g, 4750 .mu.g, 5000 .mu.g of a peptide or
agonist described herein and from 10 mg to 600 mg (e.g. 10 mg, 20
mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 120
mg, 140 mg, 160 mg, 180 mg, 200 mg, 220 mg, 240 mg, 260 mg, 280 mg,
300 mg, 320 mg, 340 mg, 360 mg, 380 mg, 400 mg, 420 mg, 440 mg, 460
mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg) of
furosemide (Lasix).
[0263] A dosage unit (e.g. an oral, intravenous or intramuscular
dosage unit) can include, for example, from 1 to 30 .mu.g, 1 to 40
.mu.g, 1 to 50 .mu.g, 1 to 100 .mu.g, 1 to 200 .mu.g, 1 to 300
.mu.g, 1 to 400 .mu.g, 1 to 500 .mu.g, 1 to 600 .mu.g, 1 to 700
.mu.g, 1 to 800 .mu.g, 1 to 900 .mu.g, 1 to 1000 .mu.g, 10 to 30
.mu.g, 10 to 40 .mu.g, 10 to 50 .mu.g, 10 to 100 .mu.g, 10 to 200
.mu.g, 10 to 300 .mu.g, 10 to 400 .mu.g, 10 to 500 .mu.g, 10 to 600
.mu.g, 10 to 700 .mu.g, 10 to 800 .mu.g, 10 to 900 .mu.g, 10 to
1000 .mu.g, 100 to 200 .mu.g, 100 to 300 .mu.g, 100 to 400 .mu.g,
100 to 500 .mu.g, 100 to 600 .mu.g, 100 to 700 .mu.g, 100 to 800
.mu.g, 100 to 900 .mu.g, 100 to 1000 .mu.g, 100 to 1250 .mu.g, 100
to 1500 .mu.g, 100 to 1750 .mu.g, 100 to 2000 .mu.g, 100 to 2250
.mu.g, 100 to 2500 .mu.g, 100 to 2750 .mu.g, 100 to 3000 .mu.g, 200
to 300 .mu.g, 200 to 400 .mu.g, 200 to 500 .mu.g, 200 to 600 .mu.g,
200 to 700 .mu.g, 200 to 800 .mu.g, 200 to 900 .mu.g, 200 to 1000
.mu.g, 200 to 1250 .mu.g, 200 to 1500 .mu.g, 200 to 1750 .mu.g, 200
to 2000 .mu.g, 200 to 2250 .mu.g, 200 to 2500 .mu.g, 200 to 2750
.mu.g, 200 to 3000 .mu.g, 300 to 400 .mu.g, 300 to 500 .mu.g, 300
to 600 .mu.g, 300 to 700 .mu.g, 300 to 800 .mu.g, 300 to 900 .mu.g,
300 to 1000 .mu.g, 300 to 1250 .mu.g, 300 to 1500 .mu.g, 300 to
1750 .mu.g, 300 to 2000 .mu.g, 300 to 2250 .mu.g, 300 to 2500
.mu.g, 300 to 2750 .mu.g, 300 to 3000 .mu.g, 400 to 500 .mu.g, 400
to 600 .mu.g, 400 to 700 .mu.g, 400 to 800 .mu.g, 400 to 900 .mu.g,
400 to 1000 .mu.g, 400 to 1250 .mu.g, 400 to 1500 .mu.g, 400 to
1750 .mu.g, 400 to 2000 .mu.g, 400 to 2250 .mu.g, 400 to 2500
.mu.g, 400 to 2750 .mu.g, 400 to 3000 .mu.g, 500 to 600 .mu.g, 500
to 700 .mu.g, 500 to 800 .mu.g, 500 to 900 .mu.g, 500 to 1000
.mu.g, 500 to 1250 .mu.g, 500 to 1500 .mu.g, 500 to 1750 .mu.g, 500
to 2000 .mu.g, 500 to 2250 .mu.g, 500 to 2500 .mu.g, 500 to 2750
.mu.g, 500 to 3000 .mu.g, 600 to 700 .mu.g, 600 to 800 .mu.g, 600
to 900 .mu.g, 600 to 1000 .mu.g, 600 to 1250 .mu.g, 600 to 1500
.mu.g, 600 to 1750 .mu.g, 600 to 2000 .mu.g, 600 to 2250 .mu.g, 600
to 2500 .mu.g, 600 to 2750 .mu.g, 600 to 3000 .mu.g, 700 to 800
.mu.g, 700 to 900 .mu.g, 700 to 1000 .mu.g, 700 to 1250 .mu.g, 700
to 1500 .mu.g, 700 to 1750 .mu.g, 700 to 2000 .mu.g, 700 to 2250
.mu.g, 700 to 2500 .mu.g, 700 to 2750 .mu.g, 700 to 3000 .mu.g, 800
to 900 .mu.g, 800 to 1000 .mu.g, 800 to 1250 .mu.g, 800 to 1500
.mu.g, 800 to 1750 .mu.g, 800 to 2000 .mu.g, 800 to 2250 .mu.g, 800
to 2500 .mu.g, 800 to 2750 .mu.g, 800 to 3000 .mu.g, 900 to 1000
.mu.g, 900 to 1250 .mu.g, 900 to 1500 .mu.g, 900 to 1750 .mu.g, 900
to 2000 .mu.g, 900 to 2250 .mu.g, 900 to 2500 .mu.g, 900 to 2750
.mu.g, 900 to 3000 .mu.g, 1000 to 1250 .mu.g, 1000 to 1500 .mu.g,
1000 to 1750 .mu.g, 1000 to 2000 .mu.g, 1000 to 2250 .mu.g, 1000 to
2500 .mu.g, 1000 to 2750 .mu.g, 1000 to 3000 .mu.g, 2 to 500 .mu.g,
50 to 500 .mu.g, 3 to 100 .mu.g, 5 to 20 .mu.g, 5 to 100 .mu.g, 10
.mu.g, 20 .mu.g, 30 .mu.g, 40 .mu.g, 50 .mu.g, 60 .mu.g, 70 .mu.g,
75 .mu.g, 80 .mu.g, 90 .mu.g, 100 .mu.g, 150 .mu.g, 200 .mu.g, 250
.mu.g, 300 .mu.g, 350 .mu.g, 400 .mu.g, 450 .mu.g, 500 .mu.g, 550
.mu.g, 600 .mu.g, 650 .mu.g, 700 .mu.g, 750 .mu.g, 800 .mu.g, 850
.mu.g, 900 .mu.g, 950 .mu.g, 1000 .mu.g, 1050 .mu.g, 1100 .mu.g,
1150 .mu.g, 1200 .mu.g, 1250 .mu.g, 1300 .mu.g, 1350 .mu.g, 1400
.mu.g, 1450 .mu.g, 1500 .mu.g, 1550 .mu.g, 1600 .mu.g, 1650 .mu.g,
1700 .mu.g, 1750 .mu.g, 1800 .mu.g, 1850 .mu.g, 1900 .mu.g, 1950
.mu.g, 2000 .mu.g, 2050 .mu.g, 2100 .mu.g, 2150 .mu.g, 2200 .mu.g,
2250 .mu.g, 2300 .mu.g, 2350 .mu.g, 2400 .mu.g, 2450 .mu.g, 2500
.mu.g, 2550 .mu.g, 2600 .mu.g, 2650 .mu.g, 2700 .mu.g, 2750 .mu.g,
2800 .mu.g, 2850 .mu.g, 2900 .mu.g, 2950 .mu.g, 3000 .mu.g, 3250
.mu.g, 3500 .mu.g, 3750 .mu.g, 4000 .mu.g, 4250 .mu.g, 4500 .mu.g,
4750 .mu.g, 5000 .mu.g of a peptide or agonist described herein and
from 0.2 mg to 10 mg (e.g. 0.2 mg, 0.4 mg, 0.5 mg, 0.75 mg, 1 mg,
1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6
mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg) of
bumetanide (Bumex.RTM.).
[0264] 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. 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.
[0265] In certain embodiments the dosage unit and daily dose are
equivalent. In certain embodiments the dosage unit and the daily
dose are not equivalent. In various embodiments, the dosage unit is
administered twenty minutes prior to food consumption, twenty
minutes after food consumption, 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
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,
six times a day.
[0266] When two or more active ingredients are combined in single
dosage form, chemical interactions between the active ingredients
may occur. For example, acidic and basic active ingredients can
react with each other and acidic active ingredients can facilitate
the degradation of acid labile substances. Thus, in certain dosage
forms, acidic and basic substances can be physically separated as
two distinct or isolated layers in a compressed tablet, or in the
core and shell of a press-coated tablet. Additional agents that are
compatible with acidic as well as basic substances, have the
flexibility of being placed in either layer. In certain multiple
layer compositions at least one active ingredient can be
enteric-coated. In certain embodiments thereof at least one active
ingredient can be presented in a controlled release form. In
certain embodiments where a combination of three or more active
substances are used, they can be presented as physically isolated
segments of a compressed multilayer tablet, which can be optionally
film coated.
[0267] The therapeutic combinations described herein can be
formulated as a tablet or capsule comprising a plurality of beads,
granules, or pellets. All active ingredients including the vitamins
of the combination are formulated into granules or beads or pellets
that are further coated with a protective coat, an enteric coat, or
a film coat to avoid the possible chemical interactions.
Granulation and coating of granules or beads is done using
techniques well known to a person skilled in the art. At least one
active ingredient can present in a controlled release form. Finally
these coated granules or beads are filled into hard gelatin
capsules or compressed to form tablets.
[0268] The therapeutic combinations described herein can be
formulated as a capsule comprising microtablets or minitablets of
all active ingredients. Microtablets of the individual agents can
be prepared using well known pharmaceutical procedures of tablet
making like direct compression, dry granulation or wet granulation.
Individual microtablets can be filled into hard gelatin capsules. A
final dosage form may comprise one or more microtablets of each
individual component. The microtablets may be film coated or
enteric coated.
[0269] The therapeutic combinations described herein can be
formulated as a capsule comprising one or more microtablets and
powder, or one or more microtablets and granules or beads. In order
to avoid interactions between drugs, some active ingredients of a
said combination can be formulated as microtablets and the others
filled into capsules as a powder, granules, or beads. The
microtablets may be film coated or enteric coated. At least one
active ingredient can be presented in controlled release form.
[0270] The therapeutic combinations described herein can be
formulated wherein the active ingredients are distributed in the
inner and outer phase of tablets. In an attempt to divide
chemically incompatible components of proposed combination, few
interacting components are converted in granules or beads using
well known pharmaceutical procedures in prior art. The prepared
granules or beads (inner phase) are then mixed with outer phase
comprising the remaining active ingredients and at least one
pharmaceutically acceptable excipient. The mixture thus comprising
inner and outer phase is compressed into tablets or molded into
tablets. The granules or beads can be controlled release or
immediate release beads or granules, and can further be coated
using an enteric polymer in an aqueous or non-aqueous system, using
methods and materials that are known in the art.
[0271] The therapeutic combinations described herein can be
formulated as single dosage unit comprising suitable buffering
agent. All powdered ingredients of said combination are mixed and a
suitable quantity of one or more buffering agents is added to the
blend to minimize possible interactions.
[0272] The agents described herein, alone or in combination, can be
combined with any pharmaceutically acceptable carrier or medium.
Thus, they can be combined with materials that do not produce an
adverse, allergic or otherwise unwanted reaction when administered
to a patient. The carriers or mediums used can include solvents,
dispersants, coatings, absorption promoting agents, controlled
release agents, and one or more inert excipients (which include
starches, polyols, granulating agents, microcrystalline cellulose,
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.
Treatment of the Side-Effects of Opioid Administration
[0273] GGC receptor agonists, e.g., GCC receptor agonist peptides
described herein, may useful in the treatment of one or more side
effects of opioid administration, e.g., opioid induced
constipation, nausea and/or vomiting. In the case of constipation,
the GCC receptor agonist peptide can be administered at a dosage to
induce laxation within a desired time (e.g., within 15 minutes, 30
minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours or 24
hours).
[0274] The GCC receptor agonist peptide can be administered to
maintain regular bowel movements in a patient who is a chronic
opioid user (e.g., a terminally-ill patient). The administration
can be via any convenient route (e.g., sublingual, parenteral,
intravenous, subcutaneous).
[0275] Thus, the peptides described herein can be administered to a
patient that is taking one or more of the following opioids:
Acetorphine, Acetyldihydrocodeine, Acetylmorphone, Alfentanil,
Allylprodine, Anileridine, Bemidone, Benzylmorphine, Bezitramide,
Buprenorphine, Butorphanol, Carfentanil/Carfentanyl, Clonitazene,
Codeine, Codeine-N-Oxide, Codeinone, Cyclazocine, Cyclorphan,
Desomorphine, Dextromoramide, Dextropropoxyphene, Dezocine,
Diacetyldihydromorphine, Diamorphine/Diacetylmorphine (Heroin),
Diethylthiambutene, Difenoxin, Dihydrocodeine, Dihydrocodeinone
Enol Acetate, Dihydroetorphine, Dihydroisocodeine, Dihydromorphine,
Dimethylthiambutene, Diphenoxylate, Dipropanoylmorphine,
Drobetabol, Ethylketocyclazocine, Ethylmorphine, Etonitazene,
Etorphine, Fentanyl, Hydrocodone, Hydromorphone, Isomethadone,
Ketobemidone, Laudanum, Lefetamine, Levallorphan,
Levo-Alphacetylmethadol (LAAM), Levomethorphan, Levorphanol,
Loperamide, Meptazinol, Metazocine, Methadone, Monoacetylmorphine,
Morphine, Morphine-6-Glucuronide, Morphine-N-Oxide, Morphinone,
MPPP (1-Methyl 4-Phenyl 4-Propionoxypiperidine), Myorphine,
Nalbuphine/Nalbufine, Nicocodeine, Nicodicodeine, Nicomorphine,
Norcodeine, Ohmefentanyl, Oxycodone, Oxymorphone, Pentazocine,
PEPAP (1-Phenethyl-4-Phenyl-4-Piperidinol Acetate (Ester)),
Pethidine (Meperidine), Phenadoxone, Phenazocine, Phenoperidine,
Pholcodeine, Piminodine, Piritramide, Prodine, Propiram,
Propoxyphene, Racemethorphan, Remifentanil, Sufentanil, Thebaine,
Thiofentanil/Thiofentanyl, Tilidine, and Tramadol. The peptide can
be co-administered with or co-formulated with any of the preceeding
peptides.
[0276] Where the GCC receptor agonist is co-formulated with an
opioid the composition may further include one or more other active
ingredients that may be conventionally employed in analgesic and/or
cough-cold-antitussive combination products. Such conventional
ingredients include, for example, aspirin, acetaminophen,
phenylpropanolamine, phenylephrine, chlorpheniramine, caffeine,
and/or guaifenesin. Typical or conventional ingredients that may be
included in the opioid component are described, for example, in the
Physicians' Desk Reference, 1999, the disclosures of which are
hereby incorporated herein by reference, in their entirety.
[0277] In addition, the composition may further include one or more
compounds that may be designed to enhance the analgesic potency of
the opioid and/or to reduce analgesic tolerance development. Such
compounds include, for example, dextromethorphan or other NMDA
antagonists (Mao, M. J. et al., Pain 1996, 67, 361), L-364,718 and
other CCK antagonists (Dourish, C. T. et al., Eur J Pharmacol 1988,
147, 469), NOS inhibitors (Bhargava, H. N. et al., Neuropeptides
1996, 30, 219), PKC inhibitors (Bilsky, E. J. et al., J Pharmacol
Exp Ther 1996, 277, 484), and dynorphin antagonists or antisera
(Nichols, M. L. et al., Pain 1997, 69, 317). The disclosures of
each of the foregoing documents are hereby incorporated herein by
reference, in their entireties.
[0278] The combination products, such as pharmaceutical
compositions comprising opioids in combination with a GCC agonist
may be in any dosage form, such as those described herein, and can
also be administered in various ways, as described herein. In a
preferred embodiment, the combination products of the disclosure
are formulated together, in a single dosage form (that is, combined
together in one capsule, tablet, powder, or liquid, etc.). When the
combination products are not formulated together in a single dosage
form, the opioid compounds and the GCC agonists may be administered
at the same time (that is, together), or in any order. When not
administered at the same time, preferably the administration of an
opioid and a GCC agonist occurs less than about one hour apart,
less than about 30 minutes apart, less than about 15 minutes apart,
and less than about 5 minutes apart. Administration of the
combination of an opioid and a GCC agonist can be, for example,
oral, although other routes of administration, as described above,
are contemplated to be within the scope of the present disclosure.
Although it is the opioids and GCC agonists may both be
administered in the same fashion (that is, for example, both
orally), if desired, they may each be administered in different
fashions (that is, for example, one component of the combination
product may be administered orally, and another component may be
administered intravenously). The dosage of the combination products
of the disclosure may vary depending upon various factors such as
the pharmacodynamic characteristics of the particular agent and its
mode and route of administration, the age, health and weight of the
recipient, the nature and extent of the symptoms, the kind of
concurrent treatment, the frequency of treatment, and the effect
desired.
[0279] Although the proper dosage of the combination products of
this disclosure will be readily ascertainable by one skilled in the
art, by way of general guidance, where an opioid compounds is
combined with a GCC agonist, for example, typically a daily dosage
may range from about 0.01 to about 100 milligrams, 0.1 to about 10
milligrams of the opioid, 15 to about 200 milligrams, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 milligrams of opioid per kilogram of patient body
weight. The opioid-GCC agonist combination product can include, for
example, from 1 to 30 .mu.g, 1 to 40 .mu.g, 1 to 50 .mu.g, 1 to 100
.mu.g, 1 to 200 .mu.g, 1 to 300 .mu.g, 1 to 400 .mu.g, 1 to 500
.mu.g, 1 to 600 .mu.g, 1 to 700 .mu.g, 1 to 800 .mu.g, 1 to 900
.mu.g, 1 to 1000 .mu.g, 10 to 30 .mu.g, 10 to 40 .mu.g, 10 to 50
.mu.g, 10 to 100 .mu.g, 10 to 200 .mu.g, 10 to 300 .mu.g, 10 to 400
.mu.g, 10 to 500 .mu.g, 10 to 600 .mu.g, 10 to 700 .mu.g, 10 to 800
.mu.g, 10 to 900 .mu.g, 10 to 1000 .mu.g, 100 to 200 .mu.g, 100 to
300 .mu.g, 100 to 400 .mu.g, 100 to 500 .mu.g, 100 to 600 .mu.g,
100 to 700 .mu.g, 100 to 800 .mu.g, 100 to 900 .mu.g, 100 to 1000
.mu.g, 100 to 1250 .mu.g, 100 to 1500 .mu.g, 100 to 1750 .mu.g, 100
to 2000 .mu.g, 100 to 2250 .mu.g, 100 to 2500 .mu.g, 100 to 2750
.mu.g, 100 to 3000 .mu.g, 200 to 300 .mu.g, 200 to 400 .mu.g, 200
to 500 .mu.g, 200 to 600 .mu.g, 200 to 700 .mu.g, 200 to 800 .mu.g,
200 to 900 .mu.g, 200 to 1000 .mu.g, 200 to 1250 .mu.g, 200 to 1500
.mu.g, 200 to 1750 .mu.g, 200 to 2000 .mu.g, 200 to 2250 .mu.g, 200
to 2500 .mu.g, 200 to 2750 .mu.g, 200 to 3000 .mu.g, 300 to 400
.mu.g, 300 to 500 .mu.g, 300 to 600 .mu.g, 300 to 700 .mu.g, 300 to
800 .mu.g, 300 to 900 .mu.g, 300 to 1000 .mu.g, 300 to 1250 .mu.g,
300 to 1500 .mu.g, 300 to 1750 .mu.g, 300 to 2000 .mu.g, 300 to
2250 .mu.g, 300 to 2500 .mu.g, 300 to 2750 .mu.g, 300 to 3000
.mu.g, 400 to 500 .mu.g, 400 to 600 .mu.g, 400 to 700 .mu.g, 400 to
800 .mu.g, 400 to 900 .mu.g, 400 to 1000 .mu.g, 400 to 1250 .mu.g,
400 to 1500 .mu.g, 400 to 1750 .mu.g, 400 to 2000 .mu.g, 400 to
2250 .mu.g, 400 to 2500 .mu.g, 400 to 2750 .mu.g, 400 to 3000
.mu.g, 500 to 600 .mu.g, 500 to 700 .mu.g, 500 to 800 .mu.g, 500 to
900 .mu.g, 500 to 1000 .mu.g, 500 to 1250 .mu.g, 500 to 1500 .mu.g,
500 to 1750 .mu.g, 500 to 2000 .mu.g, 500 to 2250 .mu.g, 500 to
2500 .mu.g, 500 to 2750 .mu.g, 500 to 3000 .mu.g, 600 to 700 .mu.g,
600 to 800 .mu.g, 600 to 900 .mu.g, 600 to 1000 .mu.g, 600 to 1250
.mu.g, 600 to 1500 .mu.g, 600 to 1750 .mu.g, 600 to 2000 .mu.g, 600
to 2250 .mu.g, 600 to 2500 .mu.g, 600 to 2750 .mu.g, 600 to 3000
.mu.g, 700 to 800 .mu.g, 700 to 900 .mu.g, 700 to 1000 .mu.g, 700
to 1250 .mu.g, 700 to 1500 .mu.g, 700 to 1750 .mu.g, 700 to 2000
.mu.g, 700 to 2250 .mu.g, 700 to 2500 .mu.g, 700 to 2750 .mu.g, 700
to 3000 .mu.g, 800 to 900 .mu.g, 800 to 1000 .mu.g, 800 to 1250
.mu.g, 800 to 1500 .mu.g, 800 to 1750 .mu.g, 800 to 2000 .mu.g, 800
to 2250 .mu.g, 800 to 2500 .mu.g, 800 to 2750 .mu.g, 800 to 3000
.mu.g, 900 to 1000 .mu.g, 900 to 1250 .mu.g, 900 to 1500 .mu.g, 900
to 1750 .mu.g, 900 to 2000 .mu.g, 900 to 2250 .mu.g, 900 to 2500
.mu.g, 900 to 2750 .mu.g, 900 to 3000 .mu.g, 1000 to 1250 .mu.g,
1000 to 1500 .mu.g, 1000 to 1750 .mu.g, 1000 to 2000 .mu.g, 1000 to
2250 .mu.g, 1000 to 2500 .mu.g, 1000 to 2750 .mu.g, 1000 to 3000
.mu.g, 2 to 500 .mu.g, 50 to 500 .mu.g, 3 to 100 .mu.g, 5 to 20
.mu.g, 5 to 100 .mu.g, 10 .mu.g, 20 .mu.g, 30 .mu.g, 40 .mu.g, 50
.mu.g, 60 .mu.g, 70 .mu.g, 75 .mu.g, 80 .mu.g, 90 .mu.g, 100 .mu.g,
150 .mu.g, 200 .mu.g, 250 .mu.g, 300 .mu.g, 350 .mu.g, 400 .mu.g,
450 .mu.g, 500 .mu.g, 550 .mu.g, 600 .mu.g, 650 .mu.g, 700 .mu.g,
750 .mu.g, 800 .mu.g, 850 .mu.g, 900 .mu.g, 950 .mu.g, 1000 .mu.g,
1050 .mu.g, 1100 .mu.g, 1150 .mu.g, 1200 .mu.g, 1250 .mu.g, 1300
.mu.g, 1350 .mu.g, 1400 .mu.g, 1450 .mu.g, 1500 .mu.g, 1550 .mu.g,
1600 .mu.g, 1650 .mu.g, 1700 .mu.g, 1750 .mu.g, 1800 .mu.g, 1850
.mu.g, 1900 .mu.g, 1950 .mu.g, 2000 .mu.g, 2050 .mu.g, 2100 .mu.g,
2150 .mu.g, 2200 .mu.g, 2250 .mu.g, 2300 .mu.g, 2350 .mu.g, 2400
.mu.g, 2450 .mu.g, 2500 .mu.g, 2550 .mu.g, 2600 .mu.g, 2650 .mu.g,
2700 .mu.g, 2750 .mu.g, 2800 .mu.g, 2850 .mu.g, 2900 .mu.g, 2950
.mu.g, 3000 .mu.g, 3250 .mu.g, 3500 .mu.g, 3750 .mu.g, 4000 .mu.g,
4250 .mu.g, 4500 .mu.g, 4750 .mu.g, 5000 .mu.g of a GCC agonist
described herein.
[0280] When provided as a single dosage form, the potential exists
for a chemical interaction between the combined active ingredients
(for example, an opioid and a GCC agonist). For this reason, the
preferred dosage forms of the combination products of this
disclosure are formulated such that although the active ingredients
are combined in a single dosage form, the physical contact between
the active ingredients is minimized (that is, reduced).
[0281] In order to minimize contact, one embodiment of this
disclosure where the product is orally administered provides for a
combination product wherein one active ingredient is enteric
coated. By enteric coating one or more of the active ingredients,
it is possible not only to minimize the contact between the
combined active ingredients, but also, it is possible to control
the release of one of these components in the gastrointestinal
tract such that one of these components is not released in the
stomach but rather is released in the intestines. Another
embodiment of this disclosure where oral administration is desired
provides for a combination product wherein one of the active
ingredients is coated with a sustained-release material which
effects a sustained-release throughout the gastrointestinal tract
and also serves to minimize physical contact between the combined
active ingredients. Furthermore, the sustained-released component
can be additionally enteric coated such that the release of this
component occurs only in the intestine. Still another approach
would involve the formulation of a combination product in which the
one component is coated with a sustained and/or enteric release
polymer, and the other component is also coated with a polymer such
as a low-viscosity grade of hydroxypropyl methylcellulose (HPMC) or
other appropriate materials as known in the art, in order to
further separate the active components. The polymer coating serves
to form an additional barrier to interaction with the other
component.
[0282] Dosage forms of the combination products include those
wherein one active ingredient is enteric coated can be in the form
of tablets such that the enteric coated component and the other
active ingredient are blended together and then compressed into a
tablet or such that the enteric coated component is compressed into
one tablet layer and the other active ingredient is compressed into
an additional layer. Optionally, in order to further separate the
two layers, one or more placebo layers may be present such that the
placebo layer is between the layers of active ingredients. In
addition, dosage forms of the present disclosure can be in the form
of capsules wherein one active ingredient is compressed into a
tablet or in the form of a plurality of microtablets, particles,
granules or non-perils, which are then enteric coated. These
enteric coated microtablets, particles, granules or non-perils are
then placed into a capsule or compressed into a capsule along with
a granulation of the other active ingredient.
[0283] These as well as other ways of minimizing contact between
the components of combination products of the present disclosure,
whether administered in a single dosage form or administered in
separate forms but at the same time by the same manner, will be
readily apparent to those skilled in the art in light of the
present disclosure.
Analgesic Agents in Combitherapy
[0284] The peptides and agonists described herein can be used in
combination therapy with an analgesic agent, e.g., an analgesic
compound or an analgesic peptide. These peptides and compounds can
be administered with the peptides described herein (simultaneously
or sequentially). They can also be optionally covalently linked or
attached to an agent described herein to create therapeutic
conjugates. Among the useful analgesic agents are: Ca channel
blockers, 5HT receptor antagonists (for example 5HT3, 5HT4 and 5HT1
receptor antagonists), opioid receptor agonists (loperamide,
fedotozine, and fentanyl), NK1 receptor antagonists, CCK receptor
agonists (e.g., loxiglumide), NK1 receptor antagonists, NK3
receptor antagonists, norepinephrine-serotonin reuptake inhibitors
(NSRI), vanilloid and cannabanoid receptor agonists, and
sialorphin. Analgesics agents in the various classes are described
in the literature.
[0285] Among the useful analgesic peptides are sialorphin-related
peptides, including those comprising the amino acid sequence QHNPR
(SEQ ID NO:), including: VQHNPR (SEQ ID NO:); VRQHNPR (SEQ ID NO:);
VRGQHNPR (SEQ ID NO:); VRGPQHNPR (SEQ ID NO:); VRGPRQHNPR (SEQ ID
NO:); VRGPRRQHNPR (SEQ ID NO:); and RQHNPR (SEQ ID NO:).
Sialorphin-related peptides bind to neprilysin and inhibit
neprilysin-mediated breakdown of substance P and Met-enkephalin.
Thus, compounds or peptides that are inhibitors of neprilysin are
useful analgesic agents which can be administered with the peptides
described herein in a co-therapy or linked to the peptides
described herein, e.g., by a covalent bond. Sialophin and related
peptides are described in U.S. Pat. No. 6,589,750; U.S. 20030078200
A1; and WO 02/051435 A2.
[0286] Opioid receptor antagonists and agonists can be administered
with the peptides described herein in co-therapy or linked to the
agent described herein, e.g., by a covalent bond. For example,
opioid receptor antagonists such as naloxone, naltrexone, methyl
nalozone, nalmefene, cypridime, beta funaltrexamine, naloxonazine,
naltrindole, and nor-binaltorphimine are thought to be useful in
the treatment of IBS. It can be useful to formulate opioid
antagonists of this type is a delayed and sustained release
formulation such that initial release of the antagonist is in the
mid to distal small intestine and/or ascending colon. Such
antagonists are described in WO 01/32180 A2. Enkephalin
pentapeptide (HOE825; Tyr-D-Lys-Gly-Phe-L-homoserine) is an agonist
of the mu and delta opioid receptors and is thought to be useful
for increasing intestinal motility (Eur. J. Pharm. 219:445, 1992),
and this peptide can be used in conjunction with the peptides
described herein. Also useful is trimebutine which is thought to
bind to mu/delta/kappa opioid receptors and activate release of
motilin and modulate the release of gastrin, vasoactive intestinal
peptide, gastrin and glucagons. Kappa opioid receptor agonists such
as fedotozine, asimadoline, and ketocyclazocine, and compounds
described in WO 03/097051 and WO05/007626 can be used with or
linked to the peptides described herein. In addition, mu opioid
receptor agonists such as morphine, diphenyloxylate, frakefamide
(H-Tyr-D-Ala-Phe(F)-Phe-NH.sub.2; WO 01/019849 A1) and loperamide
can be used.
[0287] Tyr-Arg (kyotorphin) is a dipeptide that acts by stimulating
the release of met-enkephalins to elicit an analgesic effect (J.
Biol. Chem. 262:8165, 1987). Kyotorphin can be used with or linked
to the peptides described herein.
[0288] Chromogranin-derived peptide (CgA 47-66; see, e.g., Ghia et
al. 2004 Regulatory Peptides 119:199) can be used with or linked to
the peptides described herein.
[0289] CCK receptor agonists such as caerulein from amphibians and
other species are useful analgesic agents that can be used with or
linked to the peptides described herein.
[0290] Conotoxin peptides represent a large class of analgesic
peptides that act at voltage gated Ca channels, NMDA receptors or
nicotinic receptors. These peptides can be used with or linked to
the peptides described herein.
[0291] Peptide analogs of thymulin (FR Application 2830451) can
have analgesic activity and can be used with or linked to the
peptides described herein.
[0292] CCK (CCKa or CCKb) receptor antagonists, including
loxiglumide and dexloxiglumide (the R-isomer of loxiglumide) (WO
88/05774) can have analgesic activity and can be used with or
linked to the peptides described herein.
[0293] Other useful analgesic agents include 5-HT4 agonists such as
tegaserod (Zelnorm.RTM.), mosapride, metoclopramide, zacopride,
cisapride, renzapride, benzimidazolone derivatives such as BIMU 1
and BIMU 8, and lirexapride. Such agonists are described in:
EP1321142 A1, WO 03/053432A1, EP 505322 A1, EP 505322 B1, U.S. Pat.
No. 5,510,353, EP 507672 A1, EP 507672 B1, and U.S. Pat. No.
5,273,983.
[0294] Calcium channel blockers such as ziconotide and related
compounds described in, for example, EP625162B1, U.S. Pat. No.
5,364,842, U.S. Pat. No. 5,587,454, U.S. Pat. No. 5,824,645, U.S.
Pat. No. 5,859,186, U.S. Pat. No. 5,994,305, U.S. Pat. No.
6,087,091, U.S. Pat. No. 6,136,786, WO 93/13128 A1, EP 1336409 A1,
EP 835126 A1, EP 835126 B1, U.S. Pat. No. 5,795,864, U.S. Pat. No.
5,891,849, U.S. Pat. No. 6,054,429, WO 97/01351 A1, can be used
with or linked to the peptides described herein.
[0295] Various antagonists of the NK-1, NK-2, and NK-3 receptors
(for a review see Giardina et al. 2003 Drugs 6:758) can be can be
used with or linked to the peptides described herein.
[0296] NK1 receptor antagonists such as: aprepitant (Merck & Co
Inc), vofopitant, ezlopitant (Pfizer, Inc.), R-673 (Hoffmann-La
Roche Ltd), SR-48968 (Sanofi Synthelabo), CP-122,721 (Pfizer,
Inc.), GW679769 (Glaxo Smith Kline), TAK-637 (Takeda/Abbot),
SR-14033, and related compounds described in, for example, EP
873753 A1, US 20010006972 A1, US 20030109417 A1, WO 01/52844 A1,
can be used with or linked to the peptides described herein.
[0297] NK-2 receptor antagonists such as nepadutant (Menarini
Ricerche SpA), saredutant (Sanofi-Synthelabo), GW597599 (Glaxo
Smith Kline), SR-144190 (Sanofi-Synthelabo) and UK-290795 (Pfizer
Inc) can be used with or linked to the peptides described
herein.
[0298] NK3 receptor antagonists such as osanetant (SR-142801;
Sanofi-Synthelabo), SSR-241586, talnetant and related compounds
described in, for example, WO 02/094187 A2, EP 876347 A1, WO
97/21680 A1, U.S. Pat. No. 6,277,862, WO 98/11090, WO 95/28418, WO
97/19927, and Boden et al. (J Med Chem. 39:1664-75, 1996) can be
used with or linked to the peptides described herein.
[0299] Norepinephrine-serotonin reuptake inhibitors (NSR1) such as
milnacipran and related compounds described in WO 03/077897 A1 can
be used with or linked to the peptides described herein.
[0300] Vanilloid receptor antagonists such as arvanil and related
compounds described in WO 01/64212 A1 can be used with or linked to
the peptides described herein.
[0301] The analgesic peptides and compounds can be administered
with the peptides and agonists described herein (simultaneously or
sequentially). The analgesic agents can also be covalently linked
to the peptides and agonists described herein to create therapeutic
conjugates. Where the analgesic is a peptide and is covalently
linked to an agent described herein the resulting peptide may also
include at least one trypsin cleavage site. When present within the
peptide, the analgesic peptide may be preceded by (if it is at the
carboxy terminus) or followed by (if it is at the amino terminus) a
trypsin cleavage site that allows release of the analgesic
peptide.
[0302] In addition to sialorphin-related peptides, analgesic
peptides include: AspPhe, endomorphin-1, endomorphin-2, nocistatin,
dalargin, lupron, ziconotide, and substance P.
Diabetes, Obesity and Other Disorders
[0303] Pharmaceutical compositions comprising at least two of: 1)
an agent that stimulates the production of cAMP (e.g.,
glucagon-like peptide 1 (GLP-1)); 2) an agent that inhibits the
degradation of a cyclic nucleotide (e.g., a phosphodiesterase
inhibitor); and 3) a peptide or agonist described herein useful for
treating diabetes and obesity. Such compositions may also be useful
for treating secondary hyperglycemias in connection with pancreatic
diseases (chronic pancreatitis, pancreasectomy, hemochromatosis) or
endocrine diseases (acromegaly, Cushing's syndrome,
pheochromocytoma or hyperthyreosis), drug-induced hyperglycemias
(benzothiadiazine saluretics, diazoxide or glucocorticoids),
pathologic glucose tolerance, hyperglycemias, dyslipoproteinemias,
adiposity, hyperlipoproteinemias and/or hypotensions.
[0304] The phosphodiesterase inhibitor can be specific for a
particular phosphodiesterase (e.g., Group III or Group IV) or a
non-specific phosphodiesterase inhibitor, such as papaverine,
theophylline, enprofyllines and/or IBMX. Specific phosphodiesterase
inhibitors which inhibit group III phosphodiesterases
(cGMP-inhibited phosphodiesterases), including indolidane
(LY195115), cilostamide (OPC 3689), lixazinone (RS 82856), Y-590,
imazodane (CI914), SKF 94120, quazinone, ICI 153,110, cilostazole,
bemorandane (RWJ 22867), siguazodane (SK&F 94-836), adibendane
(BM 14,478), milrinone (WIN 47203), enoximone (MDL 17043),
pimobendane (UD-CG 115), MC1-154, saterinone (BDF 8634), sulmazole
(ARL 115), UD-CG 212, motapizone, piroximone, and ICI 118233 can be
useful. In addition, phosphodiesterase inhibitors which inhibit
group IV phosphodiesterases (cAMP-specific phosphodiesterases),
such as rolipram ZK 62711; pyrrolidone), imidazolidinone (RO
20-1724), etazolate (SQ 65442), denbufylline (BRL 30892), ICI63197,
and RP73401 can be used.
Other Agents for Use in Combitherapy
[0305] Also within the disclosure are pharmaceutical compositions
comprising a peptide or agonists described herein and a second
therapeutic agent. The second therapeutic agent can be administered
to treat any condition for which it is useful, including conditions
that are not considered to be the primary indication for treatment
with the second therapeutic agent. The second therapeutic agent can
be administered simultaneously or sequentially. The second
therapeutic agent can be covalently linked to the peptides and
agonists described herein to create a therapeutic conjugate. When
the second therapeutic agent is another peptide, a linker including
those described herein may be used between the peptide described
herein and the second therapeutic peptide.
[0306] Examples of therapeutic agents used to treat heart failure
include diuretics (e.g. furesomide (Lasix), bumetanide (Bumex),
ethacrynic acid (Edecrin), torsemide (Demadex), amiloride
(Midamor), spironolactone. (Aldactone), chorthiazide (Diuril),
metolazone (Zaroxylyn)), Angiotension-Converting Enzyme (ACE)
inhibitors (e.g. captopril (Capoten), enalopril (Vasotec),
lisinopril (Prinivil, Zestril), ramipril (Altace)), Beta blockers
(e.g. carvedilol (Coreg) and Inotropes (e.g. digoxin, dobutaimine,
dopamine Milrinone).
[0307] Examples of additional therapeutic agents to treat
gastrointestinal and other disorders include:
agents to treat constipation (e.g., a chloride channel activator
such as the bicylic fatty acid, Lubiprostone (formerly known as
SPI-0211; Sucampo Pharmaceuticals, Inc.; Bethesda, Md.), a laxative
(eg. a bulk-forming laxative (e.g. nonstarch polysaccharides,
Colonel Tablet (polycarbophil calcium), Plantago Ovata.RTM.,
Equalactin.RTM. (Calcium Polycarbophil)), fiber (e.g. FIBERCON.RTM.
(Calcium Polycarbophil), an osmotic laxative, a stimulant laxative
(such as diphenylmethanes (e.g. bisacodyl), anthraquinones (e.g.
cascara, senna), and surfactant laxatives (e.g. castor oil,
docusates), an emollient/lubricating agent (such as mineral oil,
glycerine, and docusates), MiraLax (Braintree Laboratories,
Braintree Mass.), dexloxiglumide (Forest Laboratories, also known
as CR 2017 Rottapharm (Rotta Research Laboratorium SpA)), saline
laxatives, enemas, suppositories, and CR 3700 (Rottapharm (Rotta
Research Laboratorium SpA); acid reducing agents such as proton
pump inhibitors (e.g., omeprazole (Prilosec.RTM.), esomeprazole
(Nexium.RTM.), lansoprazole (Prevacid.RTM.), pantoprazole
(Protonix.RTM.) and rabeprazole (Aciphex.RTM.)) and Histamine
H2-receptor antagonist (also known as H2 receptor blockers
including cimetidine, ranitidine, famotidine and nizatidine);
prokinetic agents including itopride, octreotide, bethanechol,
metoclopramide (Reglan.RTM.), domperidone (Motilium.RTM.),
erythromycin (and derivatives thereof) or cisapride
(propulsid.RTM.); prokineticin peptides homologs, variants and
chimeras thereof including those described in U.S. Pat. No.
7,052,674 which can be used with or linked to the peptides
described herein; pro-motility agents such as the
vasostatin-derived peptide, chromogranin A (4-16) (see, e.g., Ghia
et al. 2004 Regulatory Peptides 121:31) or motilin agonists (e.g.,
GM-611 or mitemcinal fumarate) or nociceptin/Orphanin FQ receptor
modulators (US20050169917); other peptides which can bind to and/or
activate GC-C including those described in US20050287067; complete
or partial 5HT (e.g. 5HT1, 5HT2, 5HT3, 5HT4) receptor agonists or
antagonists (including 5HT1A antagonists (e.g. AGI-001 (AGI
therapeutics), 5HT2B antagonists (e.g. PGN1091 and PGN1164
(Pharmagene Laboratories Limited), and 5HT4 receptor agonists (such
as tegaserod (ZELNORM.RTM.), prucalopride, mosapride,
metoclopramide, zacopride, cisapride, renzapride, benzimidazolone
derivatives such as BIMU 1 and BIMU 8, and lirexapride). Such
agonists/modulatos are described in: EP1321142 A1, WO 03/053432A1,
EP 505322 A1, EP 505322 B1, U.S. Pat. No. 5,510,353, EP 507672 A1,
EP 507672 B1, U.S. Pat. No. 5,273,983, and U.S. Pat. No.
6,951,867); 5HT3 receptor agonists such as MKC-733; and 5HT3
receptor antagonists such as DDP-225 (MC1-225; Dynogen
Pharmaceuticals, Inc.), cilansetron (Calmactin.RTM.), alosetron
(Lotronex.RTM.), Ondansetron HCl (Zofran.RTM.), Dolasetron
(ANZEMET4), palonosetron (Aloxi.RTM.), Granisetron (Kytril.RTM.),
YM060 (ramosetron; Astellas Pharma Inc.; ramosetron may be given as
a daily dose of 0.002 to 0.02 mg as described in EP01588707) and
ATI-7000 (Aryx Therapeutics, Santa Clara Calif.); muscarinic
receptor agonists; anti-inflammatory agents; antispasmodics
including but not limited to anticholinergic drugs (like
dicyclomine (e.g. Colimex.RTM., Formulex.RTM., Lomine.RTM.,
Protylol.RTM., Viscerol.RTM., Spasmoban.RTM., Bentyl.RTM.,
Bentylol.RTM.), hyoscyamine (e.g. IB-Stat.RTM., Nulev.RTM.,
Levsin.RTM., Levbid.RTM., Levsinex Timecaps.RTM., Levsin/SL.RTM.,
Anaspaz.RTM., A-Spas S/L.RTM., Cystospaz.RTM., Cystospaz-M.RTM.,
Donnamar.RTM., Colidrops Liquid Pediatric.RTM., Gastrosed.RTM.,
Hyco Elixir.RTM., Hyosol.RTM., Hyospaz.RTM., Hyosyne.RTM.,
Losamine.RTM., Medispaz.RTM., Neosol.RTM., Spacol.RTM.,
Spasdel.RTM., Symax.RTM., Symax SL.RTM.), Donnatal (e.g. Donnatal
Extentabs.RTM.), clidinium (e.g. Quarzan, in combination with
Librium=Librax), methantheline (e.g. Banthine), Mepenzolate (e.g.
Cantil), homatropine (e.g. hycodan, Homapin), Propantheline bromide
(e.g. Pro-Banthine), Glycopyrrolate (e.g. Robinul.RTM., Robinul
Forte.RTM.), scopolamine (e.g. Transderm-Scop.RTM.,
Transderm-V.RTM.), hyosine-N-butylbromide (e.g. Buscopan.RTM.),
Pirenzepine (e.g. Gastrozepin.RTM.) Propantheline Bromide (e.g.
Propanthel.RTM.), dicycloverine (e.g. Merbentyl.RTM.),
glycopyrronium bromide (e.g. Glycopyrrolate.RTM.), hyoscine
hydrobromide, hyoscine methobromide, methanthelinium, and
octatropine); peppermint oil; and direct smooth muscle relaxants
like cimetropium bromide, mebeverine (DUSPATAL.RTM.,
DUSPATALIN.RTM., COLOFAC MR.RTM., COLOTAL.RTM.), otilonium bromide
(octilonium), pinaverium (e.g. Dicetel.RTM. (pinaverium bromide;
Solvay S.A.)), Spasfon.RTM. (hydrated phloroglucinol and
trimethylphloroglucinol) and trimebutine (including trimebutine
maleate (Modulon.RTM.); antidepressants, including but not limited
to those listed herein, as well as tricyclic antidepressants like
amitriptyline (Elavil.RTM.), desipramine (Norpramin.RTM.),
imipramine (Tofranil.RTM.), amoxapine (Asendin.RTM.),
nortriptyline; the selective serotonin reuptake inhibitors (SSRI's)
like paroxetine (Paxil.RTM.), fluoxetine (Prozac.RTM.), sertraline
(Zoloft.RTM.), and citralopram (Celexa.RTM.); and others like
doxepin (Sinequan.RTM.) and trazodone (Desyrel.RTM.);
centrally-acting analgesic agents such as opioid receptor agonists,
opioid receptor antagonists (e.g., naltrexone); agents for the
treatment of Inflammatory bowel disease; agents for the treatment
of Crohn's disease and/or ulcerative colitis (e.g., alequel (Enzo
Biochem, Inc.; Farmingsale, N.Y.), the anti-inflammatory peptide
RDP58 (Genzyme, Inc.; Cambridge, Mass.), and TRAFICET-EN.TM.
(ChemoCentryx, Inc.; San Carlos, Calif.); agents that treat
gastrointestinal or visceral pain; agents that increase cGMP levels
(as described in US20040121994) like adrenergic receptor
antagonists, dopamine receptor agonists and PDE (phosphodiesterase)
inhibitors including but not limited to those disclosed herein;
purgatives that draw fluids to the intestine (e.g., VISICOL.RTM., a
combination of sodium phosphate monobasic monohydrate and sodium
phosphate dibasic anhydrate); Corticotropin Releasing Factor (CRF)
receptor antagonists (including NBI-34041 (Neurocrine Biosciences,
San Diego, Calif.), CRH9-41, astressin, R121919 (Janssen
Pharmaceutica), CP154,526, NBI-27914, Antalarmin, DMP696
(Bristol-Myers Squibb) CP-316,311 (Pfizer, Inc.), SB723620 (GSK),
GW876008 (Neurocrine/Glaxo Smith Kline), ONO-2333Ms (Ono
Pharmaceuticals), TS-041 (Janssen), AAG561 (Novartis) and those
disclosed in U.S. Pat. No. 5,063,245, U.S. Pat. No. 5,861,398,
US20040224964, US20040198726, US20040176400, US20040171607,
US20040110815, US20040006066, and US20050209253); glucagon-like
peptides (glp-1) and analogues thereof (including exendin-4 and
GTP-010 (Gastrotech Pharma A)) and inhibitors of DPP-IV (DPP-IV
mediates the inactivation of glp-1); tofisopam,
enantiomerically-pure R-tofisopam, and pharmaceutically-acceptable
salts thereof (US 20040229867); tricyclic anti-depressants of the
dibenzothiazepine type including but not limited to
Dextofisopam.RTM. (Vela Pharmaceuticals), tianeptine (Stablon.RTM.)
and other agents described in U.S. Pat. No. 6,683,072; (E)-4
(1,3bis(cyclohexylmethyl)-1,2,34,-tetrahydro-2,6-diono-9H-purin-8-yl)cinn-
amic acid nonaethylene glycol methyl ether ester and related
compounds described in WO 02/067942; the probiotic PROBACTRIX.RTM.
(The BioBalance Corporation; New York, N.Y.) which contains
microorganisms useful in the treatment of gastrointestinal
disorders; antidiarrheal drugs including but not limited to
loperamide (Imodium, Pepto Diarrhea), diphenoxylate with atropine
(Lomotil, Lomocot), cholestyramine (Questran, Cholybar), atropine
(Co-Phenotrope, Diarsed, Diphenoxylate, Lofene, Logen, Lonox,
Vi-Atro, atropine sulfate injection) and Xifaxan.RTM. (rifaximin;
Salix Pharmaceuticals Ltd), TZP-201 (Tranzyme Pharma Inc.), the
neuronal acetylcholine receptor (nAChR) blocker AGI-004 (AGI
therapeutics), and bismuth subsalicylate (Pepto-bismol); anxiolytic
drugs including but not limited to Ativan (lorazepam), alprazolam
(Xanax.RTM.), chlordiazepoxide/clidinium (Librium.RTM.,
Librax.RTM.), clonazepam (Klonopin.RTM.), clorazepate
(Tranxene.RTM.), diazepam (Valium.RTM.), estazolam (ProSom.RTM.),
flurazepam (Dalmane.RTM.), oxazepam (Serax.RTM.), prazepam
(Centrax.RTM.), temazepam (Restoril.RTM.), triazolam (Halcion.RTM.;
Bedelix.RTM. (Montmorillonite beidellitic; Ipsen Ltd), Solvay
SLV332 (ArQule Inc), YKP (SK Pharma), Asimadoline (Tioga
Pharmaceuticals/Merck), AGI-003 (AGI Therapeutics); neurokinin
antagonists including those described in US20060040950; potassium
channel modulators including those described in U.S. Pat. No.
7,002,015; the serotonin modulator AZD7371 (AstraZeneca Plc); M3
muscarinic receptor antagonists such as darifenacin (Enablex;
Novartis AG and zamifenacin (Pfizer); herbal and natural therapies
including but not limited to acidophilus, chamomile tea, evening
primrose oil, fennel seeds, wormwood, comfrey, and compounds of
Bao-Ji-Wan (magnolol, honokiol, imperatorin, and isoimperatorin) as
in U.S. Pat. No. 6,923,992; and compositions comprising lysine and
an anti-stress agent for the treatment of irritable bowel syndrome
as described in EP01550443.
[0308] The peptides and agonists described herein can be used in
combination therapy with insulin and related compounds including
primate, rodent, or rabbit insulin including biologically active
variants thereof including allelic variants, more preferably human
insulin available in recombinant form. Sources of human insulin
include pharmaceutically acceptable and sterile formulations such
as those available from Eli Lilly (Indianapolis, Ind. 46285) as
Humulin.TM. (human insulin rDNA origin). See the THE PHYSICIAN'S
DESK REFERENCE, 55.sup.th Ed. (2001) Medical Economics, Thomson
Healthcare (disclosing other suitable human insulins). The peptides
and agonists described herein can also be used in combination
therapy with agents that can boost insulin effects or levels of a
subject upon administration, e.g. glipizide and/or rosiglitazone.
The peptides and agonists described herein can be used in
combitherapy with SYMLIN.RTM. (pramlintide acetate) and
Exenatide.RTM. (synthetic exendin-4; a 39 aa peptide).
[0309] The peptides and agonists described herein can also be used
in combination therapy with agents (e.g., Entereg.TM. (alvimopan;
formerly called adolor/ADL 8-2698), conivaptan and related agents
describe in U.S. Pat. No. 6,645,959) used for the treatment of
postoperative ileus and other disorders.
[0310] The peptides and agonists described herein can be used in
combination therapy with an anti-hypertensive agent including but
not limited to:
[0311] (1) diuretics, such as thiazides, including chlorthalidone,
chlorthiazide, dichlorophenamide, hydroflumethiazide, indapamide,
polythiazide, and hydrochlorothiazide; loop diuretics, such as
bumetanide, ethacrynic acid, furosemide, and torsemide; potassium
sparing agents, such as amiloride, and triamterene; carbonic
anhydrase inhibitors, osmotics (such as glycerin) and aldosterone
antagonists, such as spironolactone, epirenone, and the like; (2)
beta-adrenergic blockers such as acebutolol, atenolol, betaxolol,
bevantolol, bisoprolol, bopindolol, carteolol, carvedilol,
celiprolol, esmolol, indenolol, metaprolol, nadolol, nebivolol,
penbutolol, pindolol, propanolol, sotalol, tertatolol, tilisolol,
and timolol, and the like;
[0312] (3) calcium channel blockers such as amlodipine,
aranidipine, azelnidipine, barnidipine, benidipine, bepridil,
cinaldipine, clevidipine, diltiazem, efonidipine, felodipine,
gallopamil, isradipine, lacidipine, lemildipine, lercanidipine,
nicardipine, nifedipine, nilvadipine, nimodepine, nisoldipine,
nitrendipine, manidipine, pranidipine, and verapamil, and the
like;
[0313] (4) angiotensin converting enzyme (ACE) inhibitors such as
benazepril; captopril; ceranapril; cilazapril; delapril; enalapril;
enalopril; fosinopril; imidapril; lisinopril; losinopril;
moexipril; quinapril; quinaprilat; ramipril; perindopril;
perindropril; quanipril; spirapril; tenocapril; trandolapril, and
zofenopril, and the like;
[0314] (5) neutral endopeptidase inhibitors such as omapatrilat,
cadoxatril and ecadotril, fosidotril, sampatrilat, AVE7688, ER4030,
and the like;
[0315] (6) endothelin antagonists such as tezosentan, A308165, and
YM62899, and the like;
[0316] (7) vasodilators such as hydralazine, clonidine, minoxidil,
and nicotinyl alcohol, and the like;
[0317] (8) angiotensin II receptor antagonists such as aprosartan,
candesartan, eprosartan, irbesartan, losartan, olmesartan,
pratosartan, tasosartan, telmisartan, valsartan, and EXP-3137,
F16828K, and RNH6270, and the like;
[0318] (9) .alpha./.beta. adrenergic blockers such as nipradilol,
arotinolol and amosulalol, and the like;
[0319] (10) alpha 1 blockers, such as terazosin, urapidil,
prazosin, tamsulosin, bunazosin, trimazosin, doxazosin, naftopidil,
indoramin, WHP 164, and XEN010, and the like;
[0320] (11) alpha 2 agonists such as lofexidine, tiamenidine,
moxonidine, rilmenidine and guanobenz, and the like;
[0321] (12) aldosterone inhibitors, and the like; and
[0322] (13) angiopoietin-2-binding agents such as those disclosed
in WO03/030833.
[0323] Specific anti-hypertensive agents that can be used in
combination with peptides and agonists described herein include,
but are not limited to:
diuretics, such as thiazides (e.g., chlorthalidone, cyclothiazide
(CAS RN 2259-96-3), chlorothiazide (CAS RN 72956-09-3, which may be
prepared as disclosed in US2809194), dichlorophcnamide,
hydroflumethiazide, indapamide, polythiazide, bendroflumethazide,
methyclothazide, polythiazide, trichlormethazide, chlorthalidone,
indapamide, metolazone, quinethazone, althiazide (CAS RN 5588-16-9,
which may be prepared as disclosed in British Patent No. 902,658),
benzthiazide (CAS RN 91-33-8, which may be prepared as disclosed in
U.S. Pat. No. 3,108,097), buthiazide (which may be prepared as
disclosed in British Patent Nos. 861,367), and
hydrochlorothiazide), loop diuretics (e.g. bumetanide, ethacrynic
acid, furosemide, and torasemide), potassium sparing agents (e.g.
amiloride, and triamterene (CAS Number 396-01-0)), and aldosterone
antagonists (e.g. spironolactone (CAS Number 52-01-7), epirenone,
and the like); .beta.-adrenergic blockers such as Amiodarone
(Cordarone, Pacerone), bunolol hydrochloride (CAS RN 31969-05-8,
Parke-Davis), acebutolol (.+-.N-[3-Acetyl-4-[2-hydroxy-3-[(1
methylethyl)amino]propoxy]phenyl]-butanamide, or
(.+-.)-3'-Acetyl-4'-[2-hydroxy-3-(isopropylamino)propoxy]butyranilide),
acebutolol hydrochloride (e.g. Sectral.RTM., Wyeth-Ayerst),
alprenolol hydrochloride (CAS RN 13707-88-5 see Netherlands Patent
Application No. 6,605,692), atenolol (e.g. Tenormin.RTM.,
AstraZeneca), carteolol hydrochloride (e.g. Cartrol.RTM.
Filmtab.RTM., Abbott), Celiprolol hydrochloride (CAS RN 57470-78-7,
also see in U.S. Pat. No. 4,034,009), cetamolol hydrochloride (CAS
RN 77590-95-5, see also U.S. Pat. No. 4,059,622), labetalol
hydrochloride (e.g. Normodyne.RTM., Schering), esmolol
hydrochloride (e.g. Brevibloc.RTM.,Baxter), levobetaxolol
hydrochloride (e.g. Betaxon.TM. Ophthalmic Suspension, Alcon),
levobunolol hydrochloride (e.g. Betagan.RTM. Liquifilm.RTM. with C
CAP.RTM. Compliance Cap, Allergan), nadolol (e.g. Nadolol, Mylan),
practolol (CAS RN 6673-35-4, see also U.S. Pat. No. 3,408,387),
propranolol hydrochloride (CAS RN 318-98-9), sotalol hydrochloride
(e.g. Betapace AFT.TM., Berlex), timolol
(2-Propanol,1-[(1,1-dimethylethyl)amino]-3-[[4-4(4-morpholinyl)-1,2,5-thi-
adiazol-3-yl]oxy]-, hemihydrate, (S)--, CAS RN 91524-16-2), timolol
maleate (S)-1-[(1,1-dimethylethyl)
amino]-3-[[4-(4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy]-2-propanol
(Z)-2-butenedioate (1:1) salt, CAS RN 26921-17-5), bisoprolol
(2-Propanol,
1-[4-[[2-(1-methylethoxy)ethoxy]-methyl]phenoxyl]-3-[(1-meth-ylethyl)amin-
o]-, (.+-.), CAS RN 66722-44-9), bisoprolol fumarate (such as
(.+-.)-1-[4-[[2-(1-Methylethoxy)
ethoxy]methyl]phenoxy]-3-[(1-methylethyl)amino]-2-propanol
(E)-2-butenedioate (2:1) (salt), e.g., Zebeta.TM., Lederle
Consumer), nebivalol (2H-1-Benzopyran-2-methanol,
.alpha..alpha.'-[iminobis(methylene)]bis[6-fluoro-3,4-dihydro-, CAS
RN 99200-09-6 see also U.S. Pat. No. 4,654,362), cicloprolol
hydrochloride, such 2-Propanol,
14442-(cyclopropylmethoxy)ethoxy]phenoxy]-3-[1-methylethyl)amino]-,
hydrochloride, A.A.S. RN 63686-79-3), dexpropranolol hydrochloride
(2-Propanol,1-[1-methylethy)-amino]-3-(1-naphthalenyloxy)-hydrochloride
(CAS RN 13071-11-9), diacetolol hydrochloride (Acetamide,
N-[3-acetyl-4-[2-hydroxy-3-[(1-methyl-ethyl)amino]propoxy][phenyl]-,
monohydrochloride CAS RN 69796-04-9), dilevalol hydrochloride
(Benzamide,
2-hydroxy-5-[1-hydroxy-2-[1-methyl-3-phenylpropyl)amino]ethyl]-,
monohydrochloride, CAS RN 75659-08-4), exaprolol hydrochloride
(2-Propanol, 1-(2-cyclohexylphenoxy)-3-[(1-methylethyl)amino]-,
hydrochloride CAS RN 59333-90-3), flestolol sulfate (Benzoic acid,
2-fluoro-,3-[[2-[aminocarbonyl)amino]-1-dimethylethyl]amino]-2-hydroxypro-
pyl ester, (.+-.)-sulfate (1:1) (salt), CAS RN 88844-73-9; metalol
hydrochloride (Methanesulfonamide,
N-[4-[1-hydroxy-2-(methylamino)propyl]phenyl]-, monohydrochloride
CAS RN 7701-65-7), metoprolol 2-Propanol,
144-(2-methoxyethyl)phenoxy]-3[1-methylethyl)amino]-; CAS RN
37350-58-6), metoprolol tartrate (such as 2-Propanol,
1-[4-(2-methoxyethyl)phenoxy]-3-[(1-methylethyl)amino]-, e.g.,
Lopressor.RTM., Novartis), pamatolol sulfate (Carbamic acid,
[2-[4-[2-hydroxy-3-[(1-methylethyl)amino]propoxyl]phenyl]-ethyl]-,
methyl ester, (.+-.) sulfate (salt) (2:1), CAS RN 59954-01-7),
penbutolol sulfate (2-Propanol,
1-(2-cyclopentylphenoxy)-3-[1,1-dimethyle-thyl)amino]1, (S)--,
sulfate (2:1) (salt), CAS RN 38363-32-5), practolol (Acetamide,
N-[4-[2-hydroxy-3-[(1-methylethyl)amino]-propoxy]phenyl]-, CAS RN
6673-35-4;) tiprenolol hydrochloride (Propanol,
1-[(1-methylethyl)amino]-3-[2-(methylthio)-phenoxy]-,
hydrochloride, (.+-.), CAS RN 39832-43-4), tolamolol (Benzamide,
4-[2-[[2-hydroxy-3-(2-methylphenoxy)-propyl]amino]ethoxyl]-, CAS RN
38103-61-6), bopindolol, indenolol, pindolol, propanolol,
tertatolol, and tilisolol, and the like; calcium channel blockers
such as besylate salt of amlodipine (such as
3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-
-methyl-3,5-pyridinedicarboxylate benzenesulphonate, e.g.,
Norvasc.RTM., Pfizer), clentiazem maleate
(1,5-Benzothiazepin-4(5H)-one,
3-(acetyloxy)-8-chloro-5-[2-(dimethylamino)ethyl]-2,3-dihydro-2-(4-methox-
yphenyl)-(2S-cis)-, (Z)-2-butenedioate (1:1), see also U.S. Pat.
No. 4,567,195), isradipine (3,5-Pyridinedicarboxylic acid,
4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-, methyl
1-methylethyl ester,
(.+-.)-4(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedi-
carboxylate, see also U.S. Pat. No. 4,466,972); nimodipine (such as
is isopropyl (2-methoxyethyl)
1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-dicarboxylate,
e.g. Nimotop.RTM., Bayer), felodipine (such as ethyl methyl
4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate-
-, e.g. Plendil.RTM. Extended-Release, AstraZeneca LP), nilvadipine
(3,5-Pyridinedicarboxylic acid,
2-cyano-1,4-dihydro-6-methyl-4-(3-nitrophenyl)-,3-methyl
5-(1-methylethyl) ester, also see U.S. Pat. No. 3,799,934),
nifedipine (such as 3,5-pyridinedicarboxylic
acid,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester,
e.g., Procardia XL.RTM. Extended Release Tablets, Pfizer),
diltiazem hydrochloride (such as
1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,-3-
-dihydro-2(4-methoxyphenyl)-, monohydrochloride, (+)-cis., e.g.,
Tiazac.RTM., Forest), verapamil hydrochloride (such as
benzeneacetronitrile,
(alpha)-[[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimeth-
oxy-(alpha)-(1-methylethyl)hydrochloride, e.g., Isoptin.RTM. SR,
Knoll Labs), teludipine hydrochloride (3,5-Pyridinedicarboxylic
acid,
2-[(dimethylamino)methyl]4-[2-[(1E)-3-(1,1-dimethylethoxy)-3-oxo-1-propen-
yl]phenyl]-1,4-dihydro-6-methyl-, diethyl ester, monohydrochloride)
CAS RN 108700-03-4), belfosdil (Phosphonic acid,
[2-(2-phenoxyethyl)-1,3-propane-diyl]bis-, tetrabutyl ester CAS RN
103486-79-9), fostedil (Phosphonic acid,
[[4-(2-benzothiazolyl)phenyl]methyl]-, diethyl ester CAS RN
75889-62-2), aranidipine, azelnidipine, barnidipine, benidipine,
bepridil, cinaldipine, clevidipine, efonidipine, gallopamil,
lacidipine, lemildipine, lercanidipine, monatepil maleate
(1-Piperazinebutanamide,
N-(6,11-dihydrodibenzo(b,e)thiepin-1'-yl).sub.4-(4-fluorophenyl)-,
(.+-.)-, (Z)-2-butenedioate (1:1)
(.+-.)-N-(6,11-Dihydrodibenzo(b,e)thiep-in-11-yl)-4-(p-fluorophenyl)-1-pi-
perazinebutyramide maleate (1:1) CAS RN 132046-06-1), nicardipine,
nisoldipine, nitrendipine, manidipine, pranidipine, and the like;
T-channel calcium antagonists such as mibefradil; angiotensin
converting enzyme (ACE) inhibitors such as benazepril, benazepril
hydrochloride (such as
3-[[1-(ethoxycarbonyl)-3-phenyl-(1S)-propyl]amino]-2,3,4,5-tetra-
hydro-2-oxo-1H-1-(3S)-benzazepine-1-acetic acid monohydrochloride,
e.g., Lotrel.RTM., Novartis), captopril (such as
1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline, e.g., Captopril,
Mylan, CAS RN 62571-86-2 and others disclosed in U.S. Pat. No.
4,046,889), ceranapril (and others disclosed in U.S. Pat. No.
4,452,790), cetapril (alacepril, Dainippon disclosed in Eur.
Therap. Res. 39:671 (1986); 40:543 (1986)), cilazapril
(Hoffman-LaRoche) disclosed in J. Cardiovasc. Pharmacol. 9:39
(1987), indalapril (delapril hydrochloride
(2H-1,2,4-Benzothiadiazine-7-sulfonamide,
3-bicyclo[2.2.1]hept-5-en-2-yl-6-chloro-3,4-dihydro-, 1,1-dioxide
CAS RN 2259-96-3); disclosed in U.S. Pat. No. 4,385,051), enalapril
(and others disclosed in U.S. Pat. No. 4,374,829), enalapril,
enalaprilat, fosinopril, ((such as L-proline,
4-cyclohexyl-1-[[[2-methyl-1-(1-oxopropoxy) propoxy](4-phenylbutyl)
phosphinyl]acetyl]-, sodium salt, trans-, e.g., Monopril,
Bristol-Myers Squibb and others disclosed in U.S. Pat. No.
4,168,267), fosinopril sodium (L-Proline,
4-cyclohexyl-1-[[(R)-[(1S)-2-methyl-1-(1-ox-opropoxy)propox),
imidapril, indolapril (Schering, disclosed in J. Cardiovasc.
Pharmacol. 5:643, 655 (1983)), lisinopril (Merck), losinopril,
moexipril, moexipril hydrochloride (3-Isoquinolinecarboxylic acid,
2-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]-1,-
-2,3,4-tetrahydro-6,7-dimethoxy-, monohydrochloride, (3S)--CAS RN
82586-52-5), quinapril, quinaprilat, ramipril (Hoechsst) disclosed
in EP 79022 and Curr. Ther. Res. 40:74 (1986), perindopril erbumine
(such as
2S,3aS,7aS-1-[(S)--N--[(S)-1-Carboxybutyl]alanyl]hexahydro-2-indolinecarb-
oxylic acid, 1-ethyl ester, compound with tert-butylamine (1:1),
e.g., Aceon.RTM., Solvay), perindopril (Servier, disclosed in Eur.
J. clin. Pharmacol. 31:519 (1987)), quanipril (disclosed in U.S.
Pat. No. 4,344,949), spirapril (Schering, disclosed in Acta.
Pharmacol. Toxicol. 59 (Supp. 5):173 (1986)), tenocapril,
trandolapril, zofenopril (and others disclosed in U.S. Pat. No.
4,316,906), rentiapril (fentiapril, disclosed in Clin. Exp.
Pharmacol. Physiol. 10:131 (1983)), pivopril, YS980, teprotide
(Bradykinin potentiator BPP9a CAS RN 35115-60-7), BRL 36,378 (Smith
Kline Beecham, see EP80822 and EP60668), MC-838 (Chugai, see C. A.
102:72588v and Jap. J. Pharmacol. 40:373 (1986), CGS 14824
(Ciba-Geigy,
3-([1-ethoxycarbonyl-3-phenyl-(1S)-propyl]amino)-2,3,4,5-tetrahydro-2-ox--
o-1-(3S)-benzazepine-1 acetic acid HCl, see U.K. Patent No.
2103614), CGS 16,617 (Ciba-Geigy,
3(S)-[[(1S)-5-amino-1-carboxypentyl]amino]-2,3,4,-5-tetrahydro-2-oxo-1H-1-
-benzazepine-1-ethanoic acid, see U.S. Pat. No. 4,473,575), Ru
44570 (Hoechst, see Arzneimittelforschung 34:1254 (1985)), R
31-2201 (Hoffman-LaRoche see FEBS Lett. 165:201 (1984)), CI925
(Pharmacologist 26:243, 266 (1984)), WY-44221 (Wyeth, see J. Med.
Chem. 26:394 (1983)), and those disclosed in US2003006922
(paragraph 28), U.S. Pat. No. 4,337,201, U.S. Pat. No. 4,432,971
(phosphonamidates); neutral endopeptidase inhibitors such as
omapatrilat (Vanlev.RTM.), CGS 30440, cadoxatril and ecadotril,
fasidotril (also known as aladotril or alatriopril), sampatrilat,
mixanpril, and gemopatrilat, AVE7688, ER4030, and those disclosed
in U.S. Pat. No. 5,362,727, U.S. Pat. No. 5,366,973, U.S. Pat. No.
5,225,401, U.S. Pat. No. 4,722,810, U.S. Pat. No. 5,223,516, U.S.
Pat. No. 4,749,688, U.S. Pat. No. 5,552,397, U.S. Pat. No.
5,504,080, U.S. Pat. No. 5,612,359, U.S. Pat. No. 5,525,723,
EP0599444, EPO481522, EP0599444, EP0595610, EP0534363, EP534396,
EP534492, EP0629627; endothelin antagonists such as tezosentan,
A308165, and YM62899, and the like; vasodilators such as
hydralazine (apresoline), clonidine (clonidine hydrochloride
(1H-Imidazol-2-amine, N-(2,6-dichlorophenyl)4,5-dihydro-,
monohydrochloride CAS RN 4205-91-8), catapres, minoxidil (loniten),
nicotinyl alcohol (roniacol), diltiazem hydrochloride (such as
1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,-3-
-dihydro-2(4-methoxyphenyl)-, monohydrochloride, (+)-cis, e.g.,
Tiazac.RTM., Forest), isosorbide dinitrate (such as
1,4:3,6-dianhydro-D-glucitol 2,5-dinitrate e.g., Isordil.RTM.
Titradose.RTM., Wyeth-Ayerst), sosorbide mononitrate (such as
1,4:3,6-dianhydro-D-glucito-1,5-nitrate, an organic nitrate, e.g.,
Ismo.RTM., Wyeth-Ayerst), nitroglycerin (such as 2,3 propanetriol
trinitrate, e.g., Nitrostat.RTM. Parke-Davis), verapamil
hydrochloride (such as benzeneacetonitrile,
(.+-.)-(alpha)[3-[[2-(3,4
dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethoxy-(alpha)-(1-methyl-
ethyl) hydrochloride, e.g., Covera HS.RTM. Extended-Release,
Searle), chromonar (which may be prepared as disclosed in U.S. Pat.
No. 3,282,938), clonitate (Annalen 1870 155), droprenilamine (which
may be prepared as disclosed in DE2521113), lidoflazine (which may
be prepared as disclosed in U.S. Pat. No. 3,267,104); prenylamine
(which may be prepared as disclosed in U.S. Pat. No. 3,152,173),
propatyl nitrate (which may be prepared as disclosed in French
Patent No. 1,103,113), mioflazine hydrochloride
(1-piperazineacetamide,
3-(aminocarbonyl).sub.4-[4,4-bis(4-fluorophenyl)butyl]-N-(2,6-dichlorophe-
nyl)-, dihydrochloride CAS RN 83898-67-3), mixidine
(Benzeneethanamine,
3,4-dimethoxy-N-(1-methyl-2-pyrrolidinylidene)-Pyrrolidine,
2-[(3,4-dimethoxyphenethyl)imino]-1-methyl-1-Methyl-2-[(3,4-dimethoxyphen-
ethyl)imino]pyrrolidine CAS RN 27737-38-8), molsidomine
(1,2,3-Oxadiazolium, 5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-,
inner salt CAS RN 25717-80-0), isosorbide mononitrate (D-Glucitol,
1,4:3,6-dianhydro-, 5-nitrate CAS RN 16051-77-7), erythrityl
tetranitrate (1,2,3,4-Butanetetrol, tetranitrate, (2R,3S)-rel-CAS
RN 7297-25-8), clonitrate(1,2-Propanediol, 3-chloro-, dinitrate
(7CI, 8CI, 9CI) CAS RN 2612-33-1), dipyridamole Ethanol, 2,2',2'',
2'''-[(4,8-di-1-piperidinylpyrimido[5,4-d]pyrimidine-2,6-diyl)dinitrilo]t-
etrakis-CAS RN 58-32-2), nicorandil (CAS RN 65141-46-0 3-),
pyridinecarboxamide
(N-[2-(nitrooxy)ethyl]-Nisoldipine-3,5-Pyridinedicarboxylic acid,
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, methyl 2-methylpropyl
ester CAS RN 63675-72-9), nifedipine-3,5-Pyridinedicarboxylic acid,
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester CAS RN
21829-25-4), perhexiline maleate (Piperidine,
2-(2,2-dicyclohexylethyl)-, (2Z)-2-butenedioate (1:1) CAS RN
6724-53-4), oxprenolol hydrochloride (2-Propanol,
1-[(1-methylethyl)amino]-3-[2-(2-propenyloxy)phenoxy]-,
hydrochloride CAS RN 6452-73-9), pentrinitrol (1,3-Propanediol,
2,2-bis[(nitrooxy)methyl]-, mononitrate (ester) CAS RN 1607-17-6),
verapamil (Benzeneacetonitrile,
.alpha.-[3-[[2-(3,4-dimethoxyphenyl)ethyl]-methylamino]propyl]-3,4-dimeth-
oxy-.alpha.-(1-methylethyl)-CAS RN 52-53-9) and the like;
angiotensin II receptor antagonists such as, aprosartan,
zolasartan, olmesartan, pratosartan, FI6828K, RNH6270, candesartan
(1H-Benzimidazole-7-carboxylic acid,
2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]4-yl]methyl]-CAS
RN 139481-59-7), candesartan cilexctil
((+/-)-1-(cyclohexylcarbonyloxy)ethyl-2-ethoxy-[1-[[2'-(1H-tetrazol-5-yl)-
biphenyl-4-yl]-1H-benzimidazole carboxylate, CAS RN 145040-37-5,
U.S. Pat. No. 5,703,110 and U.S. Pat. No. 5,196,444), eprosartan
(3-[1-4-carboxyphenylmethyl)-2-n-butyl-imidazol-5-yl]-(2-thienylmethyl)
propenoic acid, U.S. Pat. No. 5,185,351 and U.S. Pat. No.
5,650,650), irbesartan (2-n-butyl-3-[[
2'-(1h-tetrazol-5-yl)biphenyl-4-yl]methyl]1,3-diazazspiro[4,4]non-1-en-4-o-
ne, U.S. Pat. No. 5,270,317 and U.S. Pat. No. 5,352,788), losartan
(2-N-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-y-
l)-methyl]imidazole, potassium salt, U.S. Pat. No. 5,138,069, U.S.
Pat. No. 5,153,197 and U.S. Pat. No. 5,128,355), tasosartan
(5,8-dihydro-2,4-dimethyl-8-[(2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]4-yl)me-
thyl]-pyrido[2,3-d]pyrimidin-7(6H)-one, U.S. Pat. No. 5,149,699),
telmisartan
(4'-[(1,4-dimethyl-2'-propyl-(2,6'-bi-1H-benzimidazol)-1'-yl)]-[1,1'-biph-
enyl]-2-carboxylic acid, CAS RN 144701-48-4, U.S. Pat. No.
5,591,762), milfasartan, abitesartan, valsartan (Diovan.RTM.
(Novartis),
(S)--N-valeryl-N-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]valine,
U.S. Pat. No. 5,399,578), EXP-3137
(2-N-butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidaz-
ole-5-carboxylic acid, U.S. Pat. No. 5,138,069, U.S. Pat. No.
5,153,197 and U.S. Pat. No. 5,128,355), 342%
(tetrazol-5-yl)-1,1'-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4-
,5-b]pyridine,
4'[2-ethyl-4-methyl-6-(5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-2-yl]-benz-
imidazol-1-yl]-methyl]-1,1'-biphenyl]-2-carboxylic acid,
2-butyl-6-(1-methoxy-1-methylethyl)-2-[2'-)]H-tetrazol-5-yl)biphenyl-4-yl-
methyl]quinazolin-4(3H)-one,
3-[2'-carboxybiphenyl-4-yl)methyl]-2-cyclopropyl-7-methyl-3H-imidazo[4,5--
b]pyridine,
2-butyl-4-chloro-1-[(2'-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole-carb-
oxylic acid,
2-butyl-4-chloro-1-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-1H-
-imidazole-5-carboxylic acid-1-(ethoxycarbonyl-oxy)ethyl ester
potassium salt, dipotassium
2-butyl-4-(methylthio)-1-[[2-[[[(propylamino)carbonyl]amino]-sulfonyl](1,-
1'-biphenyl)-4-yl]methyl]-1H-imidazole-5-carboxylate,
methyl-2-[[4-butyl-2-methyl-6-oxo-5-[[2'-(1H-tetrazol-5-yl)-[1,1'-bipheny-
l]-4-yl]methyl]-1-(6H)-pyrimidinyl]methyl]-3-thiophencarboxylate,
5-[(3,5-dibutyl-1H-1,2,4-triazol-1-yl)methyl]-2-[2-(1H-tetrazol-5-ylpheny-
l)]pyridine,
6-butyl-2-(2-phenylethyl)-5[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-methy-
l]pyrimidin-4-(3H)-one D,L lysine salt,
5-methyl-7-n-propyl-8-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-[1,2,4-
]-triazolo[1,5-c]pyrimidin-2(3H)-one,
2,7-diethyl-5-[[2'-(5-tetrazoly)biphenyl-4-yl]methyl]-5H-pyrazolo[1,5-b][-
1,2,4]triazole potassium salt,
2-[2-butyl-4,5-dihydro-4-oxo-3-[2'-(1H-tetrazol-5-yl)-4-biphenylmethyl]-3-
H-imidazol[4,5-c]pyridine-5-ylmethyl]benzoic acid, ethyl ester,
potassium salt,
3-methoxy-2,6-dimethyl-4-[[2'(1H-tetrazol-5-yl)-1,1'-biphenyl-4-yl]-
methoxy]pyridine,
2-ethoxy-1-[[2'-(5-oxo-2,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]met-
hyl]-1,4-benzimidazole-7-carboxylic acid,
1-[N-(2'-(1H-tetrazol-5-yl)biphenyl-4-yl-methyl)-N-valerolylaminomethyl)c-
yclopentane-1-carboxylic acid,
7-methyl-2n-propyl-3-[[2'1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidaz-
o[4,5-6]pyridine,
2-[5-[(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine-3-yl)methyl]-2-quin-
olinyl]sodium benzoate,
2-butyl-6-chloro-4-hydroxymethyl-5-methyl-3-[[2'-(1H-tetrazol-5-yl)biphen-
yl-4-yl]methyl]pyridine,
2-[[[2-butyl-1-[(4-carboxyphenyl)methyl]-1H-imidazol-5-yl]methyl]amino]be-
nzoic acid tetrazol-5-yl)biphenyl-4-yl]methyl]pyrimidin-6-one,
4(S)-[4-(carboxymethyl)phenoxy]-N-[2(R)-[4-(2-sulfobenzamido)imiduol-1-yl-
]octanoyl]-L-proline,
1-(2,6-dimethylphenyl)-4-butyl-1,3-dihydro-3-[[6-[2-(1H-tetrazol-5-yl)phe-
nyl]-3-pyridinyl]methyl]-2,1-imidazol-2-one,
5,8-ethano-5,8-dimethyl-2-n-propyl-5,6,7,8-tetrahydro-1-[[2'(1H-tetrazol--
5-yl)biphenyl-4-yl]methyl]-1H,4H-1,3,4a,8a-tetrazacyclopentanaphthalene-9--
one,
4-[1-[2'-(1,2,3,4-tetrazol-5-yl)biphen-4-yl)methylamino]-5,6,7,8-tetr-
ahydro-2-trifylquinazoline,
2-(2-chlorobenzoyl)imino-5-ethyl-3-[2'-(1H-tetrazole-5-yl)biphenyl-4-yl)m-
ethyl-1,3,4-thiadiazoline,
2-[5-ethyl-3-[2-(1H-tetrazole-5-yl)biphenyl-4-yl]methyl-1,3,4-thiazoline--
2-ylidene]aminocarbonyl-1-cyclopentencarboxylic acid dipotassium
salt, and
2-butyl-4-[N-methyl-N-(3-methylcrotonoyl)amino]-1-[[2'-(1H-tetrazol-5-yl)-
biphenyl-4-yl]methyl]-1H-imidzole-5-carboxylic acid
1-ethoxycarbonyloxyethyl ester, those disclosed in patent
publications EP475206, EP497150, EP539086, EP539713, EP535463,
EP535465, EP542059, EP497121, EP535420, EP407342, EP415886,
EP424317, EP435827, EP433983, EP475898, EP490820, EP528762,
EP324377, EP323841, EP420237, EP500297, EP426021, EP480204,
EP429257, EP430709, EP434249, EP446062, EP505954, EP524217,
EP514197, EP514198, EP514193, EP514192, EP450566, EP468372,
EP485929, EP503162, EP533058, EP467207 EP399731, EP399732,
EP412848, EP453210, EP456442, EP470794, EP470795, EP495626,
EP495627, EP499414, EP499416, EP499415, EP511791, EP516392,
EP520723, EP520724, EP539066, EP438869, EP505893, EP530702,
EP400835, EP400974, EP401030, EP407102, EP411766, EP409332,
EP412594, EP419048, EP480659, EP481614, EP490587, EP467715,
EP479479, EP502725, EP503838, EP505098, EP505111 EP513,979
EP507594, EP510812, EP511767, EP512675, EP512676, EP512870,
EP517357, EP537937, EP534706, EP527534, EP540356, EP461040,
EP540039, EP465368, EP498723, EP498722, EP498721, EP515265,
EP503785, EP501892, EP519831, EP532410, EP498361, EP432737,
EP504888, EP508393, EP508445, EP403159, EP403158, EP425211,
EP427463, EP437103, EP481448, EP488532, EP501269, EP500409,
EP540400, EP005528, EP028834, EP028833, EP411507, EP425921,
EP430300, EP434038, EP442473, EP443568, EP445811, EP459136,
EP483683, EP518033, EP520423, EP531876, EP531874, EP392317,
EP468470, EP470543, EP502314, EP529253, EP543263, EP540209,
EP449699, EP465323, EP521768, EP415594, WO92/14468, WO93/08171,
WO93/08169, WO91/00277, WO91/00281, WO91/14367, WO92/00067,
WO92/00977, WO92/20342, WO93/04045, WO93/04046, WO91/15206,
WO92/14714, WO92/09600, WO92/16552, WO93/05025, WO93/03018,
WO91/07404, WO92/02508, WO92/13853, WO91/19697, WO91/11909,
WO91/12001, WO91/11999, WO91/15209, WO91/15479, WO92/20687,
WO92/20662, WO92/20661, WO93/01177, WO91/14679, WO91/13063,
WO92/13564, WO91/17148, WO91/18888, WO91/19715, WO92/02257,
WO92/04335, WO92/05161, WO92/07852, WO92/15577, WO93/03033,
WO91/16313, WO92/00068, WO92/02510, WO92/09278, WO9210179,
WO92/10180, WO92/10186, WO92/10181, WO92/10097, WO92/10183,
WO92/10182, WO92/10187, WO92/10184, WO92/10188, WO92/10180,
WO92/10185, WO92/20651, WO93/03722, WO93/06828, WO93/03040,
WO92/19211, WO92/22533, WO92/06081, WO92/05784, WO93/00341,
WO92/04343, WO92/04059, U.S. Pat. No. 5,104,877, U.S. Pat. No.
5,187,168, U.S. Pat. No. 5,149,699, U.S. Pat. No. 5,185,340, U.S.
Pat. No. 4,880,804, U.S. Pat. No. 5,138,069, U.S. Pat. No.
4,916,129, U.S. Pat. No. 5,153,197, U.S. Pat. No. 5,173,494, U.S.
Pat. No. 5,137,906, U.S. Pat. No. 5,155,126, U.S. Pat. No.
5,140,037, U.S. Pat. No. 5,137,902, U.S. Pat. No. 5,157,026, U.S.
Pat. No. 5,053,329, U.S. Pat. No. 5,132,216, U.S. Pat. No.
5,057,522, U.S. Pat. No. 5,066,586, U.S. Pat. No. 5,089,626, U.S.
Pat. No. 5,049,565, U.S. Pat. No. 5,087,702, U.S. Pat. No.
5,124,335, U.S. Pat. No. 5,102,880, U.S. Pat. No. 5,128,327, U.S.
Pat. No. 5,151,435, U.S. Pat. No. 5,202,322, U.S. Pat. No.
5,187,159, U.S. Pat. No. 5,198,438, U.S. Pat. No. 5,182,288, U.S.
Pat. No. 5,036,048, U.S. Pat. No. 5,140,036, U.S. Pat. No.
5,087,634, U.S. Pat. No. 5,196,537, U.S. Pat. No. 5,153,347, U.S.
Pat. No. 5,191,086, U.S. Pat. No. 5,190,942, U.S. Pat. No.
5,177,097, U.S. Pat. No. 5,212,177, U.S. Pat. No. 5,208,234, U.S.
Pat. No. 5,208,235, U.S. Pat. No. 5,212,195, U.S. Pat. No.
5,130,439, U.S. Pat. No. 5,045,540, U.S. Pat. No. 5,041,152, and
U.S. Pat. No. 5,210,204, and pharmaceutically acceptable salts and
esters thereof; .alpha./.beta. adrenergic blockers such as
nipradilol, arotinolol, amosulalol, bretylium tosylate (CAS RN:
61-75-6), dihydroergtamine mesylate (such as ergotaman-3',
6',18-trione,9,-10-dihydro-12'-hydroxy-2'-methyl-5'-(phenylmethyl)-,(5'(.-
alpha.))-, monomethanesulfonate, e.g., DHE 45.RTM. Injection,
Novartis), carvedilol (such as
(.+-.)-1-(Carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propan-
ol, e.g., Coreg.RTM., SmithKline Beecham), labetalol (such as
5-[1-hydroxy-2-[(1-methyl-3-phenylpropyl) amino]ethyl]salicylamide
monohydrochloride, e.g., Normodyne.RTM., Schering), bretylium
tosylate (Benzenemethanaminium, 2-bromo-N-ethyl-N,N-dimethyl-, salt
with 4-methylbenzenesulfonic acid (1:1) CAS RN 61-75-6),
phentolamine mesylate (Phenol,
3-[[(4,5-dihydro-1H-imidazol-2-yl)methyl](4-methylphenyl)amino]-- ,
monomethanesulfonate (salt) CAS RN 65-28-1), solypertine tartrate
(5H-1,3-Dioxolo[4,5-f]indole,
7-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-,
(2R,3R)-2,3-dihydroxybutanedioate (1:1) CAS RN 5591-43-5),
zolertine hydrochloride (piperazine,
1-phenyl-4-[2-(1H-tetrazol-5-yl)ethyl]-, monohydrochloride (8Cl,
9Cl) CAS RN 7241-94-3) and the like; .alpha. adrenergic receptor
blockers, such as alfuzosin (CAS RN: 81403-68-1), terazosin,
urapidil, prazosin (Minipress.RTM.), tamsulosin, bunazosin,
trimazosin, doxazosin, naftopidil, indoramin, WHP 164, XEN010,
fenspiride hydrochloride (which may be prepared as disclosed in
U.S. Pat. No. 3,399,192), proroxan (CAS RN 33743-96-3), and
labetalol hydrochloride and combinations thereof; a 2 agonists such
as methyldopa, methyldopa HCL, lofcxidine, tiamenidine, moxonidine,
rilmenidine, guanobenz, and the like; aldosterone inhibitors, and
the like; renin inhibitors including Aliskiren (SPP100;
Novartis/Speedel); angiopoietin-2-binding agents such as those
disclosed in WO03/030833; anti-angina agents such as ranolazine
(hydrochloride1-piperazineacetamide,
N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-,
dihydrochloride CAS RN 95635-56-6), betaxolol hydrochloride
(2-Propanol, 1-[4-[2
(cyclopropylmethoxy)ethyl]phenoxy]-3-[(1-methylethyl)amino]-,
hydrochloride CAS RN 63659-19-8), butoprozine hydrochloride
(Methanone,
[4-[3(dibutylamino)propoxy]phenyl](2-ethyl-3-indolizinyl)-,
monohydrochloride CAS RN 62134-34-3), cinepazet
maleate1-Piperazineacetic acid,
4-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-, ethyl ester,
(2Z)-2-butenedioate (1:1) CAS RN 50679-07-7), tosifen
(Benzenesulfonamide,
4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]amino]carbonyl]-CAS RN
32295-184), verapamilhydrochloride (Benzeneacetonitrile,
.alpha.-[3-[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethox-
y-.alpha.-(1-methylethyl)-, monohydrochloride CAS RN 152-114),
molsidomine (1,2,3-Oxadiazolium,
5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, inner salt CAS RN
25717-80-0), and ranolazine hydrochloride. (1-piperazineacetamide,
N-(2,6-dimethylphenyl).sub.4-[2-hydroxy-3-(2-meth-oxyphenoxy)propyl]-,
dihydrochloride CAS RN 95635-56-6); tosifen (Benzenesulfonamide,
4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]amino]carbonyl]-CAS RN
32295-184); adrenergic stimulants such as guanfacine hydrochloride
(such as N-amidino-2-(2,6-dichlorophenyl) acetamide hydrochloride,
e.g., Tenex.RTM. Tablets available from Robins);
methyldopa-hydrochlorothiazide (such as
levo-3-(3,4-dihydroxyphenyl)-2-methylalanine) combined with
Hydrochlorothiazide (such as
6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide
1,1-dioxide, e.g., the combination as, e.g., Aldoril.RTM. Tablets
available from Merck), methyldopa-chlorothiazide (such as
6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide and
methyldopa as described above, e.g., Aldoclor.RTM., Merck),
clonidine hydrochloride (such as
2-(2,6-dichlorophenylamino)-2-imidazoline hydrochloride and
chlorthalidone (such as 2-chloro-5-(1-hydroxy-3-oxo-1-isoindolinyl)
benzenesulfonamide), e.g., Combipres.RTM., Boehringer Ingelheim),
clonidine hydrochloride (such as
2-(2,6-dichlorophenylamino)-2-imidazoline hydrochloride, e.g.,
Catapres.RTM., Boehringer Ingelheim), clonidine
(1H-Imidazol-2-amine, N-(2,6-dichlorophenyl)4,5-dihydro-CAS RN
4205-90-7), Hyzaar (Merck; a combination of losartan and
hydrochlorothiazide), Co-Diovan (Novartis; a combination of
valsartan and hydrochlorothiazide, Lotrel (Novartis; a combination
of benazepril and amlodipine) and Caduet (Pfizer; a combination of
amlodipine and atorvastatin), and those agents disclosed in
US20030069221.
[0324] The peptides and agonists described herein can be used in
combination therapy with one or more of the following agents useful
in the treatment of respiratory and other disorders including but
not limited to:
[0325] (1) .beta.-agonists including but not limited to: albuterol
(PROVENTIL.RTM., SALBUTAMO1.RTM., VENTOLIN.RTM.), bambuterol,
bitoterol, clenbuterol, fenoterol, formoterol, isoetharine
(BRONKOSOL.RTM., BRONKOMETER.RTM.), metaproterenol (ALUPENT.RTM.,
METAPREL.RTM.), pirbuterol (MAXAIR.RTM.), reproterol, rimiterol,
salmeterol, terbutaline (BRETHAIRE.RTM., BRETHINE.RTM.,
BRICANYL.RTM.), adrenalin, isoproterenol (ISUPREL.RTM.),
epinephrine bitartrate (PRIMATENE.RTM.), ephedrine, orciprenline,
fenoterol and isoetharine;
[0326] (2) steroids, including but not limited to beclomethasone,
beclomethasone dipropionate, betamethasone, budesonide, bunedoside,
butixocort, dexamethasone, flunisolide, fluocortin, fluticasone,
hydrocortisone, methyl prednisone, mometasone, predonisolone,
predonisone, tipredane, tixocortal, triamcinolone, and
triamcinolone acetonide;
[0327] (3) .beta.2-agonist-corticosteroid combinations [e.g.,
salmeterol-fluticasone (ADVAIR.RTM.), formoterol-budesonid
(SYMBICORT.RTM.)];
[0328] (4) leukotriene D4 receptor antagonists/leukotriene
antagonists/LTD4 antagonists (i.e., any compound that is capable of
blocking, inhibiting, reducing or otherwise interrupting the
interaction between leukotrienes and the Cys LTI receptor)
including but not limited to: zafirlukast, montelukast, montelukast
sodium (SINGULAIR.RTM.), pranlukast, iralukast, pobilukast,
SKB-106,203 and compounds described as having LTD4 antagonizing
activity described in U.S. Pat. No. 5,565,473;
[0329] (5) 5-lipoxygenase inhibitors and/or leukotriene
biosynthesis inhibitors [e.g., zileuton and BAY1005 (CA registry
128253-31-6)];
[0330] (6) histamine H1 receptor antagonists/antihistamines (i.e.,
any compound that is capable of blocking, inhibiting, reducing or
otherwise interrupting the interaction between histamine and its
receptor) including but not limited to: astemizole, acrivastine,
antazoline, azatadine, azelastine, astamizole, bromopheniramine,
bromopheniramine maleate, carbinoxamine, carebastine, cetirizine,
chlorpheniramine, chloropheniramine maleate, cimetidine,
clemastine, cyclizine, cyproheptadine, descarboethoxyloratadine,
dexchlorpheniramine, dimethindene, diphenhydramine,
diphenylpyraline, doxylamine succinate, doxylamine, ebastine,
efletirizine, epinastine, famotidine, fexofenadine, hydroxyzine,
hydroxyzine, ketotifen, levocabastine, levocetirizine,
levocetirizine, loratadine, meclizine, mepyramine, mequitazine,
methdilazine, mianserin, mizolastine, noberastine, norasternizole,
noraztemizole, phenindamine, pheniramine, picumast, promethazine,
pynlamine, pyrilamine, ranitidine, temelastine, terfenadine,
trimeprazine, tripelenamine, and triprolidine;
[0331] (7) an anticholinergic including but not limited to:
atropine, benztropine, biperiden, flutropium, hyoscyamine (e.g.
Levsin.RTM.; Levbid.RTM.; Levsin/SL.RTM., Anaspaz.RTM., Levsinex
Timecaps.RTM., NuLev.RTM.), ilutropium, ipratropium, ipratropium
bromide, methscopolamine, oxybutinin, rispenzepine, scopolamine,
and tiotropium;
[0332] (8) an anti-tussive including but not limited to:
dextromethorphan, codeine, and hydromorphone;
[0333] (9) a decongestant including but not limited to:
pseudoephedrine and phenylpropanolamine;
[0334] (10) an expectorant including but not limited to:
guafenesin, guaicolsulfate, terpin, ammonium chloride, glycerol
guaicolate, and iodinated glycerol;
[0335] (11) a bronchodilator including but not limited to:
theophylline and aminophylline;
[0336] (12) an anti-inflammatory including but not limited to:
fluribiprofen, diclophenac, indomethacin, ketoprofen,
S-ketroprophen, tenoxicam;
[0337] (13) a PDE (phosphodiesterase) inhibitor including but not
limited to those disclosed herein;
[0338] (14) a recombinant humanized monoclonal antibody [e.g.
xolair (also called omalizumab), rhuMab, and talizumab];
[0339] (15) a humanized lung surfactant including recombinant forms
of surfactant proteins SP-B, SP-C or SP-D [e.g. SURFAXIN.RTM.,
formerly known as dsc-104 (Discovery Laboratories)],
[0340] (16) agents that inhibit epithelial sodium channels (ENaC)
such as amiloride and related compounds;
[0341] (17) antimicrobial agents used to treat pulmonary infections
such as acyclovir, amikacin, amoxicillin, doxycycline, trimethoprin
sulfamethoxazole, amphotericin B, azithromycin, clarithromycin,
roxithromycin, clarithromycin, cephalosporins (ceffoxitin,
cefmetazole etc), ciprofloxacin, ethambutol, gentimycin,
ganciclovir, imipenem, isoniazid, itraconazole, penicillin,
ribavirin, rifampin, rifabutin, amantadine, rimantidine,
streptomycin, tobramycin, and vancomycin;
[0342] (18) agents that activate chloride secretion through Ca++
dependent chloride channels (such as purinergic receptor (P2Y(2)
agonists);
[0343] (19) agents that decrease sputum viscosity, such as human
recombinant DNasc 1, (Pulmozyme.RTM.);
[0344] (20) nonsteroidal anti-inflammatory agents (acemetacin,
acetaminophen, acetyl salicylic acid, alclofenac, alminoprofen,
apazone, aspirin, benoxaprofen, bezpiperylon, bucloxic acid,
carprofen, clidanac, diclofenac, diclofenac, diflunisal,
diflusinal, etodolac, fenbufen, fenbufen, fenclofenac, fenclozic
acid, fenoprofen, fentiazac, feprazone, flufenamic acid,
flufenisal, flufenisal, fluprofen, flurbiprofen, flurbiprofen,
furofenac, ibufenac, ibuprofen, indomethacin, indomethacin,
indoprofen, isoxepac, isoxicam, ketoprofen, ketoprofen, ketorolac,
meclofenamic acid, meclofenamic acid, mefenamic acid, mefenamic
acid, miroprofen, mofebutazone, nabumetone oxaprozin, naproxen,
naproxen, niflumic acid, oxaprozin, oxpinac, oxyphenbutazone,
phenacetin, phenylbutazone, phenylbutazone, piroxicam, piroxicam,
pirprofen, pranoprofen, sudoxicam, tenoxican, sulfasalazine,
sulindac, sulindac, suprofen, tiaprofenic acid, tiopinac,
tioxaprofen, tolfenamic acid, tolmetin, tolmetin, zidometacin,
zomepirac, and zomepirac); and
[0345] (21) aerosolized antioxidant therapeutics such as
S-Nitrosoglutathione.
[0346] The peptides and agonists described herein can be used in
combination therapy with an anti-obesity agent. Suitable such
agents include, but are not limited to:
11.beta. HSD-1 (11-beta hydroxy steroid dehydrogenase type 1)
inhibitors, such as BVT 3498, BVT 2733,
3-(1-adamantyl)-4-ethyl-5-(ethylthio)-4H-1,2,4-triazole,
adamantyl)-5-(3,4,5-trimethoxyphenyl)-4-methyl-4H-1,2,4-triazole,
3-adamantanyl-4,5,6,7,8,9,10,11,12,3a-decahydro-1,2,4-triazolo[4,3-a][11]-
annulene, and those compounds disclosed in WO01/90091, WO01/90090,
WO01/90092 and WO02/072084; 5HT antagonists such as those in
WO03/037871, WO03/037887, and the like; 5HT1a modulators such as
carbidopa, benserazide and those disclosed in U.S. Pat. No.
6,207,699, WO03/031439, and the like; 5HT2c (serotonin receptor 2c)
agonists, such as BVT933, DPCA37215, IK264, PNU 22394, WAY161503,
R-1065, SB 243213 (Glaxo Smith Kline) and YM 348 and those
disclosed in U.S. Pat. No. 3,914,250, WO00/77010, WO02/36596,
WO02/48124, WO02/10169, WO01/66548, WO02/44152, WO02/51844,
WO02/40456, and WO02/40457; 5HT6 receptor modulators, such as those
in WO03/030901, WO03/035061, WO03/039547, and the like;
acyl-estrogens, such as oleoyl-estrone, disclosed in del Mar-Grasa,
M. et al., Obesity Research, 9:202-9 (2001) and Japanese Patent
Application No. JP 2000256190; anorectic bicyclic compounds such as
1426 (Aventis) and 1954 (Aventis), and the compounds disclosed in
WO00/18749, WO01/32638, WO01/62746, WO01/62747, and WO03/015769; CB
1 (cannabinoid-1 receptor) antagonist/inverse agonists such as
rimonabant (Acomplia; Sanofi), SR-147778 (Sanofi), SR-141716
(Sanofi), BAY 65-2520 (Bayer), and SLV 319 (Solvay), and those
disclosed in patent publications U.S. Pat. No. 4,973,587, U.S. Pat.
No. 5,013,837, U.S. Pat. No. 5,081,122, U.S. Pat. No. 5,112,820,
U.S. Pat. No. 5,292,736, U.S. Pat. No. 5,532,237, U.S. Pat. No.
5,624,941, U.S. Pat. No. 6,028,084, U.S. Pat. No. 6,509,367, U.S.
Pat. No. 6,509,367, WO96/33159, WO97/29079, WO98/31227, WO98/33765,
WO98/37061, WO98/41519, WO98/43635, WO98/43636, WO99/02499,
WO00/10967, WO00/10968, WO01/09120, WO01/58869, WO01/64632,
WO01/64633, WO01/64634, WO01/70700, WO01/96330, WO02/076949,
WO03/006007, WO03/007887, WO03/020217, WO03/026647, WO03/026648,
WO03/027069, WO03/027076, WO03/027114, WO03/037332, WO03/040107,
WO03/086940, WO03/084943 and EP658546; CCK-A (cholecystokinin-A)
agonists, such as AR-R 15849, GI 181771 (GSK), JMV-180, A-71378,
A-71623 and SR146131 (Sanofi), and those described in U.S. Pat. No.
5,739,106; CNTF (Ciliary neurotrophic factors), such as GI-181771
(Glaxo-SmithKline), SR146131 (Sanofi Synthelabo), butabindide,
PD170,292, and PD 149164 (Pfizer); CNTF derivatives, such as
Axokine.RTM. (Regeneron), and those disclosed in WO94/09134,
WO98/22128, and WO99/43813; dipeptidyl peptidase IV (DP-IV)
inhibitors, such as isoleucine thiazolidide, valine pyrrolidide,
NVP-DPP728, LAF237, P93/01, P 3298, TSL 225
(tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;
disclosed by Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998)
1537-1540), TMC-2A/2B/2C, CD26 inhibtors, FE 999011, P9310/K364,
VIP 0177, SDZ 274-444, 2-cyanopyrrolidides and 4-cyanopyrrolidides
as disclosed by Ashworth et al, Bioorg. & Med. Chem. Lett.,
Vol. 6, No. 22, pp 1163-1166 and 2745-2748 (1996) and the compounds
disclosed patent publications. WO99/38501, WO99/46272, WO99/67279
(Probiodrug), WO99/67278 (Probiodrug), WO99/61431 (Probiodrug),
WO02/083128, WO02/062764, WO03/000180, WO03/000181, WO03/000250,
WO03/002530, WO03/002531, WO03/002553, WO03/002593, WO03/004498,
WO03/004496, WO03/017936, WO03/024942, WO03/024965, WO03/033524,
WO03/037327 and EP1258476; growth hormone secretagogue receptor
agonists/antagonists, such as NN7O.sub.3, hexarelin, MK-0677
(Merck), SM-130686, CP-424391 (Pfizer), LY 444,711 (Eli Lilly),
L-692,429 and L-163,255, and such as those disclosed in U.S. Ser.
No. 09/662,448, U.S. provisional application 60/203,335, U.S. Pat.
No. 6,358,951, US2002049196, US2002/022637, WO01/56592 and
WO02/32888; H3 (histamine H3) antagonist/inverse agonists, such as
thioperamide, 3-(1H-imidazol-4-yl)propyl N-(4-pentenyl)carbamate),
clobenpropit, iodophenpropit, imoproxifan, GT2394 (Gliatech), and
A331440, O-[3-(1H-imidazol-4-yl)propanol]carbamates
(Kiec-Kononowicz, K. et al., Pharmazie, 55:349-55 (2000)),
piperidine-containing histamine H3-receptor antagonists (Lazewska,
D. et al., Pharmazie, 56:927-32 (2001), benzophenone derivatives
and related compounds (Sasse, A. et al., Arch. Pharm. (Weinheim)
334:45-52 (2001)), substituted N-phenylcarbamates (Reidemeister, S.
et al., Pharmazie, 55:83-6 (2000)), and proxifan derivatives
(Sasse, A. et al., J. Med. Chem. 43:3335-43 (2000)) and histamine
H3 receptor modulators such as those disclosed in WO02/15905,
WO03/024928 and WO03/024929; leptin derivatives, such as those
disclosed in U.S. Pat. No. 5,552,524, U.S. Pat. No. 5,552,523, U.S.
Pat. No. 5,552,522, U.S. Pat. No. 5,521,283, WO96/23513,
WO96/23514, WO96/23515, WO96/23516, WO96/23517, WO96/23518,
WO96/23519, and WO96/23520; leptin, including recombinant human
leptin (PEG-OB, Hoffman La Roche) and recombinant methionyl human
leptin (Amgen); lipase inhibitors, such as tetrahydrolipstatin
(orlistat/Xenical), Triton WR1339, RHC80267, lipstatin, teasaponin,
diethylumbelliferyl phosphate, FL-386, WAY-121898, Bay-N-3176,
valilactone, esteracin, ebelactone A, ebelactone B, and RHC 80267,
and those disclosed in patent publications WO01/77094, U.S. Pat.
No. 4,598,089, U.S. Pat. No. 4,452,813, U.S. Pat. No. 5,512,565,
U.S. Pat. No. 5,391,571, U.S. Pat. No. 5,602,151, U.S. Pat. No.
4,405,644, U.S. Pat. No. 4,189,438, and U.S. Pat. No. 4,242,453;
lipid metabolism modulators such as maslinic acid, erythrodiol,
ursolic acid uvaol, betulinic acid, betulin, and the like and
compounds disclosed in WO03/011267; Mc4r (melanocortin 4 receptor)
agonists, such as CHIR86036 (Chiron), ME-10142, ME-10145, and
HS-131 (Melacure), and those disclosed in PCT publication Nos.
WO99/64002, WO00/74679, WO01/991752, WO01/25192, WO01/52880,
WO01/74844, WO01/70708, WO01/70337, WO01/91752, WO02/059095,
WO02/059107, WO02/059108, WO02/059117, WO02/06276, WO02/12166,
WO02/11715, WO02/12178, WO02/15909, WO02/38544, WO02/068387,
WO02/068388, WO02/067869, WO02/081430, WO03/06604, WO03/007949,
WO03/009847, WO03/009850, WO03/013509, and WO03/031410; Mc5r
(melanocortin 5 receptor) modulators, such as those disclosed in
NV097/19952, WO00/15826, WO00/15790, US20030092041;
melanin-concentrating hormone 1 receptor (MCHR) antagonists, such
as T-226296 (Takeda), SB 568849, SNP-7941 (Synaptic), and those
disclosed in patent publications WO01/21169, WO01/82925,
WO01/87834, WO02/051809, WO02/06245, WO02/076929, WO02/076947,
WO02/04433, WO02/51809, WO02/083134, WO02/094799, WO03/004027,
WO03/13574, WO03/15769, WO03/028641, WO03/035624, WO03/033476,
WO03/033480, JP13226269, and JP1437059; mGluR5 modulators such as
those disclosed in WO03/029210, WO03/047581, WO03/048137,
WO03/051315, WO03/051833, WO03/053922, WO03/059904, and the like;
serotoninergic agents, such as fenfluramine (such as Pondimin.RTM.
(Benzeneethanamine, N-ethyl-alpha-methyl-3-(trifluoromethyl)-,
hydrochloride), Robbins), dexfenfluramine (such as Redux.RTM.
(Benzeneethanamine, N-ethyl-alpha-methyl-3-(trifluoromethyl)-,
hydrochloride), Interneuron) and sibutramine ((Meridia.RTM.,
Knoll/Reductil.TM.) including racemic mixtures, as optically pure
isomers (+) and (-), and pharmaceutically acceptable salts,
solvents, hydrates, clathrates and prodrugs thereof including
sibutramine hydrochloride monohydrate salts thereof, and those
compounds disclosed in U.S. Pat. No. 4,746,680, U.S. Pat. No.
4,806,570, and U.S. Pat. No. 5,436,272, US20020006964, WO01/27068,
and WO01/62341; NE (norepinephrine) transport inhibitors, such as
GW 320659, despiramine, talsupram, and nomifensine; NPY 1
antagonists, such as B1BP3226, J-115814, BIBO 3304, LY-357897,
CP-671906, GI-264879A, and those disclosed in U.S. Pat. No.
6,001,836, WO96/14307, WO01/23387, WO99/51600, WO01/85690,
WO01/85098, WO01/85173, and WO01/89528; NPY5 (neuropeptide Y Y5)
antagonists, such as 152,804, GW-569180A, GW-594884A, GW-587081X,
GW-548118X, FR235208, FR226928, FR240662, FR252384, 1229U91,
GI-264879A, CGP71683A, LY-377897, LY-366377, PD-160170, SR-120562A,
SR-120819A, JCF-104, and H409/22 and those compounds disclosed in
patent publications U.S. Pat. No. 6,140,354, U.S. Pat. No.
6,191,160, U.S. Pat. No. 6,218,408, U.S. Pat. No. 6,258,837, U.S.
Pat. No. 6,313,298, U.S. Pat. No. 6,326,375, U.S. Pat. No.
6,329,395, U.S. Pat. No. 6,335,345, U.S. Pat. No. 6,337,332, U.S.
Pat. No. 6,329,395, U.S. Pat. No. 6,340,683, EP01010691,
EP-01044970, WO97/19682, WO97/20820, WO97/20821, WO97/20822,
WO97/20823, WO98/27063, WO00/107409, WO00/185714, WO00/185730,
WO00/64880, WO00/68197, WO00/69849, WO/0113917, WO01/09120,
WO01/14376, WO01/85714, WO01/85730, WO01/07409, WO01/02379,
WO01/23388, WO01/23389, WO01/44201, WO01/62737, WO01/62738,
WO01/09120, WO02/20488, WO02/22592, WO02/48152, WO02/49648,
WO02/051806, WO02/094789, WO03/009845, WO03/014083, WO03/022849,
WO03/028726 and Norman et al., J. Med. Chem. 43:4288-4312 (2000);
opioid antagonists, such as nalmefene (REVEX.RTM.),
3-methoxynaltrexone, methylnaltrexone, naloxone, and naltrexone
(e.g. PT901; Pain Therapeutics, Inc.) and those disclosed in U.S.
Pat. No. 6,734,188, US20050004155 and WO00/21509; orexin
antagonists, such as SB-334867-A and those disclosed in patent
publications WO01/96302, WO01/68609, WO02/44172, WO02/51232,
WO02/51838, WO02/089800, WO02/090355, WO03/023561, WO03/032991, and
WO03/037847; PDE inhibitors (e.g. compounds which slow the
degradation of cyclic AMP (cAMP) and/or cyclic GMP (cGMP) by
inhibition of the phosphodiesterases, which can lead to a relative
increase in the intracellular concentration of cAMP and cGMP;
possible PDE inhibitors are primarily those substances which are to
be numbered among the class consisting of the PDE3 inhibitors, the
class consisting of the PDE4 inhibitors and/or the class consisting
of the PDE5 inhibitors, in particular those substances which can be
designated as mixed types of PDE3/4 inhibitors or as mixed types of
PDE3/4/5 inhibitors) such as those disclosed in patent publications
DE1470341, DE2108438, DE2123328, DE2305339, DE2305575, DE2315801,
DE2402908, DE2413935, DE2451417, DE2459090, DE2646469, DE2727481,
DE2825048, DE2837161, DE2845220, DE2847621, DE2934747, DE3021792,
DE3038166, DE3044568, EP000718, EP0008408, EP0010759, EP0059948,
EP0075436, EP0096517, EP0112987, EP0116948, EP0150937, EP0158380,
EP0161632, EP0161918, EP0167121, EP0199127, EP0220044, EP0247725,
EP0258191, EP0272910, EP0272914, EP0294647, EP0300726, EP0335386,
EP0357788, EP0389282, EPO406958, EPO426180, EPO428302, EPO435811,
EPO470805, EPO482208, EPO490823, EP0506194, EP0511865, EP0527117,
EP0626939, EP0664289, EP0671389, EP0685474, EP0685475, EP0685479,
JP92234389, JP94329652, JP95010875, U.S. Pat. No. 4,963,561, U.S.
Pat. No. 5,141,931, WO9117991, WO9200968, WO9212961, WO9307146,
WO9315044, WO9315045, WO9318024, WO9319068, WO9319720, WO9319747,
WO9319749, WO9319751, WO9325517, WO9402465, WO9406423, WO9412461,
WO9420455, WO9422852, WO9425437, WO9427947, WO9500516, WO9501980,
WO9503794, WO9504045, WO9504046, WO9505386, WO9508534, WO9509623,
WO9509624, WO9509627, WO9509836, WO9514667, WO9514680, WO9514681,
WO9517392, WO9517399, WO9519362, WO9522520, WO9524381, WO9527692,
WO9528926, WO9535281, WO9535282, WO9600218, WO9601825, WO9602541,
WO9611917, DE3142982, DE1116676, DE2162096, EP0293063, EPO463756,
EPO482208, EP0579496, EP0667345 U.S. Pat. No. 6,331,543,
US20050004222 (including those disclosed in formulas I-XIII and
paragraphs 37-39, 85-0545 and 557-577), WO9307124, EP0163965,
EP0393500, EP0510562, EP0553174, WO9501338 and WO9603399, as well
as PDE5 inhibitors (such as RX-RA-69, SCH-51866, KT-734,
vesnarinone, zaprinast, SKF-96231, ER-21355, BF/GP-385, NM-702 and
sildenafil (Viagra.TM.)), PDE4 inhibitors (such as etazolate,
IC163197, RP73401, imazolidinone (RO-20-1724), MEM 1414
(R1533/R1500; Pharmacia Roche), denbufylline, rolipram, oxagrelate,
nitraquazone, Y-590, DH-6471, SKF-94120, motapizone, lixazinone,
indolidan, olprinone, atizoram, KS-506-G, dipamfylline, BMY-43351,
atizoram, arofylline, filaminast, PDB-093, UCB-29646, CDP-840,
SKF-107806, piclamilast, RS-17597, RS-25344-000, SB-207499,
TIBENELAST, SB-210667, SB-211572, SB-211600, SB-212066, SB-212179,
GW-3600, CDP-840, mopidamol, anagrelide, ibudilast, aminone,
pimobendan, cilostazol, quazinone and
N-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy-4-difluoromethoxybenzamid-
e, PDE3 inhibitors (such as ICI153, 100, bemorandane (RWJ 22867),
MC1-154, UD-CG 212, sulmazole, ampizone, cilostamide, carbazeran,
piroximone, imazodan, CI-930, siguazodan, adibendan, saterinone,
SKF-95654, SDZ-MKS-492, 349-U-85, emoradan, EMD-53998, EMD-57033,
NSP-306, NSP-307, revizinone, NM-702, WIN-62582 and WIN-63291,
enoximone and milrinone, PDE3/4 inhibitors (such as benafentrine,
trequinsin, ORG-30029, zardaverine, L-686398, SDZ-ISQ-844,
ORG-20241, EMD-54622, and tolafentrine) and other PDE inhibitors
(such as vinpocetin, papaverine, enprofylline, cilomilast,
fenoximone, pentoxifylline, roflumilast, tadalafil (Cialis.RTM.),
theophylline, and vardenafil (Levitra.RTM.); Neuropeptide Y2 (NPY2)
agonists include but are not limited to: peptide YY and fragments
and variants thereof (e.g. YY3-36 (PYY3-36) (N. Engl. J. Med.
349:941, 2003; IKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY (SEQ ID
NO:XXX)) and PYY agonists such as those disclosed in WO02/47712,
WO03/026591, WO03/057235, and WO03/027637; serotonin reuptake
inhibitors, such as, paroxetine, fluoxetine (Prozac.TM.),
fluvoxamine, sertraline, citalopram, and imipramine, and those
disclosed in U.S. Pat. No. 6,162,805, U.S. Pat. No. 6,365,633,
WO03/00663, WO01/27060, and WO01/162341; thyroid hormone .beta.
agonists, such as KB-2611 (KaroBioBMS), and those disclosed in
WO02/15845, WO97/21993, WO99/00353, GB98/284425, U.S. Provisional
Application No. 60/183,223, and Japanese Patent Application No. JP
2000256190; UCP-1 (uncoupling protein-1), 2, or 3 activators, such
as phytanic acid,
4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1-propeny-
l]benzoic acid (TTNPB), retinoic acid, and those disclosed in
WO99/00123; .beta.3 (beta adrenergic receptor 3) agonists, such as
AJ9677/TAK677 (Dainippon/Takeda), L750355 (Merck), CP331648
(Pfizer), CL-316,243, SB 418790, BRL-37344, L-796568, BMS-196085,
BRL-35135A, CGP12177A, BTA-243, GW 427353, Trecadrine, Zeneca
D7114, N-5984 (Nisshin Kyorin), LY-377604 (Lilly), SR 59119A, and
those disclosed in U.S. Pat. No. 5,541,204, U.S. Pat. No.
5,770,615, U.S. Pat. No. 5,491,134, U.S. Pat. No. 5,776,983, U.S.
Pat. No. 488,064, U.S. Pat. No. 5,705,515, U.S. Pat. No. 5,451,677,
WO94/18161, WO95/29159, WO97/46556, WO98/04526 and WO98/32753,
WO01/74782, WO02/32897, WO03/014113, WO03/016276, WO03/016307,
WO03/024948, WO03/024953 and WO03/037881; noradrenergic agents
including, but not limited to, diethylpropion (such as Tenuate.RTM.
(1-propanone, 2-(diethylamino)-1-phenyl-, hydrochloride), Merrell),
dextroamphetamine (also known as dextroamphetamine sulfate,
dexamphetamine, dexedrine, Dexampex, Ferndex, Oxydess II, Robese,
Spancap #1), mazindol ((or
5-(p-chlorophenyl)-2,5-dihydro-3H-imidazo[2,1-a]isoindol-5-ol) such
as Sanorex
.RTM., Novartis or Mazanor.RTM., Wyeth Ayerst), phenylpropanolamine
(or Benzenemethanol, alpha-(1-aminoethyl)-, hydrochloride),
phentermine ((or Phenol,
3-[[4,5-duhydro-1H-imidazol-2-yl)ethyl](4-methylpheny-1)amino],
monohydrochloride) such as Adipex-P.RTM., Lemmon, FASTIN.RTM.,
Smith-Kline Beecham and lonamin.RTM., Medeva), phendimetrazine ((or
(2S,3S)-3,4-Dimethyl-2-phenylmorpholine L-(+)-tartrate (1:1)) such
as Metra.RTM. (Forest), Plegine.RTM. (Wyeth-Ayerst), Prelu-2.RTM.
(Boehringer Ingelheim), and Statobex.RTM. (Lemmon), phendamine
tartrate (such as Thephorin.RTM.
(2,3,4,9-Tetrahydro-2-methyl-9-phenyl-1H-indenol[2,1-c]pyridine
L-(+)-tartrate (1:1)), Hoffmann-LaRoche), methamphetamine (such as
Desoxyn.RTM., Abbot ((S)--N, (alpha)-dimethylbenzeneethanamine
hydrochloride)), and phendimetrazine tartrate (such as Bontril.RTM.
Slow-Release Capsules, Amarin (-3,4-Dimethyl-2-phenylmorpholine
Tartrate); fatty acid oxidation upregulator/inducers such as
Famoxin.RTM. (Genset); monamine oxidase inhibitors including but
not limited to befloxatonc, moclobemide, brofaromine, phenoxathine,
esuprone, befol, toloxatone, pirlindol, amiflamine, sercloremine,
bazinaprine, lazabemide, milacemide, caroxazone and other certain
compounds as disclosed by WO01/12176; and other anti-obesity agents
such as 5HT-2 agonists, ACC (acetyl-CoA carboxylase) inhibitors
such as those described in WO03/072197, alpha-lipoic acid
(alpha-LA), AOD9604, appetite suppressants such as those in
WO03/40107, ATL-962 (Alizyme PLC), benzocaine, benzphetamine
hydrochloride (Didrex), bladderwrack (focus vesiculosus), BRS3
(bombesin receptor subtype 3) agonists, bupropion, caffeine, CCK
agonists, chitosan, chromium, conjugated linoleic acid,
corticotropin-releasing hormone agonists, dehydroepiandrosterone,
DGAT1 (diacylglycerol acyltransferase 1) inhibitors, DGAT2
(diacylglycerol acyltransferase 2) inhibitors, dicarboxylate
transporter inhibitors, ephedra, exendin-4 (an inhibitor of glp-1)
FAS (fatty acid synthase) inhibitors (such as Cerulenin and C75),
fat resorption inhibitors (such as those in WO03/053451, and the
like), fatty acid transporter inhibitors, natural water soluble
fibers (such as psyllium, plantago, guar, oat, pectin), galanin
antagonists, galega (Goat's Rue, French Lilac), garcinia cambogia,
germander (teucrium chamaedrys), ghrelin antibodies and ghrelin
antagonists (such as those disclosed in WO01/87335, and
WO02/08250), peptide hormones and variants thereof which affect the
islet cell secretion, such as the hormones of the secretin/gastric
inhibitory peptide (GIP)/vasoactive intestinal peptide
(VIP)/pituitary adenylate cyclase activating peptide
(PACAP)/glucagon-like peptide II (GLP-II)/glicentin/glucagon gene
family and/or those of the adrenomedullin/amylin/calcitonin gene
related peptide (CGRP) gene family including GLP-1 (glucagon-like
peptide 1) agonists (e.g. (1) exendin-4, (2) those GLP-1 molecules
described in US20050130891 including GLP-1(7-34), GLP-1(7-35),
GLP-1(7-36) or GLP-1(7-37) in its C-terminally carboxylated or
amidated form or as modified GLP-1 peptides and modifications
thereof including those described in paragraphs 17-44 of
US20050130891, and derivatives derived from GLP-1-(7-34)COOH and
the corresponding acid amide are employed which have the following
general formula:
R--NH-HAEGTFTSDVSYLEGQAAKEFIAWLVK-CONH.sub.2
wherein R.dbd.H or an organic compound having from 1 to 10 carbon
atoms. Preferably, R is the residue of a carboxylic acid.
Particularly preferred are the following carboxylic acid residues:
formyl, acetyl, propionyl, isopropionyl, methyl, ethyl, propyl,
isopropyl, n-butyl, sec-butyl, tert-butyl.) and glp-1
(glucagon-like peptide-1), glucocorticoid antagonists, glucose
transporter inhibitors, growth hormone secretagogues (such as those
disclosed and specifically described in U.S. Pat. No. 5,536,716),
interleukin-6 (IL-6) and modulators thereof (as in WO03/057237, and
the like), L-carnitine, Mc3r (melanocortin 3 receptor) agonists,
MCH2R (melanin concentrating hormone 2R) agonist/antagonists,
melanin concentrating hormone antagonists, melanocortin agonists
(such as Melanotan II or those described in WO 99/64002 and WO
00/74679), nomame herba, phosphate transporter inhibitors,
phytopharm compound 57 (CP 644,673), pyruvate, SCD-1 (stearoyl-CoA
desaturase-1) inhibitors, T71 (Tularik, Inc., Boulder Colo.),
Topiramate (Topimax.RTM., indicated as an anti-convulsant which has
been shown to increase weight loss), transcription factor
modulators (such as those disclosed in WO03/026576), .beta.-hydroxy
steroid dehydrogenase-1 inhibitors (.beta.-HSD-1),
.beta.-hydroxy-.beta.-methylbutyrate, p57 (Pfizer), Zonisamide
(Zonegran.TM., indicated as an anti-epileptic which has been shown
to lead to weight loss), and the agents disclosed in US20030119428
paragraphs 20-26.
[0347] The peptides and agonists described herein can be used in
therapeutic combination with one or more anti-diabetic agents,
including but not limited to:
PPAR.gamma. agonists such as glitazones (e.g., WAY-120,744, AD
5075, balaglitazone, ciglitazone, darglitazone (CP-86325, Pfizer),
englitazone (CP-68722, Pfizer), isaglitazone (MIT/J&J), MCC-555
(Mitsibishi disclosed in U.S. Pat. No. 5,594,016), pioglitazone
(such as such as Actos.TM. pioglitazone; Takeda), rosiglitazone
(Avandia.TM.; Smith Kline Beecham), rosiglitazone maleate,
troglitazone (Rezulin.RTM., disclosed in U.S. Pat. No. 4,572,912),
rivoglitazone (CS-011, Sankyo), GL-262570 (Glaxo Welcome), BRL49653
(disclosed in WO98/05331), CLX-0921, 5-BTZD, GW-0207, LG-100641,
JJT-501 (JPNT/P&U), L-895645 (Merck), R-119702 (Sankyo/Pfizer),
NN-2344 (Dr. Reddy/NN), YM-440 (Yamanouchi), LY-300512, LY-519818,
8483 (Roche), T131 (Tularik), and the like and compounds disclosed
in U.S. Pat. No. 4,687,777, U.S. Pat. No. 5,002,953, U.S. Pat. No.
5,741,803, U.S. Pat. No. 5,965,584, U.S. Pat. No. 6,150,383, U.S.
Pat. No. 6,150,384, U.S. Pat. No. 6,166,042, U.S. Pat. No.
6,166,043, U.S. Pat. No. 6,172,090, U.S. Pat. No. 6,211,205, U.S.
Pat. No. 6,271,243, U.S. Pat. No. 6,288,095, U.S. Pat. No.
6,303,640, U.S. Pat. No. 6,329,404, U.S. Pat. No. 5,994,554,
WO97/10813, WO97/27857, WO97/28115, WO97/28137, WO97/27847,
WO00/76488, WO03/000685, WO03/027112, WO03/035602, WO03/048130,
WO03/055867, and pharmaceutically acceptable salts thereof;
biguanidcs such as metformin hydrochloride
(N,N-dimethylimidodicarbonimidic diamide hydrochloride, such as
Glucophage.TM., Bristol-Myers Squibb); metformin hydrochloride with
glyburide, such as Glucovance.TM., Bristol-Myers Squibb); buformin
(Imidodicarbonimidic diamide, N-butyl-); etoforminc
(1-Butyl-2-ethylbiguanide, Schering A. G.); other metformin salt
forms (including where the salt is chosen from the group of,
acetate, benzoate, citrate, ftimarate, embonate,
chlorophenoxyacetate, glycolate, palmoate, aspartate,
methanesulphonate, maleate, parachlorophenoxyisobutyrate, formate,
lactate, succinate, sulphate, tartrate, cyclohexanecarboxylate,
hexanoate, octanoate, decanoate, hexadecanoate, octodecanoate,
benzenesulphonate, trimethoxybenzoate, paratoluenesulphonate,
adamantanecarboxylate, glycoxylate, glutamate,
pyrrolidonecarboxylate, naphthalenesulphonate, 1-glucosephosphate,
nitrate, sulphite, dithionate and phosphate), and phenformin;
protein tyrosine phosphatase-1B (PTP-1B) inhibitors, such as
A-401,674, KR 61639, OC-060062, OC-83839, OC-297962, MC52445,
MC52453, ISIS 113715, and those disclosed in WO99/585521,
WO99/58518, WO99/58522, WO99/61435, WO03/032916, WO03/032982,
WO03/041729, WO03/055883, WO02/26707, WO02/26743, JP2002114768, and
pharmaceutically acceptable salts and esters thereof; sulfonylureas
such as acetohexamide (e.g. Dymelor, Eli Lilly), carbutamide,
chlorpropamide (e.g. Diabinese.RTM., Pfizer), gliamilide (Pfizer),
gliclazide (e.g. Diamcron, Servier Canada Inc), glimepiride (e.g.
disclosed in U.S. Pat. No. 4,379,785, such as Amaryl.TM., Aventis),
glipentide, glipizide (e.g. Glucotrol or Glucotrol XL Extended
Release, Pfizer), gliquidone, glisolamide, glyburide/glibenclamide
(e.g. Micronase or Glynase Prestab, Pharmacia & Upjohn and
Diabeta, Aventis), tolazamide (e.g. Tolinase), and tolbutamide
(e.g. Orinase), and pharmaceutically acceptable salts and esters
thereof; meglitinides such as repaglinide (e.g. Pranidin.RTM., Novo
Nordisk), KAD1229 (PF/Kissei), and nateglinide (e.g. Starlix.RTM.,
Novartis), and pharmaceutically acceptable salts and esters
thereof; .alpha. glucoside hydrolase inhibitors (or glucoside
inhibitors) such as acarbose (e.g. Precose.TM., Bayer disclosed in
U.S. Pat. No. 4,904,769), miglitol (such as GLYSET.TM., Pharmacia
& Upjohn disclosed in U.S. Pat. No. 4,639,436), camiglibose
(Methyl
6-deoxy-6-[(2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidino]-al-
pha-D-glucopyranoside, Marion Merrell Dow), voglibose (Takeda),
adiposine, emiglitate, pradimicin-Q, salbostatin, CKD-711,
MDL-25,637, MDL-73,945, and MOR 14, and the compounds disclosed in
U.S. Pat. No. 4,062,950, U.S. Pat. No. 4,174,439, U.S. Pat. No.
4,254,256, U.S. Pat. No. 4,701,559, U.S. Pat. No. 4,639,436, U.S.
Pat. No. 5,192,772, U.S. Pat. No. 4,634,765, U.S. Pat. No.
5,157,116, U.S. Pat. No. 5,504,078, U.S. Pat. No. 5,091,418, U.S.
Pat. No. 5,217,877, US51091 and WO01/47528 (polyamines);
.alpha.-amylase inhibitors such as tendamistat, trestatin, and
A1-3688, and the compounds disclosed in U.S. Pat. No. 4,451,455,
U.S. Pat. No. 4,623,714, and U.S. Pat. No. 4,273,765; SGLT2
inhibtors including those disclosed in U.S. Pat. No. 6,414,126 and
U.S. Pat. No. 6,515,117; an aP2 inhibitor such as disclosed in U.S.
Pat. No. 6,548,529; insulin secreatagogues such as linogliride,
A-4166, forskilin, dibutyrl cAMP, isobutylmethylxanthine (IBMX),
and pharmaceutically acceptable salts and esters thereof; fatty
acid oxidation inhibitors, such as clomoxir, and etomoxir, and
pharmaceutically acceptable salts and esters thereof; A2
antagonists, such as midaglizole, isaglidole, deriglidole,
idazoxan, earoxan, and fluparoxan, and pharmaceutically acceptable
salts and esters thereof; insulin and related compounds (e.g.
insulin mimetics) such as biota, LP-100, novarapid, insulin
detemir, insulin lispro, insulin glargine, insulin zinc suspension
(lente and ultralente), Lys-Pro insulin, GLP-1 (1-36) amide, GLP-1
(73-7) (insulintropin, disclosed in U.S. Pat. No. 5,614,492),
LY-315902 (Lilly), GLP-1 (7-36)-NH2), AL-401 (AutoImmune), certain
compositions as disclosed in U.S. Pat. No. 4,579,730, U.S. Pat. No.
4,849,405, U.S. Pat. No. 4,963,526, U.S. Pat. No. 5,642,868, U.S.
Pat. No. 5,763,396, U.S. Pat. No. 5,824,638, U.S. Pat. No.
5,843,866, U.S. Pat. No. 6,153,632, U.S. Pat. No. 6,191,105, and WO
85/05029, and primate, rodent, or rabbit insulin including
biologically active variants thereof including allelic variants,
more preferably human insulin available in recombinant form
(sources of human insulin include pharmaceutically acceptable and
sterile formulations such as those available from Eli Lilly
(Indianapolis, Ind. 46285) as Humulin.TM. (human insulin rDNA
origin), also see the THE PHYSICIAN'S DESK REFERENCE, 55.sup.th Ed.
(2001) Medical Economics, Thomson Healthcare (disclosing other
suitable human insulins); non-thiazolidinediones such as JT-501 and
farglitazar (GW-2570/GI-262579), and pharmaceutically acceptable
salts and esters thereof, PPAR.alpha./.gamma. dual agonists such as
AR-H039242 (Astrazeneca), GW-409544 (Glaxo-Wellcome), BVT-142,
CLX-0940, GW-1536, GW-1929, GW-2433, KRP-297 (Kyorin Merck;
5-[(2,4-Dioxo
thiazolidinyl)methyl]methoxy-N-[[4-(trifluoromethyl)phenyl]methyl]benzami-
de), L-796449, LR-90, MK-0767 (Merek/Kyorin/Banyu), SB 219994,
muraglitazar (BMS), tesaglitzar (Astrazeneca), reglitazar (JTT-501)
and those disclosed in WO99/16758, WO99/19313, WO99/20614,
WO99/38850, WO00/23415, WO00/23417, WO00/23445, WO00/50414,
WO01/00579, WO01/79150, WO02/062799, WO03/004458, WO03/016265,
WO03/018010, WO03/033481, WO03/033450, WO03/033453, WO03/043985, WO
031053976, U.S. application Ser. No. 09/664,598, filed Sep. 18,
2000, Murakami et al. Diabetes 47, 1841-1847 (1998), and
pharmaceutically acceptable salts and esters thereof; other insulin
sensitizing drugs; VPAC2 receptor agonists; GLK modulators, such as
those disclosed in WO03/015774; retinoid modulators such as those
disclosed in WO03/000249; GSK 3.beta./GSK 3 inhibitors such as
4-[2-(2-bromophenyl)-4-(4-fluorophenyl-1H-imidazol-5-yl]pyridine
and those compounds disclosed in WO03/024447, WO03/037869,
WO03/037877, WO03/037891, WO03/068773, EP1295884, EP1295885, and
the like; glycogen phosphorylase (HGLPa) inhibitors such as
CP-368,296, CP-316,819, BAYR3401, and compounds disclosed in
WO01/94300, WO02/20530, WO03/037864, and pharmaceutically
acceptable salts or esters thereof; ATP consumption promotors such
as those disclosed in WO03/007990; TRB3 inhibitors; vanilloid
receptor ligands such as those disclosed in WO03/049702;
hypoglycemic agents such as those disclosed in WO03/015781 and
WO03/040114; glycogen synthase kinase 3 inhibitors such as those
disclosed in WO03/035663 agents such as those disclosed in
WO99/51225, US20030134890, WO01/24786, and WO03/059870;
insulin-responsive DNA binding protein-1 (IRDBP-1) as disclosed in
WO03/057827, and the like; adenosine A2 antagonists such as those
disclosed in WO03/035639, WO03/035640, and the like; PPAR.delta.
agonists such as GW 501516, GW 590735, and compounds disclosed in
JP10237049 and WO02/14291; dipeptidyl peptidase IV (DP-IV)
inhibitors, such as isoleucine thiazolidide, NVP-DPP728A
(1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrro-
lidine, disclosed by Hughes et al, Biochemistry, 38(36),
11597-11603, 1999), P32/98, NVP-LAF-237, P3298, TSL225
(tryptophyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid,
disclosed by Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998)
1537-1540), valine pyrrolidide, TMC-2A/2B/2C, CD-26 inhibitors,
FE999011, P9310/K364, VIP 0177, DPP4, SDZ 274-444,
2-cyanopyrrolidides and 4-cyanopyrrolidides as disclosed by
Ashworth et al, Bioorg. & Med. Chem. Lett., Vol. 6, No. 22, pp
1163-1166 and 2745-2748 (1996), and the compounds disclosed in U.S.
Pat. No. 6,395,767, U.S. Pat. No. 6,573,287, U.S. Pat. No.
6,395,767 (compounds disclosed include BMS-477118, BMS-471211 and
BMS 538,305), WO99/38501, WO99/46272, WO99/67279, WO99/67278,
WO99/61431 WO03/004498, WO03/004496, EP1258476, WO02/083128,
WO02/062764, WO03/000250, WO03/002530, WO03/002531, WO03/002553,
WO03/002593, WO03/000180, and WO03/000181; GLP-1 agonists such as
exendin-3 and exendin-4 (including the 39 aa peptide synthetic
exendin-4 called Exenatide.RTM.), and compounds disclosed in
US2003087821 and NZ 504256, and pharmaceutically acceptable salts
and esters thereof; peptides including amlintide and Symlin.RTM.
(pramlintide acetate); and glycokinase activators such as those
disclosed in US2002103199 (fused heteroaromatic compounds) and
WO02/48106 (isoindolin-1-one-substituted propionamide
compounds).
[0348] The peptides and agonists described herein useful in the
treatment of obesity can be administered as a cotherapy with
electrostimulation (US20040015201).
[0349] The peptides and agonists described herein can be used in
combination therapy with agents that activate soluble guanylate
cyclase, for example those described in US20040192680.
[0350] The peptides and agonists described herein can be used in
combination therapy with a phosphodiesterase inhibitor. PDE
inhibitors are those compounds which slow the degradation of cyclic
AMP (cAMP) and/or cyclic GMP (cGMP) by inhibition of the
phosphodiesterases, which can lead to a relative increase in the
intracellular concentration of cAMP and/or cGMP. Possible PDE
inhibitors are primarily those substances which are to be numbered
among the class consisting of the PDE3 inhibitors, the class
consisting of the PDE4 inhibitors and/or the class consisting of
the PDE5 inhibitors, in particular those substances which can be
designated as mixed types of PDE3/4 inhibitors or as mixed types of
PDE3/4/5 inhibitors. By way of example, those PDE inhibitors may be
mentioned such as are described and/or claimed in the following
patent applications and patents: DE1470341, DE2108438, DE2123328,
DE2305339, DE2305575, DE2315801, DE2402908, DE2413935, DE2451417,
DE2459090, DE2646469, DE2727481, DE2825048, DE2837161, DE2845220,
DE2847621, DE2934747, DE3021792, DE3038166, DE3044568, EP000718,
EP0008408, EP0010759, EP0059948, EP0075436, EP0096517, EP0112987,
EP0116948, EP0150937, EP0158380, EP0161632, EP0161918, EP0167121,
EP0199127, EP0220044, EP0247725, EP0258191, EP0272910, EP0272914,
EP0294647, EP0300726, EP0335386, EP0357788, EP0389282, EPO406958,
EPO426180, EPO428302, EPO435811, EPO470805, EPO482208, EPO490823,
EP0506194, EP0511865, EP0527117, EP0626939, EP0664289, EP0671389,
EP0685474, EP0685475, EP0685479, JP92234389, JP94329652,
JP95010875, U.S. Pat. Nos. 4,963,561, 5,141,931, WO9117991,
WO9200968, WO9212961, WO9307146, WO9315044, WO9315045, WO9318024,
WO9319068, WO9319720, WO9319747, WO9319749, WO9319751, WO9325517,
WO9402465, WO9406423, WO9412461, WO9420455, WO9422852, WO9425437,
WO9427947, WO9500516, WO9501980, WO9503794, WO9504045, WO9504046,
WO9505386, WO9508534, WO9509623, WO9509624, WO9509627, WO9509836,
WO9514667, WO9514680, WO9514681, WO9517392, WO9517399, WO9519362,
WO9522520, WO9524381, WO9527692, WO9528926, WO9535281, WO9535282,
WO9600218, WO9601825, WO9602541, WO9611917, DE3142982, DE1116676,
DE2162096, EP0293063, EPO463756, EPO482208, EP0579496, EP0667345
U.S. Pat. No. 6,331,543, US20050004222 (including those disclosed
in formulas I-XIII and paragraphs 37-39, 85-0545 and 557-577) and
WO9307124, EP0163965, EP0393500, EP0510562, EP0553174, WO9501338
and WO9603399. PDE5 inhibitors which may be mentioned by way of
example are RX-RA-69, SCH-51866, KT-734, vesnarinone, zaprinast,
SKF-96231, ER-21355, BF/GP-385, NM-702 and sildenafil
(Viagra.RTM.)). PDE4 inhibitors which may be mentioned by way of
example are RO-20-1724, MEM 1414 (R1533/R1500; Pharmacia Roche),
DENBUFYLLINE, ROLIPRAM, OXAGRELATE, NITRAQUAZONE, Y-590, DH-6471,
SKF-94120, MOTANZONE, LIXAZINONE, INDOLIDAN, OLPRINONE, ATIZORAM,
KS-506-G, DIPAMFYLLINE, BMY-43351, ATIZORAM, AROFYLLINE,
FILAMINAST, PDB-093, UCB-29646, CDP-840, SKF-107806, PICLAMILAST,
RS-17597, RS-25344-000, SB-207499, TIBENELAST, SB-210667,
SB-211572, SB-211600, SB-212066, SB-212179, GW-3600, CDP-840,
MOPIDAMOL, ANAGRELIDE, IBUDILAST, AMRINONE, PIMOBENDAN, CILOSTAZOL,
QUAZINONE and
N-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy-4-difluoromethoxybenzamid-
e. PDE3 inhibitors which may be mentioned by way of example are
SULMAZOLE, AMPIZONE, CILOSTAM IDE, CARBAZERAN, PIROXIMONE,
IMAZODAN, CI-930, SIGUAZODAN, ADIBENDAN, SATERINONE, SKF-95654,
SDZ-MKS-492, 349-U-85, EMORADAN, EMD-53998, EMD-57033, NSP-306,
NSP-307, REVIZINONE, NM-702, WIN-62582 and WIN-63291, ENOXIMONE and
MILRINONE. PDE3/4 inhibitors which may be mentioned by way of
example are BENAFENTRINE, TREQUINSIN, ORG-30029, ZARDAVERINE,
L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, and TOLAFENTRINE.
Other PDE inhibitors include: cilomilast, pentoxifylline,
roflumilast, tadalafil (Cialis.RTM.), theophylline, and vardenafil
(Levitra.RTM.), zaprinast (PDE5 specific).
[0351] The peptides and agonists described herein can be used in
combination therapy (for example, in order to decrease or inhibit
uterine contractions) with a tocolytic agent including but not
limited to beta-adrenergic agents, magnesium sulfate, prostaglandin
inhibitors, and calcium channel blockers.
[0352] The peptides and agonists described herein can be used in
combination therapy with an anti-neoplastic agents including but
not limited to alkylating agents, epipodophyllotoxins,
nitrosoureas, antimetabolites, vinca alkaloids, anthracycline
antibiotics, nitrogen mustard agents, and the like. Particular
anti-neoplastic agents may include tamoxifen, taxol, etoposide and
5-fluorouracil. The peptides and agonists described herein can be
used in combination therapy (for example as in a chemotherapeutic
composition) with an antiviral and monoclonal antibody
therapies.
[0353] The peptides and agonists described herein can be used in
combination therapy (for example, in prevention/treatment of
congestive heart failure or another method described herein) with
the partial agonist of the nociceptin receptor ORL1 described by
Dooley et al. (The Journal of Pharmacology and Experimental
Therapeutics, 283 (2): 735-741, 1997). The agonist is a hexapeptide
having the amino acid sequence Ac-RYY (RK) (WI) (RK)-NH2 ("the
Dooley peptide"), where the brackets show allowable variation of
amino acid residue. Thus Dooley peptide can include but are not
limited to KYYRWR, RYYRWR, KWRYYR, RYYRWK, RYYRWK (all-D amin
acids), RYYRIK, RYYRIR, RYYKIK, RYYKIR, RYYKWR, RYYKWK, RYYRWR,
RYYRWK, RYYRIK, RYYKWR, RYYKWK, RYYRWK and KYYRWK, wherein the
amino acid residues are in the L-form unless otherwise specified.
The peptides and agonists described herein can also be used in
combination therapy with peptide conjugate modifications of the
Dooley peptide described in WO0198324.
Methods of Treatment
[0354] A number of disorders might be prevented or treated with
GC-C receptor agonists and agents that increase cGMP levels
including the peptides and agonists described herein.
[0355] The peptides and agonists described herein can be used alone
or in combination therapy for the treatment or prevention of
congestive heart failure. Such agents can be used in combination
with natriuretic peptides (e.g., atrial natriuretic peptide, brain
natriuretic peptide or C-type natriuretic peptide), a diuretic, or
an inhibitor of angiotensin converting enzyme.
[0356] The peptides and agonists described herein can be used alone
or in combination therapy for the treatment or prevention of benign
prostatic hyperplasia (BPH). Such agents can be used in combination
with one or more agents for treatment of BPH, for example, a
5-alpha reductase inhibitor (e.g., finasteride) or an alpha
adrenergic inhibitor (e.g., doxazosine).
[0357] The peptides and 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.
[0358] The peptides and agonists described herein can be used alone
or in combination therapy for the treatment or prevention of
obesity-related disorders (e.g. disorders that are associated with,
caused by, or result from obesity). Examples of obesity-related
disorders include overeating and bulimia, hypertension, diabetes,
elevated plasma insulin concentrations and insulin resistance,
dyslipidemias, hyperlipidemia, endometrial, breast, prostate and
colon cancer, osteoarthritis, obstructive sleep apnea,
cholelithiasis, gallstones, heart disease, abnormal heart rhythms
and arrhythmias, myocardial infarction, congestive heart failure,
coronary heart disease, sudden death, stroke, polycystic ovarian
disease, craniopharyngioma, the Prader-Willi Syndrome, Frohlich's
syndrome, GH-deficient subjects, normal variant short stature,
Turner's syndrome, and other pathological conditions showing
reduced metabolic activity or a decrease in resting energy
expenditure as a percentage of total fat-free mass, e.g., children
with acute lymphoblastic leukemia. The agents described herein may
be used to reduce or control body weight (or fat) or to prevent
and/or treat obesity or other appetite related disorders related to
the excess consumption of food, ethanol and other appetizing
substances. The agents may be used to modulate lipid metabolism,
reduce body fat (e.g. via increasing fat utilization) or reduce (or
suppress) appetite (e.g. via inducing satiety). Further examples of
obesity-related disorders are metabolic syndrome, also known as
syndrome X, insulin resistance syndrome, sexual and reproductive
dysfunction, such as infertility, hypogonadism in males and
hirsutism in females, gastrointestinal motility disorders, such as
obesity-related gastroesophageal reflux, respiratory disorders,
such as obesity-hypoventilation syndrome (Pickwickian syndrome),
cardiovascular disorders, inflammation, such as systemic
inflammation of the vasculature, arteriosclerosis,
hypercholesterolemia, hyperuricaemia, lower back pain, gallbladder
disease, gout, and kidney cancer. The agents of the present
disclosure are also useful for reducing the risk of secondary
outcomes of obesity, such as reducing the risk of left ventricular
hypertrophy.
[0359] The peptides and agonists described herein can be used alone
or in combination therapy for the treatment or prevention of
gastrointestinal related disorders including: chronic intestinal
pseudo-obstruction (Ogilvie's syndrome), colonic pseudoobstruction,
Crohn's disease, dyspepsia (including functional dyspepsia or
nonulcer dyspepsia), duodenogastric reflux, functional bowel
disorder, functional gastrointestinal disorders, functional
heartburn, gastroesophageal reflux disease (GERD), gastrointestinal
motility disorders, gastroparesis (e.g. idopathic gastroparesis),
hypertrophic pyloric stenosis, Inflammatory bowel disease,
irritable bowel syndrome (IBS), post-operative ileus, and
ulcerative colitis. The peptides and agonists described herein can
be used alone or in combination therapy to patient suffering from
or susceptible to GI disorders relating to damage to the GI tract
stemming from impact or surgical intervention. The peptides and
agonists described herein can be used alone or in combination
therapy to patients at risk for or having particular diseases
associated with hypomotility (e.g. colonic inertia) or stasis in
the GI tract. For example, diabetic neuropathy, anorexia nervosa,
and achlorhydria are frequently accompanied by gastric
hypomotility. Damage to the GI tract following surgical
intervention, for instance, can result in substantial gastric
stasis. The peptides and agonists described herein can be
administered alone or in combination therapy to patients
susceptible to or having a GI disorder associated with diabetes
(e.g. diabetic gastropathy). The peptides and agonists described
herein can be used alone or in combination therapy to prevent
and/or treat GI disorders characterized by at least one of nausea,
vomiting, heartburn, postprandial discomfort, diarrhea,
constipation, indigestion or related symptoms. The peptides and
agonists described herein can be used alone or in combination
therapy to prevent and/or treat GI disorders associated with at
least one of diabetes, anorexia nervosa, bulimia, achlorhydria,
achalasia, anal fissure, haemorrhoids, irritable bowel syndrome,
intestinal pseudoobstruction, scleroderma and gastrointestinal
damage.
[0360] The peptides and agonists described herein can be used to
prevent and/or treat constipation. Constipation can be used to
describe bowel patterns which include one or more of hard, small,
infrequent stools; the sensation of difficulty in passing stool,
specifically excessive or ineffectual straining; the sensation of
incomplete evacuation. Constipation has also been described as the
passage of stool less than a certain number (e.g. 3) of times per
week. A number of conditions can be associated with constipation.
Constipation can be associated with numerous disorders and
conditions. For example, constipation can be (1) associated with
the use of a therapeutic agent (e.g. antihypertensives,
anticonvulsants, antispasmodics, analgesics, anticholinergics,
antidepressants, antipsychotics, cation-containing agents,
anticonvulsants, ganglion blockers, vinca alkaloids); (2)
associated with a muscular, neuropathic, metabolic or endocrine
disorder (including but not limited to myotonic dystrophy,
dermamyositis, systemic sclerosis, sclerodoma, amyloidosis
(neurologic or muscular), ischemia, tumor of the central nervous
system, autonomic neuropathy, Chagas disease, cystic fibrosis,
diabetes mellitus, Hirschsprung disease, hyperthyroidism,
hypocalcaemia, hypothyroidism, Multiple Sclerosis,
neurofibromatosis, Parkinson's disease, and spinal cord lesions
(for example, related to sacral nerve damage related to trauma or a
tumor or the enteric nervous system)); (3) post-surgical
constipation (postoperative ileus); (4) associated with a
structural colon alteration (for example that associated with
Neoplasm, stricture, volvulus, anorectal, inflammation, prolapse,
rectocele, or fissure); (5) associated with the a gastrointestinal
disorder; (6) associated with a systemic illness or disorder (for
example, electrolyte abnormalities, thyroid disease, diabetes
mellitus, panhypopituitarism, Addison's disease, pheochromocytoma,
uremia, porphyria); (7) chronic constipation; (8) associated with
the use of analgesic drugs (e.g. opioid induced constipation); (9)
associated with megacolon; and (10) idiopathic constipation
(functional constipation). Functional constipation can be
associated with normal transit, slow transit (e.g. one or fewer
bowel movements per week) and pelvic floor dyssynergia. Pelvic
floor dyssynergia is considered a disorder of the rectum and anus
although these patients also have abnormal contractions throughout
the colon. Patients with pelvic floor dyssynergia have abnormal
colonic pressure waves prior to defecation and present with
symptoms that may include a sensation of incomplete evacuation,
excessive straining, a need for digital disimpaction, perianal
heaviness, and tenesmus. Constipation can be associated with
bloating and abdominal pain. The peptides and agonists described
herein can be used to prevent and/or treat low stool frequency or
poor stool consistency.
[0361] The peptides and agonists described herein can be used to
treat decreased intestinal motility, slow digestion or slow stomach
emptying. The peptides and agonists can be used to relieve one or
more symptoms of IBS (bloating, pain, constipation), GERD (acid
reflux into the esophagus), duodenogastric reflux, functional
dyspepsia, or gastroparesis (nausea, vomiting, bloating, delayed
gastric emptying) and other disorders described herein. The
peptides and agonists described herein can be used to treat
flatulence.
[0362] The peptides and agonists described herein can be used to
increase intestinal motility, slow colonic transit, and to prevent
and/or treat gastrointestinal immotility and other conditions
calling for laxative or stool softener therapy. Gastrointestinal
immotility can include constipation, and also includes delayed oral
cecal transit time, irregular Taxation, and other related
gastrointestinal motility disfunction including impaction.
Impaction is a condition where a large mass of dry, hard stool
develops in the rectum, often due to chronic constipation. This
mass may be so hard that it cannot be excreted. The subjects
affected by constipation or gastrointestinal immotility can be
refractory to laxative therapy and/or stool softener therapy.
[0363] The peptides and agonists described herein can be used for
the treatment or prevention of cancer, pre-cancerous growths, or
metastatic growths. For example, they can be used for the
prevention or treatment of colorectal/local metastasized colorectal
cancer, intestinal polyps, gastrointestinal tract cancer, lung
cancer, cancer or pre-cancerous growths or metastatic growths of
epithelial cells, polyps, breast, colorectal, lung, ovarian,
pancreatic, prostatic, renal, stomach, bladder, liver, esophageal
and testicular carcinoma, carcinoma (e.g., basal cell,
basosquamous, Brown-Pearce, ductal carcinoma, Ehrlich tumor, Krebs,
Merkel cell, small or non-small cell lung, oat cell, papillary,
bronchiolar, squamous cell, transitional cell, (Walker), leukemia
(e.g., B-cell, T-cell, HTLV, acute or chronic lymphocytic, mast
cell, myeloid), histiocytonia, histiocytosis, Hodgkin's disease,
non-Hodgkin's lymphoma, plasmacytoma, reticuloendotheliosis,
adenoma, adeno-carcinoma, adenofibroma, adenolymphoma,
ameloblastoma, angiokeratoma, angiolymphoid hyperplasia with
eosinophilia, sclerosing angioma, angiomatosis, apudoma,
branchionia, malignant carcinoid syndrome, carcinoid heart disease,
carcinosarcoma, cementoma, cholangioma, cholesteatoma,
chondrosarcoma, chondroblastoma, chondrosarcoma, chordoma,
choristoma, craniopharyngioma, chrondroma, cylindroma,
cystadenocarcinoma, cystadenoma, cystosarconia phyllodes,
dysgenninoma, ependymoma, Ewing sarcoma, fibroma, fibrosarcoma,
giant cell tumor, ganglioneuroma, glioblastoma, glomangioma,
granulosa cell tumor, gynandroblastoma, hamartoma,
hemangioendothelioma, hemangioma, hemangio-pericytoma,
hemangiosarcoma, hepatoma, islet cell tumor, Kaposi sarcoma,
leiomyoma, leiomyosarcoma, leukosarcoma, Leydig cell tumor, lipoma,
liposarcoma, lymphaugioma, lymphangiomyoma, lymphangiosarcoma,
medulloblastoma, meningioma, mesenchymoma, mesonephroma,
mesothelioma, myoblastoma, myoma, myosarcoma, myxoma, myxosarcoma,
neurilemmoma, neuroma, neuroblastoma, neuroepithelioma,
neurofibroma, neurofibromatosis, odontoma, osteoma, osteosarcoma,
papilloma, paraganglioma, paraganglionia. nonchromaffin, pinealoma,
rhabdomyoma, rhabdomyosarcoma, Sertoli cell tumor, teratoma, theca
cell tumor, and other diseases in which cells have become
dysplastic, immortalized, or transformed.
[0364] The peptides and agonists described herein can be used for
the treatment or prevention of Familial Adenomatous Polyposis (FAP)
(autosomal dominant syndrome) that precedes colon cancer,
hereditary nonpolyposis colorectal cancer (HNPCC), and inherited
autosomal dominant syndrome.
[0365] For treatment or prevention of cancer, pre-cancerous growths
and metastatic growths, the peptides and agonists described herein
can be used in combination therapy with radiation or
chemotherapeutic agents, an inhibitor of a cGMP-dependent
phosphodiesterase or a selective cyclooxygenase-2 inhibitor. A
number of selective cyclooxygenase-2 inhibitors are described in
US20010024664, U.S. Pat. No. 5,380,738, U.S. Pat. No. 5,344,991,
U.S. Pat. No. 5,393,790, U.S. Pat. No. 5,434,178, U.S. Pat. No.
5,474,995, U.S. Pat. No. 5,510,368, WO02/062369, WO 96/06840, WO
96/03388, WO 96/03387, WO 96/19469, WO 96/25405, WO 95/15316, WO
94/15932, WO 94/27980, WO 95/00501, WO 94/13635, WO 94/20480, and
WO 94/26731, the disclosures of which are herein incorporated by
reference. [Pyrazol-1-yl]benzenesulfonamides have also been
described as inhibitors of cyclooxygenase-2.
[0366] The peptides and agonists described herein can be used in
the treatment or prevention of inflammation. Thus, they can be used
alone or in combination with an inhibitor of cGMP-dependent
phosphodiesterase or a selective cyclooxygenase-2 inhibitor for
treatment of: organ inflammation, IBD (e.g, Crohn's disease,
ulcerative colitis), asthma, nephritis, hepatitis, pancreatitis,
bronchitis, cystic fibrosis, ischemic bowel diseases, intestinal
inflammations/allergies, coeliac disease, proctitis, eosinophilic
gastroenteritis, mastocytosis, and other inflammatory disorders.
The peptides and agonists described herein can be used alone or in
combination therapy in the treatment or prevention of
gastrointestinal tract inflammation (e.g. inflammation associated
with a gastrointestinal disorder, gastrointestinal tract infection,
or another disorder). They can be used alone or in combination
therapy with phenoxyalkycarboxylic acid derivatives for the
treatment of interstitial cystitis, irritable bowel syndrome,
ulcerative colitis, and other inflammatory conditions, as mentioned
in US20050239902A1.
[0367] The peptides and agonists described herein can also be used
to treat or prevent insulin-related disorders, for example: II
diabetes mellitus, hyperglycemia, obesity, disorders associated
with disturbances in glucose or electrolyte transport and insulin
secretion in cells, or endocrine disorders. They can be also used
in insulin resistance treatment and post-surgical and non-post
surgery decrease in insulin responsiveness.
[0368] The peptides and agonists described herein can be used to
prevent and/or treat pulmonary and respiratory related disorders,
including, inhalation, ventilation and mucus secretion disorders,
pulmonary hypertension, chronic obstruction of vessels and airways,
acute respiratory failure, and irreversible obstructions of vessels
and bronchi. One may administer an agent described herein for
treating bronchospasm, for inducing bronchodilation, for treating
chronic obstructive pulmonary disease (including chronic bronchitis
with normal airflow), for treating asthma (including bronchial
asthma, intrinsic asthma, extrinsic asthma, acute asthma, chronic
or inveterate asthma (e.g. late asthma and airways
hyper-responsiveness), dust-induced asthma, allergen-induced
asthma, viral-induced asthma, cold-induced asthma,
pollution-induced asthma and exercise-induced asthma) and for
treating rhinitis (including acute-, allergic, hatrophic rhinitis
or chronic rhinitis (such as rhinitis caseosa, hypertrophic
rhinitis, rhinitis purulenta, rhinitis sicca), rhinitis
medicamentosa, membranous rhinitis (including croupous, fibrinous
and pseudomembranous rhinitis), scrofulous rhinitis, perennial
allergic rhinitis, seasonal rhinitis (including rhinitis nervosa
(hay fever) and vasomotor rhinitis). The peptides described herein
may also be useful in the treatment of dry eye disease and chronic
sinusitis. The peptides described herein may also be used to
prevent and/or treat disorders characterized by acute pulmonary
vasoconstriction such as may result from pneumonia, traumatic
injury, aspiration or inhalation injury, fat embolism in the lung,
acidosis inflammation of the lung, adult respiratory distress
syndrome, acute pulmonary edema, acute mountain sickness,
post-cardiac surgery, acute pulmonary hypertension, persistent
pulmonary hypertension of the newborn, perinatal aspiration
syndrome, hyaline membrane disease, acute pulmonary
thromboembolism, herapin-protamine reactions, sepsis, status
asthmaticus or hypoxia (including iatrogenic hypoxia) and other
forms of reversible pulmonary vasoconstriction. Such pulmonary
disorders also are also characterized by inflammation of the lung
including those associated with the migration into the lung of
nonresident cell types including the various leucocyte subclasses.
Also included in the respiratory disorders contemplated are:
bullous disease, cough, chronic cough associated with inflammation
or iatrogenic induced, airway constriction, pigeon fancier's
disease, eosinophilic bronchitis, asthmatic bronchitis, chronic
bronchitis with airway obstruction (chronic obstructive
bronchitis), eosinophilic lung disease, emphysema, farmer's lung,
allergic eye diseases (including allergic conjunctivitis, vernal
conjunctivitis, vernal keratoconjunctivitis, and giant papillary
conjunctivitis), idiopathic pulmonary fibrosis, cystic fibrosis,
diffuse pan bronchiolitis and other diseases which are
characterized by inflammation of the lung and/or excess mucosal
secretion. Other physiological events which are contemplated to be
prevented, treated or controlled include platelet activation in the
lung, chronic inflammatory diseases of the lung which result in
interstitial fibrosis, such as interstitial lung diseases (ILD)
(e.g., idiopathic pulmonary fibrosis, or ILD associated with
rheumatoid arthritis, or other autoimmune conditions), chronic
obstructive pulmonary disease (COPD) (such as irreversible COPD),
chronic sinusitis, fibroid lung, hypersensitivity lung diseases,
hypersensitivity pneumonitis, idiopathic interstitial pneumonia,
nasal congestion, nasal polyposis, and otitis media.
[0369] The peptides and agonists described herein can be used alone
or in combitherapy to prevent or treat: retinopathy, nephropathy,
diabetic angiopathy, and edema formation
[0370] The peptides and agonists described herein can be used alone
or in combitherapy to prevent or treat neurological disorders, for
example, headache, tension-type headache, migraines, anxiety,
stress, cognitive disorders, cerebral ischemia, brain trauma,
movement disorders, aggression, psychosis, seizures, panic attacks,
hysteria, sleep disorders, depression, schizoaffective disorders,
sleep apnea, attention deficit syndromes, memory loss, dementia,
memory and learning disorders as discussed in Moncada and Higgs
1995 FASEB J. 9:1319-1330; Severina 1998 Biochemistry 63:794; Lee
et al. 2000 PNAS 97: 10763-10768; Hobbs 1997 TIPS 18:484-491; Murad
1994 Adv. Pharmacol. 26:1-335; and Denninger et al. 1999 Biochim.
Biophys. Acta 1411:334-350 and narcolepsy. They may also be used as
a sedative.
[0371] The peptides and detectably peptides and agonists described
herein can be used as markers to identify, detect, stage, or
diagnosis diseases and conditions of small intestine, including,
without limitation: Crohn's disease, colitis, inflammatory bowel
disease, tumors, benign tumors, such as benign stromal tumors,
adenoma, angioma, adenomatous (pedunculated and sessile) polyps,
malignant, carcinoid tumors, endocrine cell tumors, lymphoma,
adenocarcinoma, foregut, midgut, and hindgut carcinoma,
gastroinstestinal stromal tumor (GIST), such as leiomyoma, cellular
leiomyoma, leiomyoblastoma, and leiomyosarcoma, gastrointestinal
autonomic nerve tumor, malabsorption syndromes, celiac diseases,
diverticulosis, Meckel's diverticulum, colonic diverticula,
megacolon, Hirschsprung's disease, irritable bowel syndrome,
mesenteric ischemia, ischemic colitis, colorectal cancer, colonic
polyposis, polyp syndrome, intestinal adenocarcinoma, Liddle
syndrome, Brody myopathy, infantile convulsions, and
choreoathetosis
[0372] The peptides and agonists described herein can be conjugated
to another molecule (e.g., a diagnostic or therapeutic molecule) to
target cells bearing the GC-C receptor, e.g., cystic fibrosis
lesions and specific cells lining the intestinal tract. Thus, they
can be used to target radioactive moieties or therapeutic moieties
(active moieties like a radionuclide, an enzyme, a fluorescent
label, a metal chelating group, a chemiluminescent label, a
bioluminescent label, a chemotherapeutic, a toxin, an inactive
prodrug, a radiosensitizing agent, a photodynamic agent) to the
intestine to aid in imaging and diagnosing or treating
colorectal/metastasized or local colorectal cancer. In addition,
they can be used to deliver antisense molecules or nucleic acid
molecules (like normal copies of the p53 tumor suppressor gene) to
the intestinal tract. The peptides and agonists described herein
can also be used to increase the number of GC-C molecules on the
surface of a cell. In some embodiments the cell is a metastasized
colorectal cancer cell. In one embodiment the peptide or agonist
described herein is therapeutically conjugated to a second agent.
In certain embodiments, the second agent can be radioactive or
radiostable. In certain embodiments the second agent can be
selected from the group consisting of a compound that causes cell
death, a compound that inhibits cell division, a compound that
induces cell differentiation, a chemotherapeutic, a toxin and a
radiosensitizing agent. In certain embodiments the second agent can
be selected from the group consisting of: methotrexate,
doxorubicin, daunorubicin, cytosinarabinoside, etoposide, 5-4
fluorouracil, melphalan, chlorambucil, cis-platin, vindesine,
mitomycin, bleomycin, purothionin, macromomycin, 1,4-benzoquinone
derivatives, trenimon, ricin, ricin A chain, Pseudomonas exotoxin,
diphtheria toxin, Clostridium perfringens phospholipase C, bovine
pancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin A
chain, cobra venom factor, gelonin, saporin, modeccin, viscumin,
volkensin, nitroimidazole, metronidazole and misonidazole. In
certain embodiments the second agent can be a cytoxic agent
selected from the group consisting of cemadotin, a derivative of
cemadotin, a derivative of hemiasterlin, esperamicin C,
neocarzinostatin, maytansinoid DM1, 7-chloromethyl-10,11
methylenedioxy-camptothecin, rhizoxin, and the halichondrin B
analog, ER-086526.
[0373] The peptides and agonists described herein can be used alone
or in combination therapy to prevent and/or treat inner ear
disorders, e.g., to prevent and/or treat Meniere's disease
(including symptoms thereof such as vertigo, hearing loss,
tinnitus, sensation of fullness in the ear), Mal de debarquement
syndrome, otitis externa, otitis media, otorrhea, acute
mastoiditis, otosclerosis, otic pain, otic bleeding, otic
inflammation, Lermoyez's syndrome, vestibular neuronitis, benign
paroxysmal positional vertigo (BPPV), herpes zoster oticus, Ramsay
Hunt's syndrome, herpes, labyrinthitis, purulent labyrinthitis,
perilymph fistulas, presbycusis, ototoxicity (including
drug-induced ototoxicity), neuromias (including acoustic neuromas),
aerotitis media, infectious myringitis, bullous myringitis,
squamous cell carcinoma, basal cell carcinoma, pre-cancerous otic
conditions, nonchromaffin paragangliomas, chemodectomas, glomus
jugulare tumors, glomus tympanicum tumors, perichondritis, aural
eczematoid dermatitis, malignant external otitis, subperichondrial
hematoma, ceruminomas, impacted cerumen, sebaceous cysts, osteomas,
keloids, otalgia, tinnitus, tympanic membrane infection,
tympanitis, otic furuncles, petrositis, conductive and
sensorineural hearing loss, epidural abscess, lateral sinus
thrombosis, subdural empyema, otitic hydrocephalus, Dandy's
syndrome, bullous myringitis, diffuse external otitis, foreign
bodies, keratosis obturans, otic neoplasm, otomycosis, trauma,
acute barotitis media, acute eustachian tube obstruction,
postsurgical otalgia, cholesteatoma, infections related to an otic
surgical procedure, and complications associated with any of said
disorders. The peptides and agonists described herein can be used
alone or in combination therapy to maintain fluid homeostasis in
the inner ear. neuronitis (including viral neuronitis),
ganglionitis, geniculate
[0374] The peptides and agonists described herein can be used alone
or in combination therapy to prevent and/or treat disorders
associated with bicarbonate secretion, e.g., Cystic Fibrosis.
[0375] The peptides and agonists described herein can be used alone
or in combination therapy to prevent and/or treat disorders
associated with bile secretion. In addition, they can be used to
facilitate or control chloride and bile fluid secretion in the gall
bladder.
[0376] The peptides and agonists described herein can be used alone
or in combination therapy to prevent and/or treat disorders
associated with liver cell regeneration. This may include
administration of the peptides and agonists to liver transplant
recipients and to patients with drug or alcohol induced-liver
damage. Furthermore, the peptides and agonists may be useful to
treat liver damage as in the case of viral mediated hepatitis. The
peptides and agonists described herein may be used alone or in
combination to prevent and/or treat liver abscess, liver cancer
(either primary or metastatic), cirrhosis (such as cirrhosis caused
by the alcohol consumption or primary biliary cirrhosis), amebic
liver abscess, autoimmune hepatitis, biliary atresia,
coccidioidomycosis disseminated, .delta. agent (hepatitis .delta.),
hemocromatosis, hepatitis a, hepatitis b, hepatitis c, or any other
acute, subacute, fulminant or chronic hepatitis of viral, metabolic
or toxic etiology, hepatocellular carcinoma, pyogenic liver
abscess, Reye's syndrome, sclerosing cholangitis, Wilson's disease,
drug induced hepatotoxicity, or fulminant or acute liver failure.
The peptides and agonists may be used in stimulating hepatic
regeneration after surgical hepatectomy.
[0377] The peptides and agonists described herein can be used alone
or in combination therapy to prevent and/or treat myocardial
infraction, coronary artery disease, nitrate-induced tolerance,
nitrate tolerance, diastolic dysfunction, angina pectoris, stable,
unstable and variant (Prinzmetal) angina, atherosclerosis,
thrombosis, endothelial dysfunction, cardiac edema, stroke,
conditions of reduced blood vessel patency, e.g., postpercutaneous
transluminal coronary angioplasty (post-PTCA), and peripheral
vascular disease.
[0378] The peptides and agonists described herein can be used alone
or in combination therapy to prevent and/or treat glaucoma.
[0379] The peptides and agonists described herein can be used alone
or in combination therapy to prevent and/or treat
immunodeficiency.
[0380] The peptides and agonists described herein can be used alone
or in combination therapy to prevent and/or treat bladder outlet
obstruction and incontinence.
[0381] The peptides and agonists described herein can be used alone
or in combination therapy to prevent and/or treat male (e.g.
erectile dysfunction) or female sexual dysfunction, dysmenorrhea,
endometriosis, polycystic ovary syndrome, vaginal dryness, uterine
pain, or pelvic pain. These peptides and agonists described herein
can be utilized as tocolytic agents that decrease or arrest uterine
contractions. The peptides and agonists described herein can be
used to prevent/treat premature/preterm labor. Premature or preterm
labor can be associated with, for example, an
illness/disorder/condition of the mother (such as pre-eclampsia,
high blood pressure or diabetes, abnormal shape or size of the
uterus, weak or short cervix, hormone imbalance, vaginal infection
that spreads to the uterus, abnormalities of the placenta, such as
placenta previa, and excessive amniotic fluid), premature rupture
of the amniotic membranes ("water breaks"), large fetus, and more
than one fetus. The peptides or agonists described herein can be
used to prevent uterine rupture. The peptides or agonists described
herein can be used treat rapid uterine contractions (for example,
associated with placental abruption wherein the placental abruption
is associated with hypertension, diabetes, a multiply pregnancy, an
unusually large amount of amniotic fluid, numerous previous
deliveries, or advanced maternal age (e.g. >40 years old). In
certain embodiments they can be used in combination with a
phosphodiesterase inhibitor. The peptides and agonists described
herein can be used alone or in combination therapy to prevent
and/or treat infertility, for example, male infertility due to poor
sperm quality, decreased sperm motility or low sperm count.
[0382] The peptides and agonists described herein can be used alone
or in combination therapy to prevent and/or treat osteopenia
disorders (bone loss disorders). "Bone loss disorders" include
conditions and diseases wherein the inhibition of bone loss and/or
the promotion of bone formation is desirable. Among such conditions
and diseases are osteoporosis, osteomyelitis, Paget's disease
(osteitis deformans), periodontitis, hypercalcemia, osteonecrosis,
osteosarcoma, osteolyic metastases, familial expansile osteolysis,
prosthetic loosening, periprostetic osteolysis, bone loss attendant
rheumatoid arthritis, and cleiodocranial dysplasia (CCD).
Osteoporosis includes primary osteoporosis, endocrine osteoporosis
(hyperthyroidism, hyperparathyroidism, Cushing's syndrome, and
acromegaly), hereditary and congenital forms of osteoporosis
(osteogenesis imperfecta, homocystinuria, Menkes' syndrome, and
Rile-Day syndrome) and osteoporosis due to immobilization of
extremitiesosteomyelitis, or an infectious lesion in bone leading
to bone loss. The peptides and agonists can be used alone or in
combination therapy to stimulating bone regeneration. The bone
regeneration may be following reconstruction of bone defects in
cranio-maxillofacial surgery, or following an implant into bone,
for example a dental implant, bone supporting implant, or
prosthesis. The bone regeneration may also be following a bone
fracture.
[0383] The peptides and agonists described herein may be used alone
or in combination therapy (for example, with other agents that
increase cGMP) to prevent or treat disorders related to an
alteration in cGMP including, but not limited to Alzheimer's
disease, psoriasis, skin necrosis, scarring, fibrosis, baldness,
Kawasaki's Disease, nutcracker oesophagus (US20050245544), septic
shock, NSAID-induced gastric disease or disorder, ischemic renal
disease or disorder, peptic ulcer, sickle cell anemia, epilepsy,
and a neuroinflammatory disease or disorder (for example as
described in WO05105765).
[0384] The peptides described herein can be used as immunogens to
create antibodies for immunoassays. The peptides described herein
that have homology to ST peptides can be used as immunogens to
treat and/or prevent one or more disease symptoms associated with
traveler's diarrhea and for vaccination against pathogens,
including but not limited to enterotoxigenic E. coli (ETEC). They
may also be used in vaccines which also comprise interleukin 18 and
either saponin adjuvant or CpG adjuvant for example as described in
WO05039634 and WO05039630. The methods described in US20040146534,
U.S. Pat. No. 4,220,584, U.S. Pat. No. 4,285,391, U.S. Pat. No.
5,182,109, U.S. Pat. No. 4,603,049, U.S. Pat. No. 4,545,931, U.S.
Pat. No. 4,886,663, U.S. Pat. No. 4,758,655, WO08402700, FR2525592,
and FR2532850 can be similarly used to create immunogens comprising
the peptides described herein. U.S. Pat. No. 6,043,057, U.S. Pat.
No. 5,834,246, U.S. Pat. No. 5,268,276, and EP368819, specifically
describe an expression system containing CTB (cholera toxin Beta
subunit) fused to an ST-like peptide under a foreign promoter for
use as a vaccine. The nucleic acids that encode the peptides
described herein may be use as genetic vaccines as described in
US20050260605 and WO0148018. The nucleic acid molecules may also be
used for the manufacture of a functional ribonucleic acid, wherein
the functional ribonucleic acid is selected from the group
comprising ribozymes, antisense nucleic acids and siRNA (as
described in WO05103073).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120040892A9).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120040892A9).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References